Naunyn-Schmiedeberg's Arch Pharmacol (1991) 343 (Suppl.): R1-R132 002812989100017F 1 CHARACTERIZATION OF THE MOUSE-MAS ONCOGENE R. Metzger, B. Bunnemann and K. Fuxe

R1

3

PROTO-

The mas on¢ogen encodes a protein with 7 transmembrane domains. Besides Its mitogenlc activity mas expression In mammalian cell culture confers anglotensin responsiveness. Therefore. the mas protein has been discussed as a possible anglotensln receptor. To study the regulatory mechanisms Involved In the production of the mas oncogene and to reveal Its primary structure, we have cloned the mouse mas gene, From a genomlc mouse library we were able to Isolate four independent positive clones by filter hybridization to human genomlc DNA. The Identification of these clones by restriction mapping and Southern hybridization revealed 3 clones with an extended 3'-region and 1 clone with a corresponding long 5'- promotor region. One clone designated Zmas 12 was subcloned into the Bluescript vector and sequenced using the dldeoxynucleotide method. The mouse mas gene has no Introns and shares 93.6% and 84.3% homology to the rat and human DNA sequences respectively. The alignment of the amino acid sequences shows high conservation of hydrophobic and high divergency in hydrophilic amlnoterminal domains. The tissue distribution of the mas protoo oncogene transcript In NMRI mice as measured by RNaseprotection assay shows high expression regions In the brain as well as In testis and kidney. Only weak but detectable amounts were found in heart, liver and adrenal gland. These results suggest a possible role of the mas oncogene as an Angiotensin receptor predomlnantely located In the central nervous system. The cloning of the mouse mas gene provides an Important precondition to Investigate the regulation of this receptorprotein under physiological and pathophlslologlcal conditions In further detail. German Institute for High Blood Pressure Research and Department of Pharmacology, University of Heidelberg, Im Neuenhelmer Feld 366, D-6900 Heidelberg, FRG

AUTOCRINE GROWTH FACTOR EXPRESSION IN C H E M I CALLY AND ONCOGENE-INDUCED HEPATOCARCINOGENESIS M. ll6hne, K. llirsch-Ernst, S. Zieroth, and G. F. Kahl kutocrine growth factor production may be involved in many processes of malignant growth. We investigated the expression of transforming growth factor-~ (TGF-~) and insulin-like growth factor I (IGF-I) as potent hepatotrophic mitogens in two model systems of hepatocarcinogenesis by Northern blot analysis and in situ hybridization techniques. In the rat hepatocyte resistance model the development of liver foci and diploid hepatocytes was induced by 2-acetylaminofluorene (AAF) treatment for 4 weeks after partial hepatectomy and diethyl nitrosamine injection. Transgenic mice carrying the SV40 early region and human growth hormone sequences as transgenes in inverse orientation driven by the metallothionein promoter develop hepatic nodules progressing to hepatocellular carcinomas. In AAF-treated rats and in transgenie mice significant expression of IGF-I restricted to the hepatocyte was observed. High TGF-~ expression was seen in some~-glutamyl transpeptidase-negative nodular areas in rat liver and in normal livers of. transgenic mice, but not in nodules or tumors. The scheme of carcinogen initiation followed by tumor promotion results in similar changes of growth factor expression as in oncogene-induced liver preneoplasia in the transgenic mouse system. IGF-I and TGF-~ may act as secondary oncogenic factors in malignant transformation of the hepatocyte. Institut f6r Pharmakologie und Toxikologie, Universit~t G6ttingen, Rober t-Koch-Strafe 40, D-3400 G6ttingen, F.R.G.

2

4

EARLY TRANSIENT DECREASE IN e-mye AND e-myb mRNA LEVELS IN ACLACINOMYCIN A TREATED FRIE/~D ERYTHROLEUKEMIA CELLS. A. Schaefer~ K. Lingelbach, C.A. Schmldt*, S. Schr~der and A. Dressel

PENTOSANPOLYSULFATE INHIBITS KAPOSI-FGF DEPENDENT TUMOR GROWTH A. Wellstein, G. Zugmaier, J.A. Califano III, S. Palk, and Marc E. Lippman

Chemical inducers of the differentiation are known to induce an early transient decrease in c-mye and c-myb mRNA levels in murlne e~ythroleukemla cells. Therefore, we investigated the early effect of the differentiationinducing anthracyclln antitumor antibiotic, aclaclnomyeln A, on n-myc and e-myb mRNA levels in Friend erythroleukemla cells, llne F4-6, using Northern blot analysis. Aclaelnomyein A, 200 ng/ml induced a rapid deerease in both e-mye and e-myb m~l~A levels during the first few hours after treatment I e-myc and e-myb m/LNA levels returned to prelncubatlon levels within 12 hours. Adriamyeln, 15 ng/ml, which is highly active at inhibiting cell proliferation in F4-6 cells without inereaslng differentlatlonj did not induce a similar early deerease in e-myc and e-myb mRNA levels. These results suggest that the transient decrease in c-myc and e-myb mRNA levels may be an early differentiationspeclfle effect of aclaelnomyeln A.

A neoangiogeulc response is critical for the unrestricted growth of solid tumors beyond a few millimeters in diameter. Release of adequate growth stimulating activity from tumor cells is obviously required for the stimulation of blood vessel growth and blockade of such stimulatory activity should repress tumor growth at microscopical level. To test this hypothesis and to study appropriate inhibitors, we used a cell line (SW-13) engineered to express Kaposi's sarcoma derived fibroblast growth factor (K-FGF). This cell construct (SW-13/K-FGF) grows into highlyvascularized subcutaneous tumors in animals due to paracrine and autocrine growth stimulation by secreted, authentic K-FGF and hence can serve as a defined tumor angiogenesis model (WELLSTEIN et al. (1990): Cell Growth and Diff 1:63-71).

*Immunbiochemisches Laboratorium, Universlt~tskllnlkum Rudolf Virchow, Berlin.

RESULTS: /n v/ire studies: Different polysulfates were studied for their selective inhibition of K-FGF-induced growth in in vitro assays. Human breast cancer ceils growing independently from FGF served as controls. Suramin and detransulfate showed slightly selective inhibition of FGF-dependent growth (3and 5-fold respectively), whereas heparin was inactive. The heparin analogue pentosanpolysulfate (PPS), however, was fully inhibitory for FGF-dependent growth and > 1,000-fold selective for this pathway. Inhibitory effects on SW13/K-FGF cells were fully reversible by an excess of added FGF. Furthermore, the concentration-response curve of PPS in human umbilical vein endothelial cells stimulated by K-FGF was indistinguishable from that in the SW-13/KFGF cells, animal studies: Daily i.p. injections of PPS (20 mg/kg) were tolerated well by athymic nude mice and prevented growth of subcutaneous SW-13/K-FGF tumor xenografts. In contrast, heparin treatment of the animals showed no effect on tumor growth.

Abt.f.Allgemelne Toxikologle, Universit~t Hamburg, und Fraunhofer Instltut f~r Toxlkologie und Aerosolforschung, Grindelallee 117, D-2000 Hamburg 13, F.R.G.

We conclude that PPS will be a useful tool to elucidate the significance of FGFs in vitro and in vivo and appears to be a prototype for the development of tumoricidal therapy based on growth factor targeted tumor therapy.

This work was supported schaft.

by Deutsche

Forsehungsgemeln-

V.T. Lombardi Cancer Center and Dep. of Pharmacology Georgetown University, Washington DC 20007

82

5 MORPHOLOGICAL ALTERATIONS I N D U C E D BY M I C R O I N J E C T I O N C. M o h r and M. L a u x

OF X E N O P U S O O C Y T E S OF p21 v ~ 4 ~ ~ ° ~

It has b e e n s u g g e s t e d that the small GTPbinding protein rho, a substrate of Clostridium botulinum C3 ADP-ribosyltransferase, is i n v o l v e d in the regulation of c y t o s k e l e t o n e l e m e n t s of m a m m a l i a n c e l l s (Paterson et al. (1990) J. Cell. Biol. Iii:i001) H e r e we r e p o r t a b o u t studies on the effects of the constitutively active recombinant p21 v a ~ 4 - r ~ o k in Xenopus oocytes. Microinjection of p21 rbo into oocytes induced dramatic morphological c h a n g e s w i t h r e d i s t r i b u t i o n of p i g m e n t s from the animal pole resulting in spotted oocytes. This e f f e c t of p21 rh° p r o t e i n was regulated by progesterone in a dosedependent manner, whereas prior ADPribosylation of the p21 r~° blocked its biological activity. About 30 min after microinjection, p21 ~b° was a s s o c i a t e d w i t h the p l a s m a m e m b r a n e . M e m b r a n e a s s o c i a t i o n of p21 rb° and its biological activity were i n h i b i t e d by l o v a s t a t i n . The d a t a i n d i c a t e that p21 ~b° is i n v o l v e d in the regulation cytoskeletal elements of Xenopus oocytes and s u g g e s t that membrane a t t a c h m e n t and b i o l o g i c a l a c t i v i t y d e p e n d on p o l y i s o p r e n y l a t i o n of the rho p r o t e i n . R u d o l f - B u c h h e i m - I n s t i t u t fur P h a r m a k o l o g i e , F r a n k f u r t e r Str. 107, D - 6 3 0 0 G i e ~ e n

7 TRANSCRIPTIONAL ACTIVATION BY CYCLIC AMP THROUGH CELL TYPE-/PROMOTER-DEPENDENT MECHANISMS W. Knepel, J. Chafitz, and J.F. Habener The 5'-flanking region of the rat glucagon gene contains, from nucleotide -291 to -298, a sequence, TGACGTCA, which mediates cAMP-responsiveness in several genes (cAMP-responsive element, CRE). However, because of non-permissive bases surrounding the CRE octamer, the glucagon CRE does not confer cAMP-responsiveness to an inert heterologous promoter in placental JEG cells that do not express the glucagon gene. This study shows that glucagon gene expression is activated by cAMP-dependent protein kinase A in a glucagon-expressing pancreatic islet cell line (InR1-G9). In transient transfection experiments using 350 base pairs of the 5'-flanking region of the rat glucagon gene fused to a chloramphenicol acetyltransferase reporter gene, cotransfection of an expression vector coding for the catalytic subunit of protein kinase A enhanced reporter activity about 4fold, whereas cotransfection of a mutant expression vector, encoding an inactive catalytic subunit, did not. Cyclic AMP induced glucagon gene transcription in another pancreatic islet cell line, HIT. By 5'-deletion, internal deletion and oligonucleotide cassette insertion this study shows that the glucagon CRE confers protein kinase A responsiveness to rat glucagon gene transcription. The glucagon CRE, at its native position, mediates protein kinase A induction as effectively and with the same sensitivity as the somatostatin or chorionic gonadotropin alpha CREs as shown by mutating the CRE contextual sequences within the rat glucagon gene 5'-flanking region. The in vitro DNase I footprinting assay revealed that the protein kinase A - dependent transactivator protein CREB-327, bacterially expressed, binds to the rat glucagon CRE. Various in vitro binding assays demonstrated multiple glucagon CRE-binding proteins in nuclear extracts of InR1-G9 cells. These data raise the possibility that the effect of cAMP on gene expression may differ depending on the cell type and/or linked promoter, a variability created at least in part by a multiplicity of CRE-binding proteins. Harvard University Medical School, Lab. Molecular Endocrinology, and Howard Hughes Medical Institute, Wellman 3, Boston, MA 02114, USA

6 LESION-INDUCED CHANGES IN EXPRESSION OF THE CHOLECYSTOKININ GENE IN RAT BRAIN CORTEX: STUDIES ON THE MECHANISM OF ACTION C. Olenik, A. Lais, A. Holland and D.K. Meyer Injuries to rat cortex cause an increase in the expression of the cholecystokinin and somatostatin genes in the ipsilateral hemisphere. The concentration of cholecystokinin-mRNA (CCK-mRNA) is significantly increased by 137% 24 hours after the injury. Forty-eight and 72 hours after the lesion enhancements by 190% are observed, while 6 days after cortex injury the levels have again reached control values. Cortex lesions have also been described to enhance the immunoreactivity of the immediate early gene (lEG) c-fos in rat cortex (Dragunow and Robertson, Brain Res. 455:295, 1988). This finding is of interest, since it has been shown that the c-fos protein can form complexes with other proteins coded for by IEGs and that the complex formed with c-jun protein can affect the transcription of the proenkephalin gene (Sonnenberg et al., Science 246:1622, 1989). In the present study, it was investigated whether a cortex injury also increased the levels of c-fos- and c-jun-mRNA and whether the timecourse of these changes corresponded to that of CCK-mRNA. One hour after a cortex injury, levels of c-fos-mRNA were enhanced by 184%. However, after 4 h levels were again in the control range. Surprisingly, a second enhancement (by 324%) was seen 24 h after cortex injury which had vanished 24 h later. In contrast, the levels of c-jun-mRNA did not increase until 24 h after the cortex lesion (by 45%), but stayed enhanced (by 104%) for the next 24 h. Thus, concentrations of both IEGs were increased 24 h after the injury, at a time at which also increased levels of CCK-mRNA were observed. The results are in agreement with the hypothesis that the complex formed by c-fos and c-jun protein may act as a "third messenger", and thus may be involved in the enhanced expression of the CCK-gene which is induced by cortex lesions. Department of Pharmacology, University of Freiburg, Hermann Herderstr. 5, D-7800 Freiburg, FRG.

8 I N F L U E N C E O F A D N A - C R O S S L I N K I N G A G E N T WITH AFFINITY TO T H E A N D R O G E N R E C E P T O R O N THE M O U S E M A M M A R Y T U M O R VIRUS H O R M O N E R E S P O N S E E L E M E N ~ IN M A M M A R Y C A R C I N O M A CELLS. F.J.BUSCH', A.B.C. C A T O ~, G. EISENBRAND" i- (2-Chloroethyl)-l-nitrosocarbamoyl- L- alanine-dihydrotestosterone-17-ester (CNC- ala-DHT) is an antitumor drug with affinity to the androgen receptor displaying high effectiveness in experimental m a m m a r y carcinomas. Interaction with the steroid hormone response element (HRE) was studied in T 47-D cells stably cotransfected with a construct containing m o u s e m a m m a r y tumor virus long terminal repeat s e q u e n c e s (MMTV-LTR) linked to the chloramphenicol acetyltranferase (CAT) g e n e and a chimeric construct containing the S V 40 promotor linked to the neomycin resistance gene. S e q u e n c e s within the LTR region of M M T V confer androgen inducibility to the M M T V - L T R promotor. CAT-activity and the ability of the c o m p o u n d to induce transcription at the M M T V - L T R region in Sl-nuclease mapping H e r e measured. After incubation with C N C - a l a - D H T (10"~M; 15 rain) there was a marked d e c r e a s e of expression at the M M T V L T R promotor in the CAT-assay. A simultaneous treatment with a n equimolar dose of DHT resulted only in a slight inc r e a s e in CAT-activity. The results obtained in the CAT a s s a y agree with m e a s u r e m e n t s of amounts of R N A accumulated at the M M T V - L T R promotor after treatment with the various compounds. Trans.qription at the M M T V LTR promotor w a s inhibited b y 10"~M C N C - a I ~ - D H T and there w a s no substantial competition with i0" M DHT. In contrast, incubation with a mixture of CNC-ala and DHT (10"~M; 15 rain) led to ove~induction of M M T V transcription in comparison to 10"~M DHT alone. ~_Iithese effects w e r e not o b s e r v e d at the S V 40 promotor which was u s e d as an internal control. Our results suggest a specific interaction b e t w e e n C N C - a l a - D H T and the LTR region or with the h o r m o n e receptor. Iun~versit~tKaisersl.,F~ Le~ensm~ittelchemie/Umwelttox., P.O.B.3049~ D-6750Kaiserslastern,FRG. ~Kernf0rsch.Zent.Krh,, Inst.f.Genetiku.T0xik01. v. Spaltst0ffen,P.0.B.3640, D 7500-Karlsruhe:FRG

R3 9 3' NONTRANSLATED REGION AFFECTS EXPRESSION OF/91 AND .82 SUBUNIT ISOFORMS OF THE MOUSE NA,K-PUMP IN XENOPUSOOCYTES S. GIoor1, S. Kr6ner2, and G. Schmalzing2

Expression of/91 subunit isoforms of the Na,K-pump of various species including mouse raises Na,K-pump activity in Xenopus oocytes (Schmalzing et al., submitted for publication). Since these cells contain (~ subunits which are nonfunctional because they lack a/9 subunit (Geering et al. AJP 257, C851, 1989), the rise of Na,K-pump activity is likely to result from the assembly of exogenous/91 subunits with excess (z subunits. Here we report that the number of Na,K-pumps of oocytes increased also after injection of cRNA specific for the/9 2 subunit isoform of the Na,K-pump of the mouse identified recently as adhesion molecule on glia (AMOG, Gloor et el. JCB 110, 165, 1990). In an attempt to examine which part of the/9 subunit is important for e//9 assembly, we injected cRNAs encoding deletion mutants into oocytes. Control cRNA for the ~e1 subunit containing 207 bases of the 3' end in addition to the 912 bases of the coding region and 35 bases of the 5' nontranslated end (cDNA hydrolyzed with Barn HI) raised the ouabain binding capacity 1.7fold. Unexpectedly, cRNA derived from cDNA hydrolyzed with Mae I containing the complete coding region but only 26 bases of the 3' end had almost no effect on ouabain binding, although the cRNA was correctly translated in the reticulocyte lysate system. Deletion of 3' nontranslated bases did not affect stability of the/91 subunit-specific cRNA in the oocyte as assessed by Northern blot analysis 4h after injection, cRNA comprising the complete coding region together with 64 or 158 bases of the 3' end (cDNA hydrolyzed with Spe I or Dra I, respectively) raised ouabain binding in oocytes approximately 1.3fold. The 3' nontranslated region was also important for the expression of the mouse/~2 subunit (AMOG) in oocytes, since deletion of 35 bases from the 3' end of the cRNA abolished its effect on ouabain binding. 1Neurobiologie, ETH ZSrich, Switzerland, and 2Max-Planck-lnstitut for Biophysik, Frankfurt/M, FRG.

10 UBIQUINONE ~ INCORPORATEDI~ PHOSPHATIDYLCHOLINE LIPOSOMES: C-METHOXYLAND H-NMREXPERIMMENTS L. Michaelis and K.E. Wirth Ubiquinone-]O is a lipid-soluble redox carrier located in the inner mitochondrial membrane. Its functioning is of vital importance for any aerobic energy transduction. The protonmotive ubiquinone cycle (respiratory chain) due to Mitchell makes specific predictions about the location and T~tion of this substance. C-NMR spectra of labelled ubiquinone in solution show only one methoxyl peak. When the ubiquinone is incorporated into sonicated liposomes two methoxyl peaks are resolved. As the portion of ubiquinone is increased from 4 molt to 12 molt the relative amplitude of the high f i e l d peak grows rapidly. The two peaks cannot simply result from the different methoxyl groups and must be due to two different environments for the ubiquinone. A series of experiments gave evidence for a power relationship between the areas. This suggests that the high-field component may originate from tetrameric ubiquinone aggregates. Similar results are seen with deuterium-labelled ubiquinone. Some trivalent Lanthanide ions influence the resonance of adjacent nuclei. Peak broadening may occur, and decreases with the inverse square of separation. Pseudo-contact shifts decrease with the inverse cube. Praseodymium ions bind to the phosphate groups of l i p i d molecules forming the outer leaflet of the bilayer. They shift the resonances from the choline N-methyl groups to the glycerol CH~. In addition, the ions alter the bulk magnetic susceptibility of the medium and hence shift the resonance position of all peaks. I t can be concluded that the ubiquinone ring is not accessible from the aqueous phase. There must be at least two different resonance positions for the ubiquinone methoxyl groups. Membrane curvature (sonication time) has a negligibly small effect on the resonance positions, Institute of Pharmacology, Heinrich-Heine-University, Moorenstr. 5, D-4000 D~sseldorf

11 LIPOPHILICITY MEASUREMENT BY CHROMATOGPJ%PHY K. Belsner and M. Pfeifer

REVERSED-PHASE LIQUID

He modified a recently published method using reversed phase HPLC for measuring the l i p o p h i l i c i t y of organic molecules (Minnick et a l . , J. Chromatograph. 461, 1989, 177). As mobile phases we used: A. 20 mM HOPS-buffer in H20, 0.15% (v/v) n-decylamine, pH 7.4 adjusted with 1M NaOH; B: 94.75Z (v/v) MeOH, 5Z (v/v) of a solution of 3~ diethylamine in water adjusted to pH 7.4 with HC], 0.25~ (v/v) n-octanol. Log(k'), the HPLC retention index is determined at different methano] concentrations by changing the relative amounts of A and B. Log(kw) is the extrapolated value of log(k') for I00~ water content of the eIuent. Extrapolation was allowed because the correlation between log(k') and the volume fraction of methanol was l i n e a r . Of 41 substances tested, 34 had a correlation coefficient r ! 0.999, for 7 r was > 0.99 and for 3 r exceeded 0.9. The HPLC data of a group of aIkylbenzenes, 4-alkylanilines, phenones, mioflazine, R 56865 (N-[l-[4-(4-fIuorophenoxy)-butyl].-4-piperidinyl]-N-methyl-2-benzothiazolamine), sabeluzole, propranolol, flunarizlne, lidoflazine, lidocain, cinnarizine, verapamil and diltiazem were measured and regressions calculted for log(PaDD) versus log(kw) (PaDD = pK. corrected octano]-water-'partition coefficient). A linear regression of log(PaDD) versus log(kw) resulted in a correlation coefficieht of r = 0.9B7 (n=30). The method is discussed as a substitute for the octanol-water p a r t l t i t i o n determination. Janssen Research Foundation, Raiffeisenstr. 8, D-4040 Neuss 21, F.R.G.

12 BINDING TO ACTIVATED CHARCOAL (AC) OF 8-ADRENOCEPTOR BLOCKING AGENTS (B-BL) CORRELATES WITH MOLECULAR WEIGHT, BUT NOT WITH HYDROPHOBICIYg{. D. Hellenbrecht and S. Eiermann _

_

Nonspecific cardiovascular toxicity of B-BL correlates with hydrophobicity (octanol-buffer partition coefficients at pH 7.0 = Papp, c.f. Hellenbrecht + Enenkel, Drug Res 34:980-83,1984). AC is given clinically as an adsorbent in the treatment of intoxications, but the structural requirements of drug binding to AC are not well understood. We measured the adsorbability of 14 B-BL (20 mg%) at pH 7.4 by constructing linear Langmuir isotherms for the monolayer binding capacity, cmax, either expressed as milligrams or as mmoles of drug bound per Ig of AC. - The log Papp of nadolol (-2.1), acebutolol (-0.70), propranolol (0.73) and of 11 amino- or ring-alkylated K~-compounds (c.f. Hellenbrecht et el, Eur J Pharmacol 29: 223-25, 1974) correlated linearly with the mg of cmax values of the 36 experiments with the B-BL (tab. IA), but not with the respective molar amounts of drug adsorption (tab. IB). Tab.l: Linear regression equations and statistical significance p y=cmax b= c= P6 A) mg/g AC = 11.0 log Papp + 171 0.000001 B) mmoles/g AC 0.0064 log Papp + 0.68 0.665 C) mmoles/g AC - 0.0024 mol wt + 1.27 0.000001 Instead, there was a very close negative relationship of mmoles of drug adsorption with the resp. molecular weights (mol wt) of the compounds (tab. IC). Binding isotherms at pH 4.0 and 1.2 revealed only 20-30% lower values of cmax. The conclusion from in vitro experiments is that B-BL adsorb to AC irrespectively of hydrophobicity and that pH of the medium is not limiting for drug adsorption onto AC. Supp by grant of Dr Paul + Cilli Weill foundation Dr med D Hellenbrecht, Centre Pharmacology, Univ Clin, Theodor Stern Kai 7, 6000 Frankfurt/M, F R G

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15

POTENTIAL ERRORS IN OONVENTIONAL SINGLE-DOSE PHARMAOOKINETIO STUDIES M. Weiss

DRUG MONITORING IN HUMAN BLOOD SERUM USING CONTINUOUS FLOW FAB/MS W.Lenhart*, Ch.Siethoff*, T.M.L6ffler #

The i n a d e q u a t e a s s u m p t i o n of i n s t a n t a n e o u s mixing o f d r u g i n a homogeneous b l o o d p o o l f o l l o w i n g bolus intravenous injection ( s a m p l i n g and e l i m i n a tion f r o m t h e same c o m p a r t m e n t ) l e a d s t o e r r o r s in the estimation of clearance (CL), steady-state volume of distribution ( V s s ) and mean d i s p o s i t i o n r e s i d e n c e t i m e (MDRT). U s i n g a p h y s i o l o g i c a l l y realistic recirculation model of drug disposition i t i s shown t h a t t h e s e e r r o r s a r e due t o ( i ) t h e neglect of the transit time of drug molecules through the sampling tissue (from the arterial to the peripheral venous site) and, (ii) the underestimation of the area under the concentration-time c u r v e as a consequence o f t h e n e g l e c t

of the initial concentration

impulse

(first

passage Of drug a t t h e r e f e r e n c e point). The effect of (i) l e a d s t o an o v e r e s t i m a t i o n o f MDRT (and c o n s e q u e n t l y of Vss) for drugs with relatively s h o r t MDRT and r e l a t i v e l y high tissue binding. The o v e r e s t i m a t i o n o f OL r e s u l t i n g from (ii) has t o be t a k e n i n t o a c c o u n t f o r a l l highly extracted drugs. While the error ( i ) can be e x c l u d e d by arterial s a m p l i n g , t h e r e a r e p r a c t i c a l constraints (experimental d e s i g n and d a t a e v a l u a in case (ii). tion) In conclusion, it appears difficult to determine the intrinsic v a l u e s o f CL and Vss ( d e f i n e d in terms of tissue v o l u m e s and e q u i l i b r i u m partition coefficients) by s i n g l e - d o s e studies u n l e s s OL/Q << 1 (O = c a r d i a c o u t p u t ) . The r e s u l t s are applied to experimental data available in the

literature. D e p a r t m e n t o f P h a r m a c o l o g y and T o x i c o l o g y , Martin Luther University, PSF 3 0 2 , FRG D-O 4010 H a l l e ( S a a l e ) ,

14 A NOTE ON THE CONCENTRATION KINETICS OF POLYHALOGENATED HYDROCARBON COMPOUNDS (PHC). J. Portig, P. Kraus, H.W. Vohland, G. Koss The group of PHC have exorbitantly high nonaqueous solvent/water partition coefficients which explain why up to 90 % of a dose given to a mammal end up in adipose tissue. However, far greater significance attaches to the added property of slow elimination as this results in accumulation over long time spans on continous exposure. As determined in mice (Han: NMRI) and rats (Han: Wistar) the mean life times of hexachlorobenzene (HCB) and the isomers (±)-alpha, beta and gamma of hexachlorocyclohexane (HCH) in several tissues (adipose, blood, brain, liver) bear no relation to partition coefficients and to solubilities in non-aqueous solvents but exhibit a strong negative correlation (r 0.99 or 1.O) with the solubilities in water. The finding underlines what would seem to be biologically plausible if not obvious that it is the strength of the interaction with body water rather than lipid that primarily determines the rates at which such chemicals may be redistributed, enzymically transformed, and excreted. Therefore, reference to "lipophilicity" alone is inadequate to denote the interdependence of concentration kinetics and physical constants in this class of compounds. Peak and steady-state levels of HCB and HCH in adipose tissue established by equal dosage are, in any case, inversely related to the solubilities in, e.g., olive oil and l-octanol. Inst. Toxikol. Pharmakol., Philipps-Univ., Pilgrimstein 2, D-3550 Marburg

Cylophosphamide and Isophosphamide are amongst the most important chemotherapeutic agents in clinical use. In the course of our studies on the detection and the chemical behaviour of Cyclophosphamide and, especially, Isophosphamide, we have developed an assay for the quantitative determination of the drugs in human blood serum based on interfacing on-line liquid chromatography to mass spectrometry; for this purpose we have chosen a combination of microHPLC with continuous flow FAB/MS (CFFAB/MS). A rather simple clean up scheme for Isophosphamide has been devised with 100% recovery and the extracts of the drug were injected as solutions in water onto the micro HPLC column (RP-18 column, 1.0x100 ram). The solvent was methanol/water/glycerol 50/40/10 (v/v/v) and a typical flow of 12 ~L/min was used to separate the drugs from contaminants and for delivery to the tip of the CFFAB probe. We have used a modified LC/MS probe of a Finnigan MAT 90 sectorfleld instrument to detect the analyte either in scanning mode or with selective ion recording 'for improved detection limits (c. 10 ng absolute or 500 n g / m l plasma). The calibration is linear over the concentration range of interest and a good day to day stability has been achieved. The procedure has been used successfully for monitoring the levels in blood serum of Isophosphamide administered to patients over an extended time period and we have found typical concentrations of 50 I~g/mL. In this paper, the procedure will be discussed in detail and the quantitative aspects as well as some typical results will be shown.

*IS-AS Institut f~r Spektrochemie, Bunsen-Kirchhoff-Str.ll 4600 Dortmund 1 #St<ische I~iniken Dortmund, Beurhausstr. 40, 4600 Dortmund 1

16 DETERMINATION OF CYCLOSPORIN A (CSA) CONCENTRATION I N TISSUE AND BLOOD AFTER TOPICAL A P P L I C A T I O N INTO R A B B I T EYE G. H O b n e r * and A. K r a u s e CSA l s a p o t e n t l m m u n o s u p p r e s s l v e d r u g used f o r inhibition of graft rejection in organ transplantations and r e c e n t l y also in ophthalmology especially for the high risk keratoplastics. To o v e r c o m e t h e known d r u g s s y s t e m i c t o x i c effects, e.g. nephrotoxicity, we h a v e i n v e s t i g a t e d the topical ocular application o f CSA. We h a v e studied the penetration o f OSA i n t o c o r n e a , conjunctiva, iris, a q u e o u s humor and b l o o d o f of CSA rabbit after droping a 2 % oily solution in rabbit eyes for 4 days. In addition the c o r n e a o f one g r o u p o f a n i m a l s w e r e t r e p a n e d . The c o n t r o l g r o u p was t r e a t e d with oil without CSA. The d i f f e r e n c e s i n t h e CSA l e v e l between the control and t h e t r e a t e d group are significant for tissue and a q u e o u s h u m o r , b u t n o t f o r b l o o d . The h i g h e s t c o n c e n t r a t i o n s were found in cornea and c o n j u n c t i v a ~ whereas the concentrations in iris and a q u e o u s humor w e r e l o w e r . A c o m p a r i son o f t r e p a n e d and u n t r e p a n e d e y e s r e v e a i s a h i g h e r CSA r e s o r p t i o n in the trepaned eyes. It was f o u n d t h a t no m e t a b o l i t e s w e r e f o r m e d . The observed tissue concentrations agree with therapeutical drug level. After topical applic a t i o n no s i g n i f i c a n t i n c r e a s e o f t h e CSA b l o o d level c o u l d be f o u n d i n d i c a t i n g a low systemic drug distribution. T h a t means t h a t t o p i c a l applic a t i o n may r e d u c e t h e t o x i c effects, whereas the local therapeutic effect remains. Division of Clinical Pharmacology, Department of P h a r m a c o l o g y and T o x i c o l o g y * and D e p a r t m e n t o f Ophthalmology, Martin Luther University, Len£nallee, D-O 4020 H a l l e

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17 EFFECT AND INTERACTION OF pH AND SUBSTANCES ON EPINEPHRINE ABSORPTION. F. Kehlbach, E. Knoll-K6hler

LOCAL

ANESTHETIC

In local anesthetic solutions epinephrine retards the systemic uptake of local anesthetic substances. Thus, the local anesthetic effect as well as the hemostatic reactions are improved and systemically the therapeutic index of the drugs hy decreasing Cmax without changing Tma x. In order to evaluate an interference of local anesthetics with the absorption kinetics of epinephrine we studied the effects of lidocaine on the absorption kinetics of epinephrine after intraoral submucosal injections in arterial blood of adrenalectomized rats. The results of these studies showed that coinjection of 2% lidocaine hydrochloride with i:I00.000 epinephrine (pH 3.5) prolonged peak time of epinephrine threefold without changing its peak level and elimination kinetics. After injection the local tissue-pH decreased and pH-values of 6.1 were measured. Basal values of 7.36 were obtained after 30 - 60 min. The onset was influenced by the individual buffer capacity of the tissue. This decrease in tissue-pH reduced the activity of COMT, which was further depressed by local anesthetic substances. The inhibition potency varied greatly in dependence on the physicochemical characteristics of the various local anesthetic drugs. From these results it can be concluded that at a constant dose level the local vasoconstrictive efficacy of epinephrine but also its systemic bioavailability - which determines untoward reaction - is mainly dependent on the pH of the local anesthetic solutions and the chemical structure of the local anesthetic substance. It is modified by the buffer capacity of the tissue into which the solutions are injected.

STEREOSELECTIVE HPLC METHOD FOR KETAMINE I T S M A J O R M E T A B O L I T E IN hl/MAN P L A S M A G. G e i s s l i n g e r , S. M e n z e l ; S o g l o w e k , W. H e r i n g * a n d H.-D. K a m p

AND

K e t a m i n e (KET) is a s h o r t - a c t i n g p a r e n t e r a l a n e s t h e t i c a g e n t t h a t has b e e n in c l i n i c a l u s e - as r a c e m a t e - for a b o u t 20 years. In c o n t r a s t to its k n o w n c l i n i c a l e f f e c t s t h e r e is only little known about the stereoselective pharmacokinetic b e h a v i o r of the KET enantiomers. We, therefore, developed a stereospecific HPLC method that utilizes a chiral column (~i acid g l y c o p r o t e i n , Grom, H e r r e n b e r g , FRG) a n d U V detection (215 nm) for the specific and sensitive determination of the KET enantiomers and its major metabolite n o r k e t a m i n e . P l a s m a s a m p l e s (i.000 ml) w e r e a l c a l i z e d (0.i00 ml 3 M NaOH) f o l l o w e d b y d o u b l e e x t r a c t i o n i n t o 5.00 ml of i c e - c o o l e d c y c l o h e x a n e b y a g i t a t i n g for 15 min. A f t e r centrifugation the c o m b i n e d o r g a n i c layers w e r e e v a p o r a t e d to d r y n e s s . The r e s i d u e w a s redissolved in an appropriate amount of mobile phase, which consisted of 2propanol/O.03 M phosphate buffer pH 7 ( 2 . 5 / 9 7 . 5 v/v). T h e u s u a l f l o w r a t e w a s 0.6 m l / m i n . T h e U V r e s p o n s e w a s l i n e a r (r>0.995) for concentrations of KET and NORKET e n a n t i o m e r s f r o m 40 n g / m l u p t o i000 ng/ml. The utility of the methodology was established b y a n a l y s i s of p l a s m a s a m p l e s from patients receiving single doses of r a c e m i c o r S-KET. *

Address: Freie Universit~t, Institut f~r Pharmakologie, Thielallee 69 - 73, W-1000 Berlin 33, Germany

Dept. of P h a r m a c o l o g y / A n e s t h e s i o l o g y , U n i v e r s i t y of E r l a n g e n , U n i v e r s i t a e t s s t r . D-8520 Erlangen, FRG

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PULMONARY UPTAKE OF PRILOCAINE, BUPIVACAINE H. Foth, D. Kietzmann , W.P. Geng, M. T. Schr6der**~and J.P. Hering ** •

*

MEPIVACAINE Ebke,

H.C.

AND

Michaelis,

Due to its unique position in the cardiovascular system the lung may play an important role as a filter between the venous and the arterial site, thereby possibly lowering the risk of local anaesthetic drug (LA)-induced cardiac or central nervous toxicity. Prilocalne, mepivacalne and bupivacaine are widely used LA for peridural anaesthesia (PDA). In patients undergoing general anaesthesia including PDA, pnlmonary-arterial and arterial plasma samples were drawn simultaneously in order to estimate the pulmonary extraction efficiency for these LA. Pulmonary elimination was also estimated in isolated perfused rat lung and liver and in anaesthetized sheep• Within 6-30 min after peridural injection peak concentrations in the pulmonaryarterial plasma of 1.6 __+0.5/Zg/ml prilocaine (200 rag; n=5; median 1.7/~g/ml), 2.2 __+0.3/~g/ml mepivacalne (200 rag; n= 11; median 2.2/~g/ml) and 0.9 + 0.1 /~g/ml bupivacaine (50 rag; n = 16; median 0.7 ,gg/ml) are observed. A remarkable uptake of LA during lung passage is found for prilocaine (median 2 to 15 rain = 37 to 14 %) and less for mepivacalne (median = 2 to 15 rain = 16 to 3 %) as well as bupivacaine (median 1.5 to 16 rain = 12 to 3 %). Similar results are obtained for the uptake of bupivacalne by the lung of sheep. In isolated rat lung the metabolic clearance of these LA, however, is relatively low: 0.5 ml/min for prilocaine, 0.2 ml/min for mepivacaine and 0.3 ml/min for bupivacaine. The mean extraction ratios of prilocaine (5%), mepivacaine (M, 2%) and bupivacalne (B, 2%) in rat lung are much lower than in liver (67 % B and 74 % M). The pulmonary first-pass elimination must be assumed to represent mainly uptake and binding to the tissue. The peak concentrations of LA in arterial plasma are not substantially lower than in pulmonary-arterial plasma and, therefore, the risk of toxicity remains unaffected, The rate of uptake by the cardiac tissue may be another important factor for the risk of cardiotoxicity for these LA. This effect is presently under investigation. Zentrum Pharmakologie und Toxikologie, * Zentrum Angtsthesiologie, Rettungsund Intensivmedizin und ** Zentrum Physiologic und Pathophysiolome Universitfit G6ttingen, Robert-Koch-Str. 40 und *'~' Humboldtallee ~23, 3710~ G6ttingen

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BINDING OF THIOPENTAL IN TISSUE-HOMOGENATES OF THE RAT IN THE PRESENCEOF HALOTHANE, ENFLURANEOR I$OFLURANE U. BOch* and J. Ch. Isenberg Previously i t was reported t h a t the thiopental concentrat i o n in the vessel-rich tissues of the rat was increased i f simultaneously an anesthesia with halothane (H) was performed (Naunyn-Schmiedeberg's Arch 1988:337:R6,No22). In rats anesthetized with enflurane (E) or isoflurane ( I ) the same phenomenon was observed, i . e . thiopental was accumulated in the well perfused tissues as compared to the control (rats without H-, E- or I-anesthesia). I t was supposed that hemodynamic effects of H, E and I cause a reduced regional blood flow and thereby an accumulation of thiopental in the tissues. Question arose whether beside these hemodynamic effects also the binding of t h i o pental in the tissue was increased in the presence of H, E or I . Thiopental-binding (O.4. mmol.l - I ) in the t i s sue-homogenate of rats ( l i v e r , brain, heart, kidney, lung, spleen and skeletal muscle) was studied by e q u i l i brium d i a l y s i s . Percentage of thiopental bound was relat i v e l y low in the homogenate of brain, lung, spleen and skeletal muscle (14-19%); i t was much higher in that of l i v e r , heart and kidney (24-27%). In the presence of H (11.8 mmol.l - I ) the %-binding of thiopental in a l l t i s sues investigated was s i g n i f i c a n t l y increased at least to a factor of 1.4 (spleen) and maximally of 2.4 (brain). The same finding of an increased thiopental-binding in the tissue-homogenate was found i f 10.3 mmol.l -I E (except skeletal muscle) or 10.2 mmol.l -I I (except kidney, spleen and skeletal muscle) were present. However, the binding-increase was s i g n i f i c a n t l y lower in the presence of the halogenated ethers as compared with H. K l i n i k f ~ r An~sthesioloyie und Intensivmedizin*, I n s t i t u t f o r Pharmakoloyie und Toxikologie, Universit~t des Saarlandes, W-6650 Hombur9

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PHARMACOKINETIC BEHAVIOUR OF ANTIBACTERIAL AGENTS IN PANCREATIC TISSUE AND JUICE B. Drewelowx, K. Kochxx, A.-K. RiethlingX and S. Liebexxx

PHARMACOKINETICS OF DEXTRAN-BOUND HIRUBIN IN RATS AND RABBITS M. Richter and P. Walsmann

Infectious ccmplications account for 80 percent of deaths from acute pancreatitis but the effectiveness of antibiotics hasn't varified yet. Until clinical studies are ~ le the choice of antibiotics must be based upon indirect criteria, especially the ability of the antibiotics to effectively penetrate into pancreatic tissue and juice (BRADLEY, E.L.; Am.J.Surg. 158 (1989) 472). We studied the l~armacokinetic behaviour of 12 antibacterial drugs in seru,, pancreatic juice and tissue after single intravenous administration in dogs. The pancreatic juice peak concentrations of all substances were measured between 20 a ~ 40minutes after application. Than the concentrations declined with the sa~e half live as in serum (except ciprofloxacin). Large differences in pancreatic juice penetration were observed. The B-lactam-antibiotics ampicillin, azlocillin, cefotiam, cefurox/m and moxalactam as well as the aminoglycosides gentamicin and amikacin showed only low penetration rates (2-10% of corresponding seru~ levels). 20% of serum concentration was measured in case of mezlocillin. In contrast chlora~phenicol, metronidazol, ofloxacin and ciprofloxacin reached in pancreatic juice levels between 50 and 162% of the serun concentrations. The pancreatic tissue penetration rates estimated 50 minutes after administration w~re nearly parallel to juice ~8rmeation but about 3,6 times higher than in pancreatic ~/~e. For the judgement of possible clinical efficiency of all tested drugs the chemotherapeutic ratios ware calculated (juice or tissue level by MIC of cfmmon pathogens in pancreatitis ). In the pharmacokinetic point of view only mezlocillin, ciprofloxacin, ofloxacin and cefoti~ seem to be suitable in the therapy of infectious complications in ~ te pancreatits. • . x . • Inst~ltute of Pharmacology and T~oxlcology , Cllnlcs o f Surgery~ and Internal M e d i c i n e ~ of the University of Rostock, Leninallee 70, ROSTOC~, 0-2500, FRG.

RECOMBINANT

Recombinant hirudin was coupled via its lysine residues to oxidized dextran of mean molecular masses of 40,000 and 70,000. The azomethinebonds were reduced to amine-bonds. The hirudindextran conjugates, isolated by chromatography, inhibited thrombin with Ki values similar to that of free hirudin. The dextran-hirudin conjugates were compared to uncoupled hirudin with respect to the pharmacokinetic behaviour following i.v. administration in rats and rabbits. The hirudin conjugates are initially distributed 2 to 4 times more slowly than ~ree hirudin~ and show significantly smaller distribution volumes in steady-state. This reduction of distribution volume may be interpreted as a possible restriction of hirudin conjugates to the circulating blood. As an essential result of the coupling of hirudin to dextran it can be seen that these products are much more slowly eliminated after reaching steady-state than free hirudin, i.e., elimination half-lives are 6 to 8 times longer. Moreover it is evident that the areas under the curves (AUC) ~ithin the time interval of 6 h after administration are ~ to l~ times larger in comparison to free hirudin. Institut f~r Pharmakologie und Toxikologie, Nedizinische Akademie Erfurt~ Nordh~user Sir. O-~OlO, FR6

74,

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ALBUMIN BINDING DOES NOT REDUCE ITRACONAZOLE ACTIVITY M. Sch~fer-Korting, R. Peuser, A. Lukacs, and H.C. Korting

PHARMACOKINETICS OF THE NOVEL RECOMBINANT PLASMINOGEN A C T I V A T O R BM 06.022 IN RATS U. Martin, J. K6hler, G. Sponer, and K. Strein

Serum protein binding up to 85% is considered to have only minor clinical impact. In case of binding exceeding 95%, however, drug activity may be reduced considerably. To evaluate the influence of protein binding on the antifungal effect of itraconazole (f~=0.999) Candida albicans was exposed in vitro to continously changing drug concentrations. The culture medium was free of protein or contained 4% human serum albumin (HSA). Using the Grasso technique, itraconazole serum levels (0-12 h) following a 200 mg oral dose (Sch~fer-Korting et al., Jo Am. Acad. Dermatol. 22, 211, 1990) were simulated. In comparison, antifungal effects of itraconazole concentrations corresponding to free drug in serum were evaluated, as well as ketoconazole (f~=0.99; 200 mg) activity. Drug free cultures served as control. Exposing C. albicans to the azoles reduced the number of colony forming units (CFU)/ml as compared to control. Due to total itraconazole ig CFU/ml was lower by 0.82±0.20 (0% HSA) and 0.65±0.23 (4% HSA). Growth, however, was not inhibited by free drug levels (0% HSA). Free ketoconazole was about as active as total drug (ig CFU/ml 1.28 and 1.45; 0% HSA). The results show a considerable contribution of albumin-bound itraconazole to antifungal activity. Infact free intraconazole levels are insufficient which contrasts ketoconazole. Pharmakolog. Institut f. Naturwiss., Universit~t Frankfurt, Theodor-Stern-Kai 7, D-6000 Frankfurt/M., and Dermatol. Klinik u. Poliklinik der Universit~t M~nchen

The novel recombinant plasminogen activator (rPA) BM 06.022 consists of the kringle 2 and protease domains of human t-PA and is unglycosylated due to its expression in E. coli cells. The pharmacokinetic properties of rPA were compared with those of alteplase in anesthetized, male rats following i.v. bolus injection over 1 min of 200 kU/kg rPA (n = 6) or alteplase (n = 7). Plasma concentrations of functionally active rPA or alteplase were measured in an indirect spectrophotometric assay using Gluplasminogen, CNBr split products of human fibrinogen, and Tos-Gly-Pro-Lys-4NA as chromogenic substrate. Euglobulin precipitation of the citrated plasma samples was performed prior to the measurement. Cma x was 2322 ± 335 IU/ml for rPA and 2175 ± 438 IU/ml for alteplase (mean ~ SEM). AUC0-1 5 h was 455 + 40 IU.h.ml -I for rPA and 90 + 15" IU-h-ml -I ~ o r alteplase. Correspondingly? Clto t was 7.6 + 0.6 ml'min-l.kg -I for rPA and 43 ± 7.8 ml-m~n-l.kg -I for alteplase. Plasma disappearance of rPA in 4/6 rats occurred with a tl/2~ of 4.3 ± 0.9 min, accounting for 49.6 ± 13.4 % of the total AUC followed by a tl/2~ of 12.3 ± 1.7 mi D . In the remaining 2 rat~, rPA had a tl/2 of 9.7 min. Alteplase disappeared + from plasma in all rats with a tl/2~ of 1.0 _ 0.i min accounting for 77.3 ± 4.9 % of the total AUC, followed by a tl/2B of 14.7 ± 3 min. We conclude that at equa~ ~oses rPA is cleared in rats about 5-times more slowly from plasma than is alteplase. Dept. GmbH,

of Pharmacology, Boehringer Mannheim Sandhofer Stra6e 116, D-6800 Mannheim 31

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INFLUENCE OF Q~II~IDINE ON PFa~RMACOKINETICS AND METABOLISM OF THE MAO-A INHIBITOR BROFi%R(~INE: ROLE OF THE DEBRISOQUINE-SPARTEINE CYTOCHRGME P450

IS P H E N A C E T I N O - D E E T H Y L A S E - A C T I V I T Y N E E D E D A S FUNCTIONAL PARAMETER FOR P450IA2 IN HUMAN

N. Feifel, K. Kucher, E. Schmidt, K.H. Antonin and P.R. Bieck The cytochrome P450IID6 (P450db-l) isozyme is responsible for the genetic debrisoquine - sparteine polymorphism of drug oxidation in humans characterized by the phenotypes 'extensive' (EM) and 'poor metabolizer' (PM). To investigate the relationship between brofaromine oxidation and the activity of this isozym we performed a study in 4 PMs and 7 EMs. One h before intake of i0 mg debrisoquine sulfate (D) or 75 mg brofaromine hydrochloride (B) i00 mg of the selective and potent (Ki = 50 nM) P450IID6 inhibitor quinidine sulfate was given. The metabolic ratio (MRD) of D was determined. B and its major metabolite O-desmethylbrofaromine (DB) were measured in plasma and urine by a capillary GC-ECD method. In the EM group the following pharmacokinetic changes occurred: during quinidine inhibition 6 out of 7 subjects converted to 'phenocopies' of the PM phenotype. The total clearence of B decreased from 192 (± 85) to 81 (± 55) ml/ min and partial metabolic clearence from 4832 (± 2278) to 1396 (± 1056) ml/min; t~ of B increased from 10.9 to 24.4 hours. The AUC(o_I2h) of DB changed from 7657 (± 2051) to 2515 (±890) [(nmol/L).h]. The Ae(o_72h) of DB was 98.7 (± 22.1) pmol before and 67.6 (±5.2) pmol after quinidine treatment, corresponding to 45.6 and 31.2 % of dose. The effects in PMs were neglectible. Pharmacokinetics in EMs with quinidine rather mimics precisely the conditions in PMs without quinidine treatment. Humanpharmakologisches Institut CIBA-GEIGY GmbH Waldh6rnlestra~e 22 W-7400 TObingen

26 VARIOUS SEBACEOUS TISSUES EXPRESS A NOVEL CYTOCHROME P450UB GENE: ISOLATION AND CHARACTERIZATION OF THE FULL LENGTH cDNA FOR EXPRESSION IN CELL CULTURE. T. Friedberg, F. Oesch, and M.A.G. Grassow. Cytochromes P450 metabolize a wide variety of endogenous and exogenous compounds. The genes coding for cytochromes P450 can be classified into various gene families according to their sequence homology (eg. P450IA, P450IIB, P450IIIA etc.). Most of these gene families contain several closely related members. The P450IIB family contains members which encode the major phenobarbital inducible hepatic cytochromes P450. Within the P450IIB family we have detected the expression of a novel P450IIB gene by oligonucleotide hybridization to mRNA of the preputial gland which is a large sebaceous gland beeing highly active in the metabolism of steroids. The expression of this protein was age and sex independent. Moreover immunohistochemistry revealed that the corresponding P450IIB protein is also localized in the meibomian gland of the eyelids which secretes a protective lipid into the tear film. The P450IIB protein was also found to be expressed in the hair shafts. The full-length cDNA of this P450IIB protein was isolated and sequenced. This sequence revealed that the cDNA should encode a functionally active cytochrome P450. We are currently in the process of expressing this cDNA in cell cultures to define the role of this novel cytochrome P450 in the metabolism of endogenous and exogenous compounds. Institute of Toxicology, University of Mainz, Obere Zahlbacherstr.67, DW 65 Mainz, Germany.

LIVER? A. Orzechowski High atTmity phenacetin O-deethylase (POD) activity, inducible b y 3-methylcholanthrene-type inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, is selectively catalyzed by cytochrome P450IA2 in rat and human liver. P450IA2 is involved in the metabolism of a variety of xenobiotics, such as N3-demethylation of caffeine and the activation of carcinogenic aromatic and heterocyclic amines. POD activity was measured in microsomes from 11 human liver samples using the mass-spectrometric method developed by Murray and Boobis (Biomed. Mass Spectrom. 13, 91-93, 1986). In this assay the reaction product paracetamol is quantified as a bistrifluoromethylbenzoyl derivative using negative chemical ionization mass spectrometric with a detection limit of about 1 pg paracetamol. POD activities were found to be in the range of 31-263 pmol/min/mg. For comparison 7-ethoxyresorufm O-deethylase activity (EROD) was measured in the same microsomal preparations. An excellent correlation (r = 0.99) with POD activity was obtained. However, no correlation was found with 7-pentoxyresbrufin O-dealkylase activity, which was found to be induced in livers from two individuals treated with phenobarbital-type inducers. Since P450IA1, catalyzing EROD in rodent liver, is not detectable in human liver samples, the results strongly suggest that both hepatic POD and EROD activities are catalyzed by human P450IA2. Consequently EROD activity may be used instead of POD activity as a selective parameter for P450IA2 with human liver microsomes. Institute of Toxicology, University of Tiibingen, Wilhelmstrasse 56, D-7400 Tiibingen, Germany

28 MODULATION OF THE COUMARIN 7-HYDROXYLASE BY N-CONTAINING HETEROAROMATIC COMPOUNDS AND COBALT

B.Hahnemann, P.Salonp~i, J.M~enp~i~i, M.Pasanen. P.Honkakoski, M.A.Lang, 0.Pelkonen and K.J.Netter The heteroaromatic compounds pyrazole, pyrazine and aminotriazole and the heavy metal cobalt selectively increase the amount of immunodetectable cytochrome P450IIA3-type II (coumarin 7-hydroxylase) in mice. As a consequence the metabolism of coumarin is increased. In contrast O-dealkylations of ethoxyresorufin and pentoxyresorufin or N-demethylations of ethylmorphine and benzphethamine remain unaffected or are decreased. This is true also for the CypIIA3-type I (steroid 15u-hydroxylase) in mice pretreated respectively. In this study we used a cDNA-probe to detect mRNA of the CypIIA3 gene after pretreatment of mice with cobalt, pyrazole and l~henobarbital (PB) in comparison to control mice. Northern-Blot analysis is able to detect an increase at the mRNA-level (2.1 kb) in DBA/2 (D2) and C57BL/6 (B6) mice. This increase is dependent on the dose of compound injected and on the time elapsed after pretreatment. The respones are much more prominent in B6 than in D2 mice, but obviously the developing of the mRNA is slower in B6 than in D2 mice. Preliminary nuclear transcription experiments suggest that the increase of the amount of mRNA is not due to an increased transcription rate of the Cyp2A3 gene in mice after pretreatment. In conclusion: (a) Pyrazole and cobalt increase the expression of the Cyp2A3-type II system by mechanisms that may differ from those involved in PB induction. (b) Since only CypIIA3-type II is preferentially affected as compared to type I both genes must be differently regulated in spite of their high homology in the amino acid sequence of the resulting protein. On the basis of these experimental findings we recommend to choose different assignments for the steroid 15u-hydroxylase and the coumarin 7-hydroxylase. Grant: Finnish Academy Medical Research Council Departments Pharm. & Tox., Lahnberge, D-3550 Marburg, FRG, Kajaanintie 52D, SF-90220 Oulu and SF-70211 Kuopio,Finland

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PATHWAY CONTROL OF BIOTRANSFORMATION BY CYTOCl4ROME P-450 ENZYMES 3. B l a n c k , O. R i s t a u , A . A . Z h u k o v , A . I . A r c h a k o v , H. R e i n , and K. R u c k p a u l

MOLECULAR BASIS OF DRUG METABOLISM INTERACTIONS: A STRUCTURAL MODEL FOR INHIBITORS OF DEBRISOQUINE HYDROXYLASE G. Strobl and T. Wolff

C y t o c h r o m e s P-450 c a t a l y s e the b i o t r a n s f o r m a t ~ o n o f s u b s t r a t e s by o x i d a t i o n via reductive oxygen splitting. T h i s enzyme a c t i v i t y ~s r e s t r i c t e d by oxidatic pathways resulting i n 0~, H~O~ and H20 generation. The p a t h w a y c o n t r o l 5y s B b ~ t r a t e s , c y t o c h r o m e s P-450 and c y t o c h r o m e b~ has been i n v e s t i g a t e d on r a b b i t l i v e r m i c r o s o ~ e s and a s e ries of benzphetamine analogues. The enzyme is r e g u l a t e d by the c o n f o r m a t i o n a l a c t i v i t y o f the s u b s t r a t a , which causes a high spin s h i f t o f the f e r r i c P-450, r e s u l t i n g in a c c e l e r a t e d e l e c t r o n t r a n s f e r and accumulation o f the f e r r o u s dioxygen complex. The l a t t e r i n termediate is s u b s t r a t a - s t a b i l i z e d towards decay by O~ and H.O. g e n e r a t i o n , cytochrome b5 d i s f a vours t h a t ~a{hway f u r t h e r by p r o v i d i n g the second e l e c t r o n f o r oxygen a c t i v a t i o n f a v o u r a b l y . The s u b s t r a t a - i n d u c e d c o n f o r m a t i o n a l change o f the enzyme, on the o t h e r hand, c o n t r o l s the branching o f the 2 - e l e c t r o n reduced i r o n - o x o i n t e r m e d i a t e i n t o s u b s t r a t a conversion and o x i d a t i c H20 f o r m a t i o n in f a v o u r o f the former patl~way. Perfluorinated uncoupling substrates underlie

Debrisoquine 4-hydroxylase is a typical activity of cytochrome P-450IIDI, a human liver enzyme catalyzing the oxidation of more than 20 drugs. The antiarrhythmic drug, quinidine, strongly inhibits the activity of this enzyme in vitro [Inaba et al. 1985, Drug Metab. Disp. 13, 443] and markedly prolongs the half life time of sev~-~al of those drugs in rive. The stereoisomer, quinine, was much less effective as inhibitor [Steiner et al. 1987, Clin. Pharmacol. Ther. 43, 575]. To explore the specificity of cytochrome P-450IIDI toward these and other inhibitors on a molecular level, we have designed a structural model by computer aided molecular modeling. First, a preliminary model for competitive inhibitors was developed. Ajmalicine, which is the strongest inhibitor in vitro and has a rigid structure was used as a template to superpose Other strongly inhibitory drugs in vitro, such as quinidine, trifluperidole, chlorpromazine, prodipine, and lobeline. The model was refined by testing more than 50 derivatives of ajmalicine, quinidine, and 4,4-diphenyl-piperidine, for their potency to inhibit bufuralol-l'-hydroxylation in human liver microsomes, a typical reaction of cytochrome P-450 IIDI, and by fitting energy-minimized conformations of these compounds into the model. Common structural elements of strong inhibitors were: (i) an extended hydrophobic region, (2) a positively charged basic nitrogen atom above this region and (3) two groups with a negative potential and the ability to accept hydrogen bonds among this region in a distance of 5 or 7 A from the nitrogen atom. This 3-dimensional structural model offers a rationale to identify drugs that can act as inhibitors of cytochrome P-450 IIDI dependent reactions in vitro and in rive.

same c o n t r o l , w h i c h f i n a l l y r e s u l t s in an inc r e a s e d H_O f o r m a t i o n . The l e a k i 6 e s s of the m o n o o x y g e n a t i c p a t h w a y to g e n e r a t e H 2 0 _ is thus i n d e p e n d e n t of s u b s t r a t e -

activation o~ t h e enzyme and does n o t oxygen-mediated tissue toxicity.

increase

Department of Biocatalysis, Institute cular Biology, Robert-R6ssle-Str. 10 0-1115 Berlin, Germany

of Mole-

3O INFLUENCE OF NOCLOPROST ON OXiGENASES OF RAT AND MAN

SOME HEPATIC MONOG.WSIfle, H.Bleyer

The

cytoprotective prostaglandin derivative (NP) w a s found active in g a s t r i c u l c e r d i s e a s e s . I n f o r m a t i o n on its e f f e c t on the h e p a t i c m o n o o x y g e n a s e s y s t e m has b e e n g a t h e r e d in a s t u d y w i t h hu/nan a n d r a t liver microsomes. G r o u p s of 18 m a l e W i s t a r r a t s (BW 2 2 5 - 2 3 5 g) were pretreated o r a l l y w i t h 0.i m g / k g NP, 5 ml/kg saline for 7 days, and 80 mg/kg p h e n o b a r b i t o n e (PB) i.p. for 3 days. M i c r o s o m e s were prepared by differential centrifugation. Further, liver microsomes from man (kidney donors) and r a t s w e r e i n c u b a t e d w i t h i0 ng/ml NP. The f o l l o w i n g p a r a m e t e r s w e r e d e t e r m i n e d : Cyt.-450, a n i l i n h y d r o x y l a s e (AH), aminopyrineN-demethylase (AD), e t h y l m o r p h i n e - N - d e m e t h y l a s e (EqV~ND), 7-ethoxyresoruf~n-O-deethylase (EROD), 7-pentoxyresorufin-O-depentylase (P~OD), and 7-ethoxycoumarin-O-deethylase (ECOD). NP d i d not b i n d to c y t . P - 4 5 0 n e i t h e r of rat nor of h u m a n l i v e r m i c r o s o m e s . P r e t r e a t m e n t of rats with PB increased cyt.P-450 content and the a c t i v i t i e s of AH, AD, EMND, PROD, and ECOD, but not of EROD. NP h a d no s i g n i f i c a n t e f f e c t s on all the parameters measured, b o t h a f t e r s u b a c u t e p r e t r e a t m e n t and a f t e r ~n vitro i n c u b a t i o n of rat and h u m a n liver microsomes.

nocloprost

Institut

f~r U m w e l t - und P r i v e n t i v m e d i z i n 38 0 - 2 2 0 0 G r e i f s w a l d , FRG

R.-Petershagen-Allee

GSF-Forschungszentrum f. Umwelt u. Gesundheit, Institut f. Toxikologie, D-8042 Neuherberg

32 CYTOCtlRONIE P-450 MEDIATED 116-14YDROXYLATION OF STEROID HORMONES G.R. 3 ~ n i g , ~d. M ~ l l e r - F r o h n e , H. R i e m e r , D. P £ e i l , N~. H e n n i n g C y t o c h r o m e s P-450 ( P - 4 5 0 ) i n t h e a d r e n a l c o r t e x catalyze different hydroxylation reactions inc l u d i n g t h e c l e a v a g e o f a C-C bond i n t h e b i o s y n t h e s i s o f s t e r o i d h o r m o n e s . The f o r m a t i o n o f g l u cocorticoids as w e l l as m i n e r a l o c o r t l c o i d s involves the llB-hydroxylation of the steroid s k e l e t o n as an e s s e n t i a l s t e p . An u n d e r s t a n d i n g o f t h e r e a c t i o n mechanism o f tl~ese b i o t r a n s f o r mations requires information on t h e s t r u c t u r a l basis of the substrata-, r e g i o - and s t e r e o s p e c i ficity. Due t o t h e r a t h e r c o m p l i c a t e d c o m p o s i tion of the mitochondrial adrenal system a microbial iiB-hydroxylating s y s t e m p r o v e d as a p p r o p r i a t e model f o r c o m p a r a t i v e s t u d i e s e s p e c i a l l y considering genetic engineering. The f u n g u s C o c h l i o b o l u s lunatus hydroxylates steroids in liB-position. Fractionation of ce11free extracts revealed llB-Iwdroxylase activity in the microsomal fraction thus indicating a memb r a n e - b o u n d s y s t e m . The s t e r o i d - i n d u c i b l e activit y i s NADPH-dependent. The l i n e a r c o r r e l a t i o n of t h e P-450 c o n t e n t w i t h t h e 1 1 ~ - h y d r o x y l a s e a c t i v i t y and i t s i n h i b i t i o n by P-450 s p e c i f i c inhibitots characterize t h e f u n g a l b y d r o x y l a s e as a P - 4 5 0 - d e p e n d e n t m o n o o x y g e n a s e s y s t e m . The P-450 w i t h a m o l e c u l a r mass o f 60 kD was p u r i f i e d 70f o l d by c h r o m a t o g r a p h i c s t e p s . Deoxycorticosterone and d e o x y c o r t i s o l are hydrox y l a t e d by t h e f u n g a l enzyme t o n e a r l y t h e same e x t e n t as by t h e a d r e n a l o n e . P r o g e s t e r o n e and 1 7 ~ - h y d r o x y p r o g e s t e r o n e are transformed I O - f o l d b e t t e r than by the adrenal P-450 116 thus i n d i c a t i n g a broader s u b s t r a t a s p e c i f i c i t y . Department o f B i o c a t a l y s i s , C e n t r a l I n s t i t u t e o f Molecular B i o l o g y , D-0-1115 B e r l i n - B u c h , FRG

R9 33 PRINCIPAL PATHWAYSOF BIOTRANSFORMATIONOF THE PROGESTAGEN DIENOGEST G, Hobe, Renate Sch~n, Petra R i t t e r , H.G. Hillesheim DIENOGEST is the p~ogestagen component of the oral contraceptive CERTOSTAT . Its chemical structure is characterizedPy a 4,9-diene-3-oxo-system in the steroid skeleton and a 17~-cyanomethyl side chain.

.~c~c~ OH

0

The t r i t i u m l a b e l l e d compound has been administered to animal species l i k e rabbit, rat and beagle-dog. DIENOGEST metabolites were excreted predom~natly in the urine, also in the rat! Urinary metabolites have been characterized by UV, IR and mass spectrometry, and by comparison with authentic reference compounds. The following biotransformation reactions could be demonstrated: Hydroxylation in d i f f e r e n t positions of the steroid molecule, among these the 11-position; aromatization of ring A; reduction of the 3-oxo-group; introduction of 2 and 4 hydrogen atoms; transformation of 17~c-CH~CN to -CH~OH; and formation of compounds with a 5(10), 9(11)-structure. Some of these reactions occur simultaneously. Some general features of 4,9-dien-3-oxo system biotransformation have been established by in v i t r o studies with rat l i v e r preparations and by microbial transformation: Hydrogenation of the 4-double bond is retarded by the 9double bond and occurs only to 5&H, not to 5wH-compounds. Thus, in plasma of animals and of man, unchanged DIENOGEST makes up a considerable part in the plasma constituents of the administered compound,.

35 INTERACTION OF REPRESENTATIVE MEMBERS F R O M TWO C L A S S E S O F A N T I M Y C O T I C S - T H E A Z O L E S AND T H E A L L Y L A M I N E S WITH N-DEMETHYLATION OF DIAZEPAM. H. Bohnemeier. W. Hecker, S. Grfine. W. Christ Azole antimycotics seem to act as inhibitors of specified enzyme systems (K. Lavrijsen etaL, Xenobiotica 1987; 1_~7:45-57). We studied the interaction with the N-demethylation of diazepam in vivo using the 14CO~ breath test. N-14CH3-diazepam was syathetized and adminis[red orally in doses ranging from 3.79 to 20 m g / k g to female Wistar-rats (250g). 14CO2-exhalation rate was continuously monitored. At the lowest dmzepam dose 13.3% (SD=2.2) of the radioactivity was exhaled within 5h. Depending on the type and dose of antimycotic, which was administered p.o. 2h prior to diazepam, the 14CO2 exhalation rate was changed. In the following table, the ratio of exhalation of pretreated to control animals is given (diazepam dose 3.79 mg/kg). Ratio Pretreated / Control-rats Dose (mg/kg) Antimycotic

100

50

25

Isoconazole 0.11 0.34' 0.50 Miconazole 0.21 0.31 0.45 Ketoconazole 0.54 0.69 0.78 Fluconazole 0.71 0.94 0.88 Itraconazole 0.95 1.05 0.99 Terbinafine 0.78 0.90 0.97 The older antimycotics of the imidazole-type are strong inhibitors of diazepam N-demethylation. In contrast, the allylarnine derivative terbinafine and the recently marketed triazoles fluconazole and itraconazole seem not to impair this metabolic pathway to a noteworthy extent. Institut fOr Arzneimittel des BGA, Seestr. 10, D-1000 Berlin 65

I n s t i t u t e for M~crobiology and Experimental Therapy, Division of Steroid Research, BeutenbergstraBe 11, 0-6900 Jena/ThSrtngen.

34 LIQUID/LIQUID-EXTRACTION OF ORGANIC TRACES FROM BIOLOGICAL MICROLITER SAMPLES Wolfgang DQnges and Bernhard Lindenthal High pressure l i q u i d chromatography is the method of choice for trace determinations of endogenous organic compounds and of drugs or toxicants from b i o l o g i c a l matrices. In many cases gas chromatography is preferable as the separation power is higher and detection is more v e r s a t i l e and sensitive, however, GC quite often can not be applied to biological samples in sub-ml amounts as appropriate sample pre-treatment operations are not available. On the basis of our own microchemical system we now propose a generally useful pre-chromatographic technique: 10 #I of whole blood is s t i r r e d slowly with 50 ~I of acetone/methyl-tert, butyl ether ( r e p e a t e d ) . After the addition of dry solvent the extract is concentrated under p a r t i a l r e f l u x to 5 #I. A v a r i e t y of v o l a t i l e (e.g. toluene) to higher b o i l i n g test compounds (e.g. 2-hydroxy-biphenyl), 10 ng each, had been added to the samples. GC/FID analyses of the dry concentrate showed recoveries for a l l 25 compounds of about 70 %, while ratios of peak areas to an internal standard corresponded to c a l i b r a t i o n s . With these operations we reinvestigated the metabolism of p-methyl-anisole, W. DOnges, Nature 243 60-61 (1973), in 15 #I incubation mixtures of rat l i v e r microsomes; phenolic metabolites in ng amounts could be identified. The reduction of the sample size opens new perspectives for biomedical investigations: microsomes from human l i v e r biopsies can be used and the need f o r laboratory animals is reduced d r a s t i c a l l y . I n s t i t u t f~r Kernchemie, Johannes Gutenberg-Universit~t Mainz, F r i t z Strassmannweg 2, D-6500 Mainz, Deutschland

Hauptwege der Biotransformation des Progestagens DIENOGESTR G. Hobo, Renate SchOn, Petra R i t t e r , H.G. Hillesheim

36 C O R R E L A T I O N OF TRAMS~DOL AND M 1 SERUM LEVELS WITH A/~TINOC I C E P T I V E A C T I V I T Y IN MICE E. Frlderichs and R. Becke~ Tramadol (T) b i o t r a n s f o r m a t l o n yields the active m e t a b o lite O - d e s m e t h y l - T (MI) (Lintz et al., A r z n e i m . - Forsch. 2ul, 1932 (1981)). TO i n v e s t i g a t e how far MI, in a d d i t i o n to intact T, is involved in analgesia, serum levels of T and M 1 were d e t e r m i n e d in mice concomitant with analgesia t e s t i n g in the mouse tail flick (p.o. 30 min, i.v. i0 min p.appl.). Immediately after the tail flick test, b l o o d was withdrawn, c o a g u l a t e d and serum levels of T and M 2 were d e t e r m i n e d by gas c h r o m a t o g r a p h y using a n i t r o g e n selective detector. TO show the analgesic effect of T alone, b i o t r a n s f o r m a t i o n was i n h i b i t e d by p r e t r e a t m e n t with the enzyme inhibitor SKF-525-A. A dose response study of i.v. O - d e s m e t h y l - T was p e r f o r m e d to c o r r e l a t e serum levels of M 1 alone with analgesic action. T i n c r e a s e d tail flick latency with an i.v. t h r e s h o l d dose of 2.15 m g / k g and m a x i m u m effective dose of 21.5 mg/ kg, leading to m e a n serum levels of 243 m g / m l T and 147 ng/ml M I at the t h r e s h o l d and 3178 ng/ml T and 909 ng/ml ~i at the m a x i m a l dose. M e t a b o l i c b l o c k a d e with 50 m g / k g ~.p. S K F - 5 2 5 - A reduced M 1 serum levels to the d e t e c t i o n limit and i n c r e a s e d T levels to 422 ng/ml and 5953 ng/ml at the b e f o r e m e n t i o n e d doses. The results o b t a i n e d after inhibition of m e t a b o l i s m show that T itself is analgesically active. Under conditions of u n i m p a i r e d metabolism, M 1 and T p l a s m a levels are within the e f f e c t i v e dose range indicating that both contribute to analgesia. Orally, only the highest dose of 46.4 m g / k g T gave an a n a l g e s i c t h r e s h o l d serum level of T whereas e f f e c t i v e levels of M 2 were reached over the whole dose range. Thus, analgesia after oral T is n e a r l y t o t a l l y dependent on M 1 and the high oral p o t e n c y is the result of firstpass metabolism. The results are typical for species with a high rate of metabolism. In humans, involvement of M 1 has been shown be less prominent. Gr~nenthal GmbH, D e p a r t m e n t of Pharmacology, Zieglerstr. 6, D-5100 Aachen, FRG.

RIO 37

39

A BALANCE STUDY OF HUMAN CARBOCYSTEINE METABOLISM C. O. Meese 1, H. Wisser2

REDUCTIVE METABOLISM OF METYRAPONE IN HUMAN LIVER MICROSOMES AND CYTOSOL E. Maser a n d T. Gebel R e d u c t i o n is a n i m p o r t a n t s t e p in t h e b i o t r a n s f o r m a t i o n of

The mucolytic agent carbocysteine (CMC, S-Carbox3nnethyl-L-cysteine) has been suggested as a probe drug for uncovering polymorphic sulphoxidatlon within a population. However, more detailed and recent studies that were based on HPLC, GC//vlS, and ~3C NMR tcchnlques [C. O. Mease et al., NaunynSchmiedeberg's Arch. Pharrnacol. 341(Suppl.),R 9, No.34,1990)] revealed a metabolic pattern that was completely different from the one inferred from the paper chromatographic assay JR. H. Warlng and S. C. Mitchell, Drug Metab. Dispos. 10, 61 (1982)]. In order to obtain a deeper insight into CMC metabolism, CMC [750rag], labelled with ~a'~4Cand ~z~4C,respectively, at the methylene group of the carboxymethylmoiety was adm~nlstered orally to two human subjects, and urine was collected in fractions up to 144h. Radioactivity was both measured in the urines and in the exhaled breath. In addition, all urine samples obtained after admlnistxation of Iz/14C-CMC (>99% ~3C) were analyzed by lmc N1VIR spectrospopy as described recently [C. O. Meese and P. Fischer, Z. Naturfor~ch. 45c, 139 (1990)]. The radioactivity measurements and the NMR method both demonstrate a biphasin kinetics (t½ - 6h and 20h) of urinary excretions. During the 0-8h period mainly unchanged CMC was eliminated, whereas after 8h metabolites of CMC, recently identified as thiodiglycolic acid, and its sulphoxide, and a novel metabolite of yet unknown structure (that accounts for about 5% of the dose administered) were determined in the samples. In addition, a slight but significant increase in radioactivity in the exhaled breath, according to 5-10% of the dose admin;stered, was registered after 8h that ceased after 24-30h. Since at least 70% of the dose administered has been recovered and no significant amounts ( > 1-2%) of cystelnyl sulphoxides were detected in the urines, a polymorphic sulphoxidation of CMC in respect to these sulpho:ddes can be excluded.

Supported by the Robert Bosch Foundation Stuttgart 1Dr. Margarmte Fiscbem-Boseh-Insfitnt fiir Klinische Phurmakologie, D-7000 Stuttgart 50, AuerbacbstraBe 112, 2Abt. Kllnische Chemle, Robert-BoschKrankenhans, D-7000 Stuttgart 50, AunrbachstraSe 110

c a r b o n y l g r o u p b e a r i n g s u b s t r a t e s in m a n y m a m m a l i a n a n d n o n m a m m a l i a n t i s s u e s . In a d d i t i o n to x e n o b i o t i c a l i p h a t i c and aromatic aldehydes and ketones the carbonyl reductases i n v o l v e d in t h i s r e a c t i o n m e t a b o l i z e a v a r i e t y of p h y s i o logical substrates including prostaglandins and steroids. In p r e v i o u s i n v e s t i g a t i o n s we p u r i f i e d a n d c h a r a c t e r i z e d a c a r b o n y l r e d u c i n g e n z y m e from m o u s e l i v e r m i c r o s o m e s u s i n g t h e k e t o n e m e t y r a p o n e (MP) a s a s u b s t r a t e . Moreover, we d e m o n s t r a t e d t h a t M P - r e d u c t i o n o c c u r s in c u l t u r e d c e l l s of c o n t i n u o u s cell l i n e s t h u s g i v i n g r i s e to t h e s u g g e s t i o n t h a t carbonyl reductases are constitutive enzymes, which play an i m p o r t a n t r o l e in n o r m a l cell p h y s i o l o g y . In t h e p r e s e n t s t u d y we c h u r a c t e r i z e d M P - r e d u c i n g e n z y m e s in h u m a n l i v e r m i c r o s o m a l a n d c y t o s o l i c f r a c t i o n s u s i n g (1) different cosubstrates, (2) d i a g n o s t i c i n h i b i t o r s a n d (3) antibodies against the MP-reductase from m o u s e l i v e r microsomes: We f o u n d NADPH to be a more p o t e n t c o s u b s t r a t e t h a n NADH in b o t h f r a c t i o n s . Of t h e i n h i b i t o r s t e s t e d o n l y d i c o u m a r o l in m i c r o s o m e s a n d i n d o m e t h a c i n in c y t o s o l c o n s i d e r a b l y decreased enzyme activity, whereas quercitrin, barbiturate a n d h e x o b a r b i t a l c a u s e d o n l y a w e a k i n h i b i t i o n . However, in microsomes 5a-dihydrotestosterone (5c(-DHT) was the s t r o n g e s t i n h i b i t o r , w h e r e a s in c y t o s o l i t h a d no e f f e c t . Immunological crossreaction w a s f o u n d in h u m a n l i v e r microsomes with the antibody against the mouse liver enzyme indicating structural homologies between the m i c r o s o m a l e n z y m e s of t h e t w o s p e c i e s . In c o n c l u s i o n , MP-reduction was s h o w n to o c c u r in m i c r o s o m e s a n d c y t o s o l of h u m a n l i v e r b e i n g m e d i a t e d b y c a r b o n y l r e d u c t a s e s . S t r u c t u r a l h o m o l o g i e s seem to e x i s t b e t w e e n t h e M P - r e d u c t a s e s from m o u s e a n d h u m a n l i v e r m i c r o s o m e s a n d 5¢(-DHT is s u p p o s e d to be t h e p h y s i o l o g i c a l s u b s t r a t e o f t h e m i c r o s o m a l enzyme, Dept. P h a r m a c o l . , P h i l i p p s - U n i v e r s i t y ,

Lahnberge, 355 Marburg

38 REVISED PROTOCOLFOR CARBOCYSTEINEPHENOTYPINGIN MAN A. Kfipfer*, F. Gerber, E. Manser, I. Turner, J.R. Idle

40

Carbocysteine (CC) exhibits polymorphic metabolism in man and the underlying deficiency of organosulfur metabolism is visible by the absence of the main CC metabol i t e (CCM) in poor metabolizer (PM) phenotypes. Several methodological d i f f i c u l t i e s have been encountered e.g, with the originally used paper chromatography (PC) and we therefore have revised the protocol for phenotype determinations as follows: CC was administered in form of capsules or syrup (750 mg p.o.) and urines were collected from 0-8 h and from 8-16 h. Two population studies (Swiss, n=106 and United Kingdom, N=60) were carried out. For CC and CCM analysis, urine was applied to silicagel TLC plates and developed in 1-butanol/ water/cone, acetic acid/butylacetate (24/10/10/4 by vol/vol). Then the TLC spots were visualized using potassium iodoplatinate solution (200 mg/L) in acetone. The Rf values for CC and CCM were 0.28 and 0.43, respectively. For peak quantification, a Shimadzu dual wavelength flying spot model CS 9000 TLC scanner (reflection mode at 510 nm) was used. The results of TLC analysis were also veryfied using the previously proposed PC method. After CC capsules and after CC syrup, the 0-8 h urine sample contained mainly CC whereas CCM was the main organosulfur compound in the 8-16 h urine collection. For statistical analysis, the CC over CCM metabolic ratio (MR) was calculated. The frequency distribution histograms showed bimodal distributions with an antimode at MR=17 in b o t h population studies. In the Swiss study, 7.5% PM (95~ confidence limits of 3.4% to 13.4%) were identified and in the British study, I0~ (g5% confidence limits of 3.8~o to 19%) were PM. Therefore, polymorphic CC metabolism occurs in Swiss and British populations and extended urine collections for proper MR calculations are required. *Dept. of Clinical Pharmacology, University of Berne, Murtenstrasse 35, CH-3010 Berne, Switzerland

In the isolated mucosa of guinea-pig intestine the conjugation of xmnobietics administered to the lumlnal or blood side of the preparation takes place in different compartments (I). Comparative experiments with p-nitroanisol (PNA) and its metebolite p-nitrophenol (PNP) formed by oxidative demethylmtioe now indiemte thmt the latter reaction is restricted mainly to the luminal compartment. I) p-Nitrophenyl sulfate (PNP-S) originating from PNA appears in the luminal solution irrespective of the side of PNA administration. However, only after luminal administration of PNP is its metabolite PNP-S released almost completely to the lumlnal side, whereas PNP-S from blood side PNP Is equally distributed to both sides. Z) For the formation of PNP-S from PNA mainly luminal 35S is used Irrespective of the sid~sof PNA administration, whereas PNP-S from PNP is formed with luminal S after luminal, and with S from both aides after bleed side PNP administration. 3) Selective damage of the luminal compartment by a hypmrtonic preincubation with 2 M sodium cyciohexanesuifamate reduces PNA metabolism to 5 % of the control value irrespective of the side of administration, whereas sulfoconjugation of bleed side PNP decreases only to 50 %. Looking for a merpholegieel correlate of theme funmtional compartments, isolated mucosae were examined histologically after luminal or blood slde hypertonic incubation. Luminal hypertoniclty resulted in s severe damage or even less of the villus epithelium while blood side hypertonicity was mainly destructive to the epithelium of the crypts suggesting a corresponding localization of the lumlnal and blood side compartment.

COMPARTMENTATO I N OF INTESTINAL DEALKYLATION AND SULFOCONJUGATION: COMPARATIVEINVESTIGATIONSWITHp-NITROANISOLANDp-NITROPHENOL F. Lauterbach,G. Ulrich-Merzenioh and G. H. RUM*

(q) La~terbach, F., Schorn, M., Sprakties, G. and Sued, R. B.; Progress in Pharmacology and Clinlcal Pharmacology 7/.__22,231 - 2~2 (1989). Supported by a grant from the M£mSstmr for W£ssensmhmft and Tmrsmh~mg des Landes Nerdrhein-Westfalmn (IV A 6 - 40102387) Arbeitsgruppe Biochemische Pharmakologie, Abtellung fur Pharmakmlogie and Toxtkolmgie and Inmt£tut for Pathologie*~ Ruhr-Univermit~t~ D-4630 BOCHUM

Rll 41

PARACETAMOL GLUCURONIDATION BY HUMAN LIVER PHENOL UDP-GLUCURONOSYLTRANSFERASE A. Forster, M. Brfick, M. El Mouelhi, S. Fournel-Gigleux', G. Siest% B. BurchelP and K.W. Bock Paracetamol glucuronidation has recently been found to be significantly increased in heavy smokers and m patients treated with phenobarbital-type inducers (Beck et al., Eur. J. Clin. Pharmacol. 31, 677-683, 1987). Three routes were followed to identify human liver UDP-glucuronosyltransferase (UGT) isozymes responsible for paracetamol glucuronidation: (I) Solubilized microsomal UGTs were separated by chromatefocusing. Three fractions exlfibiting marker UGT activities could be separated, (a) estriol UGT (pI 7.4), (b) 4-methylumbelliferone UGT (pI 6.6) and (c) 4-aminobiphenyl UGT (pI 6.2). Paracetamol UGT eluted at pI 6.6. However, due to instability of enzyme activity the isozyme could not be further purified. (2) UGT activities were studied in V79 cells containing integrated human liver phenol UGT = HLUGP1 (Harding et al. Prec. Natl. Acad. Sci. USA 85, 8381-8385, 1988). These cells expressed 4-methylumbelliferone and paracetamol UGT activities at levels comparable to those found in human liver homogenates. (3) DNA sequencing of genomic DNA fragments(350 bp from the translation start site, using primers synthesized according to the sequence of HLUGP1) indicated that human phenol UGT appears to be conserved in this region. However, some variation was observed. For example, in one DNA fragment 3 nucleotide exchanges (not leading to amino acid changes) were noted. The results suggest that human liver phenol UGT is the major enzyme responsible for paracetamol glucuronidation in human liver. Institute of Toxicology, Univers,ty of Tiibingen (D-7400 Tiibingen, WilhelmstraBe 56, Germany), 'Centre du M~dicament, Nancy (France) and bNinewells Hospital and Medical School, Dundee (UK)

42 DERMAL ESTER CLEAVAGE

IN THE ISOLATED PERFUSED

RABBIT EAR B. Henr~kus

Rabbit

and H. Kampffmeyer

ears were single-pass

protein-free

steady-state procaine, 151-1~i, following

rate

(apparent Vma x) during

and methylsalicylate 1989) was determined

19

from the effluent

acid moiety was measured by

after extraction with ethyl acetate.

The rate of hydrolysis the ectanol/perfusion

seems to be governed by buffer coefficient.

Whether the penetration the cell,

2-chloro-

(Xenobiotica,

dermal or arterial drug application.

The 4-aminobenzoic h.p.l.e,

perfused with

condition of procaine,

of the substrate

or the enzymic hydrolysis

by the lipophilicity

iDto

is dominated

will be shown in further

experiments. About

10% of the q-aminobenzoic

to be 4-acetamSdobenzoic

Walther-Straub-lnstitut Toxikologie

acid was shown

acid.

for Pharmakologie

und

der Ludwig-Maximilians-Universit~t

Nu~baumstrssse

26, 8000 M~nchen

2.

CONSTITUENTS IN THE ISOLATED PERFUSED RAT LIVER C. Egen-Sehwind, R. Eckard, and F.H. Kemper Further studies on pharmacokinetics of garlic (Allium sativum L.) constituents showed that after oral application of vinyldithiins these compounds could be detected in serum, liver, kidney, and fat tissue. Other garlic constituents or metabolites were not found after administration of fresh garlic or lyophilized garlic powder even when these preparations were administered in high dosages. Therefore the isolated perfused rat liver was used to study the metabolism of different garlic preparations as well as of garlic constituents in an isolated organ. Single pass studies were carried out using a modified KrebsHenseleit buffer as perfusion medium, after liver passage the medium was collected in fractions. As viability parameters the enzyme activities of GOT and GPT and potassium concentration in the perfusate as well as the interpretation of tissue section of the liver were used. Additionally the perfusate was analyzed by HPLC and GC-MS to find garlic constituents or metabolites. To study the .metabolism, the test substances were solved or dispersed in the pexfusion medium and infused in the flowing medium with a rate of 1 ml/min. The administration of garlic powder in different dosages revealed that allicin, the main constituent of garlic, could be detected in the perfusate only after a dose of 200 mg garlic powder~rain over 50 rain. After application of lower dosages allicin was degradated during the first liver passage. Diallyl disulfide was identified as metabolite of allicin in the perfusate as well as in the liver and in the bile which was confirmed by studies with isolated allicin. Furthermore ajoenes and vinyldithiins were investigated in this model but no metabolites could be identified. Institut ffir Pharmakologie und Toxikologie der Westffilischen Wilhelms-Universitfit Mfinster, DomagkstraBe 12, D-4400 Mfinster, FRG

44 PENICILLIN BINDING IN BETA-LACTAMASE PRODUCING STAPHYLOCOCCI: PROTECTIVE EFFECT OF POLIDOCANOL H. Keppeler, A. G~rke, B. Thielen, and W. Bruns The beta-lactamase dependent penicillin resistance of S. aureus can be greatly reduced by polidocanol (PDO), a dodecyl polyethyleneoxid ether (Bruns et al. 1985, Antimicrob. Agents Chemother. 27, 632-639). PDO does not affect the activity of beta-lactamase, but the induced synthesis of this enzyme is inhibited by PDO-concentrations which are not inhibitory for bacterial cell growth.

buffer solution.

The hydrolysis

43 STUDIES ON DIVERSE GARLIC PREPARATIONS AND GARLIC

We studied the consequences of this effect on the binding of ~H-benzylpenicillin (3H-BP) to the penicillin-binding proteins (PBPs) of resistant staphylococci. Induced cells of S. aureus S 108 grown with and without PDO were incubated with various concentrations of 3H-BP. The cytoplasmic membranes were isolated and subjected to SDS-PAGE and fluorography. Penicillin binding to the PBPs of PDO-free cells was detectable only with high concentrations of 3H-BP (100 nmol/ml). In presence of PDO (50 ~g/ml), however, binding of 3H-BP to the PBPs was about 10-fold increased. This is c o n s i s t e n t with the 90% inhibition of beta-lactamase synthesis found under these conditions. The strong PDO-effect on beta-lactamase synthesis and, on the other hand, the lacking inhibition of bacterial growth can probably be explained by preferential interaction of PDO with membrane associated protein synthesis as it has been assumed for the minocycline-inhibited beta-lactamase synthesis (Chopra and Anderson 1985, J. Antimicrob. Chemother. 16, 17-21). The results of own experiments4Point in this direction. The incorporation of I C-serine into membrane proteins was stronger inhibited by PDO than that into cytoplasmic proteins. Institut f~r Pharmakologie der Universit~t zu K~in, Gleuelerstra~e 24, D-5000 K~in 41

R12 45

47

EBSELEN INHIBITS PLATELET ACTIVATION BY MODULATION OF INTRACELLULAR CALCIUM RELEASE B. Briine

INFLUENCE OF ASPIRIN AND ESCULETIN ON THE UTILIZATION OF ASCORBIC ACID AND TOCOPHEROL DURING PLATELET AGGREGATION; R. Pella, a n d A. L u d e s

Ebselen (PZ 51; 2-phenyl-l.2-benzoisoselenazol-3(2H)-one) is a selenoorganic compound with anti-inflammatory properties. The reported glutathione peroxidase (GSH-Px)-like activity is thought to be responsible for the pharmacological activity of ebselen. We investigated the effect of ebselen on aspirin-treated platelets. Ebselen inhibits platelet aggregation caused by several commonly used agonists like thrombin or U46619 a prostaglandin endoperoxide analogue. The observed inhibitory activity followed a sigmoidal doseresponse curve with" IC50 values around 10 IxM (for both agonists; stimulated with threshold concentrations). Mobilization of intracellular calcium represents a critical parameter for platelet aggregation. Therefore the effect of ebselen on the inositol 1,4,5-trisphosphate lIP3) releasable calcium was studied. Ebselen in a concentration-dependent fashion (IC50:14±4 IxM; mean±SD, n=3) inhibits a cytosolic calcium increase in fura-2 loaded platelets after stimulation with 0.1 U/ml thrombin. In additional experiments we studied the effect of ebselen on calcium release using microsomal vesicles. Low concentrations of ebselen (1-5 IxM) added 2 min before the IP 3 stimulus lIP3; 31xM) reduces the calcium release completely. The described effects of ebselen on calcium homeostasis may contribute to the anti-inflammatory pharmacodynamic properties of this compound.

We quantitated the amounts of ascorbic acid (Vit. C) and tocopherol (Vit. E) using HPLC with coulometric electrochemical detection. Our results confirm the described high concentrations in the human platelets a n d s h o w a direct relationship between the degree of platelet aggregation and the Vit. C and E utilized by washed platelets. The mean conc e n t r a t i o n of vit. C in washed human platelets was 13.8 ± 2.4 (SEM) mg/g protein and the concentration of Vit. E 31.5 ± 3.2 (SEM) ~g/g protein. We found a significant correlation between the net loss of Vit. C and E from platelets and the degree of platelet aggregation induced by 1 ~M arachidonic acid and i0 mU/ml thrombin. In experiments with platelet rich plasma we measured that vit. C and E are not released into the plasma during platelet aggregation. Results after 1 minute with washed platelets: Vit. C (% of 0 s) control Aspirin Esculetin A r a c h i d o n i c acid 64 ± 4 92 ± ii 67 ± i0 Thrombin 74 ± 14 69 ,± 17 61 ± 6 Vit. E (% of 0 s) A r a c h i d o n i c acid 14 ± 1 65 ± 6 15 ± 2 Thrombin 24 ± 4 21 ± 8 25 ± 6 A s p i r i n is known as inhibitor of platelet cycloxygenase and esculetin of platelet lipoxygenase, i0 ~M esculetin had no effect on the reduced vitaminlevels. The inhibition of the cycloxygenase pathway by i00 ~M aspirin leads to a lowered utilization of ascorbic acid and tocopherol after arachidonic acid stimulation. Both vitamins seem to act as m o d u l a t o r s of the production of hydroperoxides and appear to serve some definite metabolic function in platelet aggregation in vivo.

Department of Biology, University of Konstanz, Universit~itsstr. 8-10, D-7750 Konstanz, F.R.G.

Department of Biology, University Postfach 5560,D-7750 Konstanz, FRG

46 EBSELEN AFFECTS THE INOSITOL (1,4,5)-TRISPHOSPHATE INDUCED CALCIUM RELEASE FROM HUMAN PLATELET MICROSOMES BY INHIBITION OF INOSITOL (I,4,5)TRISPHOSPHATE BINDING. S. Dtmmeler a n d V. Ullrlch The seleno-organie c o m p o u n d ebselen (PZ 51; 2 - p h e n y l - l , 2 benzisoselenazol-3-(2H)-one) is known to exhibit antiInflammatory activity. This property s e e m s to be related to t h e glutathtone peroxidase (GSH-Px)-like activity as well a s to t h e inhibition of lipoxygenase reactions. We have investigated t h e effect of low concentrations of ebselen on the [3H]inositol 1,4,5t r i s p h o s p h a t e lIP3) binding a n d the IP3 induced Ca 2+ release from h u m a n platelet microsomes. Using platelets m l e r o s o m e s ebselen a s well a s other SH-reagents like reactive disulfides (2,2'-dithiodipyridine), p-chloromercurobenzoyl sulfonate (CMBS) a n d N-ethylmalelmide (NEM) displaced the specific [ H]-IP 3 binding in a concentration-dependent fashion. Halfm a x i m a / inhibition w a s observed at 5.0±1.2 IxM ebselen (mean±SD; n=4). A maximally effective concentration of NEM (200 ~tM) showed 70±8.5 % (mean±SD, n=3) displacement of the specific IP 3 binding whereas 2,2'-dithiodipyridine at a concentration of 6 ~tM revealed about 40±7 % displacement (mean±SD, n=3). A commonly u s e d concentration of i mM CMBS displaced nearly all of b o u n d radioactive IP3. Since essential SH groups have been demonstrated at the IP 3sensitive c h a n n e l a n d reducing agents like dlthiothrettol (lmM), a l m o s t completely reversed the Inhibition of both, t h e IP 3 binding a s well as t h e IP 3 induced calcium release from mierosomes, we conclude a n interference of ebselen with critical sulfhydryl groups. D e p a r t m e n t of Biology, University of Konstanz, Universit&tsstr. 8-10, 7750 Konstanz, Germany.

of

Konstanz,

48 E F F E C T OF T H I M E R O S A L ON THE Ca2+-HOMEOSTASIS IN HUMAN NEUTROPHH.S A. Kottig The normal Ca2+-signal in human neutrophils is biphasic after receptor stimulation with the chemoattractant fMLP. The first phase is due to the release of intracellular calcium during the PI-response, whereas the second phase of the signal is dependent on extracellular calcium. A preincubation with the organic mercury compound Thimerosal significantly increases this second phase. We could now demonstrate by means of Mn2+-quench of the fura-2 signal that this effect of Thimerosal is due to an increased Ca2+-influx. The different receptor agonists of human neutrophils or different concentrations of one agonist differ in their ability to stimulate Ca2+-influx. Only with thos e stimuli that evoke a Ca 2+ -influx by themselves there was an amplifying effect of thimerosal on the second phase of the calcium signal. It seems that Thimerosal is only able to increase an already existing Ca2+-influx but not to activate one by itself. By means of a voltage-sensitive fluorescent dye we measured the effect of Thimerosal on the membrane potential. Thimerosal did not alter the membrane potential of resting cells but significantly reduced dep,olarisation after receptor stimulation. There are no voltage-gated Ca'~+-channels in human neutrophils. Nevertheless, the weaker depolarisation can explain the increased Ca2+-infiux, because it amplifies the free enthalpy for the entry of this divalent cation. With a superimposed artificial depolarisation by the ionophore Gramicidin it was possible to completely inhibit the Thimerosal-induced increased Ca2+-influx. University of Konstanz, LS V. Ullrich, Am GieBberg, W-7750 Konstanz, FRG

R13 49

51

THE USE OF MTT COLORIMETRIC ASSAY TO MEASURE RPE CELL PROLIFERATION N. B0tz, M.Bresgen, P.Wiedemann, and K.Heimann

ON REGIOSPECIFIC CONJUGATIONS OF VARIOUS GLUCURONOSYLTRANSFERASES

In v i t r o q u a n t i f i c a t i o n of cell p r o l i f e r a t i o n is an important step in screening cytotoxic and mitogenic substances. For this purpose c o l o r i m e t r i c assays, e s p e c i a l l y the MTT dye reduction have been shown to be a useful technique. In this study we adjusted the rapid and simple MTT c o l o r i metric assay to test the mitogenic potency of b-FGF, EGF, and TNF-alpha on r e t i n a l pigment epithelium (RPE) cells in vitro. Our results show a l i n e a r relationship between MTT formazan production and the cell number obtained by Coulter cell counting at a range from 5.000 to 100.000 RPE cells per w e l l . Increasing f e t a l c a l f serum (FCS) concentrations lead to a r i s i n g dye development r e f l e c t i n g the induced cell p r o l i f e r a t i o n . The mitogens b-FGF and EGF do not cause RPE cell p r o l i f e r a t i o n at concentrations from 0,1 to 100 ng/ml. On the contrary TNF-alpha shows a marked mitogenic a c t i v i t y for this cell line even at a concent r a t i o n of I ng/ml. Although b-FGF and EGF did not stimulate RPE cell p r o l i f e r a t i o n in this study, they may promote growth when assayed in combination with other agents. In Summary, our adaption of the MTT dye reduction technique provides a rapid, sensitive, and reproducible device for detecting mitogenic a c t i v i t y on RPE cells in vitro.

The aim of the present investigation was to characterize and to compare the glucuronidation pattern of all available phenols and of the main dihydrodiols of the polycyclic aromatic hydrocarbons (PAH) phenanthrene, chrysene, and picene. These metabolites were conjugated by five partially purified glucuronosyltransferase enzyme forms (GT) with different substrate specificities. As expected, the activity of the GTs to glucuPAH decreased with inronidate h y d r o x y l a t e d creasing number of benzene rings from phenanthrene to picene. Compared to the phenols the dihydrodiols in general w e r e poor s u b s t r a t e s or no substrates at all (picene), except of the phenanthrene-Kregion-9,10-dihydrodiol. A differing regioselective glucuronidation could be obtained with the phenanthrene and the chrysene phenols: whereas those GTs with main activity towards testosterone and androsterone preferentially conjugated the K-region phenols the GT form with main activity towards morphine preferred the non-K-region phenols 2- and 3-OHphenanthrene and 3-OH-chrysene. With picene this difference could only be seen in parts, in this case the K-region-5-OH-picene was the best substrate for all GT enzyme forms. In conclusion the specific activities of various GT forms towards different OH-derivatives of PAH show distinct regioselectivities.

Abteilung fur Netzhaut- und Glask~rperchirurgie, Universit~ts-Augenklinik K~In, J0seph-Stelzmann-Stra~e 9, 5000 K~In 41, FRG Mit UnterstUtzung der DFG (Wi 880/3-I) und der Retin0vitStiftung.

C.Augustin,

HYDROXY-PAH

BY

A.Schmoldt

Institut f~r Rechtsmedizin der Universit~t Hamburg, Butenfeld 34, 2000 Hamburg 54

50

PURIFICATION AND CHARACTERIZATION OF A RAT LIVER SULFOTRANSFERASE ACTIVATING BENZYLIC ALCOHOLS TO MUTAGENS A. C z i c h , H . - J . M a r t u s , N. Enders, H. Thomas , S. Hornhardt and H.R. G l a t t .

The m e t a b o l i c

activation

of benzylic alcohols

to

e l e c t r o p h i l i c s u l f a t e e s t e r s and t h e i r i n t e r a c t i o n w i t h c r i t i c a l c e l l u l a r n u c l e o p h i l e s such as DNA was shown in several s t u d i e s . However, very l i m i t e d i n f o r m a t i o n is a v a i l a b l e about the i n v o l v e d s u l f o t r a n s f e r a s e s . We now have p u r i f i e d and s t a b i l i z e d a sulfotransferase capable o f act i v a t i n g v a r i o u s b e n z y l i c a l c o h o l s t o mutagens. F r a c t i o n s were analyzed f o r c o n j u g a t i n g a c t i v i t y toward dehydroepiandrosterone and, in p a r t , f o r the c a p a b i l i t y o f a c t i v a t i n g b e n z y l i c a l c o h o l s in b a c t e r i a l mutagenicity assays. A r a p i d p r o cedure was developed f o r p r e p a r a t i o n o f the enzyme from l i v e r o f female Sprague-Dawley r a t , i n v o l v i n g anion exchange chromatography, concent r a t i o n w i t h aquacide I and chromatography on a h y d r o x y l a p a t i t e column. The r e s u l t i n g p u r i t y was >90~, the p u r i f i c a t i o n f a c t o r was 186 and the molecular weight amounted t o 29 kD. The enzyme activated 1-hydroxymethylpyrene, 2-hydroxymethylpyrene, 6-hydroxymethylbenzo[a]pyrene and

9-hydroxymethylanthracene

t o mutagens.

Expressed

per unit dehydroepiandrosterone-conjugating activity, o y t o s o l and p u r i f i e d enzymes showed comparable r e s u l t s . The p u r i f i e d enzyme was f u r t h e r c h a r a c t e r i z e d w i t h regard t o k i n e t i c parameters, pH optimum, and the i n f l u e n c e o f i n h i b i t o r s o r a c t i v a t o r s o f known s u l f o t r a n s f e r a s e s . A f t e r complete p u r i f i c a t i o n , an antibody was r a i s e d . U n i v e r s i t y o f Mainz, I n s t i t u t e o f T o x i c o l o g y , D6500 Mainz, FRG

52 MECHANISM BASED INHIBITION OF HUMAN LIVER CYTOCHROME P-450 BY SEVERAL SYNTHETIC STEROIDS R. B6cker and H. Lepper It could recently be demonstrated that the progestogenic gestodene inhibits the nifedipine oxidase (formation of the pyridine) as well as the hydroxylation of ethinylestradiol (EE) on human liver microsomes in vitro (F.P.Guengerich, 1990, Chem.Res.Tox.3,363). The hydoxylation of EE as the nifedipine oxidation are known to be catalyzed by closely related enzymes or by the same enzyme (F.P.Guengerich, 1988, Mol. Pharmacol. 33,500). Several synthetic steroids (including gestodene, norethisterone, desogestrel, 3-ketodesogestrel, and EE) have now been investigated on 15 different human liver microsomal preparations for their potency to inhibit the nifedipine-oxidation and the ethinylestradiol-hydroxylation in vitro. Liver microsomes were preincubated with various concentrations of the steroids for up to 240 minutes in presence of a NADPH generating system. After a tenfold dilution of the microsomal preincubation mixture nifedipine (and some other closely related dihydropyridines) and EE were used as test substrates for the determination of the residual enzymatic activity. Gestodene was the most potent inhibiting synthetic steroid of the above mentioned reactions. Preliminary experiments with the same synthetic steroids on primary rat liver hepatocytes showed a comparable inhibitory effect of the steroids at the dihydropyridine oxidation reaction. It can be concluded that by the metabolism of the synthetic steroids in vitro an inhibition is caused on the isolated microsomal P-450 fraction as on the intact hepatocytes. Lehrstuhl f0r Toxikologie und Pharmakologie Universit&tsstraBe 22, D8520 Erlangen

R14 53

55

ARE 7-DIALKYLAMINO-4-METHYL-COUMARINS SUITABLE AS SUBSTRATES OF THE MURINE COUMARIN 7-HYDROXYLASE ?

EFFECT OF SMOKING ON BIOTRANSFORMATION ENZYME ACTIVITIES IN HUMAN PLACENTA, FETAL LIVER AND KIDNEY S.J. Wallner, F. Siegers*, F. Klink* and C.-P. Siegers

T e g t m e i e r M. L e g r u m W Coumarin and its 7-alkoxy derivatives (such as herniarin, 7 - e t h o x y c o u m a r i n a n d s c o p a r o n e ) a r e w i d e l y u s e d a s model s u b s t r a t e s to m e a s u r e m o n o o x y g e n a s e a c t i v i t i e s . The O - d e a l k y l a t i o n f o r m i n g t h e f l u o r e s c e n t u m b e l l i f e r o n e is c a t a l y z e d m a i n l y b y t w o c y t o c h r o m e P - 4 5 0 i s o z y m a s . One is a h i g h m o l e c u l a r form i n d u c e d b y 8 - m e t h y l c h o l a n t h r e n e (MC) a n d s u l m a z o l e , t h e o t h e r is c h a r a c t e r i z e d b y i t s low m o l e c u l a r w e i g h t (48.5 kDa) a n d i t s i n d u c i b l i t y b y s e l e c t i v e C o h inducers such as cobalt and N-containing heteroaromates and b y p h e n o b a r b i t a l (PB). The l a t t e r i s o z y m e is c a l l e d c o u m a r i n 7-hyroxylase (Coh) or C y p I I A S - t y p e II. This study investigated 7-dimethylamino-4-methyl-coumarin and 7-diethylamino-4-methyl-coumarin a s s u b s t r a t e s of t h e coumarin 7-hydroxylase. The compounds used as laser dyes a r e known a s " C o u m a r i n 3 1 1 " a n d " C o u m a r i n 1", w h i l e t h e f r e e a m i n e is " C o u m a r i n 120". I n c u b a t i o n of t h e s u b s t r a t e (10 -4 M) w i t h c y t o c h r o m e P - 4 5 0 (10-n M) a t 87°C u p to 30 rain l e a d s to t h e m o n o a l k y l a m i n e s and 7-amino-4-methyl-coumarin as visualized by thin layer c h r o m a t o g r a p h y on s i l i c a gel 60 F=~. In t h e a c i d i f i e d a s s a y t h e m o n o a l k y l a m i n e s c a n be q u a n t i f i e d f l u o r i m e t r l c a l l y u s i n g t h e w a v e l e n g t h p a i r 3 6 5 nm a n d 4 5 5 nm f o r e x c i t a t i o n a n d emission, respectively, Experiments with microaomes of d i f f e r e n t l y p r e t r e a t e d NMRI mice r e v e a l t h a t PB d o u b l e s t h e f o r m a t i o n r a t e of t h e m o n o a l k y l a m l n e s , w h e r e a s MC d e c r e a s e s it. Among C o h - i n d u c a r s o n l y c o b a l t s l i g h t l y e n h a n c e s t h e N m o n o d e a l k y l a t l o n r a t e of t h e n e w s u b e t r a t e s . T h e s e o b s e r vations are true also for 7-diethylamino-4-CF3-coumarin ( ' C o u m a r i n 152") a s s u b s t r a t e . T h e r e s u l t s i n d i c a t e t h a t a t l e a s t t h e N - m o n o d e a l k y l a t i o n of t h e s u b s t r a t e s t e s t e d is c a t a l y z e d a t a m i n o r d e g r e e b y t h e Coh. In c o m p a r i s o n t o t h e 7 - a l k o x y c o u m a r i n s t h e s e new s u b s t r a t e s do n o t o f f e r a d e c i s i v e a d v a n t a g e w i t h r e g a r d t o t h e i r s e l e c t i v i t y i n r e c o g n i z i n g a n i n d u c t i o n of t h e Coh. Dept. Pharmacol.~ P h i l i p p s - U n i v e r s i t y ,

The influence of cigarette smoking on the glutathione (GSH) and malondialdehyde (MDA) contents as well as GSH-dependent enzymes (GSH-S-trensferase towards CDNB, GSH-peroxidase towards tbutylhydroperoxide, GSH-S-epoxide transferase towards 1,2-epoxy-3(pnitro-phenoxy)-propene)) was studied in maternal and fetal tissues of pregnant women. In smokers (n=5-10) continuing smoking during the whole pregnancy (5-40 cigarettes/die) a diminished GSH-content and GSH-S-aryltransferase activity was found in placentas at term, placentas of spontaneous abortion (week 12-28 of pregnancy), fetal livers and kidneys as compared to non-smokers (n=11-21). The GSH-peroxidase activities were not different between both groups, whereas the MDAcontents tended to be higher in fetal liver and kidney of smoking women. Concerning microsomal enzyme activities there was a 50% increase in dimethylhydrazine demethylase activities in postpartum placental tissue and 30% increase of ethoxyresorufin deethylase activities in fetal liver of smokers. The most important findings of our investigations were the decrease in the GSH-conjugating enzyme system in placenta} and fetal tissues of smoking women indicating a requirement of this defense system against the toxic constituents of cigarette smoke, in previous investigations smoking did not alter the activities of GSH-S-transferases in the placenta and fetal tissues (Drug Metab. Dispos. 9, 472, 1981; Xenobiotica 12, 543, 1982). Institute of Toxicology and Department of Gynecology and Obstetrics*, Medical University of L0beck, Ratzeburger Allee 160, D-2400 L0beck, FRG

Lahnberge, 355 Marburg

54 IN VITRO METABOLISMOF ETHYLCHLORIDE BY F-344 RAT LIVER AND B6C3FI MOUSELIVER MICROSOMES N. Fedtke, R. Hamphoff-KShler, T. Reinert, H.-J. Wiegand

56 LAURIC ACID METABOLISM IN RABBIT LUNG: MICROSOMAL KINETIC BEHAVIOR AND ANTIBODY INHIBITION Schulze, J. and Richter, E.

Two-year inhalation of 15,000 ppm ethyl chloride (ECL) induced uterine carcinomas in B6C3F1 mice but not in F-344 rats. The mechanism of this species and sex spec i f i c tumor induction is not known, but metabolic differences might play an important role. The in vitro metabolism of ECL was investigated using micresomes isolated from male and female F-344 rats and B6C3F1mice. Headspace gas chromatographywas used to determine the rate of the ECL metabolism in microsomal suspensions containing 12.5 mg microsomal protein and 250 ~mol/l ECL in the liquid phase. Under t h e s e conditions ECL was metabolized in a CO-inhibited and NADPH-dependent reaction. The micresomal a c t i v i t y of untreated animals towards ECL was about 1.5 times higher in mice than in rats (male rats 719, female rats 806, male mice 1304, female mice 1211 pmol ECL/min/mg). The gas-chromatograms obtained with complete incubation mixtures shewedan additional peak not present in the gas phase of the CO-inhibited or NADPH-lacking incubations. This peak was tentatively identified as acetaldehyde. The rate of ECL-metabolism was not enhanced comparing microsomes from untreated animals with microsomes from animals treated with methylcholanthrene (MC) or phenobarbital (PB). The data suggest that cytochrome P-450 catalyzed oxidation may be important in ECL-metabolism under in vivo conditions, in part resulting in the formation of acetaldehyde. However, the main MC- and PB-inducible cytochrome P-45D subfamilies P-450IIA and P-450IIB seem not to be involved in the microsomal ECL-metabolism. Hence, the possible induction of the ECL-metabolism by ECL i t s e l f acting upon the ethanol inducible subfamily P-450IIE is currently investigated.

Laurie acid (LA) is a medium chain fatty acid with strong detergent properties. For degradation mammals have evolved a specific and effective system of LA metabolizing enzymes (Group Cyp450IVA). In rat and rabbit liver and kidney up to 6 different isozymes have been identified, whereas in lung no data are available yet. Masters et al. (Rev. Biochem. T0xieol. 1985, p. 74) have suggested that in rabbit lung Cyp450IVBI is responsible for LA hydroxylation in 11- and 12-position. We therefore investigated the kinetic parameters of LA hydroxylation in rabbit liver, lung and kidney microsomes and tried to selectively inhibit LA metabolism in rabbit lung mierosomes by inhibitory antibodies. In all three organs tested the kinetic of LA-hydroxylation is similar. The concentration range tested was 1 - 400 umole LA; due to its detergent properties LA inhibited its own metabolism at concentrations over 100 umoles. It appears to be a one enzyme system with an apparent K M value of 10 umoles and an vmax of 15 nmoles/min/mg microsomal protein. These values were the same for all three organs within the margin of error. This result indicates that although different isozymes are present for L A metabolism they are indistinguishable by kinetic properties. In lung microsomes we tried to inhibit LA-metabolism by addition of anti-Cyp450IVB 1 (LA -concentration 20 umole). This addition showed doserelated inhibition of LA-hydroxylation; at 10 mg IgG/nmole eytochrome P450 only 40 - 60% of the control activity remained, with further reduction upon increased amounts of antibody. This effect could also be mimicked by addition of antibodies specific for other isozymes as Cyp4501A 1, IA2 andlIB4. Surprisingly the addition of preimmune serum and BSA in equivalent amounts also led to a 30 - 40% decrease in activity at protein concentrations of 20 mg/nmole cytochrome. We therefore conclude that in rabbit lung microsomes Cyp450IVB1 is only a minor contributor, if any, to LA metabolism. The apparent inhibition by antibodies in high concentration can be explained by distribution of laurie acid between the different pro-rein compartments, thus leading to a smaller substrate concentration available at the cytochrome P 450 system.

H~Is AG, Ps/Biologie-Texikologie, PB 12, P.O. Box 1320 D-4370 Marl, FRG

Supported by a grant from the Curt Bohnewand-fond. Walther Straub Institut filr Pharmakologie und Toxikologie, Nussbaumstrasse 26, 8000 M~nchen 2

R15 57

59

U P T A K E O F P O L Y C H L O R I N A T E D BIPHENYLS (PCB) IN CELL CULTURE SYSTEMS IS IMPROVED BY LIPOSOMES L. Potthoff*, J. Seedorf2, M. Dubowy s, K. Buff4, W. Leibold*

I N D U C T I O N A N D I S O E N Z Y M E P R O F I L E OF G L U T A T H I O N E T R A N S F E R A S E S IN H E P A T I C A N D E X T R A H E P A T I C C E L L LINES.

THE

S.F. R o e s c h Phospholipid-derived liposomes were reported to be suitable carriers for PCB. The transfer of lipophilic substances takes place by adsorption between liposomes and cells visualized by the model substance fluorophore 1,6-dlphenyl-1,3,5- hexatriene (DPH). Liposomes are able to distribute PCB homogenously in aqueous cell culture medium. Concentrations up to 78 m g PCB per kg liposome solution can be solved. Kinetic studies with a cell line (K 562) reveal a continous u p t a k e o f t h e PCB congener 2 , 2 ' , 4 , 4 " , 5 , 5 ' - h e x a e h l o r o b i p h e n y l (PCB No. 163) u p to 2 4 h o u r s w i t h l i p o s o m e s a s well a s w i t h m e t h a n o l a s s h u t t l e . While w i t h m e t h a n o l t h e maximum uptake of one million cells is 5 % o f 61 ~lg/kg in cell c u l t u r e medium, w i t h l i p o s o m e s m u c h h i g h e r c o n c e n t r a t i o n s a r e p o s s i b l e . A f t e r 24 h o u r s 18 % o f 5.6 m g / k g a r e i n c o r p o r a t e d in t h e cells. I n v e s t i g a t i o n s on t h e m e t a b o l i c a c t i v i t y o f l i n e s o f d i f f e r e n t c e l l - t y p e s ( J u r k a t , K 562, a n d P D e - B 1 ) m e a s u r e d b y the tetrazolium dye test documented that up to 4 mg/kg PCB No. 153 in l i p o s o m e s a d d e d f o r f o u r d a y s a r e t o l e r a ted within the acceptable range. Further investigations have to be done to determine the i n f l u e n c e o f PCB o n s e v e r a l f u n c t i o n s o f i m m u n e cells. *Immunology Unit, ZDept. of Chemistry, aDept, of Pharmacology and Toxicology, Veterinary School, 3000 Hannover, 4Research Centre for Environment and Health, 8042 Neuherberg, F R G

58 V79 CELLS GENETICALLY ENGINEERED FOR EXPRESSION OF CYTOCHROME P4501A2 AND M E T A B O L I S M OF A R O M A T I C AMINES AND C A F F E I N E C. J a n s s e n s and J. Doehmer A s p a r t o f o u r w o r k f o r the e s t a b l i s h m e n t o f a cell b a n k f o r p h a r m a c o l o g i c a l and t o x i c o l o g i c a l s t u d i e s V79 C h i n e s e h a m s t e r ceils were genetically engineered for stable expression of rat cytochrome P450IA2, which hydroxylates aromatic amines and demethylates c a f f e i n e . A c D N A l i b r a r y was m a d e f r o m A r o c l o r 1254 induced rat liver mRNA and was searched for cDNA e n c o d i n g C Y P 1 A 2 . Full l e n g t h c D N A w a s o b t a i n e d by j o i n i n g a small 5' c o d i n g c D N A f r a g m e n t and the rest of the c D N A . The c o n s t r u c t w a s v e r i f i e d by D N A s e q u e n c i n g . T h e r e c o n s t r u c t e d full l e n g t h c D N A w a s r e c o m b i n e d with a S V 4 0 e a r l y p r o m o t e r containing eukaryotic vector and transferred into V79 cells together with the selective marker gene neomycin phosphotransferase by the Ca/P coprecipitation technique. G418 resistant cell clones were checked for chromosomal i n t e g r a t i o n o f the c D N A , e x p r e s s i o n o f an a u t h e n t i c P 4 5 0 I A 2 mRNA and protein by Southern-, Northern-, Western-blotting, a n d i m m u n o f l u o r e s c e n c e s t a i n i n g o f f i x e d cells. E n z y m a t i c a c t i v i t y in P 4 5 0 I A 2 e x p r e s s i n g V79 d e r i v e d cell line X E M d - M Z was v a l i d a t e d by h y d r o x y l a t i o n o f 17B-estradiol and 2-aminofluorene. In addition, X E M d - M Z cells were s h o w n to be c a p a b l e o f specific d e m e t h y l a t i o n o f c a f f e i n e , w h i c h m a y b e u s e d as i n d i c a t o r d r u g in m e t a b o l i s m s t u d i e s (in c o l l a b o r a t i o n w i t h F u h r a n d S t a i b , K l i n i s c h e P h a r m a k o l o g i e , F r a n k f u r t a/M.). P 4 5 0 I A 2 a n d N - a c e t y l t r a n s f e r a s e e x p r e s s i n g V79 d e r i v e d c e l l line X E M d - N H was a p p l i e d f o r m u t a g e n i c i t y s t u d i e s o f a r o m a t i c a m i n e s s u c h as 2 - a m i n o f l u o r e n e and aminochrysene (in c o l l a b o r a t i o n with Glatt, Institut f. T o x i k o l o g i e , M a i n z ; W i e b e l , GSF, Neuherberg). I n s t i t u t ftir T o x i k o l o g i e , J o h a n n e s G u t e n b e r g - U n i v e r s i t ~ t Mainz, Obere Z a h l b a c h e r Str. 63, D-6500 Mainz, F R G

and S. H e s s e

H e p a t i c cell lines d e r i v e d f r o m rat H 4 I I E C 3 hep a t o m a c e l l s and the e x t r a - h e p a t i c cell lines V79, C 3 H I O T I / 2 , and WRC w e r e t e s t e d for t h e i r e x p r e s s i o n of g l u t a t h i o n e t r a n s f e r a s e (GST). G S T activities were measured using l-chloro-2,4-din i t r o b e n z e n e (CDNB) as s u b s t r a t e , m R N A e x p r e s s i o n of d i f f e r e n t GST forms, i.e. t h e i r subunits, was d e t e r m i n e d by N o r t h e r n blot a n a l y s i s e m p l o y i n g c D N A c l o n e s of i s o e n z y m e Y a ( ' l - l ' ) , Yc('2-2') and Y p ( ' 7 - 7 ' ) . All cell l i n e s t e s t e d s h o w G S T a c t i v i t i e s t o w a r d s C D N B r a n g i n g from 50-200 n m o l / m i n x mg p r o t e i n . E x p o s u r e of c e l l s to 20 ~ M b e n z ( a ) a n t h r a c e n e (BA) for 24 h does not s i g n i f i c a n t l y c h a n g e G S T a c t i v i t i e s , e x c e p t for a 1 . 5 - f o l d i n c r e a s e in the h e p a t i c lines 2 s F o u and H 4 I I E C 3 / G - . Y p - m R N A is f o u n d in all cell lines tested. U n d e r the same test c o n d i tions no s i g n a l for Y p - m R N A is s e e n in rat liver. BA d o e s not a f f e c t the e x p r e s s i o n of Ypm R N A in any of the cell lines. In c o n t r a s t to Y p - m R N A , Ya- and Y c - m R N A is d e t e c t e d o n l y in hep a t o m a c e l l s and rat l i v e r but not in the 3 ext r a h e p a t i c c e l l lines tested. BA i n d u c e s m R N A for Ya and Y c in h e p a t o m a lines 5L and H 4 I I E C 3 / T m o r e t h a n 2-fold. Cell lines e x p r e s s i n g s p e c i f i c G S T isoenzymes such as t h o s e i n v e s t i g a t e d h e r e s h o u l d be u s e f u l in a n a l y s i n g the m e t a b o l i s m of electrophilic compounds. GSF-Institut

fur T o x i k o l o g i e ,

D-8042 Neuherberg

60 1 , 6 - D I N I T R O P Y R E N E R E D U C T I O N IN P E R M A N E N T C E L L L I N E S IS M E D I A T E D BY S E V E R A L C Y T O S O L I C REDUCTASES U. H i l p e r - R e u t e r ,

O. C u m p e l i k and F.J. W i e b e l

We h a v e p r e v i o u s l y s h o w n that the h i g h l y m u t a genic diesel exhaust component 1,6-dinitropyrene (I,6-DNP) is m e t a b o l i z e d via o x i d a t i o n and red u c t i o n in m a m m a l i a n c e l l s (U. H i l p e r - R e u t e r et al. [1990] N a u n y n - S c h m i e d e b e r g ' s Arch. P h a r m a col. Suppl. 341, R23). - The p r e s e n t s t u d y was a i m e d at m e a s u r i n g the o v e r a l l c a p a c i t y of m a m m a l i a n cell lines for r e d u c t i o n of 1 , 6 - D N P and s e p e r a t i n g their c y t o s o l i c n i t r o r e d u c t a s e s by gel f i l t r a t i o n . - The r e s u l t s i n d i c a t e that the test cell lines h a v e s i m i l a r c a p a c i t i e s for r e d u c i n g 1,6-DNP. The e l u t i o n p r o f i l e s of the gel f i l t r a t i o n show s e v e r a l p e a k s c o n t a i n i n g 1 , 6 - D N P reductase activity. V79 and H4IIEC3/Gc e l l s h a v e a m a j o r p e a k of 1 , 6 - D N P r e d u c t a s e a c t i v i t y in the r e g i o n of 1 6 0 - 2 4 0 kDa. In c o n t r a s t , 5L and H e p G 2 c e l l s are d e v o i d of this p e a k but p o s s e s s a 1 , 6 - D N P r e d u c t a s e p e a k in the 46-96 kDa region. This p e a k is a l s o f o u n d in H 4 I I E C 3 / G - cells. X a n t h i n e o x i d a s e and N A D ( P ) H d e h y d r o g e n a s e ( D T - d i a p h o r a s e ) coelute at the r e g i o n s of 240 kDa and 68/78 kDa, r e s p e c t i v e l y . B e s i d e the two m a j o r 1 , 6 - D N P r e d u c t a s e peaks, a n u m b e r of m i n o r p e a k s are o b s e r v e d w h i c h h a v e not yet b e e n a s s o c i a t e d w i t h any k n o w n r e d u c t a s e form. - T h e r e s u l t s suggest, that a r e d u c t a s e f o r m of 1 6 0 - 2 4 0 kDa is r e s p o n s i b l e for the 1,6DNP t o x i c i t y w h i c h is o b s e r v e d in V79 and H 4 I I E C 3 / G - cells, but not in 5L or H e p G 2 cells. GSF-Inst.

f. T o x i k o l o g i e ,

D - 8 0 4 2 N e u h e r b e r g FRG

R16 61 RAM SEMINAL VESICLE CELL CULTURES, AN I N VITRO MODEL FOR STUDIES OF PROSTAGLANDIN H SYNTHASE (PHS) CATALYZED OXIDATION OF XENOBIOTICA. J. Foth and G.H, Degen P r o s t a g l a n d i n H s y n t h a s e ( P H S ) - p e r o x i d a s e has been s u g g e s t e d t o p r o v i d e a l t e r n a t i v e metabolic activation o f c e r t a i n x e n o b i o t i c s i n t i s s u e s low in monooxygenases (MFO), b u t so f a r i t has been difficult t o a s s e s s t h e r o l e o f PHS in m e d i a t i n g a d v e r s e e f f e c t s i n c e l l s o r in v~vo, SEMV c e l l s d e r i v e d f r o m ram s e m i n a l v e s i c l e s c o n v e r t a r a c h i d o n i c a c i d (ARA) e f f i c i e n t l y to prostaglandins but lack MFO-activity. They were used t o c o n d u c t s t u d i e s on t h e PHS-mediated m e t a b o l i s m o f e s t r o g e n s w i t h t h e goal o f r e l a t i n g t h i s t o an e n d p o i n t f o r g e n o t o x i c i t y inducible in t h i s in v i t r o model. D i e t h y l s t i l b e s t r o l (14C-DES, 2 NM) i s c o n v e r t e d t o s u l f a t e c o n j u g a t e s and t o the oxidative metabolite Z,Z-dienestrol (Z,ZDIES) by SEMV c e l l c u l t u r e s . A d d i t i o n o f ARA o r i n d o m e t h a c i n (5 #M) i n c r e a s e d and i n h i b i t e d Z,ZDIES f o r m a t i o n by 60 and 50 %, r e s p e c t i v e l y , indicating free fatty acid availability and h y d r o p e r o x i d e s as f a c t o r s i n f l u e n c i n g DES-oxidat i o n by PHS. A c o m p a r i s o n o f ARA c o n v e r s i o n t o p r o s t a g l a n d i n s and o f DES o x i d a t i o n r a t e s f u r t h e r r e v e a l s t h a t DES i s c o o x i d i z e d a l t h o u g h e n d o g e nous c o m p e t i n g c o s u b s t r a t e s f o r PHS a r e p r e s e n t , The r e s u l t s d e m o n s t r a t e t h a t SEMV c e l l s a r e a u s e f u l t o o l f o r s t u d i e s on t h e PHS-dependent o x i d a t i o n o f DES; s i n c e i n d u c t i o n o f m i c r o n u c l e i by DES has a l s o been o b s e r v e d in t h i s model i t c o u l d a l s o p r o v i d e c l u e s as t o t h e r o l e o f PHS in m e d i a t i n g g e n o t o x i c i t y o f DES and o t h e r xenobiotics. S u p p o r t e d by t h e Deutsche F o r s c h u n g s g e m e i n s c h a f t Institute o f T o x i c o l o g y and P h a r m a c o l o g y , SFB 172, V e r s b a c h e r S t r . 9, D-87OO WGrzburg

62

EXPRESSION A N D REGULATION OF PHENOL UDP-GLUC U R O N O S Y L T R A N S F E R A S E IN HEPATOCYTE CULTURES, HEPATOMA (H4IIE) CELLS A N D IN U N T R A N S FORMED A N D TRANSFORMED RAT LIVER EPITHELIAL CELLS E. R6hrdanz and P. Mfinzel UDP-Glucuronosyltransferases (UGTs) are involved in detoxication and elimination of endobiotics and of a vast array of xenobiotics including carcinogens. One isozyme of this supergene family, called phenol UGT or UGTI, is expressed at a low level in hepatocytes, but inducible by 3-methylcholanthrene (MC)-type inducers and by multiple other factors. The enzyme levels appear to be high in H41IE hepatoma cells and in rat liver epithelial cells (RLEs). In order to study mechanisms responsible for this constitutively higher expression of UGTI, a UGTI-selective DNA probe was used. Two oligonucleotide primers, prepared according to the cDNA of UGTI, and isolated rat liver DNA were used to synthesize a 2$0bp fragment (nucleotide 71350) by the polymerase chain reaction. In the presence of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) UGTI mRNA level was increased 3-fold in primary hepatocytes. Persistently increased expression (7-fold) was observed in H4IIE cells and expression was further inducible by TCDD. Regulation of UGTI was also studied in RLEs (McMahon et al. 1986, Cancer Res. ~fi, 4665). UGTI-expression depended on cell density and passage number. Expression of UGTI in RLEs of low passage number was highest at day 3 after plating whereas enzyme activity reached its maximum at day 6. Aflatoxin B~transformed RLEs and RLEs of higher passages showed even higher UGTI mRNA levels than RLEs of low passage number. The results suggest that (in contrast to primary hepatocytes) H411E cells, RLEs and transformed RLEs show high persistent expression of UGTI, a behaviour which is also found at cancer prestages (hepatocyte foci and bepatocyte nodules). The cell lines may be useful to elucidate mechanisms responsible for high constitutive expression of the isozyme.

Institute of Toxicology, University of Tiibingen, WilhelmstraBe 56 D-7400 Tiibingen, Germany

63 Biotransformation in carcinogen-induced diploid and polyploid hepatocytes separated by centrifugal elutriation J. B. Watkins III I, D. Thierau 2 and L.R. Schwarz 2 Recently it has been shown that sequential treatment of rats with partial hepatectomy, DEN and 2-acetylaminofluorene (2-AAF)('Seglen protocol') induces the emergence of diploid hepatocytes, which are thought to contain precursor cells of hepatocellular carcinomas (Saeter et al Carcinog. ~,939, 1988). It is not known whether these cells exhibit the 'toxin-resistance phenotyp' characterized by decreased activities of cytochrome P-450 dependent monooxygenaees and increased activities of several phase II enzymes. TO address the question of whether biotransformation is altered in diploid hepatocytes induced according to the 'Seglen protocol', diploid and polyploid hepatocytes were separated from hepatocytes suspensions isolated from treated rats by centrifugal elutriation. The purity of diploid and polyploid cell fractions amounted to 90 and 79 %, respectively. Benzo(a)pyrene hydroxylase, ethoxyresorufin O-deethylase (EROD) and methoxycoumarin O-demethylaee (MCOD) activities were 30-40% lower in diploid than in polyploid cells. Activities of glutathione S-transferases towards CDNB, UDP-glucuronosyltransferases towards 3-hydroxybenzo(a)pyrene(3-OHGT) or 4-hydroxybiphenyl and DT-diaphorase did not differ in the two elutriated fractions. Treatment with 2-AAF for 3 weeks induced EROD and 3-OH-GT 4-6 fold and 2-fold, respectively and administration of phenobarbital for 4 d increased EROD and MCOD activity 2- and 4-fold, 3-OH-GT activity and glutathione conjugation 2- and 1.5-fold, respectively, in both diploid and polyploid hepatocytes. In conclusion, although there is some decrease in monooxygenase activities, carcinogen-induced diploid hepatocytes do not show the typical 'toxin-resistance phenotype'. This finding is compatible with the hypothesis that proliferation and promotion of diploid hepatocytes by 2-AAF is caused by non-toxic mechanisms. 1 GSF-Institut fHr Toxikologie, D-8042 Neuberberg/MHnchen 2 Indiana Univ. School of Medicine, Bloomington, IN 47405

64 FRESHLY ISOLATED AS WELL AS CRYOPRESERVEDHEPATOCYTES ARE SUITABLE IN VITRO MODELS FOR THE BIOTRANSFORMATIONOF NEBRACETAM IN THE MOUSE, RAT, RABBIT, DOG, MONKEYAND MAN IN VIVO K.L. P l a t t , H. RuBm, E. Molitor, H. Maith, D. Utesch, B. Diener, W. Tr6ger ~, K. Mendla~, and W. Pollmann ~ The biotransformation and disposition of nebracetam (4aminomethyl-l-benzylpyrrolidin-2-one) was determined in the mouse, rat, rabbit, dog, monkey, and in man by oral administration of the carbon-14 labelled drug (2-5 mg/kg body weight) and measurement of the renal (82 - 96%) and the fecal (2 - 15%) excretion; nebracetam and i t s metabol i t e s excreted via the urine were separated by reversedphase HPLC which revealed a metabolic conversion of 35 88% and marked species differences in the metabolite profiles. Freshly isolated hepatocytes (mouse, r a t , r a b b i t ) as well as cryopreserved hepatocytes (rabbit, dog, monkey, man) were incubated with nebracetam (I0 ~M, 2 m i l l i o n c e l l s / m l ) For up to 5 h followed by HPLC separation of the metabol i t e s . The r e s u l t of t h i s in v i t r o study exhibited good q u a l i t a t i v e and s a t i s f a c t o r y q u a n t i t a t i v e agreement with the in vivo metabolism; the dog being the animal species which most closely resembles man in i t s biotransformation of nebracetam. This example demonstrates that freshly isolated as well as cryopreserved bepatocytes are suitable in v i t r o metabolism systems f o r the prediction of the hepatic metabolism of a drug in vivo. We, therefore, propose to select the r e l e vant animal species f o r chronic t o x i c i t y testing of a newly developed drug by in v i t r o metabolism studies with isolated hepatocytes. Supported by the Bundesministerium fur Forschung und Technologie I n s t i t u t f~r Toxikologie der Universit~t, Obere Zahlbacher StraBe 67, 0-6500 Mainz Abteilung Biochemie, Boehringer Ingelheim KG, D-6507 Ingelheim

R17 65 METABOLISM MEDIATED CYTOTOXICITY DETECTED BY COCULTURES OF MICROCARRIER-ATTACHEDRAT HEPATOCYTES AND BALB/C 3T3 CELLS J.U.Voss and H.Seibert The t o x i c i t y of numerous chemicals is modified by biotransformation reactions in the l i v e r . To assess the influence of xenobiotic-metabolizing pathways on the t o x i c i t y of chemicals in v i t r o , several approaches have been applied (e.g. S9-mix, freshly isolated or primary cultured hepatocytes in coculture with target cells). In an e f f o r t to improve the application of rat hepatocytes in cocultures, we developed a method to cultivate hepatocytes on microcarriers. Liver cells isolated from male rats by two-step collagenase perfusion were inoculated with collagen-coated microcarriers (Cytodex 3, Pharmacia) in Waymouth 752/I supplemented with I0% FCS, insulin, amino acids and HEPES. 24 hours after inoculation, about 34% of cells had attached to microcarriers corresponding to 0.14 mg protein/mg dry weight of microcarriers. Cells retained the a b i l i t y to metabolize xenobiotics as judged by the a c t i v i t y of 7-Ethoxycoumarindeethylase (EOD) f o r at least 48 hours. In the present study microcarrierattached hepatocytes were cocultured with BALB/c 3T3 fibroblasts as target cells. The cytotoxic effects of cyclophosphamide (CPA) were studied. Incubation conditions were examined with respect to incubation time and hepatocyte: 3T3-cell r a t i o . In the presence of hepatocytes CPA lead to a dose-dependent growth retardation of 3T3 cells as measured by protein content/ well, whereas no toxic effects on the hepatocytes were observed. Microcarrierattached hepatocytes can become a valuable tool to assess the influence of metabolism on t o x i c i t y of chemicals in cocultures. Abt. Toxikologie, Universit~t Kiel, Brunswiker Str. lO, 2300 Kiel, FRG

67 METABOHC FATE OF METHYLENE CHLORIDE IN HUMAN BLOOD R. Thier, R. Kreiling#,H. Ottenw~ilder*

An increased incidence of pancreatic tumors in humans following occupational exposure to methylene chloride has been reported by the European Community. In contrast, long-term inhalative studies with high concentrations of 2,000 and 4,000 ppm substance resulted in formation of liver and lung tumors in mice, but not in rats. This implies the existence of a species difference in either the metabolism of methylene chloride or its organ distribution. The aim of the study presented here was to investigate the distribution of radioactively labelled methylene chloride in the different components of human, since blood is the main transport vehicle for inhaled xenobiotics preceeding organ distribution. Ten human blood samples of healthy volunteers (9 ml each) were individually incubated in 22 ml head space vials with either 10 ~tl methylene chloride spiked with 14-C-labelled substance with a spec. act. of 1.3 MBq/mmol, or 0.2 ml saturated aqueous solution of the pure radiolabelled substance with a spec. act. of 925 MBq/mmol. Aliquots of 0.5 ml were drawn in 30 rain intervals and fractionated into plasma and cellular compartments by differential centrifugation. The lymphocytes were isolated by density centrifugation through a separation medium. The erythrocytes were separated from the remaining cellular components by filtration through microcristalline cellulose. The erythrocytes were lysed by addition of an equal volume of distilled water and subsequently divided into cell membranes and high and low molecular weight fractions of cytoplasm with a cutoff at 10,000 Da. Plasma differentiated likewise. Radioactivity was determined by liquid scintillation counting. The results show the existence of two distinct subpopulations among humans in terms of distribution of radioactivity between the high and low molecular weight fractions of blood plasma. In five samples, group A, radioactivity increased significantly over time in the low (< 10,000 Da) and high (> 10,000 Da) molecular weight fractions. The other samples, group B, showed no increase of radioactivity in either fraction. Radioactivity was not detectable in the erythrocytes of either group. Additional experiments showed a coincidence of the two groups with ,,conjugators" and ,,nonconjugators" for monohalogenated methanes in human erythrocytes. This suggests a common metabolic principle and an enzyme polymorphism for methylene chloride in human blood plasma. Institut ffir Arbeitsphysiologie, Ardeystr 67 4600 Dortmund 1 ~Bundesmln.f. Jugend,Frauen,Famiheund Gesundheit,Deulschherrenst~.2, 5300Bonn2 ~Fa. Hoechst, Gewerbetoxikologie, Postfach 800320, 6230 Frankfurt 80

66 ENZYME POLYMORPHISM FOR METHYL BROMIDE IN HUMAN ERYTHROCYTES MODULATES ITS GENOTOXIC RISK K.R. Schr/Sder, T. Langhof, H. Peter The genotoxic risk induced by xenobiotics can be modulated by enzyme polymorphism. Methyl bromide is conjugated in human erythrocytes with glutathione by approximately 60 % of the human population (conjugators), the rest lacking this ability (non-conjugators). The genotoxic potential of methyl bromide has been attributed to a direct alkylation of DNA; a detoxification of the substance would therefore reduce this genotoxic risk. Since blood is the first compartment and major transport vehicle for inhaled xenobiotics after passage of the lung, metabolism in erythrocytes as in the case of the ,,conjugators" could have a pronounced protective effect. In order to verify an interindividual difference in the genotoxic effect of methyl bromide, individual whole blood samples of 5 non-conjugators and 5 conjugators (3.5ml each) were incubated in 22ml head-space vials with an initial concentration of 3.6 Ixmol (5000 ppm) methyl bromide for one hour. Substrate disappearance in the gas phase of the vials was controlled by gas chromatographyon a Perkin Elmer Sigma 5 GC equipped with a 5 m 1/8" stainless steel column packed with Tenax TA 30-60 mesh, gas sample loop 0.25 ml. Following the incubation, lymphocytes were isolated by density centrifugation through a separation medium. After 24 hours of cultivation in a specific medium at 37°C, bromodesoxyuridine was added and the cells incubated for further 46 hours• Colcemide was then added for termination of mitosis; 2 hours later the cells were fixed and stained on glass slides for microscopic determination of sister cbromatid exchanges (SCE). The rate of SCE was found to be significantly higher in the case of the nonconjugators. Furthermore the rate of SCE in the lymphocytes of the conjugators was not enhanced following the exposure to methyl bromide compared to non-incubated controls. This indicates a protective effect of glutathione conjugation of methyl bromide in human blood, and the influence of the enzyme polymorphism for genotoxic risks. A further characterization of the enzymatic factor responsible for this phenomenonis therefore necessary. Preliminary experiments give evidence for the involvement of an enzyme which accepts both glutathione and cysteine as cofactor, indicating the existence of an enzyme with both glutathione transferase activity and glutathione peroxidase activity. Instimt ftir Arbeitsphysiologie, Ardeystr. 67, 4600 Dortmund 1

68 Chromium (VI) reducing capacity of human blood plasma in vitro M.Capellmann, H.M.Bolt Since chromates play a considerable role in industrial application, e.g. in welding or tanning, biological monitoring of exposed workers is important. In the metabolism of chromium(VI) the reducing capacity of human blood plasma is of importance, because reduction of Cr(VI) is regarded as a detoxification step. In the case of plasma, ascorbic acid accounts for the majority of its reducing capacity. To prove this, chromate solutions of different concentrations (20-80 ng/ml) were added to solutions containing ascorbic acid in the "physiological" concentration range (5-20 gl/ml), simulating the normal conditions in plasma (0.2 M HEPES-buffer, pH 7.4) and were also added to plasma separated from freshly drawn blood. The incubation was carried out at 37 °C. From both systems ascorbic acid and its oxidized form dehydroascorbic acid, were analyzed simultaneously by H P L C - s e p a r a t i o n and post c o l u m n d e r i v a t i z a t i o n with 1,2phenylendiamindihydrochloride. The decrease of chromate was measured by flow injection analysis using diphenylcarbazide as derivating reagent. The kinetics of the model system show a reaction of 2nd order at higher concentrations, while at lower concentrations the reaction is apparently autocatalysed. The results obtained by spiking human blood plasma are similar, as shown in a comparison between the reaction curves of the model solutions and those received after spiking human blood plasma. Institut ftir Arbeitsphysiologie an der Universit~it Dortmund Ardeystr. 67 W-4600 Dortmund 1

R18 69 METABOLISM OF MEBENDAZOLE IN A MODIFIED PERFUSED GUT SYSTEM H. Gottmanns, R. Kroker, and F.R, Ungemach*

71 ISOLATED

CHIRAL EFFECTS IN THE METABOLISM OF 1,3-BUTADIENE IN MALE B6C3F1 MICE U. Hindermeier, S. Deutschmann, D. Wistuba*

The benzimidazole anthelmintic mebendazole which is known for its inter- and intraindividual varying bioavailability had recently been investigated by an isolated perfused rat gut system for its absorption and biotransformation. Poor absorption and strongly varying metabolism under different conditions had been demonstrated (1}. However the validity of this perfusion system is limited due to the failure of a continuous flow at the serosal side for the removal of the absorbate. Thus there is no sufficient (gut wall/serosal absorbate) concentration gradient as driving force for the absorption process. Therefore in a modified system of the isolated perfused gut it has been tried to substitute the cut off of serosal blood flow. Into the flexible tube connection between the two chambers of a perfusator according to Fisher and Parsons (2l a cannula was inserted which allows to supply the serosal side of the isolated perfused gut segment with a continuous definite flow of an adequate medium. The modification of the system resulted in an increased absorption of metabolites followed by further metabolic steps with a rise of resecretion of the metabolites into the gut lumen and enhanced secretion of metabolites (especially glucurenidal into the absorbate while the absorption of the parent substance remained almost unchanged. The results indicate an increased absorption of mebendazole metabolites with different patterns under continuous serosal flow without markedly affecting the bioavailability of the active parent substance. 1) Gottmanns et al., N.S. Arch. Pharmacol. 337, 44, 1988 2l Fisher and Parsons, J. Physiol. 110, 36-46, 194-9

The plastic monomer 1,3-butadiene has been classified as a possible human carcinogen by the IARC (International Agency for Research on Cancer). Long-term inhalation studies showed a carcinogenic effect down to a concentration of 6 ppm in mice but not in rats. In the experiments presented here, male B6C3F1 mice were exposed in an open system to a constant inhalative concentration of 1,000 ppm 1,3butadiene for 8 hours. After the exposure, the animals were transferred to a closed desiccator. One hour after the transfer, 60 ml samples of the atmosphere in the desiccator were drawn with a syringe and flushed through closed 22 ml head space vials, which were subsequently frozen with dry ice. For further analysis; the vials were later thawed and subjected to complexation gas chromatography on a 25 m x 0.3 mm glss capillary colum coated with 0.125 mol nickel(1])bis[(3-heptafluorobutanoyl)-(1R)-camphorate)] on SE 30; 90 C, 1.5 bar N2. The animals were found to exhale the reactive intermediate 1,2-epoxy-3-hutene, the ratio between the S- and the Renantiomer of the epoxide beeing 74.1 to 25.9 %. This supports findings by Wistuba et al. (Chirality 1, 127-136, 1989) that the metabolism of 1,3butadiene by cytoehrome P-450 of mice and rats in vitro results in the formation of 70 % S- and 30 % R-enantiomers of 1,2-epoxy-3-butene. The results/n vitro and/n vivo suggest that chirality may play an essential role for the genotoxicity of the reactive metabolites of 1,3-butadiene and may be responsible for the observed species differences. Following 7 hour inhalative exposure to 1,3-butadiene concentrations of 10, 50, 100, 250, 500, 1,000 and 2,000 ppm, the mice were sacrificed and liver, lung and heart removed and shock frozen in liquid nitrogen. After thawing, the organs were homogenized and deproteinized with 4% aqueous sulfosalicylic acid. The glutathione content was determined with Ellman's reagent. Above concentrations of 500 ppm 1,3-butadiene, a dose-dependent depletion of glutathione was observed in all organs investigated. This indicates that in mice, glutathione conjugation of 1,2-epoxy-3-butene, the chiral reactive intermediate of butadiene, does not influence the ratio of both enantiomers of this epoxide in exhaled air.

Bundesgesundheitsamt, Postfach 330013, 1000 Berlin 33, FRG *lnstitut fur Pharmakologie u. Toxikolikologie, Freie Universitit Berlin, Koserstr.20, 1000 Berlin 33, FRG

Institut ffir Arbeitsphysiologle, Ardeystr. 67, 4600 Dortmund 1 *Inst. ffir Organ. Chemic der Univ. Tiibingen, Auf der Morgenstelle 18,. 7400 Ttibingen

7O COUMARINMETABOLISM IN ISOLATED PERFUSED RAT LIVER Th. Huwer, H.-J. Altmann, W. Grunow ........................................................ The biotransformation of coumarin (Cou) was studied using a recirculating isolated perfused rat liver system. Liver vitality was ensured by normal appearance, activity of lactate dehydrogenase in perfusate, oxygen-consumption, perfusion rate and bile flow over the experimental period of 4 hr. 14-C-Cou was added to the perfusate to yield a final concentration of 50, 150, 500 or i000 ~mol/l. Examination of the perfusate by means of HPLC revealed rapid metabolism. In the lower dose experiments (50 or 150 ~M), Cou was completely metabolized after 90 or 120 min. In the higher dose experiments the concentration of Cou at the end of experiment was 6% (500 ~M) or 15% (I000 ~M) of the applied concentration, o-Hydroxyphenylacetic acid (OHPAA) proved to be the most prominent metabolite, representing about 50% of all metabolites. Furthermore, 3-hydroxycoumarin (3OHC), o-coumaric acid (CA), ohydroxyphenyllactic acid and traces of 7-hydroxycoumarin (7OHC) were identified. In addition, several unknown metabolites were also detected. Biliary elimination of Cou-equivalents was dosedependent, ranging from 7% (I000 ~M) to 35% (50 ~M) of dose with a maximum between 30 and 60 min. The major part of radioactivity in bile was associated with unknown metabolites. Besides, we detected unchanged Cou and conjugates of OHPAA, 3OHC, CA and 70HC. Final total glutathione content of the liver ranged between 0.12 ~mol/g in the highest dose and 3 ~mol/g in the control group. 4-Glutathione and 4-mercapturic acid conjugates, as well as Cou-3-mercapturate recently isolated from rat urine, were absent in bile and perfusate. 5-7% of the applied dose remained in liver at the end of perfusion. A dose-dependent covalent binding of Coumetabolites to protein was observed. Max-von-Pettenkofer-Institut, BGA, Thielallee 88-92, i000 Berlin 33

72 SPECIES SPECIFIC PHARMACOKINETICS OF 2-NITROPROPANE IN RABBITS,ANDIN RATS P. Stei. G. Csan~v*. J.G. Fitser The solvent 2-Nitropropane (2-NP) produced liver necrosis and hepatocarciname in each of f0 rats exposed to 207 ppm for 6 months. However, in 5 rabbits exposed under identical conditions no alterations were detected (Lewis et al., J. Environ• Path. Toxicol. 2: 233, 1979). In pharmacokinetic studies with rats we could distinguish two different metabolic pathways for 2-NP, a saturable and a non-saturable one (Denk et al., Arch. ToxicoL SuppL 13: 330, 1969). Based on several further studies, we concluded that especially the non-saturable pathway would lead to hepatotoxicity and hepatocarcinogenicity in contrast to the saturable one (Denk et el., Arch. Toxicol. Suppl. 13: 330; Denk and Deml, this meeting). In order to test this conclusion we studied the pharmacokinetics of 2-NP in the rabbit strain that had proved not sensitive in the carcinogenicity study mentioned above. New Zealand White rabbits of both sexes were individually exposed in closed chambers (65 I) to atmospheric concentrations of 2-NP. Some animals were pretreated i.p. with pyrazole (320 rng/kg body weight) to inhibit cytochrome P450 mediated metabolism. Different amounts of 2-NP were administered by single injections into the systems leading to initial concentrations between 1000 and 6000 ppm. Concentrations in the atmosphere were measured by gas chromatography. Pharmacokinetic analysis of the experimental concentrationtime courses was done as previously described (Kessler et al., NATO ASI Series A: Life Sciences 159: 123,1989). The thermodynamic partition coefficient whole body/air was 170. This value was similar to that obtained in rats. Like in rats the uptake of gaseous 2-NP was 100%, its rate was limited by the alveolar ventilation which in this species is referred to be about 60000 mYh per animal of 2500 g (Guyton, Am. J. Physiol. 150: 70, 1947)• Furthermore also in rabbits two different metabolic pathways have been found but in contrast to rats no sex differences. The pathways were a saturable (Vmax = 126 [.umol/h/kg body weight]) and a non-saturable one• After pretreatment with pyrazole, only the latter pathway was still detectable. The capacity of this pathway was smaller than that of the saturable one below 300 ppm. In contrast, above 300 ppm it exceeded that of the saturable pathway• In rats however, the relative capacity of the saturable pathway was much smaller: Here, at 180 ppm (females) and 60 ppm (males) already the rates of both pathways were equal. At higher concentrations more 2-NP was metabolized via the non-saturable pathway. These findings support strongly the hypothesis formulated above concerning the different hepatotoxic and hepatocarcinogenic effectiveness of the two metabolic pathways of 2-NP. *Central Research Institute of Chemistry, Hungadan Academy of Sciences, Budapest, Hungary GSF - Institut f0r Toxikologie, Ingolst&dter Landstra6e 1, D-8042 Neuherberg

73

75

PHARMACOKINETICSOF STYRENE-7,8-OXIDEIN MOUSE AND RAT

TRACE ANALYSIS OF ETHYLENE OXIDE IN AMBIENT AND ALVEOLAR AIR M. Leutbecher, B. Marczynski*, U. FOst

X Jiang and 1~ Kessler

Styrene-7,8-oxide(SO) is a first metabolite of styrene (S). SO was mutageNc and induced tumors exclusively in the forestomach of mice and rats after oral administration. The body burden of SO generated from S depends on the

Ethylene oxide, one of the major products of the chemical industries, is an air p ollutan! emitted by various sources like exhaust fumes and cigarette

1990, Gentner Verlag, in press). Here, we present pharmacokinetic data of SO in B6C3F1 mice and in Sprague-Dawleyrats: 1. after inhalation el S and 2. after intraperitoneal (ip) and oral (po) administrationof SO. The concentration of SO in blood was directly determined (Kessler etal., J.Chromatogr. Biomed.Appl., 1990, in press). 1. In mice and rats, we found saturation kinetics for the metabolism of S w i t h Vmax=15 [p.mol/h]and atm.conc, at Vmax/2=270 [ppm],calculated tot I mouse, and with Vmax=56 [umol/hl and atm.conc, at Vmax/2=lgn [nnml c..~l~ill~t~dfor

environmental and/or endogenous sources, it is essential to detect extremely small amounts of the substance and its precursor in ambient and alveolar air. The carcinogenic potency of ethylene oxide has clearly been proven in animal experiments. In Germany, the biological monitoring concept (EKA) favors the determination of ethylene oxide in alveolar air and whole blood samples; the methods proposed by the regulatory organ have however a detection limit of 0.5 PPm.

conditions. In

rats,

SO

concentrations correlated

with

metabolic rates

of

S.

However, no such correlation was found in mice: The blood concentrations of SO increased linearly at concentrations of S between 250 and 800 [ppm]. The blood concentration of SO was 8 [ng/ml] in mice and 20 [ng/ml] in rats after exposures to 20 [ppm] of S. After exposures to 800 [ppm] of S the concentration of SO in blood of mice (4700/rig/roll) was much higher than that in rats (340 [ng/ml]). Fromthese results we expect that mice being exposed to high concentrations of S will be more sensitive than rats to effects evoked by the metabolite SO.

w,um me stomacn, investigationsm wrcosnowed mat me hair-,re or ;50 at 3/~{.; depended directly on the pH-value: In H2Obidest.(pH 6.2) half-life was 445 [min], in 1 mM HCI it was 0.4 [min]. The low amount of SO found in blood after po administration may explain the exclusive induction of forestomach tumors observed in long-term studies in both species.

GSF- Institut for roxikologie, IngolsNdter Landstr. 1, 8042 Neuherberg

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capillary column packed with GS/Q, (J & W Sci., 0.5 mm i.d.). Two different methods were developed for these experiments. In the first case, ethylene oxide was reacted in aqueous solution with sodium pipefidinodithiocarbamate, a substance that selectively reacts with epoxides. The reaction products were extracted with methylene chloride and reacted with MSTFA(N-methyl-Ntrimethylsilyl-trifiuoroacetamide) togivethe volatileTMS derivatives. This solution was analyzed by GO/MS. Alternatively, air samples were condensed into a trap cooled with liquid nitrogen, reacted with hydrogen bromide and extracted with methylene chloride. The organic phase was analysed by GO/

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........................................................

enables a quantification and differentiation of ethylene oxide resulting from either endogenous or exogenous sources or metabolized from endogenous or exogenous ethylene.

Institut fur Arbeitsphysiologie, Ardeystr. 67, 4600 Dortmund 1 * BG Krankenanstalt Bergmannsheil, Gilsingstr. 14, 4630 Bochum 1

74

76

SIMULTANEOUS DETERMINATION OF MERCAPTURIC ACIDS AND THE ENANT/OMERS OF MANDELIC ACE) IN THE URINE OF HUMANS EXPOSED TO STYRF.NF.

2-NITROPROPANE: RELATIONSHIP BETWEEN INDUCTION OF PRENEOPLASTIC FOOl IN RAT LIVER AND METABOLISM ~ n ~ t , ~,4 = n ~ t

Styrene is metabolized in humans to styrene oxide, which is genotoxic in various test systems. Detoxification of this epoxide follows two major pathways, the excretory products being mandelic acid/phenylglyoxylic acid and mercapturic acids. Since the epoxide hydrolase pathway quantitatively overweighs the GSH-transferase pathway in humans, mandelic acid has been chosen as a biomonitor for occupational exposure to styrene. Both styrene oxide and mandelic acid are chiral molecules. Therefore, the enantiomers S- and R-mandelic acid were analyzed selectively in the urine of 20 workers exposed to styrene. For this purpose, the compounds were isolated from acidified urine by solid phase extraction on Porapak Q and subsequently eluted from the adsorbent with acetonitrile. The S- and Renantiomers of mandelic acid were quantified by capillary gas chromatography on a chiral column. In a parallel analysis of the same urine samples, mercapturic acids and mandelic acid were separated on 20x20 cm silicagel tic plates with fluorescence indicator. The two metaboiites were differentiated by selective staining. The mercapturic acids used as markers were synthesized in incubations of N-acetylcysteine with the pure enantiomers of styrene oxide and further characterized by NMR spectroscopy. The ratio between the S- and R- forms was found to vary between 1:1 and 4:1. R-styrene oxide is a stronger mntagen than the S- form in the Ames test, indicating that enantioselective preference could lead to a difference in susceptibility to genotoxic effects of styrene oxide, In rats, the GSH transferase pathway has been found to play an important role in the metabolism of styrene oxide. In humans, an enzyme polymorphism hasbeen described for GSH transferase m in the liver; thiswas testedwith styrene oxide as substrate, About 40% of the population were found to be deficient in the enzyme. In our experiments, interindividual differences in the urinary excretion of the mercapturic acids by the exposed workers support the existence of the enzyme polymorphism. An individually different detoxification of styrene oxide via the GSH-transferase pathway could lead to an influence on the biological effective dose of styrene oxide and thus to a modulation of the genotoxic effects.

,'-r~uropropane ~z-t'~w) is an i n a u s m a l SOlvent. innalatlve e x p o s u r e of rats to high concentrations of about 200 ppm c a u s e d hepatotoxicity and liver carcinomas, 25 ppm h o w e v e r were ineffective (Lewis etal., J. Environ. Pathol. Toxicol. 2: 233, 1979; Griffin etal., Ecotoxicol, Environ. Safety, 4: 267,1980). For determining the concentration-response relationship we studied the metabolism of 2-NP in Sprague-Dawley rats of both s e x e s at concentrations between 0 and 250 ppm (Denk etal., Arch. Toxicol. Suppl. 13, 1989), and the induction of

Institut ffir Arbeitsphysiologie an der Universiffit Dortmund, Ardeystr. 67, 4600 Dortmund 1 * Inst. f. Arbeits- & Sozialmedizin, Uni Ttibingen, Wilhelmstr. 27, 7400 Tfibingen

p r e n e o p l a s t i c liver foci using the rat liver foci b i o a s s a y described by Oesterle and Deml (J. Cancer Res. Olin. Oncol. 105:141, 1983). In adult rats, 2-NP was metabolized via two different pathways: a saturable one of low capacity and high affinity according to Michaelis-Menten kinetics, and a non-saturable one following first-order kinetics. Striking sex differences were observed in the kinetics of metabolism: Vmax of the saturable pathway w a s higher in females, w h e r e a s first-order metabolism w a s faster in m a l e s than in f e m a l e s leading to higher rates of total metabolism. The n u m b e r s of loci were found to be related to the exposure co ncentrations of 2-NP resulting in upwards concave curves. The shapes of these curves may be explained by the interference of the saturable with the non-saturable pathway. This study indicates that no threshold d o s e in the hepatocarcinogenic effect of 2-NP can be expected. However, the risk is relatively lower at low concentrations of 2-NP. GSF - Institut for Toxikologie, Ingolstgtdter kandstr. 1 D-8042 Neuherberg

R 20 77 LIPID PEROXIDATION AS A INDUCED HEPATOTOXlCITY. M. Younes* and O. Strubelt

MECHANISM OF VANADATE-

The toxicity of sodium orthovanadate (2 mmol/I) towards isolated perfused rat livers was studied. In livers from fasted rats, vanadate led to a release of cytosolic and mitochondrial enzymes, an accumulation of calcium in the liver, a marked depletion of glutathione and an enhanced release of it into the perfusate, as well as to an augmented formation and release of thiobarbituric acid reaction material. Furthermore, a marked inhibition of oxygen consumption and a progressive decrease in perfusate flow rate due to vanadate-induced vasoconstriction were observed. Control experiments with similarly reduced perfusion rates in the absence of vanadate led to a release of cytosolic but not of mitochondrial enzymes. Also, calcium accumulation and lipid peroxidation (LPO) were not evident, indicating that reduced flow rate was only partly responsible for vanadate-induced hepatotoxicity. Feeding the animals resulting in an activation of anaerobic energy conservation reactions strongly attenuated vanadate-induced toxicity indicating that the energy status of the liver is the main target of venadate. LPO and liver damage were inhibited in parallel by allopurinol and deferrioxamine. Together with the strong cor~lation between LPO and hepatotoxicity, this suggests that LPO plays a causative role in vanadate-induced liver damage. Ommission of calcium from the perfusate did not prevent hepatotoxic responses to vanadate, indicating that calcium influx is not involved in vanadate-induced toxicity towards the perfused rat liver.

79 CELLULAR INTERACTION IN RAT HEPATOCYTE / KUPFFER CELL COCULTURE H.Desel, G.Latocha, M.MQIler, A.Steding, C.Guthardt, & C.SchrSder Damage of liver parenchymal cells (HC) in inflammatory liver disease is influenced by components of the cellular defense system. The role of liver macrophages (Kupffer cells) is not yet understood in detail. We have studied the interaction between these cell types in a hepatocyte / Kupffer cell (KC) coculture (1:1). Rat hepatocytes were cultured for 24 h. Rat KC were separated by elutriation centrifugation and added to the HC culture. Experiments were done after 48 h of coculture. Superoxide release after stimulation with zymosan or phorbol myristate acetate is strongly increased in coculture as compared to KC monoculture. - Production of malondialdehyde (MDA) in cell homogenates is enhanced in stimulated cocultures as compared to the unstimulated control experiment. Gross hepatocyte damage as determined by release of lactate dehydrogenase (LDH) is not observed with and without KC stimulation. - The toxicity of exogenous H202 to HC is' markedly enhanced by the presence of KC. MDA production in coculture is 3-10 times the production in HC monoculture. This effect can further be enhanced by KCstimulation. LDH release is also increased in coculture. H202 decomposition by HC via catalase and/or glutathione peroxidase is slowed down in the presence of KC.

-

-

Institute of Toxicology, Medical University of L~ibeck, Ratzeburger Allee 160, D-2400 L0beck, FRG. *Present address: Max von Pettenkofer-lnstitute, BGA, D-1000 Berlin 33, FRG.

These data demonstrate mutual communication between HC and KC in vitro: HC stimulate superoxide production and KC damage HC directly and sensitise HC for exogenous reactive H202. Abt. Klinische Pharmakologie, Universit&t G6ttingen, Robert-Koch-Stra8e 40, 3400 G6ttingen

78 INCREASE IN BILIARY PERMEABILITY BY ENDOTOXIN ENHANCED BY GALACTOSAMINE AND ESTRADIOLVALERATE H. Krell, R. Schuster and W. Schr6ero

8O CONCENTRATION OF 2,3,7,8-TCDD IN THE LIVER OF JUVENILE RATS AFTER CONTINUOUS EXPOSURE Elisabeth Koch, Martin Mayer, Jutta Hartmann

In order to study the mechanisms involved in the development of endotoxin-induced hepatitis and cholestasis, rats were treated with endotoxin (0.3 mg/kg), galactosamine (650 mg/kg) and estradiolvalerate (i m g / k g / w e e k for 5 weeks). In anesthetized animals, bile flow began to decrease within 2 hours when endotoxin was given in combination with galactosamine. In estradiolvalerate-pretreated rats, bile flow was lowered 5 hours after administration of endotoxin. In isolated perfused livers of treated rats, biliary clearance of 14C-sucrose or 14C-inulin was analyzed and p a r a c e l l u l a r and transcellular portions, resp., were quantitated by off-kinetics after withdrawal of the radioactive marker from the perfusate (Biochem.J. 241:635,1987). Analysis of the approximately biexponential off-kinetics of the markers in bile,C = C1e-klt+C2e -kat, revealed that the paracellular portion of the clearance (CI) accounted for the increase. Using a model to describe the p a r a c e l l u l a r access of inert solutes in terms of bile flow (F), diffusion and convection, diffusion p e r m e a b i l i t y coefficient K and reflection factor o were determined by fitting to the data the equation C~ = (I-o)F/I-O e -(~-~)F/K. An increase in p e r m e a b i l i t y by endotoxin was enhanced by pretreatment with estradiolvalerate and by combined administration of galactosamine. It is concluded that increased paracellular permeability of the bile tract is an early alteration induced by endotoxin which may be involved in pathogenesis of hepat±tis and cholestasis. Pharmakologisches Institut der Universit~t THbingen, Wilhelmstr. 56, D-7400 T~bingen.

Female Wistar rats were treated with a single loading-dose of 20, 50, 120 and 250 ng 14C-TCDD/kg s.c. and weekly maintenancedoses of 1/5 of the loading-dose. The controls were treated with the solvent. The weekly maintenance-doses were administered 21 days prior to mating and during pregnancy and the lactation period. After weaning (at 3 weeks o f age) the Fl-generation were treated with the same dose regime (loading-/maintenance-dose). Offspring from each ~oup, 4 males and 4 females, aged 4, 8 and 12 weeks were sacrificed and the hepatic TCDD concentrations (radioactivity) were measured. The table presents the 14C-TCDD concentrations as pg/g wet tissue: Age(weeks)

Dose Regime(loadiag-/mainteaance-dose) TCDD-20/4 TCDD-50/10 TCDD-120/24 TCDD-250/50

4 8 12

218-* 35 147-* 43 135-.115

544-*134 249-+ 45 243-+ 31

980_+296 534+108 514-+ 54

2171-+669 1088-+294 969-*248

The dose regime used was capable of maintaining the TCDD levels in adult rats and those older than 8 weeks. However, during the phase of rapid growth (4th to 8th week) this dosing schedule was insufficient to keep the tissue concentrations of TCDD constant. The maintenance-dose had to be increased to weekly doses of 2/5 of the loading-dose.

Supported by a grantfrom the Bundesministerium fi~rForschung und Technologie Institut ffir Toxikologie und Embryopharmakologie, Freie Universit/it Berlin, Garystr. 5, D-1000 Berlin 33

R 21

81

83

BIOLOGICAL ACTIVITIES OF MIXTURES OF POLYCHLORINATED DIBENZO-P-DIOXINS (PCDDs) AND THEIR CONSTITUENTS IN HUMAN HEPG2 CELLS H.-P. Litm and D. Scbxenk Polychlorinated dibenzo-p-dioxins (PCDDs) are environmental contaminants which cause a variety of toxic symptoms including body weight loss and thymic atrophy in rats. A good correlation has been reported between the toxic potency of various PCDDs in vivo and their potency to induce P450IA1 activities in the H4IIE rat hepatoma cell line (Safe S., Ann. Rev. Pharmacol. Toxicol. 26, 371-399, 1986). In a previous study it was found that inducing potencies of PCDDs in H4IIE ceils and in rat hepatocytes are similar. However, little is known about the inducing potency of PCDDs in human cells. Therefore, induction of P450IAl-dependent 7-ethoxyresorufin O-deethylase (EROD) was investigated in the human hepatoma cell line HepG2. The ECs0-value for the most potent PCDD, 2378-CI,DD (TCDD), determined in HepG2 cells (706 pg/plate) was much higher than those determined in rat H4IIE cells (89 pg/plate) and rat hepatocytes (37 pg/plate). The relative potency of other PCDDs was calculated from their respective EC5o values and is given as TCDD equivalents fiE). A similar rank order of TE for the 2378-substituted PCDDs was obtained in human and rat ceils, though in HepG2 cells 12378-C15DD (TE 0.75) and 123478-C16DD (TE 0.75) were nearly as potent as 2378-C1,DD. Furthermore, CIsDD did not lead to detectable EROD induction in the human cell line (TE < 0.001). For a complex PCDD mixture containing 49 congeners the experimentally obtained ECg0-value was in good agreement with that calculated from the sum of its 2378-substituted PCDDs, suggesting additive effects of the 6 most potent PCDDs. However, results obtained with mixtures containing > 50% CI~DD suggest partial antagonistic effects of ClsDD.

BIOLOGICAL ACTIVITY OF TODD, TBrDD AND THREE 2,3,7,8-MIXED HALOGENATED DIBENZO-P-DIOXINS

_

_

Institute of Toxicology, University of Tiibingen, WilhelmstraBe 56, D-7400 Tiibingen, Germany

82 BIOLOGICAL EFFECTS AND TISSUE CONCENTRATIONS OF H7CDD AND OCDD IN FEMALE WISTAR RATS Georg Golor, Maria Korte, Thomas Wiesmiiller., Wolfgang K6rner*, Hanspanl Hagenmaler* We investigated the liver enzyme induction in female Wistar rats after subcutaneous injection of 1,2,3,4,6,7,8-heptachlorodibenzo-pdioxin (H7CDD) and octachlorodibenzo-p-dioxin (OCDD) and correlated the effects with tissue concentrations of the substances (ng/g wet weight). The chemicals were disolved in a toluene/ DMSO (1+2) mixture (HTCDD) or toluene only (OCDD). A dose of 250/~g OCDD/kg body wt was injected once a week for 10 weeks. EROD (= Ethoxyresorufin O-deethylase) activity in liver microsomes and OCDD concentrations in liver and adipose tissue were determinated at the end of the treatment period and during 13 weeks after the last injection. In the second series rats were treated with a single dose of 30 ~zg HTCDD/kg body wt, EROD activity and tissue concentrations were determined three weeks after treatment. Congener Liver conc. EROD activity in liver (ng/g (pmolesx mg wet weight) prot-1x min "1) controls (n=6) HTCDD (n=4) OCDD (n=3)

0.007 (H7CDD)* 0.030 (OCDD)* 260 -+ 80 2500 + 190

77 -+ 20 990 -+ 440 320 _+ 90

* Liver concentrations in pooled material from six rats. We conclude that repeated applications of OCDD induced EROD activity, however, the inductive potency of H7CDD is clearly more than 10 times higher than that of OCDD. Studies supported by grants from the Bundesministeriumfiir Forschung und Technologie(07VDX01). Institut far Toxikologie und Embryopharmakologie, Freie UniversitiitBerlin, Garystr. 5, 1000Berlin 33 *Institutfar Organische Chemie, UniversitiitTabingen,Auf der Morgenstelle 18, 7400 Tiibingen

T h o m a s Schulz-Schalge, Karl-Heinz Schwind*, Otto Hutzinger* The combustion of brominated and chlorinated scavengers in leaded gasoline leads to the emission of polyhalogenated dibenzodioxins (PHDD) and dibenzofurans (PHDF). Considerable amounts of 2,3,7,8-PHDD were detected in the exhaust of vehicles. The toxic potency of these compounds is unkown, therefore we investigated the ethoxyresorufin-O-deethylase (I=ROD), as an indicator for Ah-receptor mediated reactions, in hepatic microsomes of adult male Wistar rats. The animals received a single subcutaneous injection of 2 nmole/kg body wt of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TODD) or 2,3,7,8tet rabromedibenzo-p-dioxin (TBrDD), 2-bromo-3,7,8-t richlorodibenzo-pdioxin (B1C3), 2,3-dibromo-7,8-dichlorodibenzo-p-dioxin (B202), 2,3,7-tribromo-8-chlorodibenzo-p-dioxin (B301). The control rats received 100/~l/kg body wt of the vehicle (dimethylsulfoxide/toluene 2+1) only. The EROD induction was investigated 7, 14, 28, 42, 56, 70, 84, 98 days after administration. The following results were obtained.

Days

TODD

B1C3

B2C2

B3C1

TBrDD

7 5210 5850 4330 2730 6740 14 3740 3180 2910 2540 3840 28 1750 1600 n.d. 922 1800 42 903 874 696 653 1410 56 401 376 232 345 801 70 285 295 275 244. 546 84 169 130 131 226 371 98 162 169 105 132 410 control 31 (N = 35) , n.£1.= not determined values in pmoles resorufin x mg protein "'x min" (mean of three animals). The inductive potency of all investigated substances was similar, only B3C1 showed only about 50% of the EROD activity of TCDD seven days after application. 98 days after administration all substances, with the exception of TBrDD, showed almost the same EROD activity. TBrDD had at this time a 2.5times higher activity. These preliminary data suggest a comparable potency of EROD induction of all investigated 2,3,7,8-PHDDs and the necessity of consideration of these compounds for the calculation of toxic equivalency factors. These studies were supported by grant Nr. 07 VDX 019 from the Bundesministerium for Forschung und Technologie (BMFT). Institut for Toxikologie und Embtyopharmakologie, Freie Universit~t Berlin, GatystraBe 5, D- 1000 Berlin 33 *Lehrstuhl for 5kologische Chemie und Geochemie der Universit&t Bayreuth, Posffach 10 12 51, D-8580 Bayreuth

84 POLYCHLORINATED DIBENZO-p-DIOXINS (PCDDs) AND ETHINYLESTRADIOL AS GROWTH MODULATORS IN RAT HEPATOCYTE PRIMARY CULTURES A. Karger 2,3,7,8-C14DD (TCDD) has been shown to act as a liver tumor promotor in female rats (Pitot et al., Cancer Res. 40, 3616-3620, 1980). To elucidate mechanisms responsible for this sex specific tumor promoting activity, the influence of 2,3,7,8-C14DD, 1,2,3,4,6,7,8-C1~DD (HCDD) and C18DD (OCDD, containing 0.5% HCDD) on EGF-stimulated DNA synthesis was studied in primary hepatocyte cultures of male and female rats in the presence of 5 % fetal calf serum. Addition of TCDD (maximal effect at 10 ~2M), HCDD (maximal effect at 10-" M), and OCDD (maximal effect at 10.9 M) increased DNA synthesis 30-50% in a strictly EGF-dependent manner, the rank order of potency suggesting involvement of the Ah receptor. Induction of P4501Al-dependent 7-ethoxyresorufin O-deethylase activity occurred at PCDD concentrations which were higher than those leading to maximal stimulation of DNA synthesis. Addition of ethinylestradiol fiLrther increased TCDD-mediated stimulation of DNA synthesis. This effect was variable. However, the hepatocyte preparations responding strongly to TCDD also responded strongest to ethinylestradiol. In the presence of 3 x 10-~2 M TCDD a 2.5-fold stimulation of DNA synthesis was obtained at 20/zM ethinylestradiol. The results indicate that PCDDs enhance EGF-stimulated DNA synthesis in rat hepatocytes in the rank order of their binding affmity to the Ah receptor. Furthermore, synergistic effects of ethinylestradiol suggest that estrogens facilitate tumor promoting actions of PCDDs. Institute of Toxicology, University of Tiibingen, Wilhehnstral~e 56, D-7400 Tiibingen, Germany

R 22 85

87

EFFECTS OF UVER TUMOR PROMOTERS ON THE ACTION OF GROWTH

INDUCTION OF E T H O X Y R E S O R U F I N - O - D E E T H Y L A S E ACTIVITY BY VARIOUS AROMATIC AMINES DOES NOT CORRELATE WITH THEIR AH RECEPTOR AFFINITY P. Cikryt and W. Muster

FACTORS IN PRIMARY RAT HEPATOCYTES AND H411ECELLS

D.Wdlfle and K.Heidelberg The influence of liver tumor promoters on the regulation of DNA synthesis was studied in primary rat hepatocytes using 2,3,7,8-tetrachiorodibenzo-p-dioxin and 3,3',4,4'-tetrachlorobiphenyl

(TODD)

(TCB), known as inducers of drug metabolizing

enzymes. The plalotropic response of these agents includes the interaction w~th receptors of a variety of growth factors. We, therefore, investigated the effects of TODD and TCB on the regulation of growth-factor stimulated DNA synthesis in serum-free culturas of adult rat hepatocytes and H411Ehepstoma cells. The maximal stimulation of DNA synthesis of hepatocytes after 2 days in culture was achieved at 10"12M TODD and 10"7M TCB. These effects were dependent on the presence of insulin (10~8M) and dexamethasone (10"8M) in the culture media. The growth stimulation of hepatocytes by epidermal growth factor or alpha 1-adrenergic agents, e,g. phenylephrine, was further enhanced by TODD or TCB. On the other hand, no significant effect of these tumor promoters was observed on the growth of H411Ecells in hormone- or serum-supplemented media. Thus, the results suggest that TODD or TCB in stimulating the DNA synthesis of normal hepatocytes interact with growth factors and that very low concentrations of these tumor promoters, i.e. several orders of magnitude below those necessary for maximal enzyme induction, are active.

Dept of Toxicology, UniversityHamburgMedical School, Grindelallee 117, D-2000 Hamburg 13, F.R.G. Suppoded by a grant from Deutsche Forschungsgemalnschaft (DFG)

The induction of a number of drug-metabolizing enzymes by polycyclic aromatic hydrocarbons seems to be mediated by a cytosolic protein: the aromatic hydrocarbon (Ah) receptor. One of the most thoroughly studied Ah receptor-mediated responses is the induction of P4501A1 which can be easily monitored by the rate of ethoxyresorufinO-deethylation (EROD).The aromatic amines 2-acetylaminofluorene (2AAF) and 4-acetylaminofluorene (4AAF) differ in their toxic and carcinogenic properties. 2AAF is a wellknown inducer of drug-metabolizing enzymes, but 4AAF has not been characterized in this respect. Recently, we have shown that 2AAF binds to the Ah receptor and 4AAF does not. The objective of this study was to compare the inducing properties of 2AAF and 4AAF on two microsomal enzyme activites, namely EROD and pentoxyresorufin-O-deethylation (PROD) in male Wistar rats. 2AAF (0.02 % in the diet) and 4AAF (0.1% in the diet) were fed for 2, 7 and 21 days. The results demonstrate that both compounds are 'mixed-type' inducers of P450 enzymes both of the '3methylcholanthrene-type' and of the 'phenobarbital-type'. The inducing capacity of 2AAF was weak compared to 4AAF. 2AAF increased both EROD and PROD activity at most 2-fold whereas 4AAF induced EROD activity 11-fold (after 2 days of treatment) and PROD activity 56fold (7 days). The specificity of P4501A1 induction by 2AAF and 4AAF was proved by Western blot with enzyme-specific antibodies. We have also tested the Ah receptor affinity of a number of compounds which, according to the literature, are inducers of EROD activity in vivo. These compounds, like the heterocyclic aromatic amines 2amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-aminodipyrido(1,2a:3',2'-d)imidazole (Glu P-2) and 3-aminodipyrido(1,2-a:3',2'd)imidazole (Glu P-3) and the 3,3'-dichlorobenzidine homologue 4,4'methylenebis(2-chloroanilin) (MOCA), do not bind to the Ah receptor in vitro, but do induce EROD activity in vivo. The mechanism of P4501A1 induction by these chemicals is not clear. In conclusion, the induction of EROD activity in vivo, especially in the case of weak inducers, does not prove an interaction with the Ah receptor in vivo. Institute of Toxicology, University of W0rzburg, Versbacher Str. 9, W-8700 W0rzburg, Germany.

86

88

EFFECTS OF THE LIVER TUMOR PROMOTER 2,3,7,8-TETRACHLORODIBENZO-

THE ROLE

P-DIOXIN (TCDD) ON THE REGULATION OF PROTEIN KINASE C AND INOSITOLPHOSPHATE METABOLISM OF RAT HEPATOCYTES IN VITRO

C.Schmu~teand E.Becker To investigate the modulation of signal transfer pathways of hepatocytes by tumor promoters in vitrq, TODD was chosen as a prototyp of polychlorinated

aromatic

hydrocarbons which bind to a cytosolic receptor protein (Ah receptor). Furthermore, TODD activates protein kinase C (PKC) in the liver (Bombick st al., Biochem. Biophys. Res. Commun. 127:296, 1985). This enzyme is known to be involved in the regulation of agonist-stimulated inosito~trisphosphate (IP3) formation. The influence of low TODD concentrations (10"12M) on the PKC activity was studied in freshly isolated rat hepatocytes and in primary hepatocytes after 24 h in serum-free culture. TODD was found to be a poor activator of PKC compared to 12-O-tetradscanoylphorbol-13acetat (TPA,10"7M). In contrast to TPA, no inhibition of vasopressin-sfimulated IP3 formation was observed after a short-term treatment (15 rain) with TODD. Long-term treatment (18 h) of primary hepatocytes with TODD (10"12M) resulted only in a small stimulation of vasopressin-mediated IP3 formation (1,5 fold) compared to TPA (2 to 3 fold). Thus, the results indicste that the effects of TODD on PKC activities and IP3 formation are different from those of TPA and may be regarded as indirect actions of TODD.

DepL of Toxicology, UniversityHamburgMedical School, Grindelallee 117, D-2000 Hamburg 13, F.R.G. Supported by a grant from Deutsche Forschungsgemeinschaft (DFG)

OF ACUTE AND CHRONIC TOXICITY OF CARCINOGENIC AROMATIC AMINES IN RAT LIVER S. Ambs and H.-G. Neumann 2-Acetylaminofluorene (AAF, initiator and promotor) is a complete rat liver carcinogen. 2-Acetylaminophenanthrene (AAP) and 4-acetylaminostilbene (AAS) are incomplete rat liver carcinogens (initiatior). We studied the role of cytotoxicity for the promoting activity of AAF. Male Wistar rats were chronically fed 0.02% AAF for up to 16 weeks. The food intake of the animals decreased. Growth stopped. Various biochemical parameters were measured in blood, but the results could not explain the pathological changes seen in the liver after 8-12 weeks. Glucose-6-phosphatase activity in liver, decreased with a minimum after 5 weeks (histological diagnosis: no focal event). Serum levels of the thyroid hormons T3 and T4 declined with a minimum after 8 weeks. Experiments with isolated perfused livers taken from animals fed AAF showed that the efflux of glucose into the perfusate and that of glutathione into the bile were lower than in controls. Menadione caused a much higher glutathione efflux in livers from control animals than in dosed animals, although the glutathione content in treated animals was higher. Oxygen consumption in livers from dosed animals was ca. 20% higher than in control animals. Therefore~ we investigated the putative role of mitochondria for the acute and chronic toxicity of AAF. The uptake of ADP into mitochondria from control animals was inhibited by the N-acetoxy derivative of AAF, but not by those of AAS and AAp. The ADP-uptake was also reduced in AAFpretreated animals with a minimum after 3-4 weeks. The phosphate uptake was impaired by all hydroxamic acid esters and by N-hydroxy-AAF. The results are consistent with the hypothesis that AAF produces acute effects which do not lead to overt toxicity, but rather represent alterations in biochemical homeostasis. If this situation is maintained it may lead to chronic toxicity. Mitochondria might be targets for such primary effects. Present address: Institute of Toxicology, University of W~rzburg, Versbacherstr.9, D-8700 W~rzburg

R 23 89 DIFFERENCES IN INITIATING AND PROMOTING ACTIVITY OF AZO DYE ISOMERS IN RAT LIVER. G. Werle-Schneider, A. Wolf*, and W. Kunz. The azo dye 4-dimethylaminoazobenzene (4-DAB) is a very potent carcinogen in rodent liver with extremely short tumor induction times at high exposure levels. We have hypothesised that this effect is due to additional "intrinsic" promoting activity of the weakly initiating agent present at high dose levels. In this study we have extended these investigations using the azo dye isomers 3'-methyl-4-DAB (3'-MeDAB) and 2-methyl-4-DAB (2-Me-DAB) designated as "complete" and "incomplete" carcinogens based on their differential carcinogenic profile. As parameters of hepatocarcinogenic response, number and volume of enzyme-altered foci (EAF) in livers of rats treated with 3'Me-DAB and 2-Me-DAB were determined. The initiating potency of the two azo dyes was tested by brief exposure of rats to the test compounds followed by continuous treatment with the promotor phenobarbital. The promoting activity was analysed by continuous azo dye treatment of rats subsequent to a limited exposure to diethylnitrosamine (DEN) as an initiator. Our results demonstrate for both compounds similar initiating potencies but considerable differences in promoting activity. While 3'-Me-DAB led to an increase in the number and especially the size of EAF initiated by DEN, 2-Me-DAB did not exhibit such promoting effects. In different strains of Salmonella typhimurium, 3'-Me-DAB and 2-Me-DAB showed qualitatively and quantitatively similar mutagenicity profiles demonstrating a correlation between mutagenic effects in vitro and initiating activity in vivo, rather than between mutagenicity and overall carcinogenity of the two azo dyes. Treatment with 3'-Me-DAB led to elevated levels of reactive oxygen in liver microsomes in vitro and in rat liver in vivo indicating that oxygen radicals may be involved in the "promoting" activity of this azo dye. *Present adress: Department of Toxicology, Sandoz Pharma Ltd., Basel, Schweiz Deutsches Krebsforschungszentrum, Institut ffir Experimentelle Pathologie, Im Neuenheimer Feld 280, D-6900 Heidelberg.

9O

DIFFERENT SENSITIVITY OF THREE RAT TOWARDS INITIATING AGENTS D. Oesterle I and B. Schlatterer 2

LIVER

FOCI

8IOASSAYS

Three different protocols for rat liver loci bioassays have been compared with respect to their sensitivity in revealing the carcinogenic potential of chemicals: The initiation/promotion schedules according to Oesterle and Deml ( A ) , according to Pereira (B), and the initiation/ selection protocol according to Tatematsu et al. (C) (Oesterle et al., Carcinogenesis, i~0, 1891, 1989). As biological parameters preneoplastic foci were evaluated histochemically. They were identified by ATPase-deficiency and by reactivation of GGTase on serial sections. We compared six initiating agents of different carcinogenic potency in two doses: 4-aminobiphenyl (4-AB; 50 and 100 mg/kg body weight), dimethylbenzanthracene (DMBA; 25 and 50 mg/kg body weight), N-nitrosomorpholine (N-NM; 50 and 100 mg/kg body weight), diethylnitrosamine (DEN; i0 and 30 mg/kg body weight) all applied one time, and 2-acetylaminofluorene (2-AAF; 20 and 50mg/kg body weight), and benzidine (BZ; i0 and 20 mg/ kg body weight), applied five times. With all protocols the .strong initiator DEN caused the highest foci incidence, followed by N-NM, DMBA, 4-AB, 2-AAF and BZ. In (B) DMBA was more effective than N-NM. With ATPase as marker, protocol (A) was found to be most sensitive in view of number and size of the foci. With GGTase as marker, protocol (B) was most sensitive for the number, except of N-NM and DEN, here the higher numbers were found with (A). For foci area protocol (C) was most effective for half of the experiments, followed by (A) and (B). A dose~dependent effect was more often observed in protocols (B) and (C). In conclusion: From the three protocols used in this comparison, protocol (A) was found to be most suitable for the detection of the initiating potency of chemical carcinogens. IGSF - Forschungszentrum fur Umwelt und Gesundheit, Institut fur Toxikologie, D-8042 Neuherberg, 2Umweltbundesamt, D-1000 Berlin, FRG

92

ENZYMES OF THE CARBOHYDRATE M E T A B O L I S M AS M A R K E R S FOR FOCI TREATMENT WITH DEN AND CLOPHEN.A 1C. Einig, IE. Eigenbrodt, 3U. G e r b r a c h t

AND XENOBIOTIC INCIDENCE AFTER 50.

The rat l i v e r foci b i o a s s a y is w i d e s p r e a d l y u s e d as an in v i v o t e s t s y s t e m for the i d e n t i f i c a t i o n of chemically induced hepatocarcinogenesis. F o l l o w i n g the two s t a g e m o d e l of carcinogenesis w h i c h i m p l i c a t e the t r e a t m e n t w i t h an i n i t i a t o r and a p r o m o t o r , the rats r e c e i v e d o n c e i0 m g / k g b.wt of diethylnitrosamine (DEN) which is a strong liver-carcinogenic chemical. After 7 days the a n i m a l s w e r e t r e a t e d twice a w e e k w i t h the p r o m o t e r C l o p h e n A 50 (10 m g / k g b.wt) for the following i0 weeks. After the e x p e r i m e n t the rats w e r e k i l l e d a n d the l i v e r c r y o s t a t s l i c e s were stainded immunhistochemically or cytochemically. The enzymes lactate-dehydrogenase (LDH) and glucose-6-phosphate-dehydrogenase (G6PDH) of the carbohydrate metabolism and glutathion-S-transferase (GST) of the x e n o b i o t i c pathway were used for the endpoint determination. The m o s t of the foci w e r e r e c o g n i z e d in the animal group which had received the complete treatment schedule in c o n t r a s t to the groups which were treated only with DEN or Clophen A 50. A l s o the r e l a t i v e size distrib u t i o n of the foci i n d i c a t e that in the g r o u p w h i c h r e c e i v e d the c o m p l e t e s c h e d u l e the i s l a n d s were significantly i n c r e a s e d . T h e m o s t loci w e r e stained with GST suggesting t h a t this e n z y m e is e x p r e s s e d d u r i n g the e a r l y s t a g e of hepatocarcinogenesis. 1 Vet. Biochemie Universit~t Gie~en Frankfurterstr. i00 6300 G i e S e n 3

91

Pharmbiodyn

Denzlingen

EFFECT OF NAFENOPIN ON CELL TURNOVER AND THE EXPRESSION OF PEROXISOMAL ENZYMES, CYTOCHROME P-452, GLUTATHIONE-TRA~SFERASE-ISOENZYMES IN WE~I[L¥ B A S O P H I L I C F O C I O F R A T LIttER B. K r a u p p - G r a s l *

Peroxisome inducing agents, s u c h as n a f e n o p i n (NAF), are h e p a t o c a r c i n o g e n s in l i f e - t i m e r o d e n t e x p e r i m e n t s , In v a r i o u s a s s a y s p e r o x i s o m e p r o l i ferators are not genotoxic or tumor-initiators. In o u r r e c e n t experiments NAF enhanced tumor f o r m a t i o n t h r o u g h p r o m o t i o n of: i. A F B l - i n i t i a t e d l o c i in l i v e r s of y o u n g r a t s a n d 2. " s p o n taneous" f o c i w h i c h a p p e a r in l i v e r s of a g i n g rats. M o s t of t h e f o c i a n d t u m o r s s e e n in N A F t r e a t e d l i v e r s w e r e of w e a k c y t o p l a s m a t i c basophilia. Their phenotype was different from that of eosinophilic-clear cell and tigroid loci. Foci were further characterized by: i. d e t e r mination of their proliferative capacity as judged by DNA-synthesis and apoptotic activity, 2. e x p r e s s i o n of N A F - i n d u c i b l e peroxisomal Bo x i d a t i o n e n z y m e s a n d c y t o c h r o m e P-452, a n d G S H transferase-isoenzymes. Like tigroid loci but unlike eosinophilic-clear cell foci, weakly basophilic loci in N A F - t r e a t e d livers do not express placentar GSH-transferase. The express i o n of o t h e r G S H - t r a n s f e r a s e - i s o e n z y m e s in all f o c i s u b t y p e s is v a r i a b l e . T h e s a m e a p p l i e s for t h e e x p r e s s i o n of p e r o x i s o m a l e n z y m e s a n d c y t o c h r o m e P-452. H o w e v e r , t h e r a t e of D N A - s y n t h e s i s and apoptotic activity is highest in w e a k l y basophilic foci suggesting an increased cell t u r n o v e r in t h i s l i k e l y t u m o r p r e c u r s o r lesion. * Present address: Institut fur TumorbiologieKrebsforschung, Borschkegasse 8a, A-1090 Wien

R 24 93 ROLE OF PRO AND M A T U R E T G F B - I IN C E L L D E A T H (APOPTOSIS) OF HEPATOCYTES Fr. Oberhammer, R. Sedivy, R. Jirtle*, R. Schulte-Hermann . . . . . . . . . . . . . . . . . . . . . . . .

!

95 METALLOTHIONEIN, COPPER AND ZINC IN HUMAN LIVER DURING PRENATAL AND POSTNATAL DEVELOPMENT D. Klein a n d K. H. S u m m e r

. . . . . . . . . . . . . . . . . . . . . . .

Administration of non-genotoxic 'compounds stimulates DNA synthesis and liver growth due to both hyperplasia and hypertrophy. Upon withdrawal of this growth stimuli the increase in organ mass is found to be reversible. Thus in a study with cyproteroneacetate about 30-40% of liver enlargement disappeared within six days after cessation of treatment; this was accompanied by cell elimination through apoptosis. As the proteins of the TGF-B family are involved in organ involution (Mullerian Duct, prostate) we investigated the occurrence of TGF-BI (pro and mature part of the protein) during this regression by immunohistochemistry. The majority of apoptoses stained positive for the pro part of TGF-B suggesting a possible role for TGF-BI in this process. Further studies with isolated cultured hepatocytes revealed that apoptosis can be induced by TGFBI. This findings indicate that TGF-BI may function as one of the signal factors during apoptosis. Institut ffir Tumorbiologie-Krebsforschung Borschkegasse 8a, A-1090 Wien *Duke University, Medical Center, Durham, NC

94 G E N E X P R E E S S I O N A S S O C I A T E D W I T H APOPTOSIS ("PROGRAMMED" C E L L DEATH)

W. Bursch I, L. Fesus 2 and M. Tenniswood 3 Apoptosis is a type of cell death involved in the maintenance of cell number homeostasis in tissues. Apoptosis is regulated by a complex interaction between extrinsic (e.g. hormones, growth factors) and intrinsic (e.g. cell cycle related) factors; disturbance of its regulation appears to be involved in teratogenesis, carcinogenesis and immunosuppression. In the present study, the expression of tissue transglutaminase (TGase) and "testosterone repressed prostate message" (TRPM-2) during apoptosis was investigated. Apoptosis occurring during regression of hormonally induced rat liver hyperplasia and in mouse lymphoma cell cultures after glucocorticoid treatment were found to be associated with TGase and TRPM-2 expression. The possible role of the gene products in the apoptotic process will be discussed. IInstitut ffir Tumorbiologie-Krebsforschung Borschkegasse 8a, A-1090 Wien 2University of Debrecen, Hungary 3University of Ottawa, Canada

The fetus a n d n e o n a t a l offspring have high d e m a n d s for Cu a n d Zn. Since little information is available o n the role of metallothionein (MT) in the homeostatic control of t h e s e m e t a l s d u r i n g development, cytosolic MT, total a n d cytosolic Cu a n d Zn, a n d the portion of MT w h i c h b i n d s Cu (Cu-load of MT) were investigated in fetal a n d n e o n a t a l h u m a n liver. Liver samples were obtained from p r e t e r m a b o r t u s e s (22, 24 a n d 32 gestational weeks) a n d from children (2-15 months) whose d e a t h was due to s u d d e n i n f a n t d e a t h syndrome. Histological a n d pathological findings of the livers were normal. MT a n d m e t a l s were d e t e r m i n e d with the recently developed thiomolybdate a s s a y (1) a n d AAS, respectively. The MT-content was higher in fetal t h a n in n e o n a t a l liver. There w a s a linear correlation (r=0.996) between cytosolic MT a n d Zn in b o t h fetal a n d n e o n a t a l liver b u t n o t between MT a n d Cu. In c o n t r a s t to fetal liver, t h e Cu-load of MT in n e o n a t a l liver seems to be determined by the Z n / C u ratio in the cytosol. Whereas the fraction of cytosolic Z n r e m a i n e d c o n s t a n t at 66°/6 of the total, i n d e p e n d e n t of the stage of development, the fraction of cytosolic Cu increased from 26% in preterm liver to a b o u t 100% within 12 m o n t h s postnatally. These results suggest t h a t MT is involved in the regulation of Cu a n d Zn metabolism d u r i n g fetal a n d n e o n a t a l development. 1) Klein et al. (1990). Anal. Biochem. 189, 35-39 GSF-Institut ffir Toxikologie, D-8042 Neuherberg

96 NEPHROTOXICITY AND MYELOTOXICITY OF NEW ANTINEOPLASTIC PLATINUM (II) AND PLATINUM (IV) COMPLEXES IN RATS U. Horn, A. H~rtl, W. Neuhaus, U. St6ckel, H.-P. Schr6er, and H. Hoffmann In comparison with cis-DDP four new platinum (II) and platinum (IV) complexes (cis-diammineplatinum(II)-lactate; cis-diammine-platinum(II)lactate,L; cis-diammine-platinum(II)-dilactate; trans-dihydroxy-cis-dichlorodiammine-platinum(IV)) were evaluated for their acute nephrotoxic and myelotoxic ~otency in male rats (Shoe:WIST) following l.v. administration of maximum tolerated doses on 5 consecutive days. Parameters for nephrotoxicity determined on day 6, 13 and 22 after the first administration of the drug included blood urea nitrogen, serum creatinine, urine volume, urinary glucose and tubule cell excretion. Parameters for myelotoxicity determined on the same days included leucocytes, platelets, hemoglobin and hematocrit. Cis-DDP was found to be the most nephrotoxic compound. The myelotoxicity of the Pt(II) complexes appeared to be similar to that of cisDDP. In contrast the myelotoxic effect of the pt(IV) complex was of minor importance. Department of Pharmacology and Toxicology, Institute of Microbiology and Experimental Therapy, Beutenbergstrasse ii, 0-6900 Jena

R 25

97

99

E F F E C T OF ARSENICALS ON GLUCOSE U T I L I Z A T I O N IN MDCK-CELLS

HEAVY METAL IONS INFLUENCE THE HISTAMINE SECRETION FROM HUMAN ADENOIDAL MAST CELLS S,Bent, W.Schmutzler

B. LieN, H. MiJckter, E. Doldea, B. Fichfl Impaired carbohydrate utilization has been demonstrated in acute arsenic poisorting. The ensuing energy depletion has mainly been attributed to an inhibition of cellnlar pyruvate dehydrogenase although effects of arsenic on other metabolic pathways may be likewise important. Cultured ceils offer a suitable experimental system to trace the utilization of extracellular carbohydrates in the presence of arsenic. We have investigated the effect of organic (oxophenylarsine; PhAsO) and inorganic (As203) arsenicals on the availability of glucose to Madin-Darby canine kidney (MDCK) cells. Following a 3 h preincubation period with glucose-free Hanks' buffer MDCK cells (adherent or in suspension) were incubated (10 - 60 min, 37°C) with PhAsO (10-7 - 10-3 mniB) or As203 (10-6 - 10-2 tool/l) in the presence of D-glucose (0.01-25 mmolB) using D-[6-14C]-glucose as tracer. Cell-associated radioactivity, dye exclusion, extracellular LDH activity and potassium release were determined. A concentration-dependent inhibition of tracer accumulation was observed with both arsenicals suggesting an impaired uptake of glucose. With As 203 cytotoxic concentrations were required for half-maximum inhibition (IC50 0.5 mmolB). On the other hand, PhAsO inhibited glucose uptake in the micromolar range (IC50 10 p-molB). At these concentrations cellular viability as assessed by dye exclusion, LDH and potassium release, and cell morphology was not affected within the experimentaltimescale. We conclude that inhibition of glucose uptake may contribute to the increased acute toxicity of organic arsenicals by further aggravating the depletion of intracellular carbohydrates.

In peritoneal rat mast ceils histamine secretion is modulated by some heavy metal ions (Wieczorek et al. Allergologie 12, 158-160, 1989). Because of the known species specific differences we tested the effects of water soluble salts of mercury (HgC12), lead [Pb(CH~COO)~], cadmium (CdSO4) and bismuth [BiO(CIO4)] on histamine release from human adehoidal mast cells. The mast cells were isolated mechanically (Schmutzler et al. Int. Archs. Allergy appl. Immun. 77, 177-178, 1985). Histamine secretion was stimulated by Concanavalin A (Con A; 50mg/1) and was quantitated by the double isotope method. Mercury increased the histamine releas~e in stimulated and unstimulated cells at a concentration of 10- " M by at least 41% of the controll values. Cadmium exerted a biphasic reaction. At 10-6 M the spontaneous and Con A induced histamine release was inhibited by 13% and 24%. At the concentration of 1044 M cadmium resembled the effects of mercury with an increased release of 25% ~nd 16%. Lead showed a different pattern: At 10T M M it enhanced the histamine release by 30% in stimulated and by 55% in unstimul~tted mast ceils. It decreased the spontaneous mediator release at 10-" M but influenced the stimulated release only marginally. Bismuth decreased the histamine release from mast cells at 10-4 M by 18% and 32%. These data show that heavy metal ions influence allergic histamine release differently. Lead increases the mediator release at concentrations measurable in human blood. The bismuth induced inhibition might contribute to its therapeutical effectiveness in gastric ulcerations. lnstitut for Pharmakologie, Medizinische Fakult~it der Aachen, D-5100 Aachen, FRG

RWTH

Walther Straub-Institut f/Jr Pharmakologie und Toxikologie der LMU, Nugbaumstr. 26, D-8000 M~inchen 2

98 INFLUENCE OF C H E L A T I N G AGENTS ON BILIARY EXCREtION AND ON TOXIC EFFECTS OF D I A L K Y L T I N C O M P O U N D S IN RATS J. Merkord x, G. K r O n i n g and G , H e n n i g h a u s e n

100

D i a l k y l t i n compounds have b e e n used as biocides, catalysts and plast stabilizer. An important toxicological p r o p e r t y of the d i a l k y l t i n compounds may be the r e a c t i o n with dithiol groups. 2,3-dim e r c a p t o - l - p r o p a n o l (BAL, dimercaprol), meso-2,3d i m e r c a p t o s u c c i n i c acid (DMSA) and 2 , 3 - d i m e r c a p t o - l - p r o p a n s u l f o n i c acid {DMPS) were compared in their potency to d i m i n i s h the b i l i a r y e x c r e t i o n of o r g a n o t i n and the o r g a n o s p e c i f i c toxicity (lesions on liver, bile duct and pancreas) of dialkyltin d i c h l o r i d e s with alkyl chains of 4-6 Catoms in rats. D i m e r c a p r o l did not influence s i g n i f i c a n t l y the o r g a n o s p e c i f i c toxic effects of d i b u t y l t i n dichloride (DBTC), d i p e n t y l t i n d i c h l o r i d e (DPTC) and d i h e x y l t i n d i c h l o r i d e (DHexTC) on liver, bile d u c t and pancreas. The b i l i a r y excretion of o r g a n o t i n after treatment of a n a e s t h e s i z e d rats with DBTC, DPTC and DHexTC was r e d u c e d to 50 % of c o n t r o l by contemporary a d m i n i s t r a t i o n of DMSA and DMPS. The toxic effects of d i a l k y l t i n compounds on liver, bile duct and pancreas were s i g n i f i c a n t l y diminished by DMSA and DMPS. These results indicate an i n t e r a c t i o n of d i a l k y l tin compounds with alkyl chains of 4-6 C-atoms with d i t h i o l compounds in vivo. Compared with d i m e r c a p r o l DMSA and DMPS are more active and both compounds may be r e c o m m e n d e d for the treatment of acute poisonings with these organotins.

M. B6hme, W. R u m m e l

Xpresent adress: Institute of P h a r m a c o l o g y and Toxicology, U n i v e r s i t y of R o s t o e k , L e n i n a l l e e 70, 0-2500 Rostock, Germany

M e c h a n i s m s of methylmercury- and HgCl2-indueed C1secretion in the rat colon

T h e action of organic and inorganic mercury on colonic epithelium was studied with the whole-cell patch-clamp technique and the Ussing chamber. Methylmercury induced an increase of m e m b r a n e outward current in enterocytes of isolated crypts patched from the basolateral side. This action was inhibited by a CF channel blocker and a K + channel blocker indicating an increase of both the CI- and the K + conductance. In contrast, HgC12 did not affect m e m b r a n e current. In m u c o s a preparations, both compounds increased the shortcircuit current (Isc) and the tissue conductance (Gt) indicating an activated CI" secretion. The effect on Gt was m o r e pronounced, w h e n the H g compounds w e r e given to the mucosal side, whereas Isc response was m o r e sensitive to the H g compounds applied to the serosal side. Tetrodotoxin but not atropine suppressed the effect of serosally applied H g c o m p o u n d s on Isc indicating the m e d i a t i o n by non cholinergic s e c r e t o m o t o r neurons. Inhibition by i n d o m e t h a c i n gave evidence for the participation of neuronally acting prostaglandins. Addition of dithiothreitol reversed the actions of H g - c o m p o u n d s suggesting a reaction with SH-groups. Institut ftir P h a r m a k o l o g i e und Toxikologie, Universitiit des Saarlandes, D-6650 Homburg/Saar, F.R.G. Supported by SFB 246, project C2.

R 26 101 EFFECT OF HEAVY METAL IONS ON THE PHAGOCYTOSIS OF HUMAN BLOOD MONOCYTES G. Zwadlo-Klarwasser and C. G6ttsch Heavy metal ions are reported to decrease many macrophage functions e.g. antigenpresentation and killing of parasites and tumor cells. Their influence on the phagocytosis however is discussed controversially. We therefore studied the effect of the metals cadmium (CdS04), mercury (HgC12), lead (Pb(CHACOO)2) and bismuth (BiO (CIO4)) on the latex phagocytosis o f - h u m a n blood monoeytes. Ceils were isolated with Ficoll-Paque centrifugation and incubated for 90 min at 37" C with the latex particles and the metals in concentration ranges from 10 -4 to 10-8 M. Results were expressed as the relative number of monocytes (Mo) ingesting latex particles versus the total Mo population (% positive Mo) and as the particle number/positive Mo a~ parameter of phagocytic aetivty. Concentrations of 10-" M of bismuth, cadmium and lead and of 10 -5 M of mercury reduced the viability of the Mo from >95% to about 80% and decreased the phagocytic activity. Cadmium, lead and mercury at 10-~ M also diminuished the % positive Mo probably due to toxic effects. By contrast bismuth did not i n f l u e n c e l h e number of phagocytic Mo at 10-4 M and eve~ increased it at 10 - 6 M. Cadmium in concentrations lower than 10T M M had only weak effects while mercury strongly reduced the number and activity of phagocytic Mo up to 10 -8 M. Lea~l depressed the phagocytosis of Mo at 10 -6 M, but increased it at 1 0 - ° M . These results show that heavy metal ions affect the phagocytosis of blood monocytes differently. Mercury and lead inhibit the phagocytosis in concentrations only twofold higher than those found in human blood. Bismuth seems to activate Mo in terms of an increased phagocytosis. This activation may lead to an enhanced wound healing and may therefore contribute to the beneficial effects of bismuth on gastrointestinal lesions. Institute of Pharmacology, RWTH Aachen, Wendlingweg 2, 5100 Aachen, F.R.G.

103 RENAL TOLERANCE OF THE COMBINATION TREATMENT WITH FUROSEMIDE-CEFPIROME OR GENTAMICIN-CEFPIROME IN RATS C. Cojocel, H. H. Donaubauer, H. Fuchs, K. H. Langer and D. Mayer The present study was designed to investigate the morphological and functional effects on the rat kidney after i . v . treatment of female Wistar rats with cefpirome (CPO) (700 mg/kg/d) or with the combination of furosemide (FU)-cefpirome (20 + 700 mg/kg/d) or gentamicin (GNT)-cefpirome (20 + 700 mg/kg/d) for 30 days. CPO was administered i h after FU or GNT administration. In control groups, rats were given saline, CPO, FU or GNT alone. Morphological and functional changeswere monitored at the end of the treatment period by standard histology and measurements of the plasma concentrations of Na~, K÷, Ca++, C l , P042-, bilirubin, glucose, uric acid, creatinine, urea (BUN), GOT, GPT and alkaline phosphatase. The a b i l i t y of renal cortical slices from male rats to accumulate p-aminohippurate (PAH) and tetraethylammonium (TEA) or to synthesize glucose (gluconeogenesis) was also investigated after 5, 10 or 15 days of similar treatment. Except a marginal increase in BUN, there were no changes of the clinical chemistry parameters in rats treated with CPO alone or with the combinations FU-CPO or GNT-CPO. Treatment of rats with FU or GNT alone induced the expected calcification of tubular cells or tubule cell necrosis/atrophy, respectively. The combination treatments FU-CPOor GNT-CPOdid not potent i a t e the tubule damage induced by FU or GNT alone but rather showed a distinct protective effect. Furthermore, protective effects of the combination therapy were also observed on the a b i l i t y of the rat renal cortex to accumulate organic ions or to synthesize glucose. Hoechst AG, Postfach 800320, D-6230 Frankfurt/M 80, FRG

102 SMALL-SCALE EXPOSURE SYSTEM FOR MERCURY-(Hg-203) VAPOR. R. Fiohtner and S. Halbach.

104

The investigation of the toxicokinetics of mercury upon inhalation of the vapor (Hg °) requires the use of an exposure system for small animals with specific performances: rapid development and stability of preselected Hg ° concentrations, continuous operation over variable exposure times, easy monitoring of [Hg °] in air and ready determination of Hg in organs and carcass. These .~pecifications can be realized by generating Hg from the reduction of Hq ++ that had been labeled with radioactive Hg-203 ÷+. The commonly used reducing agents SnCI 2 or NaBH 4 gave unsatisfactory results, i.e. the time period for the buildup of the Hg ° concentration was too 10ng and stability could not be maintained. Therefore, the reducing properties of hypophosphoric acid (HPHo02) were tested. Continuous measurement with AAS ~howed that rise time of [Hg °] was below i0 min and the plateau was stable and higher than with the other reductants. The height of the plateau, i.e. [Hg °] in air was linearily correlated to the [Hg +÷] in solution. Estimation of Hg-203 in the wasted solution gave concentrations lower than 2% of that of the initial Hg ++ solution, i.e. vaporization of Hg was nearly complete. The time to attain 90% of the plateau [Hg °] in the exposure chamber can be calculated to be 3.7 min, which is in accordance with 4.6 min actually measured. Body burden and organ distribution of Hg were determined after exposure to 0.5, 1.0 and 2.0 mg Hg°/m 3 for i, 2 and 3 h. Under these conditions Hg uptake was linearily correlated to exposure time or concentration.

p-Aminophenol is nephrotoxic in rats and causes necrosis of the pars recta of the proximal tubules. Aminophenol is oxidized by hepatic and renal enzymes to a reactive quinone imine which is presumed to react with glutathione, y - G l u t amyltranspeptidase-dependent uptake of t h i s glutathione S-conjugate, which is structurally related to toxic S-conjugates formed from bromoquinone, into the kidney may explain nephrotoxicity. We have investigated the biosynthesis and in vitro nephrotoxicity of aminophenol-derived Sconjugates in rats and rat kidney cortex cells. After i.p. application of aminophenol, l-amino-3glutathione-S-yl-4-hydroxybenzene (AP-SG) was identified as a biliary metabolite by thermospray mass spectrometry and by its pH-dependent electronic spectra. AP-SG was cytotoxic in rat kidney cortex cells; cell killing by AP-SG was significantly reduced by inhibition of ~-glutamyltranspeptidase and by inhibition of cysteine conjugate 6-1yase. Moreover, inhibition of cytochrome P-450 by SKF-525A and prostaglandin synthase by indomethacin also protected the cells from AP-SG induced toxicity. These results suggest that biosynthesis and renal metabolism of AP-SG contributes to p-aminophenol nephrotoxicity. Institut f~r Toxikologie, Universit~t W~rzburg, Versbacher Str. 9, D-8700 W~rzburg, F.R.G.

GSF-Institut

f. Toxikologie,

8042 - Neuherberg

AMINOPHENOL GLUTATHIONE

M. Koob,

NEPHROTOXICITY: CONJUGATES

C. Klos,

BIOSYNTHESIS

OF T O X I C

C. Kramer and W. Dekant

R 27 105

107

I N V E S T I G A T I O N S ON POSSIBLE A C T I V A T I O N P A T H W A Y S OF THE B L A D D E R C A R C I N O G E N N - N I T R O S O BU T Y L - 3 - C A R B O X Y P R O P Y L A M I N E [BCPN] D. J a c o b u n d C. J a n z o w s k i

ARE REACTIVE RADICALS FORMED AS A CONSEQUENCE REPERFUSION OF THE ISCHEMIC HEART ?

Activation of N-nitrosobutyl-3-carboxypropylamine [BCPNI b y c~- or ~ - o x i d a t i o n m a y b e i n v o l v e d i n t h e mechanism of i t s o r g a n o t r o p i c e f f e c t to t h e u r i n a r y b l a d d e r . T h e r e f o r e i n - v i t r o m e t a b o l i s m of BCPN a n d i t s ~-oxidized metabolite N-nitrosobutyl-2-oxopropylamine [BOPN] w a s s t u d i e d . BOPN w a s d e b u t y l a t e d a t h i g h r a t e s w h e n i n c u b a t e d with rat liver microsomes, whereas pig urinary bladder m i c r o s o m e s w e r e m u c h l e s s a c t i v e . BCPN w a s d e a l k y l a t e d a t b o t h a l k y l g r o u p s to a l o w e x t e n t in t h e s e s y s t e m s . It a l s o w a s f o u n d to b e n o n g e n o t o x i c in N a m a l v a c e l l s , i r r e s p e c t i v e of t h e m o d e of a c t i v a t i o n . In c o n t r a s t , B O P N i n d u c e d D N A s i n g l e s t r a n d b r e a k s [SSB] w h e n i n c u b a t e d w i t h P B - i n d u c e d r a t l i v e r m i c r o s o m a l f r a c t i o n . N - N i t r o s o b u t y l u r e a [BNU], N - n i t r o s o - 3 ~ carboxypropylurea [CPNU] a n d N - n i t r o s o - 2 - o x o p r o p y l u r e a [OPNU] w e r e t e s t e d a s d i r e c t l y a c t i n g m o d e l compounds yielding the same ultimate electrophiles as BCPN a n d BOPN. O P N U i n d u c e d h i g h r a t e s of D N A SSB in N a m a l v a c e l l s a n d u r i n a r y b l a d d e r c e l l s w i t h n o i n d i c a t i o n for r e p a i r w i t h i n 4h. BNU s h o w e d t h e s a m e effect only at lO-fold higher concentrations. DNA damage w a s r e p a i r e d to a s i g n i f i c a n t e x t e n t [4hi, CPNU s h o w e d v e r y w e a k SSB i n d u c i n g a c t i v i t y . T h e r e s u l t s s u g g e s t t h a t ~ - o x i d a t i o n of BCPN m i g h t b e a p o t e n t i a l a c t i v a t i o n pathway. Department of F o o d Toxicology, University,

Chemistry and Environmental D-6750 Kaiserslautern

106 S T U D I E S ON THE M E C H A N I S M OF THE P A N C E E A T I C BC E L L T O X I C A C T I O N OF A L L O X A N S. L e n z e n A n e w i n d i c a t o r s y s t e m for e l u c i d a t i o n of a l l o x a n B - c e l l t o x i c a c t i o n was used: the i n h i b i t i o n of s p e r m i n e - i n d u c e d c a l c i u m u p t a k e by m i t o c h o n d r i a in p e r m e a b i l i z e d p a n c r e a t i c B - c e l l s f r o m o b / o b m i c e as m e a s u r e d w i t h a specially designed calcium minielectrode. The g l u c o k i n a s e is the s i g n a l r e c o g n i t i o n e n z y m e of the p a n c r e a t i c B - c e l l for i n i t i a t i o n of g l u c o s e - i n d u c e d i n s u l i n s e c r e t i o n . B o t h a l l o x a n and the s u g a r m a n n o h e p t u l o s e i n h i b i t glucose-induced insulin secretion through i n h i b i t i o n of this enzyme. However, o n l y a l l o x a n is B - c e l l t o x i c due to t o x i f i c a t i o n of a l l o x a n as a r e s u l t of the i n t e r a c t i o n w i t h the g l u c o k i n a s e . A l l o x a n not o n l y i n h i b i t s this e n z y m e but at the s a m e time y i e l d s d i a l u r i c a c i d t h r o u g h r e d u c t i o n of alloxan. T h r o u g h r e d o x c y c l i n g b e t w e e n a l l o x a n and d i a l u r i c a c i d c y t o t o x i c free r a d i c a l s are g e n e r a t e d , w h i c h are r e s p o n s i b l e for p a n c r e a t i c B - c e l l death. The half m a x i m a l i n h i b i t o r y c o n c e n t r a t i o n for i n h i b i t i o n of c a l c i u m u p t a k e in p e r m e a b i l i z e d o b / o b m o u s e B - c e l l s was 3.4 m M for a l l o x a n and 5.8 m M for d i a l u r i c acid. G l u c o s e p r o t e c t e d a g a i n s t the e f f e c t of a l l o x a n but not of d i a l u r i c a c i d a p p a r e n t l y t h r o u g h o c c u p y i n g the g l u c o k i n a s e g l u c o s e b i n d i n g site and t h e r e b y p r e v e n t i n g t o x i f i c a t i o n of alloxan. I n s t i t u t f~r P h a r m a k o l o g i e und T o x i k o l o g i e , Universit~t G6ttingen, D-3400 G6ttingen

H. N o h l

OF

and K. S t o l z e

R e p e r f u s i o n i n j u r y of i s c h e m i c o r g a n s is s u g g e s t e d to r e s u l t f r o m m e t e b o l i c d e r a n g e m e n t s initia t i n g an i m b a l a n c e d f o r m a t i o n of free o x y g e n radicals. M o s t i n v e s t i g a t o r s in this f i e l d have u s e d the s p i n t r a p 5 , 5 - d i m e t h y l p y r r o l i n e - l - o x i d e (DMPO) to s t a b i l i z e these short lived radicals and m a k e t h e m v i s i b l e b y m e a n s of E a R t e c h n i q u e . EaR-signals obtained from intravascular DMPO w e r e r e p o r t e d to i n d i c a t e the f o r m a t i o n of free O H ' - r a d i c a l s and in some c a s e s also c a r b o n cent e r e d r a d i c a l s . We w e r e u n a b l e to c o n f i r m these findings. C a r b o n c e n t e r e d r a d i c a l s w e r e not obtained irrespectively of conditions studied while oxygen centered DMPO-adducts could only be d e t e c t e d in r e a s o n a b l e a m o u n t s w h e n iron was add e d to the p e r f u s a t e . U n d e r t h e s e c o n d i t i o n s an ascorbyl-related EaR-signal c a m e hp w h i c h was also p r e s e n t w i t h o u t the a d d i t i o n of iron. However, the i n t e n s i t y of the latter varied with the a m o u n t of iron r e l e a s e d into the perfusate. Although our r e s u l t s do not exclude oxidative s t r e s s as the p a t h o g e n e t i c m e c h a n i s m of postisc h e m i c o r g a n injury, i n t r a v a s c u l a r f o r m a t i o n of o x y g e n - c e n t e r e d D M P O - a d d u c t s is u n l i k e l y to result f r o m free o x y g e n r a d i c a l s of the p e r f u s a t e . T h i s p o i n t w i l l b e d i s c u s s e d in t e r m s of b i o c h e mical and physicochemical considerations.

I n s t i t u t e for P h a r m a c o l o g y and T o x i c o l o g y , V e t e r i n a r y U n i v e r s i t y of V i e n n a , L i n k e B a h n g a s s e II, A-1030 Vienna

108 ALTERED OXYGEN-HEMOGLOBIN DISSOCIATION RATES IN SMOKERS S.Kunkel*, P.Ever *~ The importance of smoking as a possible risk factor in in coronary heart disease (CHD) may be related to the effect of carbon monoxide (C0) on oxygen exchange in hemoglobin (Hb). Therefore we have examined the kinetics of this process in vitro using a fast-reaction technique (stopped-flow). Blood from 14 smokers and 13 non-smokers was diluted (approx. 60umol/l Mb) with bis-tris buffer (pH 7.4; 0.2mol/]) containing 2 mmol 2,3-diphosphoglycerate and mixed with 20mmol/l sodium dithionite/bis-tris as oxygen acceptor in the reaction chamber of an Aminco-Dasar stopped-flow apparatus. The change in oxyhemoglobin absorption was followed at 576/593nm. The initial reaction (loss of the first oxygen molecules I) proceeded with a halflife of 14.2 ! 0.7 ms in non-smokers but in smokers this process was slower (ti/2 = 16.5 ± 0.7 ms; p < 0.05). In the later phase of the reaction (occuring over the period of 14-30 ms) the rate of oxygen release in smokers was also reduced (tl/2 = 9.1 t 0.7 ms versus 8.4 ~ 1.13 ms; p= 0.065). These changes are apparently due to the presence of CO (8.4% ~ 2.4%) is smokers since in subsidiary experiments the halflife of oxyhemog]0b~n in the late reaction phase increased from 7 to 21ms when the HbCO saturation was raised from 0.8% to 45%. These results support the view that CO modulates the release of oxygen from hemoglobin in smokers under normal conditions and that this phenomenon can impair oxgyen availability in CHD patients. i. Dalziel and O'Brien (1960) Biochem. J.,78,236-245. * Dept.Clin.Pharmacology, University Hospital Frankfurt **Inst. Pharmacology and Toxilogy, University of M~nchen

R 28 109 DINITROBENZENE DERIVATIVES INHIBIT PLATELET AGGREGATION BY INCREASING INTRACELLULAR CYCLIC GMP LEVELS F. v. Appen Preincubation of washed human platelets with 1-chloro-2,4dinitrobenzene (CDNB) or with 1,3-dinittobenzene leads to dose- and timedependent inhibition of agonist-induced platelet aggregatiom This inhibitory effect is correlated with an impaired agonist-induced intracellular calcium release. Using radioimmunoassays a steady increase of intracellular cyclic GMP level is observed during treatment of intact platelets with CDNB or dinitrobenzene. Compared with control cells a 6 to 10 fold increased activity of guanylate cyclase is detected in the eytosolic fraction of CDNB-pretreated platelets. However, CDNB and 1,3-dinitrobenzene are also potent activators of soluble guanylate cyclase activity in cell free assay systems. The presence or absence of low molecular weight thiols does not influence the activation of soluble guanylate cyclase by the dinitrobenzenes. With heme-free enzyme preparations, however, the stimulating effect of the dinitrobenzenes is abolished. The presented data provide evidence for an activation of soluble guanylate cyclase by dinitrobenzenes or their metabolites via interaction with its heine group.

111 UV-PHOTODEGRADATION OF QUINOLON~S - BINDING OF PHOTOPRODUCTS TO HUMANSKIN E.M. Tiefenbacher and H. Kurz Various 6- fluoroquinolones absorb radiation energy" from the UVrange of the electromagnetic spectrum. Phototoxic or photoallergic effects have been reported for some of these substances. Exact mechanisms of these reactions are unknown so far. We investigated the influence of UVA on aqueous solutions of ciprofloxacin, ofloxacin and fleroxacin, HPLC - chromatograms of the irradiated substances (UVA dose= 100 J / c m z ) revealed photodegradation and formation of several photoproducts. Binding of activated photoproducts to human cells is discussed as mechanism for phototoxic rea'ctions. We are interested in the binding of ciprofloxacin and its photoproducts to human skin. Skin was obtained from human thighs, reduced with N2 and l¥ophilisized to a homogenous powder. Binding was determined by equilibrium dialysis with ciprofloxacin in phosphate buffer (pH 7.4, 10-3 - 10-6 tool/l) or a similar, but irradiated solution (10 -4 tool/l, 100 J/cm 2) at 4° C for 20 h. In the range from 10-3mol/l to 10-6mol/l, ciprofloxacin binding is concentration dependent;10- 3tool/1 are bound to 14~3+1.8%, 10-6tool/1 to 82.0 + 3.0%. Irradiated ciprofloxacin is bound to the same extent as non-irradiated. After dialysis of irradiated 10-4 tool/1 ciprofloxacin solutions none of the photoproducts could be detected as free fraction. Using 10- 3 tool/1 solutions, one photoproduct, more polar than ciprofloxacin and binding to 47.3%, and two less polar photoproductm binding both to 75.0%, could be detected. Some photoproducts seem to bind to a higher degree to human skin than ciprofloxacin itself. It is not known so far whether binding of these substances to human skin is specific or non-specific~ _

Walther-Straub-Institute of Pharmacology and Toxicology, Ludwigs-Maximilians-University, NuSbaumstr. 26, D-8ooo Miinchen 2

Department of Biology, University of Konstanz, Universit~tsstr. 8-10, D-7750 Konstanz, F.R.G.

110 The long-term effect of the rodenticide brodifacoum on blood coagulation and on the hepatic vitamin K metabolism in rats J.J. Mosterd* and H.H.W. Thijssen Brodifacoum is a potent anticoagulant rodenticide which acts as a vitamin KI antagonist. An abnormal vitamin K1 metabolism was reported for factory workers for at least 18 months after accidental exposure to brodifacoum. However, clotting factor activity at that time was normal, suggesting a dissociation between the coumarin effect on vitamin K metabolism and clotting factor synthesis (Park et al, Br J Clin Pharmacol 21, 289, 1986). We investigated the long-term effects of brodifacoum on vitamin K metabolism in rats. Rats received a single dose of brodifacoum (0.2 mg/kg, p.o.). The anticoagulant effect, the hepatic vitamin K cycle and the microsomal warfarin binding were assayed during 30 days. Ex-vivo microsomal vitamin K epoxide reductase and microsomal warfarin binding remained inhibited for at least 30 days. Clotting factor synthesis, however, was restored from beyond day 7. The in-vivo vitamin K metabolism was also disturbed: accumulation of vitamin K epoxide in the liver following a pharmacological vitamin K dose was still manifest at day 30. Liver tissue and liver microsomal brodifacoum concentrations remained almost stable and no brodifacoum could be detected in the circulation. In conclusion, brodifacoum is a persistent rodenticide with high binding affiriity for the liver microsomal warfarin binding site, resulting in a long lasting suppression of the vitamin K cycle. Apparently, a partly operating vitamin K cycle suffices for normal clotting factor synthesis. In vivo, the vitamin K cycle can be uncoupled from the vitamin K dependent earboxylation reaction. Dept. of Pharmacology, University of Limburg, P.O. Box 616, 6200 MD maastricht, The Netherlands "Supported by the Dutch Heart Foundation

112 METALLOTHIONEIN IN HUMAN EPIDERMAL KERATINOCYTES IN CULTURE H. Kappus* and Ch. Reinhold'* Human skin is exposed to d r u g s , chemicals, irradiation etc. which induce toxicity. Some of the mechanisms involved include oxidative s t r e s s which leads to cellular damage of lipids, p r o t e i n s and DNA r e s u l t i n g in cell death, metabolic d i s t u r b a n c e s , mutagenicity and carcinogenicity. We studied the effects of organic peroxides in c u l t u r e d human k e r a t i n o c y t e s . In c o n t r a s t to other cells lipid peroxidation was h a r d l y inducible, although cellular toxicity o c c u r r e d (Kappus and A r t u c , Bioelectrochem. Bioe n e r g . 1 8 , 263, 1987; A r t u c et al., Arch. Dermatol. Res. 281, 49, 1989). Because metallothionein has been shown to t r a p h y d r o x y l radicals, we wondered w h e t h e r this molecule is protective a g a i n s t oxidative s t r e s s and w h e t h e r it is p r e s e n t in the keratinocytes u s e d . Keratinocytes were isolatted from various f r e s h human skin samples. Cell cult u r i n g was carried out according to s t a n d a r d methods. After removal of the cells from the dishes t h e y were homogenized and cytosol p r e p a r e d b y u l t r a c e n t r i f u g a t i o n . Metallothionein was determined b y the C d - s a t u r a t i o n method (Klein et a l . , Anal. Biochem. 189, 35, 1990). In f r e s h l y isolated keratinocytes 0.1 - 0.3 p.g metallothionein was measured p e r mg cytosolic p r o t e i n . In keratinocytes cult u r e d for 2-4 weeks the metallothionein content increased to 0.6 - 2.0 ~tg/mg p r o t e i n , p r o b a b l y due to culture condit_ions. When we c u l t u r e d keratinocy-tes in the p r e s e n c e of Zn ions the metallothlonein content increased u p to 15 fold compared to r e s p e c t i v e controls. These r e s u l t s indicate that metallothlonein is p r e s e n t in human epidermal cells (keratinocytes) and that it is inducible in culture b y a n u m b e r of f a c t o r s . T h u s , keratinocytes might be a suitable tool to s t u d y the protective role of metallothionein in oxidative s t r e s s and toxicity. * I n s t i t u t ffir Landstrafie 1, ** Hautklinik, D-1000 Berlin

Toxikologie, GSF, Ingolst~idter D-8042 N e u h e r b e r g FU, A u g u s t e n b u r g e r Platz 1, 65

R 29 113 INFLUENCE OF rHIRUDIN ON NICROTNROMBOSI$ INDUCED BY RUSSEL'$ VIPER VENOM (RVV) G. Nowak, E. Bucha and +J, N e i e r

The a d m i n i s t r a t i o n o f small amounts of RVV ( I 0 25 pg/kg x h) was followed by formation o f microthrombi in r a t lungs. Previous a p p l i c a t i o n o f 1 2 ~ I - f i b r i n o g e n (3.7 MBQ, I-2 h before the experiment) and 111-In-labelled platelets (I MSq, 2-24 h before the experiment) served to measure the microthrombosis continuously by means of a gamma-scintillation probe. Much lower venom doses were required for the induction of p l a t e ] e t deposition than for fibrin deposition in rat lungs, The a d m i n i s t r a t i o n of

rhirudin before the application of venom prevented the p l a t e l e t accumulation and the deposition o f f i b r i n o g e n , rasp. Already very low doses o f h i r u d i n w e r e able t o prevent f i b r i n d e p o s i t i o n whereas much higher doses were necessary to i n h i b i t p l a t e l e t d e p o s i t i o n . Preliminary LDso experiments with and without r h i r u d i n pretreatment resulted in the f o l l o w i n g f i n d i n g s : h i r u d i n proved t o have a b e n e f i c i a l effect on the animal blood coagulation system by reducing the l e t h a l i t y caused by RVV (4.3 f o l d lower LDs0 values a f t e r pretreatment with rhirudin). Present address: Institute of Pharmacology and Toxicology, Medical Academy Erfurt, Nordh~user Str. 74, O-5010 Erfurt, FRG; ÷ P e n t a p h a r m AG Lid, Basel, Switzerland

115 CYTOSKELETAL CHANGES IN ENDOTHELIAL CELLS F O L L O W I N G A P P L I C A T I O N OF C.NOVYI A L P H A TOXIN A. Oksche, R. N a k o v and E- H a b e r m a n n Alpha toxin of C.novyi type A is a single-chain cytotoxin of about 200 kDa. The toxin (0.02 2pg/ml) induces retraction of the cell body and occasional membrane blebbing of cultured porcine p u l m o n a r y artery endothelial cells in a concentration dependent manner. Cells treated with the highest concentration for up to 6h remain adherent to their p r i m a r y support as shown by scanning electron microscopy. Cells pretreated with alpha toxin (125 ng/ml) for 24h and then passagad still adhere to their secondary support. However, such cells show neither spreading, nor sprouting, nor mitosis w h i c h indicates an effect of alpha toxih on their cytoskeleton. Changes in the arrangement of m i c r o f i l a m e n t s have been shown d i r e c t l y with fluorescent p h a l l o i d i n as a marker specific for F-actin. A l r e a d y in early stages of alpha toxin treatment (2pg/ml, 2h) stress fibers disintegrate an~ F-actin is enriched around the nucleus whereas microtubules and intermediate filaments are not altered. The amount of F-actin but not of total actin decreases when m e a s u r e d by DNAase inhibition and calculated as the fraction of total protein. It is concluded that alpha toxin alters the c y t o s k e l e t o n which then leads to retraction of endothelia and, finally, oedema formation. Since the morphological changes resemble those described for C.difficile cytotoxins A and 'B, the three toxins may share a common mode of action. However, the primary target of the toxins is still unknown.

R u d o l f - B u c h h e i m - I n s t i t u t f~r Pharmakologie der Universit~t Gie~en, Frankfurter Str. 107, D-6300 GieSen

114

116

THE CHROMAFFIN CELL: A SUITABLE MODEL FOR INVESTIGATING THE ACTIONS AND THE METABOLISM OF TETANUS AND BOTULINUM A NEUROTOXINS P. Marxen and H. Bigalke

RESTORATION OF NORADRENALINE RELEASE FROM TETANUS TOXIN-TREATED CHROMAFFIN CELLS F. Bartels

Tetanus (Tetx) and botulinum A neurotoxins (BoNtx) block the exocytotic release of catecholamines from bovine chromaffin cells, provided they are able to reach the cell interior, Chromaffin cells, being excellent models for the study of neurosecretion, are inherently deficient in polysialogangliosides, the binding sites for the toxins. Basically, there are three different ways of translocating the toxins into the cells: 1) Enrichment of cell membranes with polysialogangliosides enables the toxins to bind to the plasma membranes and hence to accumulate in the cells. 2) Poration of the cell membrane by cytolysins or digitonin allows the toxin molecules to diffuse into the cells. 3) Exposure to a strong electric field results in the formation of transient pores in the cell membrane through which the toxins may enter the cytosol.-To develop full inhibitory action in chemically permeabilized chromaffin cells, the toxins have to be applied in their reduced form. The use of radioiodinated toxins, introduced into chromaffin cells by electroporation, allowed the observation of the intracellular processing of these substances over a period of several days. The toxins and their derivatives were extracted, separated by SDSPAGE and detected by autoradiography. Increasing amounts of the toxins were split into two chains and further degraded to small fragments within a period of 5 days. The block of exocytosis, nonetheless, outlasts the intracellular survival of both toxins. This leaves room for speculations that a hitherto unidentified component in the exocytotic machinery, knocked out by the toxins, may have to be resynthesized to restore exocytosis, or that the capacity to block exocytosis resides in a small fagment of the toxin molecules. Furthermore, it cannot be excluded that an amount of toxin too small to be detected may suffice to maintain the block of exocytosis. Medizinische Hochschule Hannover, Abteilung Toxikologie, 3000 Hannover 61, FRG This work was supported by the DFG (Bi:274/4-1)

Tetanus toxin blocks carbachol-stimulated noradrenaline release from ganglioside-preloaded chromaffin cells. The block persists for several days. Specific anti-tetanus toxin antibodies, applied to digitonin-permeabilized chromaffin cells at a time when the block is fully evident, bind to intracellular tetanus toxin without being able to restore .exocytosis (Marxen et al., 1990). When tetanus toxin-treated cells are exposed to an electric field, pores open up in the plasma membrane. The pores are large enough for the toxin (and cytosolic substances) to move out of the ceils. In spite of this, the cells fail to resume the secretion of noradrenaline for at least 6 days, although in toxin-untreated cells, the exocytotic machinery has recovered from poration long ago. When specific anti-tetanus toxin antibodies are present during electropermeabilization, the intracellular interaction of tetanus toxin with its antibody restores exocytosis within 72 hours. It is concluded that tetanus toxin irreversibly alters a component essential for exocytosis and that, after the intracellular neutralization of tetanus toxin by its antibody, a slow recovery is achieved, probably by resynthesis of its as yet unidentified target. Marxen P., Ahnert-Hilger G., Wellh6ner H.H., and Bigalke H. (1990) Toxicon 28:1077-1082 Medizinische Hochschule Hannover, Abteilung Toxikologie, 3000 Hannover 61, FRG This work was supported by the DFG (Bi:274/4-1)

R 30 117

119

POINT HUTATIONS IN CYTOTOXIN GENE OF PSEUBOIqON~S

IN VITRO TOXICITY OF LITHIUM: EFFECTS, TISSUE

4ERUGII~A

CONCENTRATIONS - NO PROTECTIVE EFFECT OF MYO-INOSITOL Stephan Khig, Tetsuji Nagao, Mike Collins

G. Xiong and F. Lutz

Pseudomonas aerugino~a is a patho~nic organism causing l i f e threatening dise~ses by pr(w~ucing several toxic factors. One of the path~enic factors is an acidic protein of 28 kDa. The gene of that has ~)een cloned into pSN3, sequenced and expressed in E. c o l i in t h i s laboratory [ 1 ] . By research of deletion and point mutants, three dowains on the cytotoxin gene were found to c(~3nect with the cytotoxin toxic activities. Activity paramel~rs used were binding to rabbit ghosts and ~ r m e a b i l i t y increase of human granul~ytes. Severe1 point mutants on a domain of bp 700 - 858 were prel:kared. The r e s u l t s showed t h a t bp 745 - 778 a r e connected with biolc~ical functions. Both a c t i v i t i e s became very low when point mutants occured in that r e g i o n . Whereas the f u n c t i o n s were not changed when p o i n t mutations were c ~ t r u c t e d i n I~o 700 - 744 and 779 - 858. There are two cys t h e c y t o t o x i n p r o t e i n are two cys (c(~ons a t bp 68 - 70 and 64~ - 645) i n t h e c y t o t o x i n p r o t e i n . When bp 68 - 70 were d e l e t e d o r / a n d bp 645 - 645 were changed t o g l y c i n e , the c y t o t o x i n p r o t e i n sh(~ed low b i n d i n g a c t i v i t y and no g r a n u l o c y t e s w e l l i n g activity. I t seemed t h a t t h e $-S b r i ~ may be very i m p o r t a n t t o keep t o x i n conformation and biolcw3ical activities. [ 1 ] O r l i k - E i s e l G, Lutz F, E i s e l U, Struckmeier H, Kr~uter ~, Niemann H (1770) Arch Hicr(Y~iol 15~:5~1-5~8.

Lithium was tested at various concentrations using a rat wholeembryo culture system in order to establish a concentrationresponse relationship (c.f. table). CR Somites Score Abn (#g/ml) (ram) (n) (%) Control 5O 100 150 2OO

4.68 3.72* 3.48** -3.36** 2.28**

28 27* 27* 27* 24**

38 36* 34** 31"* 23**

60 100

CR = crown-rump length; Abn = frequency of abnormal embryos; • = 0.01 _ p _<0.05; "* = p _<0.01 While LiC1 did not interfere with normal development of the yolk sac at a concentration of 50 or 100 t~g/ml in the culture medium, it did significantly interfere with embryonic growth and development at these concentrations. Therefore, we consider this a direct embryotoxic effect of lithium and not a membrane-mediated effect. This conclusion is supported by the fact that significant concentrations of Li + (1.51-+0.51 mrnole/1) are transferred to the embryo under culture conditions (150 ~zg/LiC1/ml = 3.5 rnmole Li+/1). The dismorphogenie development observed in vitro (eg. absent eye cup, retardation of limb buds and ear vesicles) seems to correlate relatively well with the effects observed in vivo. There is evidence that lithium acts by inhibition of inositol phosphate metabolism. For this reason we supplemented the culture medium with varying concentrations of myo-inositol in order to protect against the lithium-induced toxicity, however, no protective effect of myo-inositol could be found.

I n s t i t u t ffir PharmaKolc~ie und Toxikolo~ie der Universit~t, Frankfurter Sir. 107, D-6300 GieBen, FR~

Insdtut far Toxikologie und Embtyopharmakologie, Freie UniversitiitBerlin, Garystr. 5, D-IO00 Berlin 33

118

120 CYTOTOXICITY TEST USING BLASTOCYST DERIVED EUPLOID EMBRYONAL STEM CELLS: A NEW APPROACH TO IN VITRO TERATOGENESIS SCREENING H. Splelmann and G. Laschinski

RISK ASSESSMENT OF EFFECTS OF COMBINATIONS OF TERATOGENS AT LOW DOSE LEVELS Ibrahim Chahoud, Renate Thiel Generally, man is exposed to multiple teratogens, therefore, it is important to assess the extent and type of synergism which may result from such exposure. The determination of effects at a low dose level and the evaluation of the combination of xenobiotica at this level is a key issue of ultimate dsk assessment. We carried out a study with two well-known teratogens, hydroxyurea (HU) and 6-mercaptopudne riboside (6-MPR). Five groups of rats were treated with a single dose on day 11 of pregnancy after the following treatment regime: (1) a non-teratogenic dose (250 mg/kg) of HU only, (2) a iust-teratogenic dose (10 mg/kg) of 6-MPR only, (3) both doses simultaneously, (4) both doses but HU 4 hrs previous to 6-MPR or (5) the opposite sequence. The table shows some results of the evaluation of the fetal skeletons on day 21 of pregnancy. Anomaly Fetuses (n) Radius (bent) Ulna (bent) Ulna (short) Hindpaw (polydactyly) Tibia (short)

Frequency (%) of gross-structural anomalies HU 6-MPR HU+6-MPR HU+6-MPR simultan. 4 hrs prey, (154) (348) (225) (174) 0 3.4 0.4 1.1 0 2.3 t .4 0 0 2.3 0.4 2.9

6-MPR+HU 4 hrs prev. (141) 7.1 4.3 12.0

0 0

12.0 7.1

5.5 4.9

0.9 0.4

4.0 0.6

The data show that the application of the non-teratogenic dose of HU, simultaneously or 4 hrs previously, decreased (antagonism) the frequency of anomalies produced by the just-teratogenic dose of 6-MFR. The application of the same dose of HU 4 hrs affer application of 6-MPR increased (overadditive) the teratogenic effect. The study shows that the effect of a combination of two teratogens depends on the sequence and the timing of the application of the substances. The data indicate that the risk of combinations of teratogens may not be simply additive, but may obey more complex rules. Suppotledbya grantfrom the Deutsche Forschungsgemeinschaffgiven ~ 8FB 174.

Institutfor Toxikologieund Embryopharrnakologie,FUSedin,Garystf.5, ~10(~)~din 33

To develop a mammalian in v i t r o system For t e r a t o g e n i c i t y testing, cytotoxicity o f x e n o b i o t i c s was evaluated in pluripotent e u p l o i d embryonal stem c e l l s (ESC) d e r i v e d From mouse b l a s t o c y s t s . ESC are a b l l e to d i f f e r e n t i a t e i n t o a v a r i e t y o f embryonal t i s s u e s , r e t a i n e u p l o i d c h r o mosome c o n s t i t u t i o n , and p r o l i f e r a t e v e r y r a p i d l y . The d i m e t h y l t h i a z o l - d i p h e n y l t e t r a z o l i u m bromide (MTT) assay was a more a p p r o p r i a t e t e s t system f o r c y t o t o x i c i t y det e r m i n a t i o n in ESC than both the the Neutral Red uptake and t h e Kenacid Blue p r o t e i n s t a i n i n g method. Only co~pounds which do not r e q u i r e metabolic a c t i v a t i o n , were selected From the data base f o r v a l i d a t i o n o f in v i t r o t e r a t o g e n e s i s assays recommended by Smith et a l . ( T e r a t . Carcinog. Mutag. 3,461, 1983). Results obtained w i t h ESC were cempared to corresponding data From F i b r o b l a s t s Fro~ 14-day mouse embryos t o d e t e c t d i f f e r e n c e s in s e n s i t i v i t y between u n d i f f e r e n t i a t e d c e i l s (ESC) and d i f f e r e n t i a t e d c e l l s ( F i b r o b l a s t s ) . I n Fact, ESC showed a higher s e n s i t i v i t y a g a i n s t known teratogens than F i b r o b l a s t c u l t u r e s , which allows c a l c u l a t i o n o f a s e n s i t i v i t y r a t i o of a d u l t c e l l s ( d i f f e r e n t i a t e d F i b r o b l a s t s ) versus embryonal c e l l s ( u n d i f f e r e n t i a t e d ESC) in a mammalian system s i m i l a r t o the hydra assay. Although some x e n o b i o t i c s had to be c l a s s i f i e d False n e g a t i v e in t h i s t e s t , t h e ESC c y t o t o x i c i t y assay hold promise as a new in v i t r o screening assay in teratology. ZEBET ( Z e n t r a l s t e l l e z u r Erfassung und Bewertung yon E r s a t z - und Erg~nungsmethoden zum T i e r v e r s u c h ) , I n s t . Veter i n a r y Medicine, BUNDESGESUNDHEITSAMT, Postfach 33 O0 13, 1000 BERLIN 33, GERMANY Supported by a ~ r a n t from the German Dept. of Research and Technology (BMFT, Bonn).

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121 DEVELOPMENT OF ZEBRAFISH EMBRYOS UNDER THE INFLUENCE OF p-BENZOQUINONE, CHLOROACETALDEHYDE, AND CYCLOHEXANOL (3. Qroth, V. Herdt, and K. J. Freundt Fertilized eggs (n = 12 / concentration) from zebrafish (Brachydanio retie) were used to explore in vitro possible actions of p-benzoquinone (BC), chloroacetaldehyde (CA), or cyclohexanol (CH) on embryogenesis. The experiments (application of chemicals) were started at the morula stage (~--16 cells). The eggs were observed up to hatching (about /4 days) using a microscope. Morphological deviations from the normal development were registered and the results were evaluated as reported previously (Naunyn-Schmiedeberg's Arch Pharmac 3/41 Suppl, 1990, No 82, R 21). Addition of 0./4 mg BC/I incubation media (/4 h exposure) resulted in a 40 % reduction of the hatching ratel this BC concentration caused skeleton malformations - especially atrophy of the tail - in 90 % of the monitored eggs. A concentration of 0.35 mg BC/I was without influence on the physiological development of the fish larva. A reduction in blood circulation following deformation of the heart (formation of a tube) was caused in about 10 % of the embryos treated with 2 mg CA/1 (continuous exposure during the experimental period); the same changes were observed in 70 % of the embryos treated with /4 mg CAll. Fifty percent of the embryos died before hatching after application of 3 mg CA/I. A dosage of 0.8 mg CH/ml (continuous exposure) resulted in a 40 % reduction of the hatching rate, The same CH concentration caused a definite malformation of the heart in about 80 % of the embryos treated and malformations of the skeleton (especially of the tail) in 50 % of the embryos treated. The normal separation of the head from the yolk sack was disturbed in all embryos treated with 0.6 mg CH/ml. Conclusion: CH was most active. The fish egg model allows to easily differentiate between types of embryogenesis lesions caused by chemicals. Institute of Pharmacology and Toxicology, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Maybachstr. 14 - 16~ D-6800 Mannheim 1

122 COMPARATIVE APPROACHES FOR LOW DOSE PRENATAL TOXIC RISK ESTIMATION OF THE ALKYLATING MODEL C O M P O U N D EMS IN MICE Thomas Piatzek*, Ute Rahm, Gerd Bochert The prenatal toxic risk of low doses of ethylmethanesulfonate (EMS) in NMRI mice was assessed using different approaches. (1.) A "safe" dose was calculated using the no-effect-level(NOEL)/risk factor approach. The NOEL was determined by evaluating skeletal abnormalities. Under our experimental conditions a NOEL dose of 100 mg/kg body wt was determined. By use of the safety factor 10 a "safe" dose of 10 mg/kg was calculated, the use of the safety factor 30 resulted in a "safe" dose of 3.3 mg/kg EMS. (2.) The lowest effective dose of teratogenicity was 150 mg/kg EMS (5.6% gross-structural abnormalities). Using the Abbott procedure, the additional risk over background was calculated (= 5.0%). The doses corresponding to incidences of 1% and 0.1% were calculated using linear extrapolation to zero. The values were: 1% = 29.8 mg/kg, 0.1% = 3.0 mg/kg EMS. (3.) Low dose risk estimation was performed using probit analysis. Based on a previous doseresponse study in our laboratory, the doses corresponding to 1O~ (152.5 mg/kg) and 0 . 1O~ (138.6 mg/kg EMS) were estimated. (4.) In a tentative approach we estimated low dose risks based on target dosimetrv using the initial adduct rate of oe-ethylguanine in the embryos as dosimeter. Assuming arbitrarily a linear relationship also in the low dose range between teratogenicity and adduct rate and based on an exponential dose adduct function the doses 102 and 99 mg/kg EMS were calculated as being correlated to a risk of 1% and 0.1%, respectively. Our results clearly demonstrate pronounced differences between the various methods used for risk estimation. Studies supported by grants from the Deutsche Forschungsgemeinschaft awardedto the Sfb 174 (Risikoabsch~,tzungvorgeburtlicherSch&digungen). Institut for Toxikologie und Embryopharmakologie, Freie Universit'dt Berlin, Gatystr. 5, D-IO00 Bedin 33 * present address: Max von Pettenkofer-lnstitut, BGA

123 BLOOD PROTEIN ADDUCTS AS BIOMONITOR FOR EXPOSURE TO XENOBIOTICS USING STYRENE OXIDE AND ETHYLENE OXIDE AS TEST SUBSTANCES A. Mfiller, H.W. Goergens* Exposure to suspected carcinogens is usually monitored by measuring the concentration in ambient air or by quantification of the substance or its metabolites in blood or urine. The genotoxic effect can however not be monitored by these methods, since interindividual differences such as enzyme polymorphisms modulate metabolism and toxicity. The biological effective dose can be monitored by the determination of binding products of the alkylating agent with DNA in lymphocytes, or as surrogate with blood proteins. In pharmacology, albumin ,,binding" of pharmaceutics is well investigated, an implementation for exposure monitoring in toxicology is however not yet established. This study presents initial experimental results for the development of the use of protein adducts in toxicology. The reactive epoxides ethylene oxide and both enantiomers of styrene oxide were added to a.5 ml aqueous solution of 10 mg/ml human hemoglobin or human serum albumin and incubated for four hours in 22 ml head space vials under constant rolling at 37 ° C, then shock frozen in liquid nitrogen and lyophilized to remove unreacted epoxide. The proteins were redissolved in 5 ml buffer (10 mmol Tris, 100 mmol sodium acetate, 0.1 mmol CaCI2, pH 7.4) and 0.5 ml aliquots drawn for heat denaturation of proteins (95°C for 30 rain and subsequent cooling in ice water) prior to tryptic digestion with subsequent pe~ptide mapping through capillary electrophoresis on a P/ ACE 2000 system+~. The alkylated amino acids were then characterized by an HPLC-mass spectrometry analysis. HPLC was performed with a RP 18 microbore column with methanol/H20/1% trifluoroacetic acid and glycerine for elution. Fast Atom Bombardment (FAB) mass spectrometry analysis was done on a MAT 90 spectrometer by Finnigan, Bremen. Alkylation products in human blood proteins were determined by both methods, peptide mapping and mass spectrometry following HPLC. These methods are therefore suited for further development as monitors for exposure to alkylating agents or xenobiotics with alkylating metabolites. #Acknowledgement: We thank Beckman Co., Munich, for capillary electrophoresis measurements in their application laboratory. .Institut ffir Arbeitsphysiologie~ Ardeystr. 67, 4600 Dortmund 1 Arbeitsmed. Zentrum des TOV Rheinland, Friedrich-Engels-Altee 346, 5600 Wuppertal 2

124 DETECTION OF DNA ADDUCTS IN RAINBOW TROUT EXPOSED TO BENZO[a]PYRENE

T.M. Grether and W.P. Watson The objectives of the current studies are to develop techniques for monitoring potential exposure to polycyclic aromatic hydrocarbons (PAHs) and to improve the assessment of environmental risk associated with such exposures. With these aims 32p-postlabelling techniques are being applied to study the formation of DNA adducts in rainbow trout (Oncorhynchus mykJss) exposed to benzo[a]pyrene (BP), a representative carcinogenic PAH. Such techniques allow determination of the biologically effective dose, i.e. the target dose, which takes account of such factors as metabolism and disposition. The present studies have investigated the relationship between exposure dose of BP and the formation of BP-DNA adducts in livers of trout. Following single i.p. injections of BP (25 mg/kg), adducts could not be detected after 1 or 8 days exposure. These results suggested that trout are relatively resistant to the genotoxi¢ effects of BP. Therefore, in order to determine whether an induction period was necessary, fish were exposed via two consecutive injections (20 mg and 40 mg/kg) at an interval of 8 days and for a total of 16 days. After excision of livers~r,,om sacrificed fish, DNA was extracted and then analysed by the ~Ppostlabelling-TLC assay (Gupta, Cancer Res.,4_.@.,5656,1985). The DNA from exposed fish showed predominantly a single major add,u~t which co-chromatographed with an authentic sample of (+)-N (7R,8S,9R-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyreneol 0S-yl) 2'deoxyguanosine-3',5'-bisphosphate. These results indicate that trout can metabolize BP to its ultimate genotoxic form, the (+)7R,SS,9R,10R-diol-epoxidebut the rate and disposition are effected by lower body tempe~;~u=reand lower levels of cytochrome P-450, relative to mammals. The P-postlabelling technique is thus applicable for the detection of potential exposure of aquatic species to carcinogenic PAHs. These techniques may ultimately lead to improved environmental risk estimates for exposure resultidg from PAHs in aquatic environments. Shell Research Ltd., Sittingbourne Research Centre, Sitttingbourne, Kent ME9 8AG, England

R 32 125 M O D U L A T I O N OF GENOTOXICITY IN HUMANS BY INTERINDIVIDUAL DIFFERENCES IN METABOLISM B. Gansewendt, E. Hallier Differences in metabolism due to enzyme polymorphism can result in an individual disposition for the genotoxic effects of xenobiotics. Such differences influence both toxifying and detoxifying pathways. Slow and fast acetylators for aromatic amines is a classic example for such an enzyme polymorphism; the former bear a higher risk for bladder tumors than the latter. In the study presented here, 8 bladder tumor patients with a history of occupational exposure to aromatic amines were investigated both with dapsone and caffeine. Dapsone and its monoacetylated metabolite were determined in blood plasma, whereas caffeine and its metabolites AFMU (5-acetylamino-6-formylamino-3-methyluracil) and A A M U (5acetylamino-6-amino-3-methyluracil) were quantified in urine, both by HPLC. The results show that caffeine is superior to dapsone as a test substance for slow and fast acetylators in occupational toxicology, due to its relative lack of side effects and the use of urine instead of blood samples. In further experiments, radiolabelled ethylene oxide and methylene chloride were incubated in 22 ml head space vials with 3.5 ml whole blood from healthy human donors. The distribution of radioactivity in the cellular and plasmatic fractions showed the existence of two distinct subpopulations with different metabolic activity for these xenobiotics. The two groups fully coincided with the ,,conjugators" and ,,non-conjugators" previously described for the metabolism of methyl chloride, methyl bromide and methyl iodide. Incidentally, an enzyme polymorphism for glutathione transferase ~t in human liver has been reported and confirmed with styrene oxide as substrate. A common enzyme polymorphism must therefore be postulated. In contrast to aromatic amines, the tumorigenicity of these substances for humans is uncertain. In order to determine which genetic group in terms of enzyme activity bears the higher genotoxic risk, test systems with genetic endpoints such as binding products with lymphocyte DNA, alkaline elution of lymphocyte DNA and determination of sister chromatid exchanges and/ or chromosomal aberrations must substitute for the observation of manifest tumors. For biomonitoring purposes, binding products with serum albumin and/or hemoglobin can serve as a surrogate for alkylation products in DNA in occupational toxicology.

127 THE USE OF A GENOTOXICOLOGICAL TEST BATTERY FOR INVESTIGATING WASTE PRODUCTS OF THE SEMICONDUCTOR INDUSTRY l.Wolff, N.Werner, S.Bauer, S.Reissig, G.Jenennchefi, and P.Hoffmann For investigations of solid and gaseous waste products of the semiconductor industry a genotoxicological test battery including the Ames test, the Drosophila somatic mutation and recombination test (SMART) and the chromosome aberPation and SCE assay with bone marrow cells of rats was established; i.e. organisms of different levels of organization and several genetic endpoints were considered. Analytical determinations revealed that the solid and gaseous waste products are complex mixtures of halogenated hydrocarbons, nitrogenous substances and inorganic compounds. Our test program included solid samples from different parts of the gas pipe behind the vacuum pump as well as the emitted gases of the etching reactors. Single oral administration of solid specimens did not result in an inreased chromosome aberration frequency in rats whereas in the SMART well defined and reproducible genotoxic effects were found. The findings were correlated with the results of th~ Ames test. After a 4-week inhalation period not only the chromosome aberration frequency but also the SCE rate were significantly increased compared with spontaneous rates. Our results demonstrate a genotoxic hamard of the tested wastes, which could be clearly described by means of the applied battery of tests.

Institute of Industrial Toxicology, Martin Luther University Halle, Franzosenweg la, 0-4020 Balle, FRG.

Institut ffir Arbeitsphysiologie, Ardeystr. 67, 4600 Dortmund 1

126 MONITORING THE GENOTOXIC EFFECTS OF THE OCCUPATIONAL EXPOSURE OF METAL WORKERS • ~,~ J. Fuchs, J. Burg, H. G. Bienfait , G. Hiltl*, U. Bolm-Audorff # To monitor the genotoxic effects of a complex mixture of occupationally used chemicals directly in men the alkaline filter elution of DNA was applied. This test acting as a biological effectiveness test can also be used in cases where the exposure is unknown. Metal workers are occupationally exposed to different ingredients of the cutting fluids including Nnitrosodiethanolamine, which is known as a moderatly potent rodent carcinogen. 65 male metal workers from different small to medium-size enterprises were investigated. In order to quantitate the genotoxic injury the number of single strand breaks of the DNA was determined. DNA was isolated from mononuclear cells of I0 ml of the venous blood of the donors. The single strand break rate of the total group of metal workers as well as the rates of its smoking and non-smoking subgroups showed on the average only a slight increase compared to the corresponding control groups. However in the group of metal workers, which had an occupational exposure to cutting fluids more than 4.5 h/d the quantity of single strand breaks was clearly increased compared with workers with a shorter exposure. In the group of non-smoking workers the longer exposed men showed an increase of the mean single strand break rate of more than 40% (p ~0.01). The extent of the genotoxic injury was correlated to an environmental dose dependent parameter. *Hessisches Sozialministerium, Dostojewskistr. 4, D-6200 Wiesbaden, FRG #Amt f~r Arbeitsschutz, Hamburgerstr. 47, D-2000 Hamburg, FRG Institut f~r Toxikologie, Universit~t Mainz, Obere Zahlbacherstr. 67, D-6500 Mainz, FRG

128 MUTAGENICITY OF CHLOROPRENE (2-CHLORO- 1,3 -BUTADIENE) IN THE AMES-TEST AND IN HUMAN LYMPHOCYTES G. Westphal, E. Turner, U. Jorritsma Chloroprene (2-chloro-l,3-butadiene) is widely used as a plastic monomer. The substance has not yet been classified in terms of carcinogenicity to humans by the IARC (International Agency for Research on Cancer). Since the chemically related substance 1,3-butadiene is carcinogenic in animal experiments, an investigation of the mutagenic effects of chloroprene was performed. The substance is an industrial intermediate and therefore not commercially available in pure form. It was purchased from JohnsonMatthey Co., FRG, as a 50% solution in toluene. Since pure chloroprene is very unstable, it was extracted prior to the experiments by distillation and the purity (99,8 %) controlled by NMR spectroscopy and gas chromatography. In the Ames test with Salmonella typhimurium TA 100, chloroprene showed a dose dependent increase of revertants in a concentration range of 1 to 10 Iamol,.both with and without metabolic activation by rat liver microsomes induced with Arochlor 1254. An addition of reduced glutathione resulted in an enhancement of cytotoxicity but a reduction of the mutagenicity of chloroprene. When aminooxyacetic acid, an inhibitor of 13-1yase, was added to the test system, cytotoxicity was lower than in the incubations with glutathione but higher than in the samples with rat liver S 9 mix alone. This indicates the formation of a reactive intermediate through glutathione conjugation. Human lymphocytes were isolated from from freshly drawn blood samples of healthy donors by density centrifugation through a separation medium. After 24 hours of cultivation in a specific medium at 37°C, chloroprene was added at different concentrations. 30 minutes later, bromodesoxyuridine was added and the ceils incubated for further 46 hours. Colcemide was then added for termination of mitosis; 2 hours later the cells were fixed and stained on glass slides for microscopic determination of sister chromatid exchanges (SCE). At higher concentrations, chloroprene showed a marked cytotoxicity for the human lymphocytes. At lower concentrations, a significant increase of SCE was observed. The results indicate a cytotoxic and mutagenie effect of chloroprene both in Salmonella typhimurium and in human lymphocytes. The cytotoxic effect on human lymphocytes could explain an observed immunosuppressive effect of the substance. Institut flJr Arbeitsphysiologie an der Universit~it Dortmund, Ardeystr. 67, 4600 Dortmund 1

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129 Formation of DNA adducts by the hydroxyanthraquinone lucidin

131 DNA-BINDING BY HETEROCYCLIC AROMATIC AMINES FROM THE DIET I. FaShauer, D. Wild and D. Henschler

Poginsky,B., Westendorf,J., BI6meke,B., Dominiak,M., and Marquardt,H. Lucidin (1,3-dihydroxy-2-hydroxymethylanthraquinone)is the genotoxic principle of madder roots (Ruble tinctorum L.). To investigatewhether lucidin, indeed, interacts covalentlywith DNA, we incubatedthe compound (200.ug/ml)for 3 hr with DNA or polydG*polydC and $9 mix and examinedthe reisolated nucleic acids by 32p. poatlaballing.Similaradductpatterns,containingup to 5 spots,were obtainedon PEIcellulose tic with DNA or polydG*polydC. Formation of DNA adducts was also observed after treatmentwith lucidin (40/zg/ml) of primary rat hepatocytesin culture. Finally, DNA adducts were detected in liver, kidney, duodenum and colon of male Parkas micethat had beentreatedorallyfor 4 days with lucidin (2rag/d), its glycoside lucidinprimeverosids (10rag/d) or Ruble TeepR (1/2 tablet/d), a pharmaceutical preparationcontainingthe two compounds.Theseresultssuggestthat the therapeutic use Oflucidinmay constitutea carcinogenicrisk. Dept. of Toxicology, UniversityHamburgMedical School, Grindelallee 117, D-2000 Hamburg 13, F.R.G.

Supportedby a grantfrom DeutscheForschungsgemeinschaft,Bonn and Erich and Gertrud Roggenbuck-Stiftungzur Krebshilfe,Hamburg

Numerous heterocyclic aromatic amines have been detected in heated protein-containing materials such as fried fish, fried beef and beef extract. They are highly mutagenic in Salmonella and those tested are carcinogenic in mice, rats and macaques. We are studying the mechanisms of these genotoxic effects. Here we report on the formation of modified DNA-bases ("adducts") by four heterocyclic aromatic amines (IQ, MelQ, MeIQx, PhIP) in various rat organs. Rats were given a single oral dose (150, 15 or 1.5 #mole/kg) and after 12-72 h DNA was prepared from liver, stomach, intestine, pancreas, lungs and kidneys. The 32p_ postlabelling technique was used to detect and quantitate the adducts. Each amine induced the same characteristic set of adducts in all organs studied; the adduct frequencies varied widely with amine and organ. Results (adducts/108 bases) are given for high dose animals 12 h after treatment : . Amine Liver Stem. Intest. Pancr. Lungs Kidney ... IQ 130 4 10 10 MelQ 780 230 165 330 464 MelQx 110 9 8 12 14 PhlP 4 10 27 50 12 10 The liver was the main target organ of IQ, MelQ and MelQx whereas pancreas and intestine were the main target organs of PhlP. In addition, data on the repair of adducts will be reported. The DNA-binding potencies of the heterocyclic aromatic amines appear to be correlated with their mutagenic potencies in Salmonella. This study clearly shows the potent DNA-binding activity of heterocyclic aromatic amines in several organs of the rat; this genotoxic effect is very likely closely related to tumour initiation. Institute of Toxicology, University of WOrzburg, D-8700 Werzburg, Fed. Republic of Germany.

130 SYNTHESIS, MUTAGENICITY AND DNA ADDUCT FORMATION OF DIASTEREOMERIC FJORD-REGION DIHYDRODIOL EPOXIDES OF BENZO[c]PHENANTHRENE, BENZO[g]- AND BENZO[c]CHRYSENE. A. Seidel 1, T. Steinbrecher1, A. Hewer2 and D.H. Phillips2 Both diastereomeric fjord-region dihydrodiol epoxides of benzo[c]phenanthrene combine high mutagenic and tumorigenic activity with low chemical reactivity. In order to further investigate the molecular basis of the surprisingly high biological activity within this specific type of dihydrodiol epoxides, the synthesis of the syn-and anti-diasteromeric fjord-region dihydrodiol epoxides of benzo[g]- and benzo[c]chrysene has been undertaken. These compounds as well as both diastereomeric fjord-region dihydrodiol epoxides of benzo[c]phenanthrene and the bay-region dihydrodiol epoxides of benzo[a]pyrene were investigated for their half-lives in a physiological buffer and for their mutagenicity in S. typhimurium and in V79 Chinese hamster cells (in collaboration with H.R. Glatt, Institute of Toxicology, University of Mainz). The extent of formation of DNA adducts in V79 cells was measured by 32p-postlabelling. All six of the investigated fjord-region dihydrodiol epoxides showed high stability in physiological buffer at 37 °C (TI/2 > 2h) and extraordinarily high mutagenicity in four strains of S. typhimurium (TA97, TA98, TA100 and TA104) and in V79 cells. In V79 cells, the anti-dihydrodiol epexide of benzo[c]- chrysene was 8.6 and 12 times more active than the anti-dihydro- diol epoxides of benzo[c]phenanthrene and benzo[a]pyrene, respectively. The potent mutagenicity of the fjord-region dihydrodiol epoxides in V79 cells is due to the high frequency with which they form DNA adducts, rather than due to formation of adducts with greater mutagenic potential (Supported by the Deutsche Forschungsgemeinschaff (SFB 302)).

l Institute of Toxicology, University of Mainz, Ober¢ Zahlbacher Str. 67, D-W-6500 Mainz, Germany 2 Institute of Cancer Research, Cotswold Road, Belmont, Sutton, Surrey SM2 5NG, UK.

132 E F F E C T O F D N A A L K Y L A T I O N B Y N I T R O S O U R E A S O N SEMIC O N S E R V A T I V E D N A SYNTHESIS B Y P R O K A R Y O T I C D N A POLYMERASE C. Pfeiffer, A. F u c h s M 13 mp 18 p h a g e D N A w a s incubated with a series of homologue alkylnitrosoureas to study the effect of alkylation o n the propensity of single stranded D N A to act as a template for D N A synthesis using E coli polymerase Klenow fragment (KF polymerase). T r e a t m e n t with N-Methyl-N-nitrosourea (MNU) resulted in a clearly concentration-dependent inhibition of D N A synthesis. T h e sequencing gel revealed that polymerase stops introduced by methylation occured 1 b a s e 3' to template adenine residues. In contrast to methylation, alkylation b y longer chain nitrosoureas resulted in m u c h weaker effects on polymerase activity detectable as b a n d s on the sequencing gel. Moreover, no concentration-dependency w a s detectable as w a s the c a s e with MNU. From ethyl- to n-butylnitrosourea, the introduction of polymerase stops shifted from adenine to one b a s e 3' of template thymine residues. T h u s effects of alkylation on D N A elongation by K F polymerase can be discriminated with respect to the alkylating agent used. Universit~t K a i s e r slautern, FR Lebensmittelchemie/Umwelttoxikolgie, PoO.B. 3049, D - 6 7 5 0 Kaiserslautern, FRG.

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135

COVALENT BINDING OF QUINOID METABOLITES TO TUBULIN: A POSSIBLE MECHANISM FOR ANEUPLOIDY INDUCTION BY CARCINOGENIC ESTROGENS U. Harttig I, M. Metzler 2 and B. Epe I

MUTATIONAL ACTIVATION OF THE c-Ha-RAS GENE IN LIVER TUMORS OF VARIOUS RODENT STRAINS WITH DIFFERENT SUSCEPTIBILITY TO HEPATOCARCINOGENESIS A. Buchmann~. A. Luz2, N. Drinkwater 3 and M. S c h w a r z . _ ' _ _

Neoplastic cell transformation of Syrian hamster embryo (SHE) cells by diethylstilbestrol (DES) is most probably not caused by DNA damage and point mutations but involves the induction of mitotic aneuploidy. As metabolic activation appears to be important, too, we have tested the hypothesis that covalent binding of DES metabolites to certain proteins (rather than to DNA) might cause aneuploidy and thereby initiate carcinogenesis. When SHE cells are exposed to14c-labelled quinoid metabolites of DES, tubulin is one of the cellular proteins found to be covalently labelled. Experiments under cell-free conditions indicate that the covalent binding of DES metabolites to tubulin is associated with a pronounced inhibition of microtubule assembly and could therefore represent the chemical basis of chromosomal non-disjunction and aneuploidy. After reaction of 14C-DES metabolites in SHE cells, however, most label is found in an unidentified low-level protein of 27.4 kDa. In leukemic HL-60 cells, which have a high potential for peroxidative metabolism, the pattern of covalent protein labelling observed after incubation with DES is the same as with quinoid metabolites of DES. Interestingly, no labelling of a 27.4 kDa protein is found in these cells. IInstitute of Toxicology, University of Wdrzburg, Versbacher Sir. 9, D-8700 W~rzburg, Germany 2Dep. of Food Chemistry and Environmental Toxicology, University of Kaiserslautern, D-6750 Kaiserslautern, Germany

The frequency and pattern of mutations at codon 61 of the c-Ha-ras gene have been analyzed in liver tumors and precancerous liver lesions from various rodent strains with differing susceptibility to hepatocarcinogenesis. The animal strains used were the highly susceptible C3H/He, CBA, B6C3F1, and CFI mice, the insensitive C57BL/6J and Balb/c mice and the comparatively resistant Wistar rat. Using the polymerase chain reaction and allele-specific oligonucleotide hybridization, C - > A transversions at the first base and A - > T transversions or A - > G transitions at the second base of c-Ha-ras codon 61 were detected in 20 - 60% of spontaneous or carcinogen-induced liver tumors of the four sensitive mouse strains but not in liver tumors of the two insensitive mouse strains or the Wistar rat. Further analyses of c-Ha-ras codon 12 mutations in liver tumors from the insensitive rodent strains also failed to give any positive results. In early precancerous liver lesions, c-Ha-ras codon 61 mutations were found in 13 - 14% of lesions 6f the sensitive C3H/He and B6C3F1 mouse strains, whereas no such mutations could be detected in precancerous lesions of the insensitive C57BL/6J mouse. Taken together, our results indicate a close correlation between the mutational activation of the c-Ha-ras gene in liver tumors of the different rodent strains and their susceptibility to hepatocarcinogenesis, whereby the mutations appear to provide a selective growth advantage, leading to a clonal expansion of the mutated liver cell population, only in livers of sensitive but not of insensitive strains. ~Institute of Toxicology, University of Tiibingen, 7400 Tiibingen, Germany, 2Institute of Pathology, GSF, 8042 Neuherberg, Germany, 3McArdle Laboratory for Cancer Research, Madison, USA, 'Institute of Experimental Pathology, DKFZ, 6900 Heidelberg, Germany

134

136

INTERRELATIONSHIP OF MICRONUCLEUS FORMATION, GENOMIC

MUTATIONS IN THE HA-RAS PROTO-ONCOGENE IN LIVER TUMORS OF THE C3H MOUSE OCCURRING SPONTANEOUSLY OR AFI'ER TREATMENT WITH TUMOR PROMOTING AGENTS. R. Bauer-Hofmann, S. Kress, and J. Mahr

LOSS AND CELL TRANSFORMATION INDUCED BY DIETHYLSTILBESTROL (DES) D. Schiffmann, H. Stopper, P. Lenz, U. De Boni* and B. K. Vig** Certain nongenotoxic carcinogens (e.g.some estrogens like DES) cause mitotic disturbances resulting in displacement, nondisjunction and subsequent loss of chromatin elements. DES induces micronuclei (MN) and neoplastic transformation in Syrian hamster embryo (SHE) flbroblasts. However, the key events at the early onset of this multistage process are still under debate. Our previous results show that DES induced MN contain kinetochores (K+) suggesting the presence of whole chromosomes/chromatids. We now have analyzed the meta-anaphase ring arrangement of chromosomes in SHE cells. After DES treatment, a threefold increase in the number of chromosomes displaced from the ring was observed. In mouse LA 9 cells we found a similar increase, with a maiority of K+ chromosomes . Most likely, these displaced chromosomes can give rise to the observed K+-MN. Furthermore, a 10% subpopulation of these MN exhibits chromatin compaction (electron microscopy). Since such MN will not be reintegrated, genomic loss will occur, possibly depending on the type of chromosome / chromatid enclosed. In addition, the frequency of DES-mediated SHE cell transformation also is 10% of the one for MN formation. Further investigations will show, whether these phenomena are related to the loss of tumor suppressor genes. Institute of Toxicology,University of Wfirzburg,87 Wiirzburg, Germany *Dept. of Physiology, University of Toronto, Canada, **Dept. of Biology, University of Nevada Reno, USA.

In the present study we have analysed the frequency and pattern of point-mutations in codon 61 of the Ha-ras proto-oncogene in liver tumors of male C3H/He mice that occurred spontaneously or after treatment with 500 ppm phenobarbital or I0 ppm dieldrin in their diet. The liver tumor prevalence at 52 weeks after start of treatment was 41% (15/37) in control mice, and 63% (10/16) and 67% (10/15) in mice treated with phenobarbital or dieldrin, respectively, indicating that the two promoting agents led to an enhancement of the liver tumor frequency. Serial frozen sections were performed through the entire liver of each animal and stained for glucose-6-phosphatase activity. Liver lesions characterized by a loss in this enzyme activity ranging between 0.5 and 3.0 mm in diameter were punched out from the liver sections. For mutation analysis we used the method of in vitro amplification of DNA via the polymemse chain reaction combined with selective oligonucleotide hybridisation. Our preliminary results demonstrate the presence of codon 61 mutations both in spontaneous tumors and in tumors induced by prolonged treatment with phenobarbital and dieldrin. The mutation frequencies, however, showed striking differences between the three treatment groups examined. While approximately 60% (11 out of 17) of the spontaneous liver tumors contained point mutations in codon 61, the mutation frequency at this gene locus was lowered to a value of only 25 % in tumors occurring during treatment with either phenobarbital (4 out of 16 tumors) or dieldrin (6 out of 24 tumors). These results suggest that mutations in the Ha-ras gene in liver tumors of promotortreated mice represent background mutations not related to treatment and, secondly, that tumor promotors such as phenobarbital and dieldrin also confer a selective growth advantage on those initiated mouse hepatocytes that do not contain an activated Ha-ras gene. Deutsches Krebsforschungszentrum, Institut fiir Experimentelle Pathologie, Im Neuenheimer Feld 280, D-6900 Heidelberg.

R 35 137

139

DETECTION OF GENOMIC ALTERATIONS IN CARCINOGENINDUCED MOUSE LIVER TUMORS BY DNA FINGERPRINT ANALYSIS. O. Mtiller

COMPARISON OF PARTICULATE GUANYLYL CYCLASE FROM BOVINE ADRENAL CORTEX WITH A CLONED ANF-SENSITIVE GUANYLYL CYCLASE. J.-M. Helm, H.-J. Ftille, S. Singh , and R. Gerzer

According to the present understanding of the carcinogenic process, the transition of a normal cell to a tumor cell is mediated by sequential genetic alterations in critical cell regulatory genes such as protooncogenes or tumor suppressor genes that may lead to either the activation or the inactivation of these genes. Molecular correlates are structural changes of the genome ranging from specific point mutations to loss of entire chromosomes. Evidence for structural alterations in the genome of tumor cells has classically been accumulated from cytogenetic studies. In the present investigation DNA fingerprint analysis was used to study structural abnormalities in the genome of mouse liver tumor cells. Liver tumors were induced in three different strains of mice by single injection of 20 I.tg/g body wt. diethylnitrosamine on day 15 after birth. Liver tumors were removed 30 to 45 weeks later. Tumor DNA was isolated, digested with restriction enzymes and hybridized on Southern blots with wild-type bacteriophage M13 DNA as probe. The resulting fingerprints of tumor DNA were compared with those of D N A from normal liver tissue. Genomic aberrations were detected in two out of 68 tumors analyzed, one stemming from a C57BL/6J and the other from a C3H/He mouse. In spite of the fact that the frequency of genomic alterations observed in this study was relatively low, DNA fingerprint analysis has the advantage that, in contrast to classical cytogenetic analyses, frozen tissue can be used and analyses can be performed without selection of metaphase chromosomes.

Recently, an ANF-sensitive guanylyl cyclase (GC-A) has been cloned from a rat brain cDNA library (Nature 338:78-83 (1989)). A rat glioma cell line (C6) was permanently transfected with rat GC-A cDNA with the lipofectin method. Stable clones were selected with geneticin. The following characteristics of this cloned ANF-sensitive guanylyl cyclase were evaluated: stimulation of cyclic GMP accumulation as well as binding characteristics were measured in confluent monolayers, whereas guanylyl cyclase activity was measured with membranes from homogenized cells (100 000 x g; 30 rain). ANF, BNF, atriopeptin 1 (AP 1) as well as urodilatin, a recently described N-terminally elongated ANFanalog, were used. The characteristics of GC-A were compared with those of membrane guanylyl cyclase prepared from bovine adrenal cortex (BAC). In C6 cells expressing GC-A, ANF and BNF stimulated cyclic GMP accumulation to the same extent (ED5~'I nM), whereas AP 1 and urodilatin were less effective (EDs~,-10 nM). The analogs tested showed the same order of potency when g'uanylyl cyclase activity was evaluated with a membrane fraction from C6 cells expressing GC-A. In contrast, binding characteristics were comparable for ANF and the analogs BNF and urodilatin. In contrast to these results obtained with GC-A, AP 1 did not stimulate guanylyl cyclase activity in membranes from BAC, while urodilatin was as effective as ANF and BNF. In BAC membranes, binding of ANF and BNF was comparable. These results suggest that GC-A is different from the ANF-sensitive guanylyl cyclase in BAC membranes. Since the binding characteristics for the tested ANF analogs in C6 cells expressing GC-A were comparable with those on BAC membranes, while the order of potency for activation of guanylyl cyclase differed, signal transduction mechanisms in response to various natriuretic peptides may be distinct in BAC and GC-A.

Deutsches Krebsforschungszentrum, Institut fiir Experimentelle Pathologie, Im Neuenheimer Feld 280, D-6900 Heidelberg.

Supported by the Deutsche Forschungsgemeinschaft (Ge 399/3-3) Medizinische Klinik, Klinikum Innenstadt Ziemssenstr.1, D-8000 Miinchen 2, FRG

Universit~tt,

~o

138 I~AND EXHIBIT BINDING

der

I~ oGMP-DEPENDENT PROTEIN DIFFERENT PHOBPHOTRANSFERASE ACTIVITIES

W.Landgraf,

P.Ruth,

KINASE AND

B.May, A.Keilbach,

(oGK) oGMP

F.Hofmann

The enzymatic properties of recombinant type I~and I$ cGK were studied. Recombinant Iu- and I# cGK were transiently expressed by t r a n s f e c t i o n of the cloned cDNAs into COS-7 cells. Cytosolic extracts contained about 1 ~g of Iu- or I~ cGK per i0 u cells. The recombinant cGKs were indistInguishable in molecular weight from cGK purified either from bovine lung or tracheal s m o o t h muscle, The extracts were m e a s u r e d for phosphotransferase activit~ at 3 0 " C in the presence of an synthetic inhibltor p e p t i d e for cAMP-dependent protein kinase and the substrate peptide GRTGRRNSI. In the presence of cGMP or 8-Br-cGMP the catalytic rat~s w~re 5.0±.04 (3) and 4 9 ±.03 (3) ~mol.m~n'~.mg ~ for Iu- and I@ cGK, respectively. These catalytic rates are similiar to those obtained for the Iu- and I~ cGK purified from bovine tracheal smooth muscle. For the Iu cGK, half maximal stimulation of the phosphotransferase activity was obtained at 0.i ~M cGMP or 0.3 ~M 8-Br-cGMP. In contrast, the K_ values of the I~ cGK for cGMP and 8-Br-cGMP ~ere increased 13-fold and 41-fold, respectively. A similar shift in the activation curve was f o u n d between the purified bovine tracheal Iu- and I~ cGK. Equilibrium binding of cGMP at 4°C d e m o n that both isoforms bound 2 moles cGMP/ strated mol subunit. For the Iu cGK a high and low affinity cGMP binding site was detectable whereas for the IB cGK only a low affinity binding site was calculated. These results were confirmed by measuring the dissociation of cGMP at 4°C. For the Iu cGK a slowly and rapidly cGMP exchanging site was obtained, whereas two rapidly cGMP exfor I@ cGK. These changing sites were m e a s u r e d results show that the p h o s p h o t r a n s f e r a s e activity and the cGMP binding o f I ~ - and I~ cGK are regulated by its different aminoterminal domains. Inst.f. Pharmakologie und Toxikologie der Techn. Universit~t, Biedersteiner Str.29,W-8 M ~ n c h e n 40

EXPRESSION OF E . C O L I ~ F U N C• T I O N A L

THE HUMAN INTERACTION §

~z-ADRENOCEPTOR WITH 2 FORMS §

E.Selzer , S.Marullo" A.D.Strosberg

IN O F .. G ~

and W.Schutz

The coupling of the human B2-adrenoceptor expressed in E.coli was investigated using 2 splice variants of the ~-subunit of G s synthetized in E.coli (rG s ~ - ~ and rGs~ . " L ) and the . B~-subunit . . . .purified from bovlne braln. In competition blnd~ng experiments with (-) [125I]iodocyanopindolol (ICYP) and isoproterenol (ISO), rG s~- . ~ and. rGs~L.S~ recon. . o . • stltuted guanine nucleot1~e-sensltlve, hlgh-afflnity agonist binding. Titration of the B2-adrenoceptor at a fixed concentration ratio of ICYP/ISO (60pM/20nM) showed• that rG S ~ - ~ and. rG ~. were equi~L , , potent (EC~o-3nM) in reconstltut~ng ~ g h - a f f ~ n ~ t y binding. S~ecificity was also probed using rGs~PT , a mutant of rG.s -L with an altered carboxyterminus, a n d rG.~1 , w h l ~ were -20- and -200-fold less potent, ~ s p e c t ~ v e l y . A comparison of the S2-adrenoceptor expressed in E.coli with the receptor in $49 cyc-membranes revealed a similar affinity of rG s~-s and rG sa- for the recombinant and native . receptor. I n c ~ a t l o n of rGs~.s.B[ with partially solubilised E.coli membranes followed by dilution results in the stable incorporation of G s into the membranes as assessed by Western blots. Incorporated rGs¢-s is capable of coupling to the recom• • blnant receptor as determined not only by ICYP/ ISO competition curves but also by ISO-mediated acceleration of the rate for [35S]guanosine 5 ~-(3O-thio) triphosphate binding. These results show that (i) receptor-G protein coupling can be reconstituted in E. coli using recombinant components and that (ii) the ~2-adrenoceptor does not .discriminate between splice variants of Gs~. * I n s ¢ i t u t e of Phar~co[ogy, U n i v e r s i t y of V i e ~ a W~hcJnger SOt. 13a, A-lOgO Vienna § Ins~iout Cochin ~ G~n~ti~e ~ t 4 c u t a i r e , 22 rue M4chain, F-75014 Paris suppor¢~ ~ a grant f r ~ the "Fo~s zur F6rderung de~ wissenschafttichen Forschung" (P~22)

R 36

141 PERTUSSIS TOXIN ABOLISHES THE INHIBITORY ACTIVITY OF A SERUM FACTOR ON 8-ADREN~GICALLY IN-~JCED CYCLIC kMPPRODUCTION IN RAT GLIC~4A C6-BUrl CELLS A. Sarem-~slani and B.Hamprecht* A ser~n factor (SF) inhibits 8-adrenergically induced cyclic AMP-production in rat glioma cells C6-BU-I cells (v~n Calker et al., Hoppe Seyler's Z. Physiolog. chen. 1977;358:1188). Recent studies have shown a pertussis toxin (PTX) sensitive GTPase activity of fetal calf ser~n (FCS) on this cell line (Milligan, Biochem. J. 1987;245:501-505). We investigated the effect of PTX on the inhibitory activity of SF, SF was partially purified by extraction of lyophilized bovine serum with methanol followed by chrcr~tography on a silicagel-col~m~. The inkibitory activity was enhanced by addition of fatty acid free BSA to aqueous solutions of SF. C6-BU-I cells were cultured in the absence of FCS. Cyclic AMP levels are given in p~nol/r~ protein (means ± SD, n = 3). additions

without PTX 26

±

PTX (50 n g / m l )

1

27 ~

2

Isoproterenol (400 nM)

799 ± 90

832 ±

55

Isoproterenol (400 r~) + SF (i:i000)

402 ± 23

801 ± 119

These results indicate that SF inhibits the 8adrenergically induced cyclic AMP-preductic~ through a PTX-se~nsitive substrate, probably a C~-protein. Since Rireceptors have not been found in C6-BU-l-cells, SF could be a valuable tool to investigate Gi-protein-mediated signal transduction in this cell modell. The physiological role and the chemical structure of SF rerm~in to be clarified. Abteil~gf~rK]inischeCh~mie,Robert-Bosch-Kraken~,D-7000Stuttgart50 ~Physio[wisch-Ch~isch~l~itu~der~iversit~f,D-7400~bin~,~RG

142 C Y C L I C AMP M E D I A T E S U P - R E G U L A T I O N PROTEIN ALPHA-SUBUNITS I N CHICKEN

~

=

~

OF I N H I B I T O R Y G HEART MUSCLE C E L L S

..........................................

In a d d i t i o n to a d e c r e a s e i n t h e number o f s t i m u l a t o r y r e c e p t o r s , a d e n y l y l c y c l a s e s t i m u l a t o r y hormones have been shown to i n c r e a s e t h e l e v e l o f t h e a l p h a subunits of the inhibitory G-protein of adenylyl c y c l•a s e (G• i a ). The p u r p o s e o f t h e p r e s e n t s t u d y was t o l n v e s t l g a t e w h e t h e r hormonal u p - r e g u l a t l o n o f Gia i s a c y c l i c AMP-mediated p r o c e s s and w h e t h e r i t i s correlated with receptor down-regulation. Exposure of chicken heart muscle cells for 3 days w i t h t h e f i - a d r e n o c e p t o r (fi-AR) a g o n l s t i s o p r o t e r e n o l (lOuM) d e c r e a s e d t h e number o f fi.-AR a s d e t e r m i n e d by b i n d i n g s t u d i e s w i t h (3H)CGP ]2177 by a b o u t 60%. Q u a n t i t a t i o n of G. by p e r t u s s i s t o x i n - c a t a i y z e d ( 3 2 p ) A D P - r i b o s y l a ~ o n showed an i n c r e a s e i n t h e l e v e l o f Gi a by a b o u t a f a c t o r o f 2 i n membranes o f i s o p r o terenol-treated c e l l s . S i m i l a r to i s o p r o t e r e n o l ~ ~he receptor-dependent adenylyl cyclase stimulator p r o s t a g l a n d i n E 1 (lOuM) and t h e r e c e p t o r - i n d e p e n d e n t a d e n y l y l c y c l a s e s t i m u l a t e r f o r s k o l i n (lOuM) i n o f Gia by a b o u t a f a c t o r of t w o c r e a s e d the level and d e c r e a s e d t h e number of ~I-AR by 40 and 50%, respectively. In c o n t r a s t , t r e a t m e n t o f t h e h e a r t m u s c l e c e l l s i n t h e p r e s e n c e of t h e fi-AR a n t a g o n i s t w i t h weak p a r t i a l a g o n i s t i c a c t i v i t y c e l i p r o l o l (lOuM) d e c r e a s e d t h e number of 8I-AR by 20% b u t had no e f f e c t on t h e l e v e l o f G i a - p r o t e i n s . Conclusion: The d a t a s u g g e s t t h a t hormonal u p - r e g u ~-~-~. . i s m e d i a t e d b y. c y c l i c A~P. T h i s i n a c r e a s e in t ~ e l e v e l o f Gi a ~s n. o t s t r i c t l y correlat e d w i t h r e c e p t o r d o w n - r e g u l a t i o n . The ~-AR downr e g u l a t i o n i n d u c e d by p a r t i a l ~-AR a g o n i s t s seems to be m e d i a t e d . b y a c y c l i c A ~ P - i n d e p e n d e n t mechanism. M e d i z i n i s c h e K l i n i k I , U n i v e r s i t ~ t M~nchen, K l i n i k u m Grofihadern M a r c h i o n i n i s t r o 15~ D-8000 MHnchen 70~ F.RoGo

143 B-ARRESTIN AND 6-ADRENERGIC RECEPTOR KINASE MEDIATE HOMOLOGOUS DESENSITIZATION OF B-ADRENOCEPTORS M.J, Lohse Stimulation of B-adrenoceptors by agonists causes a specific loss of receptor responsiveness that is termed homologous desensitization. This process has been suggested to be mediated by a kinase that specifically phosphorylates agonist-oecupied B-receptors,termed B-adrenergic receptor kinase (BARK)• However, in a reconstituted system using the purified components B-receptor, Gs and BARK, BARK-mediated phosphorylation has only minor effects on B-receptor function, suggesting that additional factors we required for homologous desensitization• A protein that is proposed to represent such a factor was cloned and was termed B-arresfin. B-An'estin is similar to the retinal protein arresfin that has been implicated in inhibition of signal transmission from rhodopsin to Gt. Both proteins, B-arrestin and arrestin, were overexpressed in COS-cells and were purified from the cytosol to apparent homogeneity using sequential ammonium sulfate precipitation, gel filtration, and chromatography on hydroxylapatite and Mono-Q. In a. reconstituted system, both proteins were capable of inhibiting signal transduction from B-receptors to Gs, with B-arrestin being about 100-fold more potent. The reverse specificity was found in the rhodopsin-Gt-system: arrestin was much more potent than B-arrestin. Phosphorylafion of B-receptors by BARK resulted in, a 50-fold increase in the potency of B-arrestin. Under these conditions, half-maximal inhibition of B-receptor function was achieved at a ratio of B-arrestin to receptors of 1:1. B-Arrestin seems to compete with Gs for binding to the receptors. Thus, homologous desensitization appears to occur in the following steps: Agonist-occupied receptors are phosphorylated by BARK. This promotes the binding of g-arrestin, which prevents the interaction between receptor and Gs, and thus causes the desensitized state of the receptors. To determine the rate limiting step, stable cell lines were created overexpressing either BARK or B-arrestin, and different concentrations of B-receptors. Alterations of receptor function were observed only in cells expressing high levels of B-receptors: in these cells overexpression of BARK attenuated receptor signalling and caused increased homologous desensitization• This suggests that BARK-mediated phosphorylation is the rate-timiting step in homologous desensitization of B-receptors. Laboratory of Molecular Biology, University of Munich, Max-PlanckInstitute of Biochemistry, 8033 Martinsried, FRG•

144 CHOLERA TOXIN AND THE PHORBOL ESTER TPA SYNERGISTICALLY STIMULATE ADENYLYL CYCLASE IN PPJMARYASTROCYTE CULTURES P.J.Gebicke-Haerter, A Schobert, G. Hertting Cholera toxin (Ctx) ADP-ribosylates the a - s u b u n i t of G s , the stimulatory G protein of adenylyl cyclase (AC), which results in prolonged activation of the enzyme. It is shown here, that pretreatment of cultured rat astroglia with Ctx both markedly increased levels of intracellular cAMP and resulted in a change of astrocyte morphology into a stellate, ramified form. These effects were observed with as little as 1 ng Ctx/ml media. A significant rise of intracellular calcium by the action of the calcium ionophore A23187 had no influence on AC activities in both untreated or Ctx-treated cells. The phorbol ester TPA, however, synergistically enhanced cAMP levels in the presence of Ctx. This effect was abolished when cells were preincubated with T P A for 18 h to deplete protein kinase C (PKC). Pretreatment of cells with pertussis toxin (Ptx) and subsequent stimulations with A23187 or T P A had no effects on cAMP levels whatsoever. Simultaneous incubations with Ptx and Ctx and subsequent additions of stimuli revealed characteristics typical for Ctx alone. From these data it is concluded, that phorbol esterstimulated PKC either indirectly (via pp60 c'src) or directly phosphorylates the catalytic unit of AC. The described synergism arises from the concomitant activation of G s by Ctx. Institute of Pharmacology, University of Freiburg, HermarmHerder-Str. 5, D-7800 Freiburg i.Br.

R 37 145 RECONSTITUTION OF THE A~-ADENOSINE RECEPTOR WITH DEFINED G PROTEIN ~-SUBUNITS REVEALS A SELECTIVIT Y• F O R r G ~ - o ~

M~chael

Frelssmuth

and Maurine

L. Linder §

The ability of the bovine brain A~-adenosine receptor to discriminate between different G protein subtypes was tested using ~-subunits synthetized in E.coli (rG~-subunits). When combined with a three-fold molar excess of purified 5[-subunit and used at high concentrations, all three subtypes of rG~ and r G ~ were capable of reconstituting guanine nucleotide-sensitive, high-affinity binding of the agonist (-)~-3[~sI](iodo-4-hydroxyphenylisopropyl)adenosine (IHPIA) to the receptor. Titration of the receptor with increasing amounts of rGi~ I, rGi~2, rGi~3 and r G ~ revealed a -10-fold highe~ affinity fo~ rG~.~. This selectivity was also observed in the absence of the 6~subunit Other ~-subunits (rG sQ ' $ , rGs., rG S ~ PT, ~'L and rGz~ ) dld not interact with the receptor. In N-ethylmaleimide-treated bovine brain membranes, rG~=~ was the only rG. a -subunit capable of recon• , stltutlng high-affinity agonist binding. Similarly, r G ~ . competed potently with [~-~2P]GDP-prelabeled r ~ ~ for activation ,by the agonist-liganded At-adenosine receptor, whlle a ~50-fold molar excess of r G ~ was required to quench the receptormediated release of [~-32P]GDP from rG~.~. Hence, in spite of the extensive homology between o-subunits of the G~/Go-group, the At-adenosine receptor appears to discriminate between the subtypes. •

,

* Institute of Pharmacology, University of Vienna W~hringer Str. 13a, A-1090 Vienna § Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 supported by a grant from the "Fonds zur F6rderung der wissenschaftlichen Forschung" (P7622)

146 PERTUSSIS TOXIN-SENSITIVE AND -INSENSITIVE INCREASES IN CYTOSOLIC CALCIUM IN HUMAN ERYTHROLEUKEMIA (HEL) CELLS I. Schwaner The pluripotent human erythroleukemia cell line, HEL, possesses thrombocytic, erythrocytic and macrophage-like properties. ~-Adrenergic agonists increase cytosolic Ca2+ ([Ca2+]i) in HEL-cells via pertussis toxin (PT)-sensitive guanine nucleotide-binding proteins (G-proteins) of the Gi family (Michel MC et al. (1989) J Biol Chem264:4986). We studied the increases in [Ca2÷]i mediated by various receptor agonists and focussed on their PT-sensitivity. Thrombin, platelet-activating factor (PAF), prostaglandins E1 and E~ (PGEI and PGE~), the PGEz-analogue, sulprostone, the stable analogue of prostaglandin I2, iloprost, ATP and UTP increased [Ca2+]i through transient mobilization of Ca~+ f r o m intracellular stores and sustained influx from the extracellular space. ADP was less effective than ATP and UDP; UMP and AMP were ineffective. The kinetics of the Ca2+ signals induced by nucleotides and eicosanoids were (luite different. PT completely inhibited the rises in [CaZ÷]i stimulated by PGEz and sulprostone and p a r t i a l l y inhibited the one mediated by PGEI. Rises in [Ca2+]i induced by the other agonists were PT-insensitive. All increases in [Ca2*]i were desensitized in a homologous manner. PGE2 and sulprostone did not desensitize the effect of iloprost and vice versa. PGEI blunted the responses of the other eicosanoids but not vice versa. Our data suggest that (I) HEL cells possess PGEz- and PGI~-specific receptors and PGEI mimicks the effects of PGE2 and iloprost. ( I I ) HEL cells possess purinoand pyrimidinoceptors with properties similar to those of macrophages but not of thrombocytes. (III) PT-sensitive a n d - insensitive mechanisms play very different roles in the rises in [Ca2+]i mediated by various types of agonists. I n s t i t u t f(ir Pharmakologie, Freie Universit~t Berlin, Thielallee 69/73, D-IO00 Berlin 33, F.R.G.

147 ANTISENSE OLIGONUCLEOTIDES HYBRIDIZING WITH CODING SEQUENCESOF G-PROTEINe-SUBUNITS REDUCETHE HORMONALINHIBITION OF VOLTAGE-DEPENDENTCa2+ CURRENTSIN GH3 CELLS. C. Kleuss*, C. Ewel*, J. Hescheler, G. Schultz, and B. Wittig* While the number of known G-proteins is rapidly increasing, the identity of G-proteins involved in functional coupling between receptors and modulated effectors is not known for most systems. We tried to inhibit the expression of G-protein e-subunits by intranuclear injection of oligonucleotides into rat pituitary GH3 cells. Oligonucleotides were synthesized, which can either hybridize with a coding sequence common to known G-protein e-subunits or a coding sequence found in the two forms of the GO ~-subunit (~oi and eo2). Intranuclear injection of either oligonucleotide markedly reduced the inhibition of voltage-dependent Ca2÷ currents by somatostatin or carbachol. The reduction of Ca2+ current inhibition was observed 12 hours after injection; a maximum was reached between 24 and 48 hours. Antisense ol.igonucleotides hybridizing with coding sequences for either the eol- or the :o2-subunit selectively reduced the Ca2+ current inhibition induced by carbachol or somatostatin, respect i v e l y . Oligonucleotides complementary to coding sequences of e-subunits of Gs and the Gi family and sense oligonucleotides were ineffective. These results are consistent with the involvement of Go-subtypes in the inhibitory Ca2+ current modulation and establish intranuclear injection of antisense oligonucleotides as a tool for the identification of G-protein functions. * I n s t i t u t fur Molekularbiologie und Biochemie, Freie Universit~t Berlin, Arnimallee 22, D-tO00 Berlin 33 Institut fur Pharmakologie, Freie Universit~t Berlin, Thielallee 69-73, D-tO00 Berlin 33

148 PEPTIDE ANTIBODIES DISTINGUISH BETWEEN SUBTYPES OF GO ~-SUBUNITS K. Spicher, F.-J. K]inz, B. N~rnberg, I. Tychowiecka, W. Rosenthal Go is a highly abundant pertussis toxin-sensitive G-protein in neuronal, neuroendocrine and pituitary cells, in which i t may be involved in the inhibitory modulation of voltage-dependent Ca2+ channels. Its e-subunit (eo) exists in multiple forms. This diversity is at least in part explained by alternative splicing of a single transscript, yielding the two products, eol and eo2 (Hsu et a l . , J. Biol. Chem. 265, 11220-11226, 1990; Strathmann et a l . , Proc. Natl. Acad. Sci. USA 87, 6477-6481, 1990). The proteins encoded by eol and ~o2 mRNAdiffer in their Nterminal thirds. Antibodies against synthetic peptides corresponding to confined regions of Go e-subunits were raised in rabbits. Recombinant proteins (obtained by expression of ~oI and eo2 cDNA in E. coli) and membranes of insulin-secreting cell lines (SV 40-transformed pancreat i c B-cells from hamster, HIT; rat insulinoma cells, RINm5F) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. An antibody (AS 6) raised against a peptide common to both eol and Co2 (one-letter code C-NLKEDGISAAKDVK) recognized eol and eo2 fusion proteins and 39 and 40 kDa proteins in membranes of HIT and RINmSF cells. An antibody (AS 244) raised against a peptide specific for eol (one-letter code SKNRSPNKEIYCHM) recognized the %1 but not the eo2 fusion protein and 40 but not 39 kDa proteins in membranes of HIT and RINm5F cells. An antibody (AS 201) raised against a peptide specific for eo2 (one-letter code C-GPSAFTEAVAHIQGQY) recognized the mo2 but not the eol fusion protein and 39 but not 40 kDa proteins in membranes of HIT and RINm5F cells. The data indicate that 39 and 40 kDa GO e-subunits are encoded by %2 and %1 mRNA, respectively. Supported by the Deutsche Forschungsgemeinschaft. I n s t i t u t fur Pharmakologie, Freie T h i e l a l l e e 69/73, D-IO00 Berlin 33

Universit~t

Berlin,

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C O M P L E M E N T A T I O N OF FORMYL PEPTIDE RECEPTORM E D I A T E D SIGNAL T R A N S D U C T I O N IN XENOPUS OOCYTES P. Gierschik, P. Stannek, S0 Gierschik, M. Voigt*, and P. Schultz

Formylmethionine-containing peptides (e.g. fMetLeu-Phe) stimulate a v a r i e t y of neutrophil functions by interacting with specific cell surface receptors which are coupled via a G-protein(s) to stimulation of p h o s p h o l i p a s e C (PLC). To characterize the formyl peptide r e c e p t o r - m e d i a t e d signal transduction at the molecular level, we have cloned the receptor cDNA and expressed the receptor protein in ~enopus laevis oocytes. R e c e p t o r a c t i v i t y was determined e l e c t r o p h y s i o l o ~ i c a l l y by measuring f M e t - L e u - P h e - e l i c i t e d opening of C a ~ ÷ - d e p e n d e n t C]- channels. Injection of pure formyl peptide receptor mRNA did not lead to p e p t i d e - d e p e n d e n t changes in m e m b r a n e current. In contrast, m a r k e d alterations of membrane currents were o b s e r v e d in response to fMet-Leu-Phe when the receptor mRNA was s u p p l e m e n t e d with poly(A) ÷ RNA isolated from undifferentiated human leukemia (HL-60) cells. Injection of the latter RNA alone did not lead to e l e c t r o p h y s i o l o g i c a l responses to fMetLeu-Phe. These results d e m o n s t r a t e that Xenopus oocytes injected with pure formyl peptide receptor mRNA lack a specific component which is essential for formy] peptide receptor-mediated signal t r a n s d u o t i o n and is present in immature granulocytes. Identification of this factor most likely a G-protein subunit or a PLC isoenzyme - will provide important information about the molecular mechanisms by which G-protein-coupled receptors stimulate phospholipase C. Pharmakologisches Institut and *Zentrum f6r Molekulare Bio]ogle, U n i v e r s l t ~ t Heidelberg, Im Neuenheimer Feld 366 and *280, D-6900 H e i d e l b e r g

A D P - R I B O S Y L A T I O N OF THE G E L S O L I N ACTIN C O M P L E X BY CLOSTRIDIAL TOXINS M. Wi!lel t A. Weqner ~ and K. Aktories *,~ C. botulinum C2 toxin and C__.perfrinqens iota toxin A D P - r i b o s y l a t e monomeric but not polymsrized actin. The actin-binding protein gelsolin which forms dimeric (GA) and trimeric (GAA) complexes with actin, severs, nucleates and caps filamentous actin thereby regulating the dynamic transition between monomeric and p o l y m e r i z e d actin. Here we studied the ADPr i b o s y l a t i o n of actin in the gelsolin actin complex. GA and GAA were isolated from the lysate of human platelets or leukocytes by DNAse I-affinity c h r o m a t o g r a p h y or by using anti-gelsolin antibody and were characterized by gel filtration on HPLC. Iota toxin ADP-ribosylated GA and GAA as effectively as G-actin. The time course and the NAD dependence of iotatoxin induced A D P - r i b o s y l a t i o n of actin and GA were identical. In contrast, C2'toxin ADP-ribosylated GA less effectively than actin. GA bound A D P - r i b o s y l a t e d actin (AN) to form the GAAr complex with an about 3-fold higher affinity than unmodified actin. Also A D P - r i b o s y l a t e d GA~ formed the GArA complex with actin. The nucleation activities of GA and of GAr were identical. In contrast, the nucleation activity of A D P - r i b o s y l a t e d GAAr was largely decreased compared with unmodified GAA. The data show that GA and GAA are substrates of ADPribosylating toxins and indicate that the two actin molecules of the GAA complex are functionally different. I R u d o l f - B u c h h e i m - I n s t i t u t f~r Pharmakologie der U n i v e r s i t ~ t Gieaen, D-6300 Gie~en, ~Institut f~r Physiologische Chemic, RuhrUniversit~t, D-4630 Bochum, SPharmakologisches Institut des Universit~tsklinikums Essen, D-4300 Essen

150

152

COUPLING TO ACTIN

OF fMLP-RECEFTORS FILAMENTS

Karl-Norbert Jesaitis

Klotz,

Dan

FROM HUMAN NEUTROPHILS

Siemsen,

Algirdas

J.

Desensitization of formyl peptide induced responses of human neutrophils like superoxlde p r o d u c t i o n has been shown to be a c c o m p a n i e d by a simultaneous segregation of f M L P - r e c e p t o r s into an actin- and fodrin-rich domain of the plasma m e m b r a n e w h i c h is d e p l e t e d in guanyl n u c l e o t i d e b i n d i n g proteins (G proteins). It is not known, however, w h e t h e r the receptors only co-localize w i t h or also bind to actin. Receptors solubilized from neutrophil membranes with o c t y l g l u c o s i d e as a d e t e r g e n t sediment as a 7 S form in a 5-20% sucrose gradient. In the presence of GTP, w h i c h u n c o u p l e s the receptors from G proteins, they sediment as a 4 S form. If receptors were s o l u b i l i z e d with Triton X--100, however, the f M L P - r e c e p t o r s sediment to the pellet along w i t h unsolubilized material in the presence and absence of GTP. This d e m o n s t r a t e s that the receptors were linked to complexes w h i c h are d i s r u p t e d by o c t y l g l u c o s l d e but p r e s e r v e d in Triton X--100. Since Triton X--100 ~oes not solubilize the c y t o s k e l e t o n release of receptors from the pellet was attempted in the presence of 600 mM KCl, a condition which specifically d i s r u p t s actin filaments. S o l u b i l i z a t i o n of the neutrophil membranes in Triton X--100 under this condition released the receptors from the pellet. This result suggests that fMLP-receptors are linked to actin filaments which may play a direct role in the regulation of receptor function. D e p a r t m e n t of Chemistry, M o n t a n a State University, Bozeman, MT 59717, USA

A NOVEL C3-LIKE A D P - R I B O S Y L T R A N S F E R A S E PRODUCED BY CLOSTRIDIUM LIMOSUM I. Just and G. Schallehn* Clostridium botulinum exoenzyme C3 ADPribosylates the small G T P - b i n d i n g proteins rho and rac. Here we report on the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a C3-1ike A D P - r i b o s y l t r a n s ferase (ADP-r) p r o d u c e d by a C. limosum (C.1.) strain isolated from a human lung abscess. The C.1. ADP-r (Mr 25,000) was p u r i f i e d from the culture supernatant by ammonium sulfate precipitation, gel filtration, anion exchange c h r o m a t o g r a p h y and, finally, by elution from SDS-PAGE. As known for C3 exoenzyme, the C.I. ADP-r m o d i f i e d the small G T P - b i n d i n g proteins rho and rac w i t h a K. value of about 0,4 ~M for NAD. rho/rac p r e v i o u s l y A D P - r i b o s y l a t e d by C3 was d e - A D P - r i b o s y l a t e d by C.I. Thus, the exoenzymes apparently A D P - r i b o s y l a t e d the same amino acid. Both enzymes showed similar pI (C3: 10.6 and C.l: 10.3) and proteolytic peptide analysis. Furthermore, they were immunologically related. In contrast to C3, the C.I. ADP-r was auto-ADP-ribosylated. These data indicate that C.I. produces an exoenzyme which belongs to the novel class of C3-1ike ADP-ribosyltransferases. Pharmakologisches Institut des Universit~tsklinikums Essen, Hufelandstr. 55, D-4300 Essen I, FRG *Institut f~r Mad. M i k r o b i o l o g i e und Immunologic der U n i v e r s i t ~ t Bonn, Sigmund-Freud-Str. 25, D-5300 Bonn, FRG

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I N H I B I T I O N OF THE C L O S T R I D I U M B O T U L I N U M EXOENZYME C3-INDUCED A D P - R I B O S Y L A T I O N OF r h o / r a c PROTEINS BY M A S T O P A R A N A N D COMPOUND 48/80 G. Koch and B. Habermann* The small G T P - b i n d i n g p r o t e i n s r h o and r a c are A D P - r i b o s y l a t e d by Clostridium botulinum C3 A D P - r i b o s y l t r a n s f e r a s e . Mastoparan, a peptide toxin from wasp venom, and other histamine liberators have been reported to activate h e t e r o t r i m e r i c G proteins (G~, Ge) d i r e c t l y w i t h o u t receptor interaction (Higashijima T., J. Biol. Chem. 265, 14176, 1990). Here we inv e s t i g a t e d the effects of m a s t o p a r a n and other histamine liberators on the low molecular weight GTP-binding proteins rho and rac (Mr 21 kDa). C 3 - - i n d u c e d A D P - r i b o s y l a t i o n of 21 kDa human p l a t e l e t membrane proteins was inhibited by c o m p o u n d 48/80, m a s t o p a r a n and melittin half-maximally at 15, 20 and 22 DM concentrations, respectively and maximally (>90%) at 100, 100 and 50 DM concentrations, respectively. I n h i b i t i o n of A D P - r i b o s y l a t i o n was e n h a n c e d by GTP[S]. In contrast, apamin, a bee venom without histamine-liberating activity, had no effect. M a s t o p a r a n inhibited C3-catalyzed ADP-ribosylation of a porcine brain r h o p r o t e i n p r e p a r a t i o n but not ADPr i b o s y l a t i o n of r e c o m b i n a n t r h o expressed in E. coll. In control experiments no influence of c o m p o u n d 48/80 on C3 N A D - g l y c o h y d r o l a s e a c t i v i t y was found. The data suggest that histamine liberators not only activate h e t e r o t r i m e r i c G proteins but also interact with small G T P - b i n d i n g proteins.

CHARACTERIZATION OF GUANINE NUCLEOTIDE-BINDING PROTEINS IN INTRACELLULAR, GOLGI-ASSOCIATED VESICLES FROM RAT ADIPOCYTES A. Schiirmann-Bartsch and H.G. Joost Glucose transporters in insulin-sensitive cells exhibit a characteristic subcellular distribution between plasma membranes and an intracellular, vesicular compartment from which they are translocated to the plasma membrane in response to insulin. We have previously proposed that guanine nucleotide-binding proteins participate in the regulation of glucose transport activity through a direct interaction with the carrier protein. In the present study we investigated the subcellular localization of guanine nudeotide-binding proteins in relation to glucose transporters. Isolated adipocytes were incubated in the presence or absence of insulin, homogenized, and fractionated by dkfferential centrifugation. Total [~sS]GTPrS-binding, [Z~S]GTPrS-binding to 23-28 kDa G-proteins, and immunoreactivity of G-protein c~ and fl-subunits were determined in plasma membranes and a Golgi-enriched fraction of intracellular microsomes (lowdensity microsomes). Total GTPrS-binding and 23-28 kDa G-proteins were evenly distributed between plasma membranes and low-density microsomes. In contrast, levels of ~-subunits of Gs and G± were ten-fold lower in the low-density microsomes than in plasma membranes;/3-subunits were not detectable in the low-density microsomes. Insulin gave rise to the characteristic translocation of glucose transporters but failed to alter the subcellular distribution of any of the G-proteins. Further separation of the low-density microsomes on a discontinuous sucrose gradient revealed that none of the GTP-binding proteins copurified with glucose transporters. Moreover, the ~-subuults of G~ and G~ as detected with antiserum against a common peptide sequence (c%omon) could be separated by this procedure. These data indicate that adipocytes contain considerable amounts of 23-28 kDa G-proteins, and moderate amounts of Gz and G~ in intracellalar, Golgi-associated vesicles other than those containing the glucose transporter. Thus, the presumed regulatory interaction of glucose transporters and G-proteins does not involve a dose spatial relationship. The functional significance of the presence of G-protein a-subunits in distinct, intracellular vesicles remains to be elucidated. Abteilung Pharmakologle und Toxikologie I, Institut fiir Pharmakologie und Toxikologie der Universit~it G/Sttingen, Robert-Koch-Str. 40, D-3400 G6ttingen, FRG.

* R u d o l f - B u c h h e i m Institut f~r Pharmakologie, J u s t u s - L i e b i g U n i v e r s i t ~ t Gie~en, Frankfurter Str. 107, D-6300 Gie~en. P h a r m a k o l o g i s c h e s Institut, U n i v e r s i t ~ t Essen, Hufelandstr. 55, D-4300 Essen.

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ACTIVATION OF HUMAN N E U T R O P H I L S BY M A S T O P A R A N J. Norqauer, M. Eberle and H.D. Lemke*

C R O S S - T A L K BETWEEN P H O P H O L I P A S E A2 AND PHOSPHOLIPASE C IN HUMAN GRANULOCYTES M. Camps and E. Strohmaier

Mastoparan (MP), a peptide toxin from wasp venom has been r e p o r t e d to activate regulatory G-proteins without receptor interaction. Here we studied the effects of MP on human neutrophils. MP induced biphasic changes in the o r g a n i z a t i o n of the cytoskeleton with rapid Factin p o l y m e r i z a t i o n followed by a slow and sustained d e p o l y m e r i z a t i o n below the initial baselevel F-actin content. Incubation of neutrophils with pertussis toxin (PT) inhibited M P - s t i m u l a t e d F-actin polymerization, however did not prevent sustained depolymerization of F-actin. Analysis of phospholipids performed in parallel revealed that MP stimulated rapid formation of p h o s p h o i n o s i t o l 3,4,5-trisphosphate (PIP3) and consumption of phosphoinositol-4,5-bisphosphate. PT treatment blocked MP-induced formation of PIP3. Furthermore, MP stimulated release of p r i m a r y granules in a manner sensitive to intracellular Ca z÷ . Cytochalasin B (CB) enhanced M P - s t i m u l a t e d secretion. PT treatment of neutrophils r e d u c e d M P - s t i m u l a t e d secretion in the presence of CB but not in its absence. MP also triggered superoxide anion production and induced complement type 3 receptor up-regulation. The M P - i n d u c e d superoxide anion production was enhanced by CB. The data indicate that beside PT-sensitive Gproteins other mechanisms are involved in the activation of human neutrophils by MP. ~udolf-Buchheim-Institut f~r Pharmakologie, Frankfurter Str. 107, D-6300 Gie~en and *Enka AG, D-8753 Obernburg.

Phospholipase C (PLC)-mediated hydrolysis of phosphatidyl inositol 4 , 5 - b i s p h o s p h a t e (PIP2) is a major m e c h a n i s m by which extracellular mediators regulate the functions of their target cells. Human granulocytes contain a soluble PLC that is stimulated b y a soluble OTP-binding protein(s). We have p r e v i o u s l y d e m o n s t r a t e d that this soluble PLC is also a c t i v a t e d by exogeneous G-protein 8~-subunits and that this stimulation is additive to stimulation of PLC by the endogeneous GTP-bindlng protein. Here, we present evidence that soluble granulocyte PLC is m a r k e d l y s t i m u l a t e d by products of phospholipase A2 (PLA~) and identify the s t i m u l a t o r y principle as arachidonic acid (AA). S t i m u l a t i o n of PLC by AA was not due to formation of AA metabolites as the effect of AA was not p r e v e n t e d by several cyelooxygenase or lipoxygenase inhibitors. Stimulation of PLC was also seen with other long (n~]8) chain cJs-unsaturated fatty acids, but was not observed with short chain (n~16) cischain unsaturated, trans-unsaturated, or saturated fatty acids. Most importantly, however, the abllity of AA to stimulate inosito] phosphate formation did not appear to be related to its amphiphilic nature, since its effect was fully additive to the effect of deoxycholate, a well known amphiphilic stimulator of PLC. These results suggest that AA g e n e r a t e d by PLA2 may be d i r e c t l y involved in stimulating p h o s p h o l i p a s e C, which would represent a novel m e c h a n i s m for cross-talk between these important transmembrane signalling systems. Pharmakologisches Institut, Univers~t~t Heidelberg, INF 366, D-6900 Heidelberg

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THE SOLUBLE P H O S P H A T I D Y L I N O S I T O L 4,5-BISPHOSPHATE P H O S P H O M O N O E S T E R A S E OF HL-60 G R A N U L O G Y T E S ~. Hou Cytosolic fractions of human HL-60 granulocytes contain two enzymatic activities that hydrolyze phosphatidylinositol 4,5-bisphosphate (PIPs) : phospholipase C (a phosphodiesterase) and a p h o s p h o m o n o e s t e r a s e (PME) that hydrolyzes PIP2 to PIP and Pi • The hydrolysis of PIP~ to PIP and Pi was investigated using HL-60 cytoso] and sonicated phospholipid vesicles containing [~H]PIPz as substrate. PIPs hydrolysis was markedly reduced in the presence of 2,3-diphosphog]ycerate (2,3-DPG). Half-maximal and maximal inhibition were o b s e r v e d at ~ 5 and 30 mM, respectively. Neither 2- nor 3-monophosphoglycerate affected PIP~ hydrolysis. Control experiments performed under the same conditions but using [zH]PIP as substrata did not reveal PIP p h o s p h o r y l a t ~ o n or PIP hydrolysis. Thus, HL-60 cytosol contains a PIP~-speciflc PME that is specifically inhibited by 2,3-DPG. We suggest that 2,3-DPG acts by mimicking the two vicinal phosphates present in the 4,5-posltlon of PIPs. In the presence of 2,3-DPG, the n o n h y d r o l y z a b l e GTP-analogue GTP[S] (I00 ~M) m a r k e d l y stimulated the formation of PIP from PiPs. This effect was not m i m i c k e d by ATP[S], and was also seen in the presence of 3 mM ATP. As PIP p h o s p h o r y l a t i o n is not evident under the conditions used in th5s study, GTP[S] is likely to stimulate PIPs PME rather than inhlb~ting PIP kinase. Taken together, the results show the existence in HL-60 cytosol of a P I P ~ - s p e c i f i c PME which is m a r k e d l y stimulated by GTP[S], p r e s u m a b l y via a c t i v a t i o n of a soluble GTP-binding protein present in HL-60 cytosol. Identification of this protein is subject to further investigation. Pharmakologisches Institut, Universit~t Heidelbergi INF 366, D-8900 Heidelberg

STIMULATION AND

158 A SYNTHETIC LIPOPEPTIDE INDUCES EXPRESSION OF NADPHOXIDASE IN HL-60 LEUKEMIC CELLS THROUGHPERTUSSIS TOXINSENSITIVE AND -INSENSITIVE MECHANISMS R. Seifert

INHIBITION OF HUMAN

PLATELET ADENYLYL

CYCLASE BY TRANSDUCIN B~-SUBUNYIS M. Ronzani, C. Stannek and K.H. Jakobs The influence of 13~-
stimulatory effect on basal AC

activity. Stimulation was half-maximal and maximal (about 10-fold) at approximately

1 nM

and 10 nM TDBr-GTP[,S], respectively. Furthermore, TD~-GTP[,rS] exerted an inhibitory influence on forskolin-stimulated AC activity. The effect was half-maximal and maximal (about 60 %) at approximately

1 nM and 10 nM TDB~--GTP[rS], respectively. At the same

concentrations, the gr-dimers isolated from TD eluted with either GTP or GppNHp did not increase basal or decrease forskolin-stimulated AC activity. The effects of TDBr-G'/T[rS] on basal and forskolin-stimulated AC were quantitatively similar to those of GTP[rS] or GppNHp added directly to the membranes. Furthermore, the effects of TDBr-GTP[~-S] and GTP[rS] added together to the membranes were non-additive. Finally, similar to GTP['rS], preincubation of the mebranes with TDBr-GTP[rS]resulted in persistent stimulation (basal) or inhibition (furskolin-stimulated) of AC. On the other hand boiling and treatment with hydroxylamine caused a loss of AC regulation by TDBr-GTP['rS], but not by GTP['rS]. The data indicate that TDBr isolated from TD eluted with GTP[rS] potently and efficiently activates G s and G i in human platelet membranes, resulting in stimulation and inhibition of

AC, respectively, and furthermore suggest that the observed effects of TDBr-GTP[rS] are due to a covalent modification, most likely a thiophosphorylation, of TDSr by GTP[~'S]. Pharmakologisehes Institut der Universit~tt Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, F R G

160 INFORMATION TRANSFER BY TRANSDUCIN 13r-SUBUNITS: FORMATION OF GTP[rS] FROM THIOPHOSPHORYLATED TRANSDUCIN I~" T.Wieland

The synthetic lipopeptide, N-palmitoyl-S-[2,3-bis(palmi-

We have recently reported that the/~subunit of the retinal G protein transducin (TD) can

toyloxy)-(2RS)-propy]]-(R)-cysteinyl-(S)-seryl-(S)-lysyl(S)-lysyl-(S)-lysy]-(S)-lysine (Pam3Cys-Ser-(Lys)¢), ac-

be thiophosphoD'lated

tivates human neutrophils through pertussis toxin (PTX)sensitive and -insensitive mechanisms (Seifert R et al. (1990) Biochem J 267:795-802). We studied the role of Pam3Cys-Ser-(Lys)4 in the differentiation of HL-60 leukemic cells. The lipopeptide induced expression of NADPH oxidase in HL-60 cells in a time- and concentration-dependent manner. The effect of the lipopeptide was potentiated by dibutyryl cAMP (BtzcAMP), dimethyl sulfoxide (MezSO), retinoic acid (RA), 1,25-dihydroxy vitamin D3 (I,25(OH)zDa) , interferon-~ (IFN-y) and tumor necrosis factor-~ (TNF-~). PTX but not it3 B-oligomer p a r t i a l l y inhibited expression of NADPHoxidase induced by Pam3CysSer-(Lys)¢ as measured by phorbo] myristate acetate (PMA)- and arachidonic acid (AA)-induced superoxide (Oz-) formation, whereas differentiation induced by Bt2cAMP ~as not affected by PTX. In Pam3Cys-Ser-(Lys)4-differentiated HL-60 cells, PMA-induced 0~- formation was less sensitive to inhibition by PTX than the one induced by AA. Pam~CysSer-(Lys)4 did not increase cytosolic'Ca~+ in undifferentiated HL-60 cells. Our data suggest that (I) Pam3CysSer-(Lys)4 induces differentiation of HL-60 cells through a mechanismwhich does not depend on a rise in cytosolic Ca2+ and is different from that of Bt~cAMP, Me,SO, RA, 1,25(OH)zD3, IFN-~ and TNF-~, that ( I I ) the signal transduction pathways by which Pam~Cys-Ser-(Lys)4 induces the expression of NADPHoxidase involve pertussis toxin-sensitive and -insensitive mechanisms and that ( I l l ) in systemic bacterial infections, bacterial lipoproteins and endogenous substances may interact synergistically to induce expression of NADPHoxldase in myeloid cells. Institut f~r Pharmakologie, Freie Universitat Berlin, Thielallee 69/73, D-tO00 Berlin 33, F.R.G.

by GTP[rS] in the presence of rod outer segment membranes.

Furthermore, thiopbosphorylated

TD 13r-subunits (TDB~-.PS) added to membranes of

differentiated HL-60 ceils mimicked in an apparently GDP-dependent manner the effects of GTP[~-S] added directly to these membranes on formyl peptide receptor-G protein interactions. Therefore, thiophosphate transfer from TD~--PS to GDP leading to formation of GTP[-rS] was studied. Formation of [3H]GTP[~'S] from [3H]GDP and TD~--PS occurred without apparent lag phase and required divalent cations at millimolar concentrations, with Mg 2+ and Mn 2+ exhibiting similar efficiencies and Ca 2+ , Ba 2+ and Sr2+ having little or no effect. Using various concentrations Of TDB'r-PS and [3H]GDP as substrates, a m~ximal transfer ratio of one mote of thiophosphate from one mole of TDI~r-PS to GDP was observed, suggesting that the stoichiometry of thiophospborylation of TDBz-PS was one. Most important, thiophosphate transfer from TDBr-PS to GDP was absolutely dependent on the presence of a HL-60 membrane component(s) which exhibited saturation. When HL 60 membranes were pretreated with 10/~M G'IT[rS], formation of [3H]GTP[rS] due to TDI}r-PS was reduced by about 70 %. Moreover, TDB~--PS-dependent formation of [3H]GTP[rS] from [3HJGDP was inhibited by coincuhation

with various guanine

nueleotides but not by adenine nucleotides. In addition, using [3H]ADP as substrata, formation of [3H]ATP[zS] was not detected. The data indicate that G protein Br-subenits can serve as thiophosphorylated

intermediates in G protein activation by GTP[~-S] and

furthermore suggest that the thiophosphate transfer from TDBr-PS to GDP leading to formation of GTP['rS] occors at the guanine nucleotide-binding ~-subunit of G proteins. Pharmakologisebes Institut tier Oniversitat Heidelberg,Ira Neuenheimer Feld 366, D-6900 Heidelberg, FRG

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GTP['rS]-INDLrCED RELEASE OF G PROTEINS FROM HL 60 CELL MEMBRANES

~ C H R O M A T O G R A P H Y O F A LOW-AFFINITY A D E N O S I N E BINDING P R O T E I N (ADENOTI2q) F R O M H U M A N PLATELETS

J. Kreiss In binding studies of the labelled G'I~ analog [35S]GTP[rS] to membranes of differentiated

T. Fein and U. Schwabe

HL 60 cells, it was found that binding measured with nitrocellulose filters (NC, pore size 0.45 ~*M) was higher than binding measured with glass microfibre filters (GF/C). This difference was small at early time points and more than two-fold when incubation was prolonged (~ 90 rain at 30°C). Since these data sogessted that some of the GTP[rS]-binding proteins present in the membrane preparation are released during incubation, membranes were preincubated (3 to 4 hr at 30°C) and high speed supernatants (2 hr at 163,000 g) were prepared. Preineubation alone did not release [35S]GTI?[-rS] binding proteins from the membranes. On the other hand, when [35S]GTP[~-S] was present during preincubation, about 50 % of total [35S]GTP[~-S] binding determined with NC filters was found in the supernatant. GDP decreased [35S]GTP[~-S] binding and its appearence in the supernatant. This efr~ct of GDP was counteracted by the formyl peptide receptor agonist fMet-Leu-Phe, resulting in an increased release of protein-bound [35S]GTP[rS] from the membranes. The supernatant [35S]GTP[~-S]-binding protein appeared as single peaks in gel filtration chromatography (Ultrogel AeA 34) and after sucrose density gradient centrifugation. From these experiments, a molecular weight of about 73,000 was calculated for the released [35S]GTP[rS]-binding protein(s). The data indicate that membrane-associated GTP[rS]binding proteins can be released from the membranes by binding of the guanosine tripbosphate in a time-dependent process. The mechanism underlying this release as well as the nature of the released protein(s) are under study. The molecular weight of the released protein(s) suggests that it may not be a monomeric G protein ~-subunit but is most likely

Several tissues such as human platelets, human placenta, rat liver and guinea pig lung have been shown to contain A 2 adenosine receptors as well as non receptor binding sites, which can be measured with the radioligand 5-N-Ethyl,e3~boxamldo[3H]adenosine ([3H]-NECA). The pharmacological profile of the [ H]-NECAbinding deviates from the activation of the .A~ adenosine receptors coupled to adenylate cyelase. A low-affinity adenosine bmding protein has recently been separated from the A.2 receptor and partially purified from human platalets. Purification by gelftltration, ion-exchange and hydroxyapatite-chromatography revealed a polype~ptide with an apparent molecular mass of 104 kDa. More recently, a similar [°H]-NECA binding protein (adenotln) was purified from human placenta and shown to have a high percentage of homology with mamalian stress proteins (Hutchinson et al.: Biochemistry 29: 5138-5144, 1990). In this study we have developed a NECA-affitfity column for a more effective purification of the NECA-binding protein from human platelets. The coupling of adenosine-5'-earboxylie a d d and an amino terminal agarose gel leads to a NECA-agarose with a 4-atom hydrophifie spacer arm. Platelet membranes were solubilized with the zwlttarionie detergent CHAPS (3 [3-(cholamldopropyl)-dimethylammonlo]-l-propanesalfonate) and the solub'fllzed extract subjected to gel filtration, which is the l~'st step of separation of A 2 adenosine receptors from adenotin. The adenotin containing fractions were complefly adsorbed to the NECA-afiinlty column and were rapidly and almost completly eluted by the addition of 4 mM N E C A in the presence of :tOOmM NaCI. Following the removal of NECA, the purified adenotin bound with affinities similar to the crude soluble preparation. The NECA-af£mlty chromatography may permit the complete determination of the structure of adenotin and the further study of its physiological function. Pharmakologisches Institut der Universit~it, I m Nanenttelmer Feld 366, D-69OO Heidelberg, F R G

released from the membranes with another protein. Pharmakologisches Institut der Universit~t Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, FRG

162

164

RECEPTOR-STIMULATED AND GUANOSINE TRIPHOSPEI_ATE-DEPENDENT

PHARMACOLOGICAL CHARACTERIZATION OF A NOVEL A D E N O S I N E BINDING SITE F R O M BOVINE BRAIN

RELEASE OF G PROTEIN-BOUND GTP[rS] IN I-IL60 CELL MEMBRANES C. Kupprion

A. Lorenzen, S. Griin and H. Vogt

It is generally assumed that binding of the hydrolysis-resistent GTP analog GTP[rS] to G proteins is essentially irreversible and that GTP[rS]-boand G proteins are dissociated from and thus do not interact with agonist-liganded receptors. The reversibility of GTP[rS] binding to G proteins was studied in membranes of differentiated HL 60 cells which contain formyl pepdde receptors interacting with pertussis toxine-sensitive G i proteins. Addition of unlabelled GTP[~-S] to HL 60 membranes prelabelled with [35S]GTP[rS] caused a rapid dissociation of bound [35S]GTP['rS], measured either as fall in [35S]GTP[rS] bound to the membranes or as free [35S]GTP[rS] appearing in the supernatant. The effect of unlabelled GTP[rS] was time-kinetically biphasic, was also seen witb other nucleotkies (GTP, GppNHp, GDP[fiS], GMP and ATP) and was largely independent of the concentration of [35S]GTP[rS] used in the labelling period. Most interestingly, activation of the formyl peptide receptor by its agonist fMet-Leu-Phe, having by itself nn effect, enhanced the nucleotide-induced dissociation of bound [35S]GTP[~-S]. This effect of fMet-Leu-Phe required the presence of Mg2+ and a guanosine tripbosphate (GTP[~'S] > GTP > GppNHp), while with other nucleotides only small (GDP[fiS]) or no effects (GMP, ATP) of fMet-Leu-Phe were observed. Finally, the fMet-Leu-Phe-induced release was not seen in membranes of pertassis toxin-treated HL 60 cells and was also not seen at high ( > 5 nM) GTP[rS] concentrations present in the prelabelling period. The data indicate (1) that binding of GTP['rS} to membrane G proteins is not irreversible and (2) that agonistactivated receptors can even interact, either directly or indirectly, with GTP[rS]-liganded G proteins leading to release of bound guanine nucleotide. Pharmakologisches Insfitut tier Universitat Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, FRG

5'-N-Ethylcarboxamido[3H]adenosine ([3H]NECA) has been shown to bind to A t and A x adenosine receptors and also to non-receptor binding sites. In this study, we describe a novel binding site for adenosine that can be detected in solubilized extracts from bovine brain membranes. Bovine striatal membranes were solubilized with CHAPS (3-[3-(cholamidopropyl)dimethylammonio]-lpropanesulfonate), and the soluble extract was subjected to gel filtration on a Sepharose CL-6B-cohimn. [3H]NECA-binding activity was eluted in three separate peaks. The first peak was eluted with the void volume and contained appro]dmately 10% of the total [3H]NECA binding sites. According to its pharmacological profile it was identified as the A 2 adenosine receptor. The second peak contained 40% of the [3H]NECA binding sites with pharmacological properties typical of the non-receptor NECA-binding protein adenotin. In the third peak a [3H]NECA-binding site was eluted that could clearly be distinguished from A 2 adenosine receptors and from adenotin by a characteristic pharmacological profile. The radiofigand [3H]NECA showed a saturable binding to these fractions with a Braax value of 12,900 fmol/mg protein and a K d value of 22 riM. In competition experiments, adenosine, NECA, NAD, inosine, 5'-AMP, and SAH (S-adenosyl-L-homocysteine) were the most potent ligands (K i values 15, 23, 57, 71, 82 and 82 nM, respectively). In contrast to adenosine A 2 receptors, this site did not bind the A2-selective agonist CGS 21,680 (2-[p-(2-carboxyethyl)- phenethylamino]-5'-N-ethylcarboxamido adenosine). R-N 6-phenylisopropyladenosine (R-PIA) and S-N 6-phenylisopropytadenosine (S-PIA) were almost equipotent (K. values 3.6 and 2.2/~M respectively). A f f i .. 1. ' . nttles of adenosine receptor antagomsts were 100 to 1,oo0fold lower at th~s new binding site compared to A 2 adenosine receptors. A binding site with an identical pharmacological profile could be detected in soluble extracts from bovine cortical membranes. These results suggest the existence of a novel high affinity binding site for adenosine of unknown function in bovine brain membranes. Pharmakologisches Institut dar Universit~t, Im Neuenheimer Feld 366, D-6900 Heidelberg, FRG.

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EXTRACELLULAR ADENOSINE 5'-TRIPHOSPHATE (ATP) ANTAGONIZES THE EFFECTS OF POTASSIUM CHANNEL OPENERS IN GUINEA-PIG HEART PAPILLARY MUSCLE *,zp. Illes a n d *A. B e r t

EXCITATORY EFFECTS OF THE ADENOSINE ANALOGUES, PIA AND NECA, MEDIATED BY A NON-A~ ADENOSINE RECEPTOR

P a p i l l a r y m u s c l e s w e r e i s o l a t e d from t h e r i g h t v e n t r i c l e of guinea-pig heart and stimulated with electrical pulses. The e v o k e d a c t i o n p o t e n t i a l s (AP) w e r e r e c o r d e d b y i n t r a c e l l u l a r microelectrodes; the simultaneously elicited contractions w e r e m e a s u r e d i s o m e t r i c a l l y . We u s e d c r o m a k a l i m a n d Re 31-6930 [2- (6-eyano-2,2-dimethyl-2H1-benzopyran- 4-yl)pyridine 1-oxide]; these compounds open potassium chann e l s , w h i c h a r e s e n s i t i v e to i n t r a c e l l u l a r ATP (KAte c h a n n e l s ) . T h e a p p l i e d c o n c e n t r a t i o n s of c r o m a k a l i m a n d Re 3 1 6 9 3 0 m a r k e d l y s h o r t e n e d t h e AP d u r a t i o n a n d d e p r e s s e d t h e c o n t r a c t i l e f o r c e , b u t did n o t c h a n g e e i t h e r t h e r e s t i n g m e m b r a n e p o t e n t i a l , or t h e a m p l i t u d e a n d t h e m a x i m u m r i s e v e l o c i t y of t h e AP. G l i b e n c l a m i d e c l o s e s K a ~ c h a n n e l s ; t h i s s u b s t a n c e w a s i n a c t i v e on i t s own, b u t r e d u c e d t h e e f f e c t s of c r o m a k a l i m a n d Re 3 1 - 6 9 3 0 . ATP a n d i t s e n z y m a t i c a l l y s t a b l e a n a l o g u e a , l ~ - m e t h y l e n e ATP ( a d ~ - m e A T P ) a c t e d in a s i m i l a r m a n n e r a s g l i b e n c l a m i d e . G l i b e n c l a m i d e a n d a,13meATP s h i f t e d t h e c o n c e n t r a t i o n - r e s p o n s e c u r v e of c r o m a k a l i m in a p a r a l l e l w a y to t h e r i g h t . N e i t h e r t h e P~p u r i n o c e p t o r a n t a g o n i s t s u r a m i n n o r t h e P , - (A~-) p u r i n o ceptor antagonist 8-cyclopentyl-l,3-dipropylxanthine (DPCPX) c o u n t e r a c t e d a,l~-meATP. T h e c a l c i u m c h a n n e l b l o c k e r n i t r e n d i p i n e a l s o s h o r t e n e d t h e AP d u r a t i o n a n d depressed the contractile force. However, both glibenclamide a n d a,l~-meATP f a i l e d to i n t e r f e r e w i t h n i t r e n d i p i n e . In c o n c l u s i o n , ATP m a y a l t e r t h e e f f e c t s o f p o t a s s i u m c h a n n e l openers both at lntra- and extracellular sites. However, e x t r a c e l l u l a r ATP d o e s n o t a c t v i a t h e k n o w n s u b t y p e s of P~-purinoceptor.

Angela Ameri In a previous study it was shown that the adenosine analogue, PIA, at a concentration of 2.5 ~mol/l either reduced or increased the excitability of hippocampal CA1 neurones. The inhibitory effect was abolished by the selective A1 receptor antagonist, DPCPX, and thus was mediated by the adenosine A1 receptor. To further characterize the receptor involved in producing excitatation, intracellular recordings were made from CA1 neurones in slices of the rat hippocampus. Drugs were applied by superinsion. The slices were pretreated with DPCPX (0.5 tzmol/l) to block A~ receptor-mediated responses. PIA was then applied at different concentrations in solutions containing DPCPX (0.5 ~nol/l). DPCPX alone produced a depolarization, an increase of membrane resistance, and spontaneous discharges of action potentials. PIA (1 - 5 #tool/l) caused a concentration-dependent excitation of all neurones tested. It further depolarized the neurones, increased their membrane resistance and spontaneous impulse discharges. Also NECA (5 ~mol/l), which inhibited neuronal excitability when applied alone, produced an excitation during block of the A1 receptors by DPCPX. The excitatory effects of PIA and NECA disappeared during superfusion of a medium containing theophylline in combination with DPCPX and the agonists. Forskolin failed to change the membrane properties of the CA1 neurones so that an involvement of cAMP in the excitatory effects of the adenosine agonists described could be excluded. The results provide evidence ~ a t PIA and NECA increase neuronal excitability by binding to an adenosine receptor other than that of the A1 type. This receptor is probably of the A2 type, because the excitatory effects were abolished theophylline. This study was supported by the SFB 246. Institut fiir Pharmakologie und Toxikologie der Universitat des Saarlandes, D-6650 Homburg/Saar.

~ D e p a r t m e n t o f P h a r m a c o l o g y , Byk G u l d e n P h a r m a c e u t i c a l s , B y k - G u l d e n - S t r a s s e 2, D - 7 7 5 0 K o n s t a n z a n d Z D e p a r t m e n t of P h a r m a c o l o g y , U n i v e r s i t y of F r e i b u r g , H e r m a n n - H e r d e r S t r a s s e 5, D - 7 8 0 0 F r e i b u r g , F.R.G.

166

168

ADENOSINE RECEPTOR ACTIVATION REDUCES ISOPROTERENOLSTIMULATED ACTIVITY OF PHOSPHATASE INHIBITOR-I IN INTACT GUINEA PIG VENTRICLES J. Neumann ], R. C, G u p ~ , A. M~ Watanab~o The adenosine agonist (-)-N -phenylisopropyladenosine (PIA) attenuates the isoproterenol (Iso)-induced increase in force of contraction and protein phosphorylation in perfused hearts. These dephosphorylations can only in part be explained by a reduction in cAMP. However, it is conceivable that an activation of protein phosphatases is involved. The activity of phosphatases could be regulated by the protein phosphatase ir~hibitor-I which is only inhibitory after phosphorylation by protein kinsse A. Therefore, we measured the activity of inhlbitor-i (I-l) from isolated pe~fused electrically stimulated (3Hz) guinea-pig ventricles. The inhibitory effect of I-I was measured against the catalytic subunit of rabbit skeletal muscle type 1 phosphatase and expressed as % of maximal inhibitory activity. Under these conditions Iso (I0 ruM, 1 min) stimulated I-I activity from 30.5 ± 3.8 % to 48.6 ± 4.5 %. Additionally applied PIA (I ~M) reduced I-I activity to 36.2 ± 4.3 % (p
STUDIES ON THE POST-RECEPTOR ADENOSINE RECEPTORS INHIBITING R E L E A S E IN H I P P O C A M P U S C. A l l g a i e r and R. G r e b e r

I Abteilung Allgemeine Pharmakologie, Universit~ts-Krankenhaus Eppendorf, Universit~t Hamburg, MartinistraSe 52, D-2000 Hamburg 20, FRG, 2Department of Medicine, Indiana University School of Medicine, 46202 Indianapolis, aLilly Research Laboratories, Lilly Corporate Center, 46285 Indianapolis, USA.

MECHANISMS OF NORADRENALINE

Presynaptic adenosine receptors which inhibit depolarization-induced n o r a d r e n a l i n e (NA) r e l e a s e in h i p p o c a m p u s belong to the A 1 subtype, as i n d i c a t e d by the a g o n i s t a f f i n i t 9 and the pA 2 value of the selective Al-adenosine receptor antagonist 1,3-dipropyl 8-cyclopentylxanthine (9.25; a g a i n s t R-PIA). The p o s t - r e c e p t o r m e c h a n i s m s of p r e s y n a p t i c A 1 r e c e p t o r s are still u n d e r investigation. Particularly, in r e s p e c t to the involvement of p e r t u s s i s toxin (PTX)-sensitive G proteins and a d e n y l a t e cyclase controversial d a t a h a s b e e n reported. [3H]NA r e l e a s e from r a b b i t h i p p o c a m p a l slices was elicited with electrical field stimulation. Nethylmaleimide (NEM) and PTX w e r e used to inves t i g a t e the c o u p l i n g of A 1 r e c e p t o r s to P T X - s e n s i t i v e G proteins. S t i m u l a t i o n w i t h 4 p u l s e s / l o 0 Hz w a s p e r f o r m e d to e x c l u d e an i n d i r e c t e f f e c t on ~2-adrenoceptor activation. N E M (30 mM; 30 min) a n d PTX (8 ~g/ml; 18 h) s i g n i f i c a n t l y d i m i n i s h e d i n h i b i t i o n of [3 H ] N A r e l e a s e c a u s e d by the p r e f e r e n t i a l A 1 a g o n i s t R-PIA. C y c l i c A M P p h o s p h o d i esterase (PDE) inhibitors and the adenylate c ~ c l a s e (AC) a c t i v a t o r f o r s k o l i n i n c r e a s e d e v o k e d [~H]NA release. 1,9-Dideoxy-forskolin, which fails to a c t i v a t e a d e n y l a t e cyclase, s h o w e d no effect. 8 - B r - c A M P only s l i g h t l y a f f e c t e d t r a n s mitter release. However, the agonist-induced i n h i b i t i o n of [3H]NA r e l e a s e was i m p a i r e d by AC a c t i v a t i o n , PDE i n h i b i t i o n and p r e s e n c e of 8-Brc A M P in a s i m i l a r manner. T h e s e r e s u l t s s u g g e s t a role of PTX-sensitive G proteins and AC in modulation of NA release via presynaptic A1 receptors Inst. Pharmacol., H . - H e r d e r - S t r . 5 , D-78 F r e i b u r g

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EFFECTS OF ADENOSINE 5'-TRIPNOSPHATE (ATP) ON RAT LOCUS COERULEUS NEURONES L. Harms a n d M. T s c h 6 p l

INTERACTIONS BETWEEN NITRIC OXIDE AND PROSTACYCLIN ISOLATED RABBIT HEARTS DURING I S C H A E M I A AND R E P E R F U S I O N

T h e e f f e c t s of ATP and its a n a l o g u e s a , ~ - m e t h y l e n e ATP ( u , ~ - m e A T P ) a n d 2 - m e t h y l t h i o ATP w e r e t e s t e d on c e n t r a l n o r a d r e n e r g i c n e u r o n e s of t h e n u c l e u s locus c o e r u l e u s in a b r a i n slice p r e p a r a t i o n . T h e s p o n t a n e o u s f i r i n g r a t e was i n c r e a s e d b o t h by a , ~ - m e A T P a n d 2 - m e t h y l t h i o ATP, b u t n o t b y ATP i t s e l f . In t h e p r e s e n c e of t h e P , - p u r i n o c e p t o r a n t a g o n i s t 8 - c y c l o p e n t ¥ 1 - 1 , 3 - d l p r o p y l x a n t h i n e (DPCPX), ATP also e n h a n c e d t h e firing; t h e r a n k o r d e r of p o t e n c y of t h e a g o n i s t s was a , ~ - m e A T P > 2 - m e t h y l t h i o ATP > ATP. T e t r o d o t o x i n did not i n f l u e n c e t h e e f f e c t of a , ~ - m e A T P , b u t t h e P u - p u r i n o c e p t o r a n t a g o n i s t s u r a m i n c o u n t e r a c t e d it. In t h e s u b s e q u e n t e x p e r i m e n t s , t h e n e u r o n e s were h y p e r p o l a r i z e d by c o n t i n u o u s c u r r e n t i n j e c t i o n in o r d e r to m e a s u r e c h a n g e s of m e m b r a n e p o t e n t i a l a n d a p p a r e n t i n p u t r e s i s t e n c e . Both ATP a n d a , ~ - m e A T P p r o d u c e d a t r a n s i e n t h y p e r p o l a r i z a t i o n followed b y d e p o l a r i z a t i o n ; t h e s e r e s p o n s e s w e r e a c c o m p a n i e d b y a d e c r e a s e a n d i n c r e a s e of t h e i n p u t r e s i s t a n c e , r e s p e c t i v e l y . Some ceils s h o w e d only d e p o l a r i z a t i o n . On t h e s e c o n d a p p l i c a t i o n of t h e P ~ - p u r i n o c e p t o r a g o n i s t s t h e h y p e r p o l a r i z a t i o n was no l o n g e r p r e s e n t , b u t t h e d e p o l a r i z a t i o n r e m a i n e d s t a b l e or e v e n i n c r e a s e d . T h e d e p o l a r i z i n g r e s p o n s e was d i m i n i s h e d a t h i g h e r m e m b r a n e p o t e n t i a l s , b u t did n o t d i s a p p e a r c o m p l e t e l y e v e n a t a b o u t t h e e x p e c t e d potassium equilibrium potential. The depolarization was markedly depressed, when the external potassium concent r a t i o n w a s r a i s e d fourfold, or t h e r e c o r d i n g e l e c t r o d e was filled with c e s i u m chloride. T h e r e was no r e s p o n s e a t all in a h i g h m a g n e s i u m low c a l c i u m c o n t a i n i n g medium or in t h e p r e s e n c e of c o b a l t . In c o n c l u s i o n , t h e a c t i v a t i o n of P~p u r i n o c e p t o r s a t locus c o e r u l e u s n e u r o n e s m a y r e d u c e a c a l c i u m - s e n s i t i v e potassium conductance and thereby enhance the spontaneous firing rate.

Langendorff hearts of rabbits were perfused at constant volume and subjected to 2 h of low-flow ischaemia (MI) followed by 30 min of reperfusion (RP). NO (oxyhaemoglobin-technique) and PGI2 (6-oxo-PGFla, RIA) were measured continously in the coronary effluent. 6-oxo-PGF1, release was significantly enhanced during early RP up to 77 ± 6 pmol/min (2. min of RP) as compared to 15 ± i pmol/min under non-ischemic conditions. In contrast, enhanced NO formation (above basal levels) was observed in the late phase of RP (30. min). Both effects were inhibited by addition of L-NNitroarginine (L-NNA, i00 pM), L-N-Monomethylarginine (LN M ~ , 30 ~M) or indomethacin (IND, 3 pM) to the perfusion buffer before MI:

IN

I. Woditsch

CONTROL

NO (nmol/l) 6-oxo-PGFla (pmol/min)

60 ±

L~NNA

L-NMMA

6

14 ± 4*

16 ± 2*

77 ± 63

2 ± 3*

28 ± 2*

IND

37

± 6*

1.8 ± 0.4*

*p < 0.05, (treatment vs control), mean ± SEM, n = 5 - 12 These findings suggest interactions of the two endothelium derived relaxing agents NO and PGI2 in the intact coronary circulation during ischaemia and reperfusion. Institut ffir Pharmakologie, Heinrich-Heine-Universit~t, Moorenstr. 5, W-4000 Dfisseldorf i

D e p a r t m e n t of P h a r m a c o l o g y , U n i v e r s i t y of F r e i b u r g , H e r m a n n - H e r d e r - S t r a s s e 5, D - 7 8 0 0 F r e i b u r g , F.R.G.

170

172

PROSTAGLANDINS IN THE SPINAL CORD FACILITATE TRANSMISSION OF NOCICEPTIVE INFORMATION FROM PERIPHERY TO THALAMUS I. J u r n a

THE CARDIOPRCrI~ ~'~'EC~f OF VASODILATOR A~ RELATIONSHIP WITH THE EIOOSANOID SYSTEM P. Mentz v K.E. Pawelskiv Ch. Giessler

E v i d e n c e is a c c u m u l a t i n g in f a v o u r of a c e n t r a l a n a l g e s i c a c t i o n of n o n s t e r o i d a n t l i n f l a m m a t o r y d r u g s (NSAIDs). This a c t i o n m i g h t be due to an i n h i b i t i o n of t h e f o r m a t i o n of p r o s t a g l a n d i n s in t h e s p i n a l cord. To t e s t t h i s h y p o t h e s i s , e x p e r i m e n t s were c a r r i e d out on r a t s u n d e r u r e t h a n e a n a e s t h e s i a in which n o c i c e p t i v e a c t i v i t y was e v o k e d by s u p r a m a x i m a l e l e c t r i c a l s t i m u l a t i o n of t h e i p s i - or c o n t r a l a t e r a l s u r a l n e r v e and r e c o r d e d from s i n g l e n e u r o n e s in t h e d o r s o m e d i a l p a r t of t h e v e n t r a l n u c l e u s of t h e t h a l a m u s , P r o s t a g l a n d i n s were d i s s o l v e d in e t h a n o l or DMSO and a d m i n i s t e r e d by i n t r a t h e c a l i n j e c t i o n to t h e l u m b o s a c r a l s p i n a l cord. All p r o s t a g l a n d i n s t e s t e d (PGEz, PGEI, PGFI~, PGD2; dose r a n g e 5 - 1 0 0 I ] g / r a t ) c a u s e d a d o s e - d e p e n d e n t i n c r e a s e in t h e n u m b e r of C f i b r e - e v o k e d impulse d i s c h a r g e s in t h a l a m i c n e u r o n e s . It is c o n c l u d e d t h a t p r o s t a g l a n d i n s in the spinal cord facilitate impulse transmission from n o c i c e p t i v e a f f e r e n t s to n e u r o n e s s e n d i n g t h e i r a x o n s to t h e brain, a n d t h a t NSAIDs r e d u c e pain s e n s a t i o n by i n h i b i t i n g p r o s t a g l a n d i n f o r m a t i o n in t h e s p i n a l cord. S u p p o r t e d by t h e S c h w e r p u n k t 'Nociception a n d Schmerz' of t h e DFG. I n s t i t u t ffir P h a r m a k o l o g i e a n d Toxikologie d e r U n i v e r s i t f i t des S a a r l a n d e s , D - 6 6 5 0 H o m b u r g / S a a r , FRG

DRUGS

Experiments in isolated heart preparations of guinea pigs and anaesthetized rabbits indicated a close relation between cardiac function and the fornlation of prostanoids in the heart. Prostacyclin (PGI2) was determined by an inhibition of the ADP-induced platelet aggregation and 6-oxoPGEI~ or thrc~boxane B~ (T~B~) by a specific and highly -~ , , ~ z sensltlve enzyme ~mmunoassay. The vasodilator drugs dipyridamole, nitroglycerin and oxyfedrine induced a significant increase of the myocardial prostanoid formation. Cardiac injury by ischemia, hypoxia, aortic stenosis, application of PAF (platelet activating factor) or anaphylaxis was linked with a marked decrease of contractile force, heart rate, coronary flow and blood pressure. Accordingly the changes of the cardiac function induced a 2-I 0 fold increase of the prostanoid release frc~ the isolated hearts or the incubated myocardium. The injury-mediated cardiac depression w-as significantly inhibited by the xrasodilator drugs. Concomitantly the enhanced PG-formation could be suppressed. The results indicate that pathophysiological changes in the heart are followed by a stimulated prostanoid biosynthesis and that the cardioprotective effect of the vasodilator clrugs includes inhibition of both phencmena. Department of Pharmacology and Toxicology, Martin-LutherUnversity, Leninallee 4, 0-4020 Halle/S.

R 44 173

175

THE P~0STAGLANDIN D2 (PGD2) ~ALOGUE ZK 118.182 INCREASES ~YOCA~DIAL CONT~ACTILITYIN THE ANAES~ETIS~D ~AT. F.M. McDonald,B. Maa~, D. Wehrmann,B. MQller. ZK 118.182 ([5Z,13E]-[9R,llR,15S]-9-chlor-3-oxa-15-cyclohexyl-ll,15-dihydroxyacid) is a chemically and 16,17,18,19,20-pentanor-5,13-prostadienoic metabolically stable, 0rally-activePGD~ analogue which binds selectivelyto DP-recept0rs (humanplateletme~brane)wlth twicethe affinityof 3H-PGD2. Intravenous infus~0n of ZK 118.182 over 20min to pent0barbital-anaesthetised rats (n =7 per group) led to d0se-dependentreductions in mean arterial blood pressure which were ~aximal 2-3~in after the start of the infusion but tended to return towards control values despite continuing drug administration.Left ventricular dP/dtmax was initiallyreduced, but then increased above control values duringfurther infusionof ZK 118.182 (seeTable) % of pretreatmentvalue (x) ~se (~glkglmln) ~ ~Idtmax ~ min 20' min 20' 20' Solvent 97 i01 97 97 98 ZK 118.182 0.i 96 99 95 i01 i01 0.3 83 97 89 126 106 1.0 73 85 83 99 i00 3.0 71 84 81 121 109 I0.0 57 80 70 138 113

RESPONSES TO DEFIBROTIDE IN HUM~ PLATELETS LO~JgED W I T H THE F L U O R E S C E N T CALCIUM INDICATOR INDO-IAM I. Baumann, J. Baumann and H. Strobach Defibrotide (DEF), a p o l y d e o x y r i b o n u c l e o t i d e of m a m m a l i a n origin has been shown to exhibit antit h r o m b o t i c activities in various models in vivo and in vitro. This study investigates the influence of DEF on cytoplasmic free calcium _,(Cai2+) in w a s h e d human platelets under control conditions and after stimulation by calcimycin (CAL, 3 ~M). Concomitantly, thromboxane (TX) generation was d e t e r m i n e d by RIA of TXB~. Cellular ~a2+ .. ~ -Klnetics were m e a s u r e d in a Sbimadzu RF5000 fluorimeter using INDO-12LM (1.5 ~M). Stimulation of Ca2+-levels in platelets by C_%L was s u b s t a n t i a l l y d e c r e a s e d when platelets w e r e preincubated (2 min, 37°C, T y r o d e - H E P E S buffer, pH 7.35-7.40) with DEF (0.i mg/ml). Removal of extracellular Ca 2+ resulted in reduced CALinduced increase in Ca~ 2+. P r e t r e a t m e n t with DEF antagonized both b a s a ~ and CJtL-stimulated Ca 2+ levels:

Following pretreatmentwith oapt0pril 10mg/kgi.v. or pr0pran0101img/kg i.v., the ZK llS.182-inducedpositive in0tr0pic effect was depressedby 40 and 30% respectively. BW A868C (3-benzyl-5-(6-cathoxyhexyl)-l-(2-cyclabexyl-2hydr0xyethylamin0)-hydant0in,Wellc0me Research Lab.), a selectiveDP-recept0r antagonist, at a dose of 10mg/kg i.v. abolishedboth the reduction in blood pressure and the initial fall in dP/dt~ax seen with ZK i18.1~2. The secondary increase in dP/dt~aw due to ZK 118.182 however was completely ~affected by pretreatmentwith B~ A868C (antagoniststudies: n = 5-7 per group). In conclusion, the PGD2 analogue ZK 118.182 induced increases in myocardial c0ntra~ility in the anaesthetisedrat which were 0nly m0d~ately attenuatedby ACE inhibition or 5-adren0cept0r blockade. The absence of effect of the selective DP-recept0rantagonistBW A868C on the positive in0tr0piceffect of ZK 118.182 impliesthat the receptormediatingthis effect is not identicalto the classicalvsscular/plateletDP-recept0r.

Exte~al

ICe 2+] n DEF: 4-6 CAL: 4-6 DEF + CIJ~: 4-6

"0" Change - 41 +203 + 85

mM 2 [%] (control ± 2 - 13 ± 29 +420 ± 25 +101

~ = 100%) ± 2 ± 38 ± 9

Similar changes were obtained using throlmbin (0.5 IU/ml) as stimulus. There was also a tend e n c y for r e d u c e d T X - f o r m a t i o n in C~tL-stimulated samples after DEF treatment. These results suggest that reduced cellular 2+ Ca - m o b i l i z a t i o n and, eventually, i n h i b i t i o n of TX-formation, are involved in the a n t i t h r o m b o t i c a c t i o n of DEF.

Research Laboratoriesof SoberingAG, Berlin, M~llerstr. 170-178:~i000 Berlin 65~ FRG

Institut f~r P h a r m a k o l o g i e der H e i n r i c h - H e i n e Universit~t, Moorenstr. 5, W-4000 D~sseldorf

174

176

AGONIST-INDUCED THROMBOXANE FOR!MATION IN PI~TELET RICH PI~ASMA DEPENDS ON THE ~ / ~ T I C O A G U I ~ T E. Br~ggener*, E. Glusa*, K. Schr~r

High density lipoprotein from different species ~ n h i b ± t s t h e t h r o m b o x a n e A2 g e n e r a t i o n capacity of platelets from clotting whole blood A. B e i t z , J. Beitz, Ch. G l e s s l e r , B. K u k l i n s k i l , G. H ~ l l e r 2 and H . - J . M e s t

In o r d e r to c l a r i f y the possible c o n t r i b u t i o n oZ platelet-derived thromboxane (TX) A 2 to aggregation and secretion, human platelet rich plasma (PRP) was a n t i c o a g u l a t e d w i t h the thrombin a n t a g o n i s t r - h i r u d i n (3.6 mmol/l) or acidic citrate d e x t r o s e (ACD) ( 6 . 8 ntmol/l) and stimulated by ADP (4 ~mol/1) or collagen (2.5 ~g/ml). In c o m p a r i s o n w i t h A C D - t r e a t e d PRP, a n t i c o a g u l a t i o n by hirudin y i e l d e d at a p p a r e n t l y u n c h a n g e d aggregation and ATP-secretion significantly less TXB2:3.1 ±1 2.003± vs. 25.4 ± 6.0 ng/ml (~JDP) and 68 ~ 6 vs; l0 ng/ml (collagen) (n = 4-5, P < 0.05)~ P r e t r e a t m e n t of the PRP w i t h the thromboxane receptor antagonist daltroban (0.1 - 100 ~mol/l) resulted in comparable inhibitions of collagen-induced aggregation, secretion a n d TXB 2 formation (n = 4-6). D e t e r m i n a t i o n of the free Ca++-concentration in A C D - p l a s m a yielded 46±4 ~mol/l Ca ++ w h e r e a s in r - h i r u d i n plasma 626 ± 14 ~mol/l Ca ++ w e r e m e a s u r e d (n=6). C o - i n c u b a t i o n of PRP w i t h hirudin and A C D prior to s t i m u l a t i o n by ~JDP e n h a n c e d TXB 2 g e n e r a t i o n to levels found in A C D w h i l e s u p p l e m e n t a t i o n w i t h 10 ~Lmol/l Ca ++ s u p p r e s s e d this effect completely. The data show that T X - g e n e r a t i o n in PRP is Ca ++d e p e n d e n t and c o n s i d e r a b l y lower at p h y s i o l o g i cal C a + + - c o n c e n t r a t i o n s than in citrated PRP. This does, however, not n e c e s s a r i l y affect the i n h i b i t i o n of platelet a g g r e g a t i o n and secretion to thron~boxane recepto r antagonists, such as daltroban. Institut f~r Pharmakologie, Heinrich-Heine-Universit~t D~sseldorf, Moorenstr. 5, W-4000 D~sseldorf i, present a d d r e s s : * I n s t i t u t f~r Pharmakologie und Toxikologie, Medizinische ~kademie Erfurt, N o r d h ~ u s e r Str. 74, O-5010 Erfurt

In epidemlologlcal, c11nlcal and e x p e r i m e n t a l s t u d l e s was d e m o n s t r a t e d t h a t an e n h a n c e d l e v e l 1 1 p o p r o t e i n (HDL) can p r o t e c t of high density agalnst the development of atherosclerosls. Additionally it is accepted that the progression of atherosclerosls i s a c c o m p a n i e d by m o d u l a t i o n i n t h e t h r o m b o x a n e A2 (TXA2) g e n e r a t i o n of platelets. The a i m o f t h e p r e s e n t p a p e r was t o investigate whether causal connections exist b e t w e e n HDL and TXA2 f o r m a t i o n . We p r e p a r e d p l a s m a fractions r i c h i n HDL f r o m b l o o d o f humans, dogs and rabbits, respectively by a p r e c i p i t a t i o n m e t h o d . The effect o f t h e s e HDL f r a c t i o n s on TXA2 g e n e r a t i o n by p l a t e l e t S was i n v e s t i g a t e d in blood from healthy versus atherosclerotic p e r s o n s and HDL, t a k e n f r o m r a b b i t s , in blood f r o m normo - v e r s u s h y p e r l i p e m i c rabbits. The results demonstrate that the addltlon o f HDL t o clotting blood inhibits t h e TXA2 f o r m i n g c a p a c ± t y of platelets in dependence of the origin o f ~DL. HDL t a k e n f r o m p l a s m a o f human b l o o d had a s t r o n g e r inhibitory effect on TXA& g e n e r a t i o n t h a n HDL i s o l a t e d f r o m dog b l o o d . D e p a r t m e n t o f P h a r m a c o l o g y and T o x i c o l o g y , Martin Luther University Halle-Wittenberg, School of Med±cine, P . O . Box 3 0 2 , 0 - 4 0 1 0 H a l l e and 1Medical Clinic, K a r l Marx U n i v e r s i t y Leipzig 2Medical Clinic, MLU H a l l e - W i t t e n b e r g

R 45 179

177 PHARMACOLOGICAL PROFILE OF KO-286011, A N T A G O N I S T OF PAF IN V I T R O AND IN V I V O G. Ostermann, B. Hofmann, J. Greiner, R.SchOtz, H.-P. Kertscher, and U. Till

A

NEW

Racemic l-O-hexadecyl-2-O-ethyl-glycero-3phosphoric acid 4~(N,N-dimethylamino)pyridiniumbutylester (KO-286011) was proved for P A F - i n h i b i t o r y actions. A n t i - P A F effects w e r e d e m o n s t r a t e d on platelets in v i t r o by means of aggregation, s e c r e t i o n and b i n d i n g assays. The inhibition was concentration-dependent, specific for PAF and of c o m p e t i t i v e type. The pA 2 for inhibiting the P A F - i n d u c e d platelet aggreg a t i o n in h u m a n plasma was 6.44. [3H]PAF binding studies p r o v i d e d e v i d e n c e that KO-286011 exerts ~ts i n h i b i t o r y action at the PAF-receptot level. A f t e r injection of 0.5 m g / k g to rabbits the ex vivo P A F - i n d u c e d p l a t e l e t agg r e g a t i o n was s e l e c t i v e l y influenced by KO286011. M a x i m u m inhibition was o b s e r v e d 5 m i n after a d m i n i s t r a t i o n and t h e r e a f t e r d e c l i n e d over a p e r i o d of 90 min. G i v e n intravenously to g u i n e a - p i g s 5 min b e f o r e PAF (i00 ng/kg) at dosages of 10 and 25 Bg/kg, KO-286011 reduced the peak level of p l a s m a t h r o m b o x a n e B 2 by 46 and 80 %, respectively. I.v. a d m i n i s t r a t i o n of 5 ~g/kg to rats d i m i n i s h e d the P A F - i n d u c e d (i ~g/kg) hemoconcentration, leukopenia and increase of f i b r i n o l y t i c a c t i v i t y b y 78, 56 and 38 %, respectively. Taken together it is conc l u d e d that KO-286011 represents a n e w potent and selective antagonist of PAF w h i c h is directed not only towards p l a t e l e t s but also to leukocytes and the v a s c u l a r endothelium.

METABOLISM OF CYSTEINYL LEUKOTRIENES BY THE ISOLATED PERFUSED RAT KIDNEY

Joachim Fauler, Andreas Wiemeyer*, Mamoru Yoshizawa, and Hans-Joachim Schurek* The metabolism of cysteinyl leukotrienes by the isolated perfused rat kidney was investigated. For this LTC4, LTD4 or LTE4 were studied in separate experiments. The isolated perfused rat kidney metabolized all cysteinyl leukotrienes to the final metabolite N-acetyI-LTE4. In the presence of 5% albumin 50% of LTC4 was metabolized to LTD4 (22%), LTE4 (15%) and N-acetyI-LTE4 (13%) within 60 min. Excretion of radioactivity into urine was less than 1%. In contrast, in the absence of albumin, LTC4 was completely metabolized within 45 min to NacetyI-LTE4, the sole and final metabolite of LTC4 found in the perfusion medium as well as in urine. After 60 min 19% and 42% of total radioactivity were found in the perfusion medium and in urine, respectively. Isolated glomeruli metabolized LTC4 to LTD4 and to LTE4 but not to N-acetyI-LTE4 at a rate comparable to the rate observed by the isolated perfused kidney. This suggests that other structures than glomeruli must be involved in the acetylation of LTE4. The present study shows that the isolated perfused rat kidney metabolizes cysteinyl leukotrienes to the sole and final metabolite N-acetyI-LTE4. In the presence of albumin metabolism is slowed down and excretion of N-acetyI-LTE4 into urine is prevented. Departments of Clinical Pharmacology and Nephrology*, Hannover Medical School, Hannover Germany

Institute of Pathological Biochemistry, Medical Academy, N o r d h & u s e r StraSe 74, D-5010 Erfurt, FRG

178

180

A MONOCLONAL ANTIBODY AGAINST CYSTEINYLLEUKOTRIENES LTC4, LTD4, LTE4 AND ITS APPLICATION AS A LT-RECEPTOR ANALOG. D.Bickel, T. R6der*, H.J. Bestmann*, K. Brune

5-LIPOXYGENASE METABOLITES OF SELENIUM D E F I C I E N T R B L 1-CELLS

A monoclonal antibody (1A-LDR1) raised against cysteinylleukotrienes (LT) was characterized by inhibition assays in standard fluid phase RIA. For this purpose different LTderivates, LT-fragments and LT-receptor-antagonists were incubated with the mAb in the presence of radioactive LTE4. The mAb showed a nearly identical binding constant of 1-4 nM for the naturally occuring LT (LTE4, LTD4, LTC4), the deuter/zed LTE4 and the N-acetyl-LTE4. Steric modifications (LTE4(S,S), LTE4(20-OH), LTE3 aminiothiophenol- LTE4 and -LTE3, LTA4(S,S), LTA4(S,R), acetyl- LTA4) however dimi-nished the sensitivity. Crossreactions with fragments of the LT-molecule (L-cysteine, gintathione, arachidorfic acid) were not detectable. The different LT-receptor-antagonJsts showed differential reactions depending on their structural relationship to the LT-molecule. Since the spectrum of the reactivity of the mAb was very sirrglar to that of the natural LT-receptor, we produced olyclonal anti-idiotypic antibodies against mAb-Fab, raised ~Tb~toS'threeil~kal~i'ia~d~l°pt~P~]ucan:~lb°ads~nlnln~a~bi~ad e twheestbeir~l~il~g t from a protein-extract of macrophages, seperated by SDS gel electrophoresis. Thiswork was supportedby the DFG, grant No. MO 340/3-2 Institut fiir Pharmakologie und Toxikologie, UniversitAt Erlangen-N~irnberg Universit~itsstr.22, 8520 Erlangen. * Instltut fiir Orgartische Chemie, Henkestr.42, 8520 Erlangen

Frank Weitzel Rat basophile leukemia (RBL 1)- cells were cultured for 5 days on a selenium deficient (Se') medium or, alternatively on a medium cogtaining 0.5 pars per million selenium as Na2SeO 3. 5 x 10 cells/ml were harvested and washed, prestimulated for 5 min with 1raM CaC12, then stimulated with 2/~M ionomycine in the presence of Y0/~M arachidonic acid. HPLC analysis of 5-1ipoxygenase products showed a 6 fold increase in Se" cells. When cell homogenates were subjected to analogous stimulation a shift in the products in favour of increased H P E T E formation was observed in Sehomogenates. The data show increased hydroperoxide metabolites and increased 5-1ipoxygenase activity in GSHperoxidase (GSH-Px) deficient cells. Biochemical Pharmacology, Faculty of Biology, University of Konstanz, D-7750 Konstanz.

R 46 181 INVOLVEMENT OF EXTRACELLULAR CALCIUM IN LEUKOTRIENE BIOSYNTHESIS Monika Schatz-Munding Leukotriene B4 (LTB4) the most potent proinflammatory mediator produced by human polymorphonuclear leukocytes (PMN) is synthesized by the action of 5-1ipoxygenase after liberation of arachidonic acid by phospholipase A2. Our work refers to the question why human PMN generate leukotrienes in high yields only after stimulation by a Ca2+-ionophore and not after stimulation by receptor-agonists e.g. fMLP, LTB4 or PAF. By using different concentrations of ionomycin we established that for the liberation of arachidonic acid a pronounced threshold level of intracellular free Ca2+ exists below which no leukotrienes could be detected. In contrast 5-1ipoxygenase activity increases with rising concentration of intracellular Caz+ and saturates at about this threshold level. For liberation of arachidonic acid a permanently elevated intracellular Ca2+-Ievel is necessary since complexation of extraeellular Ca2+ immediately stops the generation of 5-1ipoxygenasemetabolites. However 5-1ipoxygenase under certain conditions is active with Ca2+ provided solely from intracellular stores alone. Upon stimulation of human PMN with receptor agonists a Ca2+transient is generated which only touches the threshold level necessary for arachidonic acid liberation after ionomycin stimulation. Hence the rate-limiting step after stimulation by receptor-agonists seems to be the liberation of arachidonic acid after receptor stimulation. Small amounts of arachidonic acid may be provided by other mechanism than direct activation of phospholipase A2. University of Konstanz, LS V. UIIrich, Am GieBberg, W-7750 Konstanz, FRG

183 NEUROKININ A - INDUCED CHLORIDE SECRETION IS INHIBITED BY A CHLORIDE CHANNEL BLOCKER IN CANINE TRACHEAL EPITHELIUM M. Hropot, H.J. Lang, H.G. Alpermann, and P. Hainz Furosemide inhibits bronchoconstriction induced by distilled water, exercise, allergen, adenosine and sodium metabisulfite in asthmatic subjects. Furosemide was reported to reduce neurally mediated contractions of airway smooth muscles. It has been shown that neurokinin A (NKA) stimulates the chloride secretion in tracheal epithelium probably by increasing production of intracellular cAMP. The aim of this study was to investigate if the NKA-induced chloride secretion can be influenced by chloride channel blockers or by Na+2CI-K + cotransport blockers of furosemide type. For this purpose the membranous portion of the dog trachea was dissected and mounted in the Ussing chamber. These tissues were bathed on both sides by Ringer bicarbonate solution circulated and oxygenated at 37°C. Short-circuit current (ISC), transepithelial potential difference and conductance were measured. Furosemide (10 -4 tool/I) caused a pronounced decrease of NKA-induced chloride secretion (ISC control 1.3 + 0.3 vs. furosemide 0.4 ± 0.04/~Eq.cm-2.h-1), only when applied to the serosal side. The sulfonic acid derivative S 80 5116 (N-benzyl-4-cyclohexylsulfonyl-5sulfamoyl orthanilic acid), structurally related to furosemide, exerted a significant decrease of ISC when added to the mucosal side, ICso being 10 -6 mol/l. The chloride channel blocker S 84 9103 (5-nitro-2-(3-phenylpropylamino) benzoic acid) decreased the NKA-induced chloride secretion when applied to the mucosal side of tracheal tissue to a significantly greater extent than the other agents. In conclusion, these findings suggest that the chloride channel blocker S 84 9103 is a more specific inhibitor of NKA-induced chloride secretion than conventional Na~2CI-K + cotransport blockers. This compound might be a potential agent in bronchial asthma therapy. Hoechst AG, POB 800320, D-6230 Frankfurt/M. 80

182 "PORIN 31HL" MIGHT FORM PART OF THE CHLORIDE CHANNEL COMPLEX WHICH IS THOUGHT TO BE AFFECTED IN CYSTIC FIBROSIS F.P. Thinnes, A. Schmid*, R. Benz*, and N. Hilschmann Purified "Porin 31HL" from human B-lymphocytes was reconstituted into lipid bilayer membranes where it formed defined voltage-dependent channels. Five minutes preincubation with 100 ~M 4,4'-Diisothiocyanatostilbene -2,2'-disulfonate (DIDS), potent inhibitor of chloride transport, altered the channel-forming properties of the protein, now showing small irregular channels instead of distinct steps. Furthermore, the voltage-dependence of the channel was lost by the action of DIDS(1). "Porin 31HL", with known primary structure, is an integral constituent of the plasmalemm of human B-cells. The molecule might be homologous to a protein from epithelial cells of porcine kidney which is supposed to be responsible for the chloride uptake in reconstituted liposomes and which can also be ±nhibited by DIDS. If so, "Porin 31HL" might be part of the chloride channel complex the opening of which is defective in cystic fibrosis. A flip-flop-model of chloride channel complex regulation will be discussed. l)Thinnes, F.P., mann, N. (1990) 1047-1050.

Schmid, A., Benz, R. & HilschBiol. Chem. Hoppe-Seyler 371,

Max-Planck-Institut ffir experimentelle Medizin, Abteilung Immunchemie, Hermann-Rein-StraSe 3, W-3400 G6ttingen *Lehrstuhl f. Biotechnologie, Universit~t Wfirzburg, R~ntgenring ii, W-8700 W~rzburg

184

Phospholipase C-induced anion secretion in the rat colonic mucosa M. Diener and C. Egl%me Phospholipase C (PLC) from clostridium difficile induced a biphasic increase in short-circuit current (Isc) in the rat colon. Isc rose rapidly to a transient peak, before it increased again to a plateau lasting for several h. Ion replacement experiments and sensitivity to furosemide or a C1- channel blocker indicated that PLC induces a C I secretion. The first peak was suppressed by indomethacin indicating the mediation by prostaglandins. In contrast, the plateau phase was only partially sensitive to the cyclooxygenase blocker. The long-time action of PLC was dependent on intra- and extracellular Ca 2+, although PLC did not induce an increase in the intracellular Ca 2+ concentration. The effect of PLC was blocked by the protein kinase inhibitor, staurosporine. Carbachol, when added during the plateau phase of the PLC response, induced a paradox change in Isc: a rapid, transient increase in Isc was followed by a long lasting decrease. This inhibition of the PLC response was more pronounced after elevation of the external Ca 2+ concentration. Also a Ca 2+ ionophore, ionomycin, and a Ca 2+ channel activator, BAY K 8644, inhibited the PLC response. The results suggest a dual dependence of action of PLC on the intracellular Ca 2+ concentration. Institut fiir Pharmakologie und Toxikologie, and Institut ffir medizinische Biochemie, Universit/it des Saariandes, D-6650 Homburg/Sear, F.R.G. Supported by SFB 246, project C2

R 47

185 ORGANICANIONTRANSPOR~IN THEGUINEAPIG JEJUNUM:STUDIESWITH(14C)p-AMINOBENZ01CACID IN THE PRESENCEOF PROBENECIDANDOTHERORGANICACIDS C. Bolz In the isolated mucmsa of guinea pig jejunum (1) net secretion of aminebeezotc acid (PABA) and the release of about 90 % of its acetylated metabolite (Ac-PADA) to the administration side mere described earlier (2). For getting further information on the processes of organic anion transport, the effect of some organic acids (~0 mM) on the absorption, secretion, tissue content (to) and metabolism of 10 pM PABA within 45 min incubation in iris-buffered medium of pH ?.4 was studied. After lum~nal coadm[ntstrution of PABA and probenecid (P), selicyHc acid (S), dicloxac~11in (D), amoxic[11~n (A) or benzoic acid (B) an increased permeation from Iumen to blood (P 2~5 %, S 208 %, D 148 %, A 162 %, B 14~ % of control) Bed an increased tc (P 21? %, S 2~ %, D 208 %, A I ~ %, B I~4 % of control) were observed. If the orBanic acids were administered in the counterseIution ( i . e . the bloods[de), on1~ P, Sand D increased lumen-to-blood-permeation (P 4~9 %, S 255 %, D 160 % of control) and tc (P 255 %, S 25? %, D 199 % of control), while there was no signif$cant effect of A or B. Lactic acid (L) caused a decreased tc and absorptive flux ~rrespect~ve of the s~de of administration. For blood side administration of PABA~ there was a decreased secretion with an unchanged tc when P and S were coadministered with PABA (P 4B %, S 44 % of control), and with an increased tc when added to the countersolution ( P secretion 55 %; tc I?6 %: S secretion 54 ~, tc 154 % of control). Neither co- nor counteradministration of D~ A: B or L showed a significant effect on to or secretion of PABA. An increased total tc was mostly accompanied by an increase of the amount of Ac-PABA in the tissue and a decrease of its amount on the side of administration. The results are discussed in terms of different transport mechanisms for organic anions in the brush border and basoIateral membranes. (I) Lautarbaoh, F., Naunyn-Schmiedeberg% Arch. Pharmaco1. 29_.~2~ 201 -

212 (1977). (2) Sprakties, G., Neunya-Schmiedeberg's Arch. Pharmacol. 32.~9~ R 43

(1985). Arbeitsgruppe Biechemische Phormakologie, Abteilung fur Pharmakolog~e und Toxikolog[e, Ruhr-Universit~t, D-46}0 BOCHUM

186 THE EFFECTS OF ARYLOXYACETIC ACID ANALOGUES ON THE TRANSPORT OF DIVALENT ANIONS ACROSS THE HUMAN ERYTHROCYTE MEMBRANE A . G . R . V . B e r g h o u t , E. Tz~mog~anni Aryloxyacetic a c i d a n a l o g u e s a r e f o u n d among d r u g s t h a t l o w e r plasma c h o l e s t e r o l in humans by a f f e c t i n g l ~ p o p r o t e ~ n m e t a b o l i s m ( J . Sheph e r d and C . J . P a c k a r d , T r e n d s P h a r m a c o l . S c i . 9 : 3 2 6 , 1988) and among h e r b i c i d e s t h a t p r e v e n t a c y l l~p~d and membrane s y n t h e s i s by i n h i b i ting the acetyl-CoA carboxylase of susceptible p l a n t s ( J . L . Harwood, T r e n d s B~ochem. S c i . 13:330, 1988). Besides these actions, these a g e n t s a r e a l s o known t o b l o c k c h l o r i d e ion c h a n n e l s in s t r i a t e d muscle (G. B e t t o n ~ e t a l . , J. Med. Chem. 3 0 : 1 2 6 7 , 1987) and t o i n h i bit the chloride-bicarbonate exchange across t h e r e d b l o o d c e l l membrane (G.F. Fuhrmann and K. R u d o l p h , , N a u n y n - S c h m ~ e d e b e r g ' s A r c h . P h a r macol. 337:R47, 1988). In t h e p r e s e n t c o n t r ~ b u t i o n , t h e ~ n h i b ~ t ~ o n by a range of aryloxyacetic acid analogues of the sulfate equilibrium e x c h a n g e a c r o s s t h e human erythrocyte membrane was s t u d i e d . The c o n c e n tration o f t h e s e compounds r e s u l t i n g in 50 % ~nh~b~tion of sulfate transport v a r i e d between I - 1000 m i c r o m o l a r . For b e z a f L b r a t e and cLprofibrate a v a l u e o f 411 and 362 ~M, r e s pectLvely, was d e t e r m i n e d . P r e l i m ~ n a r y s t u d i e s s u g g e s t t h a t t h e s e a g e n t s ~ n t e r f e r e w~th t h e b i n d i n g s l t e o f H~DIDS, a s p e c i f i c inhibitor of the erythrocyt~ anion exchanger. D e p a r t m e n t o f P h a r m a c o l o g y and T o x i c o l o g y , Philipps Un~verslty, School o f M e d i c i n e , K a r l von-Frisch-Strasse, D - 3550 M a r b u r g , F.R.G.

187 Inhibition of Bile Acid Uptake into ~epatocytes by Bumetanide Derivatives. E. Petzinger,

W. F6llmann,

S. Schulz, P.W. Feit

The loop diuretic bumetanide is transported into hepatocytes by bile acid carriers (E.P. et al. Am.J.Physiol. 256:G78-G86, 1989). Bumetanide inhibits bile acid uptake competitively. We investigated the structure-activity relationship among 21 bumetanide derivatives and their affinity to the uptake systems for conjugated and non-conjugated bile acids. The conclusions are: No relationsship between the diuretic effect of bumetanide derivatives (and hence their inhibition of the renal Na~K÷CI ion cotransporter) and the inhibition of the hepatic bile acid transporter exists. We assume diff- fRo~_//--~-~_n ~H-(CH2)3-CH3 erent structural requi- ' ~ k~_/ v y A ~ rements which are essenH2N02-~-~C00Hv tial for its affinity to Bumetanide the hepatic organic anion transporter versus the renal ion, transporter. An amino-group at C1 yields less effective inhihitors of bile acid uptake. An amino-group at C3 abolish all inhibitory activity. Substitution of the carboxylic-group by a sulfonic acid yields compounds which preferentially inhibit uptake of conjugated bile acids (taurocholate). An additional side chain at the phenoxy part of the molecule (R") gives compounds which selectively inhibit the uptake of unconjugated bile acids (cholate). Minimal energy calculation of the spatial structure of the bumetanide molecule suggests a core maintained by hydrogen bonds which may fit to the steroid recognizing part of the bile acid transporter. Institut f~r Pharmakologie & Toxikologie, FB 18, Frankfurter Str. 107, W 6300 Giessen.

188

CYTOSTATIC ACTIVITY OF ACLACINOMYCIN A CONSERVED DESPITE MULTIFACTORIAL DRUG RESISTANCE. S.Bielack, M.MUnchmeyer, F.Gieseler, G.Looft, K.D.Erttmann, R.Erttmann, K.Winkler The otherwise very active anthracyclines are a c l a s s i c a l t a r g e t of p-glycoprotein mediated p l e i o t r o p i c multidrug resistance (mdr). Antileukemic therapy could b e n e f i t .notably from anthracyclines t h a t r e t a i n t h e i r c y t o s t a t i c p o t e n t i a l in spite of mdr. Therefore, we investigated the d i f f e r e n t i a l s e n s i t i v i t y of the doxorubicin induced, multidrug r e s i s t a n t murine erythroleukemia c e l l l i n e F4-6/R and i t s s e n s i t i v e parent l i n e F4-6 to various anthracyclines, inculding daunorubicin,idarubicin and the 9 - a l k y l a t e d aclacinomycin A. In contrast to the F4-6 wild type, F4-6/R expresses the p-glycoprotein and shows the c l a s s i c a l pattern of resistance due to mdr. A d d i t i o n a l l y , a reduced DNA topoisomerase I I level (demonstrated in the pBR 322 plasmid r e l a x a t i o n assay) causes cross resistance to such substances as mitoxantrone, etoposide and amsacrine. In comparison to the s e n s i t i v e F4-6, the 50% i n h i b i t o r y dose of anthracyclines in F4-6/R was found to be higher by a f a c t o r of 51 f o r doxorubicin, 35 f o r daunorubicin and 12 f o r i d a r u b i c i n , but only 3 f o r aclacinomycin A. The values f o r other drugs were calculated at 19 ( v i n c r i s t i n e ) , 17 (etoposide), 16 (mitoxantrone) and 11 (amsacrine). Conclusion: The 9 - a l k y l substituted anthracycline aclacinomycin A is capable o f r e t a i n i n g much of i t s c y t o s t a t i c e f f i c a c y despite the presence of mdr in the m u l t i f a c t o r i a l l y r e s i s t a n t F4-6/F4-6/R model. Abteilung f u r p~diatrische H~matologie und Onkologie, U n i v e r s i t ~ t s k i n d e r k l i n i k , M a r t i n i s t r . 5 2 , D - 2 0 0 0 Hamburg 20, Germany; Medizinische P o l i k l i n i k WUrzburg, Germany; Bernhard-Nocht-lnstitut f u r Tropenmedizin, Hamburg, Germ. Supported by: F~rdergemeinschaft zur Heilung und E r f o r schung von Krebskrankheiten im Kindesalter e.V. Hamburg

R 48 189 INTESTINAL ABSORPTION OF B-LACTAMANTIBIOTICS: FUNCTIONAL AND STEREOSPECIFIC RECONSTITUTION OF THE INTESTINAL PEPTIDE TRANSPORTSYSTEM W. Kramer

191

The uptake of orally active ~-amino-B-lactam antibiotics and pligopeptides into small intestinal cells occurs by a H~-dependent transport system. An integral glycosylated membrane polypeptide of M. K 127000 was identilied as a component of the peptlde transporter from rabbit small intestinal brush border membrane vesicles (BBMV) by photoaffinity labeling with photosensitive Blactam antibiotics and dipeptides. With nonionic detergents such as n-octylglucoside, Triton X-tO0 or CHAPS this protein could be solubilized and was purified using wheat germ lectin a f f i n i t y chromatography and subsequent FPLC ion exchange chromatography on Mono S columns. By solubilisation the a b i l i t y for specific binding of 8lactam antibiotics determined by direct photoaffinity labeling with [~H]benzylpenicillin was lost indicating a denaturation of the binding protein. After incorporation either of solubilized BBMV-proteins or the purified 127 KDa protein into asolectin proteoliposomes the specific binding a f f i n i t y was reconstituted; a high and specific photolabeling of the 127 KDa protein occured which was inhibited by 8-1actam antibiotics or oligopeptides. Dcephalexin, a substrate of the intestinal peptide transporter, was e f f i c i e n t l y taken up by proteoliposomes from reconstituted BBMV in contrast to pure liposomes. The uptake was stimulated by an inwardly directed H+gradient and was stereospecific for D-cephalexin whereas the L-diastereomere only showed a very slow uptake. These findings of successful reconstitution of stereospecific transport a c t i v i t y of the intestinal peptide transporter greatly support our hypothesis that a ]27 KDa membrane protein of the brush border membrane is a constituent of this transport system. Hoechst Aktiengesellschaft, Postfach 80 03 20 D-6230 Frankfurt am Main 80

P h l o r e t l n m o l e c u l e s e x h i b i t k e t o - e n o l t a u t o m e r i s m . The p K v a l u e of t h i s r e a c t i o n w a s d e t e r m i n e d s p e c t r o s c o p i c a l l y to be 7 . 2 6 + 0 . 0 6 u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s . P h l o r e t i n is a n e f f e c t i v e i n h i b i t o r of g l u c o s e e f f l u x , b u t n o t of g l u c o s e i n f l u x . T h e K i - v a l u e of g l u c o s e e f f l u x i n h i b i t i o n is pH d e p e n d e n t . A t pH 6.5 we m e a s u r e d a K i - v a l u e of 0.36 and a t pit 9 of 22,7 pM. By p l o t t i n g a f f i n i t y a g a i n s t pH a c u r v e was obtained which completely matched the keto-enol t a u t o m e r y c u r v e . It c a n be c o n c l u d e d , t h a t only the n n c h a r g e d k e t o n i c form of p h l o r e t i n is t h e i n h i b i t o r of glncose transport. By u s i n g c o m p u t a t i o n a l chemistry a g e o m e t r i c o p t i m i s a t i o n of t h e k e t o n i c a n d e n o l i c form s h o w s completely different shapes of t h e molecules. Several o v e r l a p p i n g s t r u c t u r e s c a n be d r a w n of t h e k e t o n i c form with beta-D-glucopyranose. B i n d i n g s t u d i e s of p h l o r e t i n to human erythrocytes s h o w e d a pH depe, n d e n c e w h i c h is p r o p o r t i o n a l to t h e k e t o n i c form of t h e molecule. A t pH 6.5 a n d 0.2 pM p h l o r e t i n d i f f e r e n c e s h a v e b e e n f o u n d in b i n d i n g with respect to the glucose gradient. Subtraction of u n s p e c i f i c b i n d i n g as m e a s u r e d u n d e r p h l o r e t i n i n s e n s i t i v e g l u c o s e i n f l u x c o n d i t i o n s from t o t a l b i n d i n g d u r i n g e f f l u x r e v e a l a p p r o x i m a t e l y 200 000 s e n s i t i v e c a r r i e r s i t e s p e r e r y t h r o c y t e . T h i s r e s u l t p o i n t s s t r o n g l ~ to s p e c i f i c e f f e c t s of p h l o r e t i n in t h e k e t o n i c form a t a n a l l o s t e r i c g l u c o s e c a r r i e r p r o t e i n s i t e in h u m a n e r y t h r o c y t e s .

190

192

IDENTIFICATION AND PARTIAL ISOLATION OF BINDING PROTEINS FOR PEPTIDE D R U G S IN ISOLATED NASOLATERAL RAT LIVER PLASMA MEMBRANES [blPm]. U.Wenzel and K.Ziegler

Funotional and tissue epeolfi¢ expression of the high voltage aotlvated (L-T~pe) oal¢lum channels from ¢ardlao and smooth mus¢le E. Bosse*, M. Biel +, R. Hullin +, V. Flockerzi*

Three non-ionic detergents (Triton-X-100, Nonidet P 40 and Octylglycoside) and one zwitterionic detergent (CHAPS) were tested for their suitability in affinity-chromatography with regard to identification and isolation of binding proteins for peptlde drugs in isolated blPm. The ligand was a hydrophobic cyclohexapeptide, the somatostatin-analogue 008. Octylglycoside and CHAPS solubilized plasma membrane proteins, in contrast to those treated with Triton-X-100 and NP 40 , show no non-specific binding to the gel matrix and a better selectivity of specific eluted proteins in SDS-PAGE concerning especially the range between 43 and 67 kDa. The main candidates of a bile acid transport system, to which 008 has a high affinity (Ki-value of competitive bile acid uptake inhibition ~3~M), are located in just this molecular weight [MW] range. Several different elution steps of the 008-affinltychromatography-column were carried out to obtain an effective enrichment of proteins in SDS-PAGE. A KSCN-gradient elutes proteins with a MW of 58, 54 and 48 kDa and a taurocholategradient elutes proteins with a MW of 48 and 43 kDa, if the membranes are solubillzed with Octylglycoside. CHAPS-solubilization results in a 48 kDa protein in the KSCN-gradient. These proteins are enriched if extrinsic proteins are removed by EDTA-extraction of the membranes and may be accessible to further isolation and purification. Institut fur Pharmakologie und Toxikologie der Veterin~rmedizin, Frankfurter Str. 107, 63 Giessen, FRG

PHLORETIN KETO-ENOL TAUTOMERISM AND INHIBITION OF GLUCOSE TRANSPORT IN H U M A N ERYTHROCYTES G. F. F u h r m a n n . S. D e r n e d d e a n d G. F r e n k i n g *

D e p a r t m e n t of P h a r m a c o l o g y a n d T o x i c o l o g y a n d D e p a r t m e n t of C h e m i s t r y * , P h i l i p p s - U n i v e r s i t ~ i t M a r b u r g , Karl y o n Frisch-Str., 3550 Marburg

Recently, we cloned the cDNA of a L-type calcium channel from smooth muscle (1). Using polymerase chain reactions we now cloned the cDNA of a cardiac L-type calcium channel. Both calcium channels arise by differential splicin~ of a common primary transcript. They are identlcal in their coding and non-coding nucleotide sequences except for: (a) the 5"ends encoding the aminotermini, the predicted transmembrane segments IS6 (b) and IVS3 (c) and (d) an insert of 75 nucleotides encoding 25 aminoacids within the cytoplasmic loop between domain I and II of the smooth muscle channel. Northern blot experiments and polymerase chain reactions demonstrate that the calcium channel cloned from cardiac mRNA is exclusively expressed in heart whereas the smooth muscle calcium channel is expressed in heart, lung, trachea, aorta and brain, presumably in vascular and alrways smooth muscle cells which are present in these tissues. Both channels were functionally expressed in Xenopus ooc~tes and fibroblasts. The latter system will facilitate studies on the physiological regulation of the native channels by other protelns, hormones and drugs. 1)Biel M. et al FENS Letters 251,191-196(1990). *)Institut f~r Medizinische Biochemie der +,Universit~t des Saarlandes, 6650 Homburg und ;Institut f~r Pharmakologie und Toxikologie Technische Universit~t M~nchen, 8000 M~nchen

R 49 193 ANION AND CATION DEPENDENCE OF THE{ PARTIALLY PURIFIED MITOCHONDRIAL DIHYDROI:YRIDINE Ca 2+ ANTAGONIST RECEPTOR G.Zernig and N.Reider The inner mitochondrial membrane contains specific 1,4-dihydropyridine (DHP) Ca2÷ antagonist binding sites which are associated with an inner mitochondrial membrane anion channel (Zernig et al., Mol. Pharmacol. 38:362, 1990). By use of anion exchange chromatography, sucrose density gradient centrifugation and cation exchange chromatography (-+)-[3H]nitrendipine binding to the solubilized mitochondrial DHP receptor was enriched 23-fold (digitonin extract 0,12 -+ 0.04 pmol/mg protein at 1 nM total radioligand concentration; peak fractions after cation exchange chromatography 2.75 _+ 0.84 pmol/mg; means ± SD of 3 purifications). As already demonstrated for particulate preparations (Zernig and Glossmann, Biochem. J. 253:49, 1988), (-+)-[3H]nitrendipine binding to the solubilized and partially purified mitochondrial DHP receptor showed marked ion sensitivity. The rank order for maximal (+_)-[3H]nitrendipine binding stimulation was F" > CI" > I- (4.74 > 2,50 > 0.70 pmol/mg protein at lnM total radioligand concentration; potassium salts). This rank order very closely resembled Eisenman's binding selectivity sequence VII for monovalent anions (Eisenman, Biophys. J. 2(2):259, 1962) and clearly differed from the diffusion coefficient rank order for hydrated monovalent anions (i.e. I" > CI" > F'). The rank order for monovalent cations was K+ > Na+ > Li + > Cs+ (2.50 > 2.26 > 1.44 > 0.88 pmol/mg; chloride salts) which is also clearly distinct from their diffusion coefficient rank order (Le, Cs + > K+ > Na + > Li+). The relevance of these rank orders for the estimation of the anion and cation binding domain size of the mitochondrial DHP receptor complex is discussed. Supported by FWF grants P7492-MED and K0042-MED and Dr.Legerlotz foundation. Institut for Biochemische Pharmakologie, P.Mayr-Str.1,A-6020 Innsbruck

195 COS CELLS AS A MODEL FOR STUDYING DRUG EXPORT BY PGLYCOPROTEIN (PGP): STEREOISOMERS OF CALCIUM ANTAGONISTS BLOCK CELLULAR VINBLASTINE EFFLUX WITH EQUAL EFFICACY. E. Schwarz, M. Kouba, G.Vogt and V. H611t

PGP is a multidrug transporter encoded by the MDRI gene. Enhanced expression of PGP is responsible for the c e l l u l a r efflux of several drugs used in cancer therapy leading to multidrug resistance. In addition, other drugs, such as calcium channel antagonists , have been shown to be transported by PGP and to overcome multidrug resistance in cancer patients. We now report that COS-7 monkey kidney tumor cells express high levels of both MDR1 mRNA and PGP as revealed oby Northern b l o t and Western b l o t a n a l y s i s . Moreover, ~ H - v i n b l a s t i n e (VBL) is effectively export#d by COS-7 c e l l s . The c e l l u l a r accumulation of ~H-VBL was inhibited by several a n t i c a n c e r drugs and by o t h e r drugs known to be s u b s t r a t e s o f PGP. Moreover, t r a n s f e c t i o n of COS c e l l s w i t h a v e c t o r expressing MDR1 ant~sense RNA r e s u l t e d in an increased accumulation o f ~H-VBL. A s e r i e s o f stereoisomers w i t h phenylalkylamine s t r u c t u r e (verapamil, devapamil, emopamil) and dihydropydine structure (nimodipine~ f e l o d i p i n ~ i s r a d i p i n e , n i t r e n d i p i n e and n i g u l d i p i n e ) increased ~H-VBL accumulation in COS c e l l s at micromolar concentration. The rank order of potency in COS c e l l s was s i m i l a r t o t h a t in PGP overexpressing doxorubicin-resistant mouse leukemia c e l l s (D89-065). There was no e s s e n t i a l d i f f e r e n c e between the v a r i o u s steroisomers in increasing VBL accumulation in both c e l l t y p e s . The n i g u l d i p i n e enantiomers belong to the most potent drugs. Since ( - ) - n i g u l d i p i n e displays an about 40f o l d lower a f f i n i t y f o r calcium channel binding s i t e s than ( + ) - n i g u l d i p i n e , but is about e q u a l l y p o t e n t in i n h i b i t i n g c e l l u l a r e f f l u x of VBL, i t may be a useful drug f o r reversing multidrug resistance in cancer patients. Department of Physiology, University of Munich, P e t t e n k o f e r s t r . 12, D-8000 MQnchen 2, FRG;

194

196

SIALIC ACID MAY PLAY A FUNCTIONAL ROLE WITHIN THE CALCIUM CekHA~NEL G. Werner, K. Addicks , and M. Sarram

PRE3/ENTION OF VERATRIDINE INDUCED INTI~J~SYNAPTOSOMAL [Ca2+]i INCREASEBY R 56B65 B.A. Osikowska-Evers, F. Tegtmeier, T. Peters Calcium overload occurring during ischemialhypoxia influences functional and structural changes in neurons which f i n a l l y lead to cell injury or death. Calcium entry blockers, by lowering neuronal calcium increase, protect brain from ischemiclhypoxic injury. Synaptosomes depolarized by veratridine are a useful tool to mimic hypoxiclischemic insults on the nerve terminal i~n vitro. ~_~ V~vo brain studies show that R 56B65 (N-[l-[4-(4-fluorophenoxy)butyl]-4-piperidinyl] -N-methyl-2-benzothiazolamine) is protective in cerebral Ischemia (1). Therefore, the effect of R 56865 on veratridine induced [Ca2+li increase was studied in rat cor~ical synaptosomes, using fluorimetric measurement of [Ca~+]1 with Fura-2.

Cellular calcium metabolism may be influenced by the glycocalyx component sialic acid (Langer etal., Pharmacol Ther 16: 331-376, 1982). We here report on the influence of this anionic sugar on the myocardial and vascular activity of calcium antagonists. Sialie acid removal from the external matrix of isolated gmnea pig left atrial strips supramaximall}, stimulated at 2 Hz (resting force 4,9 mN) or rabbit thoracic aortic ring segments (resting tension 2,0 g) was performed by incubation with neuraminidase (2,0 units/nil; 2h; Type V, Sigma) in carbogen-saturated Tyrode (30°C) or KrebsHenseleit solution (37o C) following an equilibration period of lh and was controlled electron microscopically. After frequently washing out the enzyme, aortic rings were precontracted by 130 mM KC1. The action of different calcium antagonists was evaluated in cumulative dose-response-curves (0,1 nM-100 #M) by measuring myocardial contractile force isometrically and vascular tension isotonieally using a strain gauge. Control experiments with untreated organs were conducted in parallel. Neuraminidase treatment resulted in a sialie acid release of 62,0---2,2% (S.E.M.; atria; n = 3) and 57,3 +-1,3% (aorta; n = 5), as determined according to the thiobarbituric acid method of Warren (J Biol Chem 234: 1971-1975, 1959). Total sialic acid content following 0,1 M H2SO 4 was (nmol/mg wet weight): 1,30--,0,03 (atria), 1,70--.0,05 (aorta). [:Ialf-maximal negative inotropic effects (ICsI/, in /~M+-S.E.M.; p < 0 , 0 5 ; n=6-9) by the calcium antagonistff~studied in control experiments and neuraminidase pretreated guinea.pig atria, were obtained with nisoldipi.n,e at 0,5--0,1 and 0,2 ± 0,04 , gallopamil ~t 0,5-+0,1 and 0,3±0,02, diltiazem at 8,7-+1,2 and 5,4---0,8, fendiline at 24,3+-3,0 and 24,3±2,7. Similar results, however, ICs0 being in the nanomolar concentration range, were obtained In rabb~t aorta. Thus, sialic acid may play a functional role in calcium channels, possibly facilitating calcium influx. Institut ftir Pharmakologie der Universit~it zu K61n, Gleueler Str. ~4, D-5000 K61n 41, FRG }Zentrum der Anatomie der Universit~it zu K61n, Joseph-Stelzmann Str. 9, D-5000 K61n 41, FRG

Veratridine (l x lO-5 M) increased [Ca2+]i in control synaptosomes from If3 18 (SD) to 368M± BO (~2~,~ nM (n - lO) within 2 min. mR 56B65 (l x lO-B - l x 5 M; n = B) inhibited strongly (9 ± 9 % (SD) and B7 ± 3 % (SD~; respectlvely) veratridine induced intrasynaptosomal [Ca~+]i increase in a dose dependent manner with an IC 50 of l x lO-6 M. Possibly, Ca2+ entry into synaptosomes occurs through voltage-gated Ca2+ channels opened by veratridine induced membrane depolarization. However, Ca2+ influx directly through the veratridine sensitive Na+ channel, and/or Na+ICa~+ exchanger working in a reverse mode, can not be excluded. I t remains to be elucidated which of these pathways is influenced by R 56865. Nevertheless, above data f u l l y support _ i _Bn vivo findlngs indicating that cerebroprotective effects of R 56865 (1) are partly due to its calcium-antagonistic properties (2). (1) Scheller et a l . , Naunyn-Schmied. Arch. Pharmacol., Suppl 339, R I07 (19BY) (2) Koch et a i . , Cardiovasc. Drug Rev. B, 238-254 (1990) JANSSEN RESEARCHFOUNDATION, D-4040 NEUSS21

R 50 197 THE RELATIONSHIP OF INOSITOL 1,4,5-TRISPHOSPHATEINDUCED CALCIUM RELEASE TO VARIOUS CALCIUM TRANSPORT MECHANISMS IN SUBCELLULARFRACTIONS FROM RAT LIVER H.-P. Bode and M. Trautmann* In contrast to the endoplasmic reticulum calcium pump, the calcium transporters present in some organelles of the vacuolar apparatus (chromaffin granules and the inositol 1,4,5-trisphosphate (IP3)-sensitive calcium store of exocrine pancreas c e l l s ) as well as the plasma membrane calcium pump appear to be calcium proton exchanging ATPases, with obligatory coupling of calcium and proton transport. Examining vesicular calcium uptake in subcellular f r a c t i o n s , the l a t t e r mechanisms of calcium transport therefore can be i d e n t i f i e d using agents that affect the i n t r a v e s i c u l a r proton concentration. To investigate whether vacuolar organelles or the plasma membrane are targets of IP 3 in rat l i v e r c e l l s , we have examined the effect of the vacuolar proton pump i n h i b i t o r bafilomycin and the potassium proton exchanging ionophore nigericin on vesicular calcium transport in subcellular fractions from r a t l i v e r . Bafilomycin had no effect on calcium uptake by any of the f r a c t i o n s . Purified Golgi and endosomal fractions showed no calcium pumping a c t i v i t y . Nigericin p a r t l y inhibited calcium uptake in an early sedimenting microsomal f r a c t i o n , but did so only in the presence of an outside to inside potassium concent r a t i o n gradient across the vesicular membranes, suggesting that this effect was due to a l k a l i n i z a t i o n of the vesicular lumen. Nigericin did not a l t e r the amount of calcium releasable by IP 3 from the same f r a c t i o n . We conclude that n i g e r i c i n acts on calcium pumping inside-out plasma membrane vesicles, which are devoid of IP3-receptors. In summary, no evidence favoring association of the IP3-receptor with an organelle d i s t i n c t from endoplasmic reticulum in subcellular fractions from rat l i v e r was found.

199 T H E DIIq,,Y,D R O P Y R I D I N E ( _ + ) - N I G U L D I P I N E I N H I B I T S TT Y P E C a z'" C U R R E N T S IN A T R I A L M Y O C Y T E S . K. Seydl The whole cell tight seal recording technique v~as used to investigate the effects of the ~ovel drug niguldipine on Ca 2+ currents in guineapig atrial cells. C£ z+ currents were separated into T-type and L-type components by an appropriate stimulus protocol, i. e. first, T-type C~z~" currents were evoked by depolarizing voltage pulses from_ a holding potential of -90 mV to -40 mV and then, L-type Cg z+ currents were activated by a subsequent depolarization to 0 mV. Fast sodium currents were eliminated by substituting choline for Na and outward currents were suppressed by the use of Cs instead of K in both extracellular and intracellular solution. T- and L-type current characteristics were further confirmed by a selective inhibition by 40 /~M Ni and by 1 #M_ ~-+)-PN 200-110, respectively. Based on this characterization of C~z ÷ current components we tested for the effects (-+)-niguldipine (N/G), Extracellular application of 1 ~M NIG resulted in a potent blockade of T-type C£z + current~ ,(to 7-+4 % of control, n=7) and in a weaker inhibition of L-t3)pe Ca z ÷ currents (to 22-+9 % of control, n=6). Current to voltage relationships clearly showed that both Ca 2+ currents are blocked at all voltages examined (-60 to -40 mV). Inhibition of T- and L-type currents occurred in a slow fashion. At a concentration of 1 # M NIG half maximal reduction in T-type current a,lnplitude was observed within tl/,, = 52 sec. The ICCn for T-type CUz ÷ current inhibition was estimate8 to be 0.13 ~M. TH%~IC50 for NIG on L-type Ca z+ currents could not be clearly resolved~tue to the slow ~.~set of NIG action, which is obscured by a concomitant L-type Ca 2 current run-down. This study clearly indicates a T-type Ca 2+ current blocking activity of NIG with a high relative affinity, NIG appears, however, not to be selective among Tand L-type Cd 2+ currents. (Supported by Austrian Research Funds, project S 45-03) Institute for Biophysics, University of Linz, Altenbergerstr. 69, A-4040 Linz, Austria

Inst.f.Pharmakol., K.v.Frisch S t r . , Lahnberge und *Zentr.f.lnnere Med., Klinikum d. Univ., D-3550 Marburg

198 R E V E R S A L OF A N T H R A C Y C L I N E A C C U M U L A T I O N DEFICITS IN M U L T I D R U G R E S I S T A N T F R I E N D L E U K E M I A CELLS B Y T H E DIHYDROPYRIDINE B859-35, THE R-ENANTIOMER OF NIGULDIPINE. A. Reymann*, C. Woermann*, and M. Dietel+

M u l t i d r u g r e s i s t a n c e (HI)R) i s a ma3or o b s t a c l e i n c a n c e r c h e m o f h e r a p y . The use o f t h e e x p e r i m e n t a l c h e m o s e n s i t i z e r v e r a p a m i l , known t o b l o c k a n t i c a n c e r e x t r u s i o n from MOl~-ma]ignancies i n v i t r o , i s h a m p e r e d b y i n vivo cardiovascular toxicity observed at concentrations r e q u i r e d £or t h e r e v e r s a l of MDR ( D i c k s o n R.B. a n d G o t t e s m a n n ~.N., T r e n d s P h a r m a c o l Sci 11, 305, 1990). The d i h y d r o p y r i d i n e B859-35 ( R - e n a n t i o m e r of n i g u l d i pine), a n a g e n t w i t h m i n o r c a r d i o v a s c u l a r e f f e c t s , was studied in an a n t h r a c y c l l n e accumulation assay in routine leukemia cells strain F4-6RADR e x p r e s s i n g p g l y c o p r o k e i n . Cells w e r e p r e i n c u b a t e d w i t h B859-35 (i0 rain, d i m e t h y l s u l f o x i d e i% v / v a s s o l v e n t ) f o l l o w e d b y 60 min i n c u b a t i o n i n t h e p r e s e n c e of r a d i o l a b e l l e d c y t o s t a t i c s (i ~mol/l), and c e n t r i f u g e d t h r o u g h a s i l i cone o i l l a y e r i n t o t r i c h l o r o a c e t i c a c i d (10% w/v). I~859-35 e l e v a t e d low a c c u m u l a t i o n o f d o x o r u b i c i n , d a u norubicin (DNR) or mitoxantrone in resistant F 4 - 6 R A D R cells to levels measured in drug-sensitive F4-6P precursor cells. Concentration-response studies on D N R accumulation revealed that B859-35 was m o r e potent than verapami] (ECs0 0.73 //mol/l vs. 5.4 //tool/l). In parallel to increased D N R contents (pmol/mg protein, F4-6RADR, solvent: 303+27; B859-35 3,3 ~mol/l: 1067-+174; sensitive F4-6P, solvent: 948+110; n=8-9, SEM), the amount of D N R tightly b o u n d after washing of the pellet obtained from F 4 - 6 R A D R (i.e. protein, DNA, RNA), was increased 3.9-fold to levels observed in F4-6P. The results indicate that B859-35 is a potent chemosensitizer, with regard to in-vitro accumulation of cytostatics and improved d r u g access to binding sites in F 4 - 6 R A D R leukemia cells of the M D R phenotype. (Supported b y the DFG, SFB 232,C3 and Di 276/i-3.) *Abteilung Allgemeine Pharmakologie, Unlversit~tsKrankenhaus Eppendorf, Universitat Hamburg, Martin~stra~e 52, D-2000 H a m b u r g 20, FRG. (+) Institut fur PathologJe, Universik~t Kiel, Michaelisstra~e Ii, D2300 Kiel I, F E G

2O0 EFFECTS OF A CARDIODEPRESSANT FACTOR ON ACTION POTENTIALS AND IONIC CURRENTS iN ISOLATED GUINEAPIG CARDIOMYOCYTES. B.Koidl*, S.Hallstr6m**, U.M~ller***, K.Werdan***, and G.Schlag** A cardiodepressant factor (CDF) was isolated from the plasma of dogs post hypovolemic-traumatic shock by column chromatography. The CDF-fraction contains a hydrophilic peptide ( M W < I O 0 0 ) . By means of photometric measurement of cell pulsation and patch-electrode recording of membrane potential and whole cell ionic current its influence on excitation and contraction was studied in isolated guinea pig cardiomyocytes. CDF reduced dose dependently down to a pathophysiologically relevant concentration of 1.84 ml shock plasma aliquot/ml the amplitude of contraction in electrically stimulated cells. This effect could be rapidly reversed by wash-out and could be abolished within seconds by elevation of the calcium concentration within the bath from 1.8 to 10 mmol/I. Action potential measurements showed a distinct reduction of the plateau-phase and an increase in the duration at 50 and 90 percent of repolarization. Voltage-clamp measurements revealed an inhibition of the calcium current and a change in the N-shaped current voltage relationship of the potassium current suggesting an increase in IK1 and a decrease in the delayed rectifier component IK. The negative inotropic effect as well as its rapid reversibility by elevation of calcium in the the bathing solution indicate a similar point of attack of CDF and calcium antagonists. The effects on IK are not necessarily in contradiction with this interpretation. The rapid reversibilty of the effect however suggests a very different access of the substance to the receptors of the calcium channel. *lnst.f.Med.Physik u. Biophysik d. Univ.Graz, A-8010 Graz * * L u d w i g Boltzmann-lnstRut f. Exp. u. Klin.Traumatologie, Wien, * * *Med.Klinik I d.Univ.M(Jnchen, Klinikum GrolShadern

R51 201

203

Increasing and d e c r e a s i n g effects of extracellular m a g n e s i u m on inward currents in heart ventricular cells W. Vierling, A. Dichtl

DILTIAZEM AND (+)-TETRANDRINE INCREASE THE CA2+ AFFINITY OF THE L-TYPE ~Az + CHANNEL: STEREOSELECTIVE REVERSAL B Y A NOVEL CA2+ - ANTAGONIST R. Staudinger and H. Glossmann

The influence of an increase in the extracellular m a g n e s i u m c o n c e n t r a t i o n on inward currents of isolated m y o c y t e s from g u i n e a - p i g hearts was investigated in whole cell v o l t a g e - c l a m p experiments. From a h o l d i n g potential of -80 mY in a c o n d i t i o n i n g clamp step to -40 mV the sodium current was inactivated. The slow calcium inward current was e l i c i t e d in a second step to 0 mV. M a g n e s i u m c o n c e n t r a t i o n - d e p e n d e n t l y and reversibly decreased this current (by about 40% after an increase from 1.2 to 9.6 mmol/l, taking the spontaneous run down into account). C o n s i d e r i n g the v o l t a g e - c u r r e n t r e l a t i o n s h i p of the calcium inward current by changing the second clamp step, m a g n e s i u m decreased the maxim a l l y available current, but a d d i t i o n a l l y shifted the r e l a t i o n s h i p by about 10 mY to more positive potentials. A l t e r i n g the c o n d i t i o n i n g dep o l a r i z a t i o n to -55 mV increased an early current component at the second d e p o l a r i z a t i o n (probably an incompletely inactivated sodium current) which was further strongly augmented after addition of magnesium. This can be explained by a shift of the dependence of the inward current on the potential of the first depolarization (by ca. +5 mV). It is concluded that magnesium in g u i n e a - p i g ventricular heart exerts an inhibiting effect on calcium inward current, but a d d i t i o n a l l y influences voltage-current relationships by inc r e a s i n g the "effective" m e m b r a n e potential.

Positive heterotropic allosteric regulators of dihydropyridine (DHP) blndlng2+(( + )-cis-diltiazem, ( + )-tetrandrme," trans-diclofurime) to L- tv_oe Ca channels increase the affinity of the channel for C£Z+ [ Ca"z+ site I] (Vitamins and Hormones 44, 155-328). BM 20.1140 (BM, ethgl.2,2-diphenyl-4-(1-pyrrolidino)-5-(2-picolyl)oxy-valerate)) is a novel Ca ~+- antagonist (pA 2 for (-)-BM= 8.64; for (+)-BM= 7.29; taenia caeci). (-)-BM but noi (+)-BM effects were antagonized by DHPchannel activators. (-)-BM is one of the most potent negative heteroropic allosteric regulators of (+)-cis-diltiazem binding to skeletal muscle ~l-SUbunits (K05 = 1.12 4aM). (-+)-BM, (-)-BM and (+)-BM accelerat4 association 'of (+)-[aH]-isradipine, (+)-BM accelerates isradipine dissociation from 0.024 min "1 to 0.093 min "1. (+)-BM and ()-BM are noncompetitive inhibitors for the phenylalkylamine site. (-)BM is a potent stimulator of equilibrium DHP binding, depending on channel subtype; (+)-BM is without effect, but reverses the positive allosteric effect of (-)-BM with a Ki= 3.3 nM. Likewise (+)-BM reverses the effects of (+)-tetrandrine, ~' + )-cis-diltiazem, fostedil and trans-diclofurime. The high- affinity Ca 2"+ site i on the el- subunit is formed •upon binding . of DHP's (K05 . = 315 . . nM [Ca2+),tree ) • (+)tetrandrme (10 #M) increases the channel affimty 8- fold (Kn ~ = 40 nM [Ca2+]lf.r~^) perhaps by increasing the number of Ca 2~: c~rdinating carbonyl oxygens. (+)-BM (10 #M) prevents the colfformatmnal change induced by (+)-tetrandrine (-K , ~ = 236 nM [Ca2+]fr^~ , , ( + ) - c.i s • . ~J.D it~c dlltmzem and the DHP's act from the outer face of the elJSUbumt (J. Gen. Physiol. 95, 1-27), whereas the phenylalkylamine binding site is at the intracellular fac9 (Striessnig et al., Proc. Natl. Acad. Sci. U.S.A., in press) close to Caz÷ site II (Nature 346, 321-322). The location of Ca z÷ 'site I is not yet known. Supported by FWF (S-4501) Institut fiir Biochemische Pharmakologie, Peter Mayrstr.1, A-6020 Innsbruck, Austria

Institut f~r P h a r m a k o l o g i e und Toxikologie der T e c h n i s c h e n Universit~t M~nchen, Biedersteiner Str. 29, D-8000 M ~ n c h e n 40

202

2O4

FENDILINE ACTS AS A DIRECT BLOCKER OF L-TYPE CALCIUM CHANNELS IN GUINEA-PIG VENTRICULAR CELLS W. Schreibmayer and O. Tripathi*

SYNTHESIS AND SAR STUDY OF THE C a - A N T A G O N I S T I C PROPERTIES OF A SERIES OF N E W D I P H E N Y L A L K Y L ~ I N E S P. Caldirola, P. Zandberg and H. Timmerman

The modulation of the L-type calcium currents of guinea-pig ventricular myocytes by fendlline ( ),-[.-Phenyl-N-(1-phenylethyl)benzenepropanamine) was investigated with the patch-clamp method. The Ltype calcium current is Necked directly by fendiline with an IC50 of 17.0 -+ 2.4~M. Its kinetics is modulated in a specific way: the availability curve of c'alcium channels is shifted to negative membrane potentials and inactivation time constants are shortend. Additional administration of the agonistic dihydropyridine compound (4R,4S)-Bay K 8644 (racemic Methyl-l,4-dihydro-2,6-dimethyl-3-nitro4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate) at a concentration of 1/~M in the presence of 30~uMfendiline resulted in a further block of calcium channels instead of the expected increase in the cardiac calcium current. Neither nifedipine, diltiazem nor verapamil resulted in a similar paradoxic effect in combination with (4R,4S)-Bay K 8644. Instead in all cases the expected agonistic behaviour of (4R,4S)-Bay K 8644 was observed. The most likely explanation for diphenylalkylamine action on the voltage sensitive calcium channel is, thus, binding to a receptor site that is physically different from the sites for dihydropyridines, phenylalkylamines and benzothiazepines.

A c c o r d i n g t ~ binding studies the following subgroups of Ca ++ can be differentiated: Ia: dihydropyridines; Ib: diphenylalkylamines; II: verapamil; III: benzothiazepines. SAR studies for dip h e n y l a l k y l a m i n e s are currently missing. Thus we have synthesized and tested the C a - a n t a g o n i s t i c p o t e n c y of d i p h e n y l a l k y l a m i n e s of the following structure: R1~

This work was supported by the Austrian Research Fund ($4504B) *:Physiology Division, Central Drug Research Institute, Lucknow 226001, India; Department of Medical Physics and Biophysics, University of Graz, Harrachgasse 21/IV, A-8010 Graz, Austria

c--x R2

/ R4

The compounds are rather simple m o l e c u l e s of ~,~ich the skeleton is fit for rational manipulation. By considering: the length of the chain b e t w e e n the benzhydryl m o i e t y and the nitrogen; including an ether function (X= O;S), saturation and unsaturation of the chain (X= CH2; CH) ; the substitution of the n i t r o g e n (R3; R4) , a series of amines is obtained. The compounds have been tested in a 3Hnitrendipine binding assay and in a number of in vitro systems such as smooth m u s c l e r e l a x a t i o n using d i f f e r e n t contractants and an a n t i p l a t e l e t a g g r e g a t i o n test. These compounds are not strong C a + + - e n t r y blockers (Ki is 10-6); their pharmacological properties can be explained by interference w i t h the Ca ++ homoeostasis via other ways. The synthesis will be presented together w i t h b i o l o g i c a l data and SAR investigations. D e p a r t m e n t of P h a r m a c o c h e m i s t r y , Vrije U n i v e r s i teit, De Boelelaan 1083, NL-I081 HV A m s t e r d a m / The N e t h e r l a n d s

R 52 205 MYOCARDIAL AND VASCULAR ACTIVITY OF SOME AGONISTIC 1,4-DIHYDROPYRIDINES IN VITRO. R. Ratke, W. Klaus and U. Fricke Calcium agonists of the 1,4-dihydropyridine (DHP) type are positive inotropic and cause vasoconstriction by facilitating the calcium inward current into cardiomyocytes and smooth muscle cells through voltage-dependent channels (Schramm et. al. Arzneim.-Forsch./Drug Res. 33 (1983) 1286; Alonso et. al., Gen. Pharmac. 20, 827 1989). Because of their pronounced vasoconstrictor activity, currently known calcium agonists cannot be used clinically as cardiostimulants. We here describe the action of (-)-S-Bay K 8644 [(-)-S-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2trifluoromethylphenyl)-pyridine-5-carboxylate] and some new related compounds on porcine right ventricular trabeculae and coronary arteries: Bay W 5035 [2-aminoethyl ester derivative], Bay P1974 [6-hydroxyhexyl ester derivative]. Porcine ventricular trabeculae were stimulated with 1 Hz in an oxygenated KrebsHenseleit-solution (resting tension 5raN; 30°C). Porcine right coronary artery segments were examined in the same solution after a testcontraction with 60 mmol/l KC1, washout and moderate depolarization with 15 retool/1 KCI (resting tension 20 raN; 37°C). Alldrugs were added cumulatively. At porcine trabeculae Bay W 5035 (IC50 1,46_+0,16 xl0 "° mol/1) displays a bell shaped dose response curve with omuch lower affinity than (@S-Bay K 8644 (ICso 1,35- 0,11 xlo -~ mol/1) but comparable activity. In contrast, Bay W 5035 showed no detectible_constriction of the coronary arteries at a concentration up to 10-0 mol/l. Bay P 1974 is slightly positive inotropic and vasoconstrictive. Interestingly, Bay K 8644 is able to enhance the 60 mmol/1 KCl-tone of the artery while Bay W 5035 does not affect the state of contraction and Bay P 1974 acts like an antagonist. This behaviour may be explained by a different affinity of the agonistic resp. antagonistic enantiomers of these compounds. From our experiments ~t seems likely that the cardioselectivity of DHP calcium agonists can be improved by molecular variations. Institut ffir Pharmakologie der Universit~it zu K61n, Gleueler Strage 24, W-5000 K61n 41

207 C H A R A C T E R I Z A T I O N OF C A L C I U M A N T A G O N I S T I C E F F E C T S O F T H R E E M E T A B O L I T E S OF T H E N E W A N T I H Y P E R T E N S I V E AGENT NAFTOPIDIL: (NAPHTHYL)HYDROXY-NAFTOPIDIL, (PHENYL)HYDROXY-NAFTOPIDIL, AND O-DESMETHYLN A F T O P I D I L . M. G r u n d k e and H.M. H i m m e l T h e a n t i h y p e r t e n s i v e d r u g n a f t o p i d i l b l o c k s ~la d r e n o c e p t o r s and i n h i b i t s Ca2 + e n t r y v i a p o t e n t i a l - d e p e n d e n t c h a n n e l s in v a s c u l a r and cardiac m u s c l e (Himmel et al. 1991, J C a r d i o v a s c P h a r m a col, in press). N a f t o p i d i l u n d e r g o e s e x t e n s i v e b i o t r a n s f o r m a t i o n in vivo. W e t h e r e f o r e h a v e s t u d i e d t h e p o s s i b l e p h a r m a c o l o g i c a l e f f e c t s of its m e t a b o l i t e s ( n a p h t h y l ) h y d r o x y - , (phenyl)hydroxy-, o - d e s m e t h y l - n a f t o p i d i l (NHN, PHN, DMN) in p a p i l l a r y m u s c l e s and i s o l a t e d m y o c y t e s of t h e g u i n e a - p i g heart. In p a p i l l a r y muscles, 30 ~ M of all c o m p o u n d s r e d u c e d the f o r c e of cont r a c t i o n (PD2-values = 5.5) and decreased the A P D 2 0 and APDs0. A P D g 0 w a s s l i g h t i y i n c r e a s e d by PHN, h a r d l y a f f e c t e d by DMN, and s h o r t e n e d by NHN. The A P a m p l i t u d e and V m a x w e r e s l i g h t l y r e d u c e d b y PHN, but little i n f l u e n c e d b y DMN and NHN. In v o l t a g e - c l a m p e d v e n t r i c u l a r cells, the c a l c i u m c u r r e n t Ica w a s c o n c e n t r a t i o n - d e p e n d e n t ly depressed b y the t h r e e c o m p o u n d s in t h e r a n g e of i-I00 ~M. M e m b r a n e c u r r e n t s m e a s u r e d w i t h s l o w r a m p p u l s e s w e r e m a r k e d l y r e d u c e d by i00 # M of e i t h e r N H N or P H N i n d i c a t i n g an i n h i b i t o r y e f f e c t on K + - c o n d u c t a n c e . Our d a t a s h o w t h a t NHN, PHN, a n d DMN are e f f e c t i v e as C a 2 + - a n t a g o n i s t s in vitro. Thus, the m e t a b o l i t e s are exp e c t e d t o c o n t r i b u t e to the a n t i h y p e r t e n s i v e a c t i o n of t h e i r p a r e n t c o m p o u n d n a f t o p i d i l . P h a r m a k o l o g i s c h e s Institut, U n i v e r s i t ~ t - G H S Essen, H u f e l a n d s t r . 55, 4300 E s s e n i, G e r m a n y

206 THE CONTRIBUTION OF a- AND CA++-CHANNEL BLOCKING PROPERTIES TO THE VASODILATING EFFECT OF NAFTOPIDIL

208

W. Bartsch, A. Schbffel, M. Kohler, B. Eberwein, R.G. Hooper and L. Kling Previous experiments have provided evidence that the antihypertensive effect of naftopidii is attributable not only to its s-blocking property but also 1o Ca~-antagonism. To evaluate the importance of these properties investigations were performed to calculate the pA~.values of different vasodilating compounds against noradrenaline (NA) and CaCI~ as contracting agents. METHOD: Rabbit aortic spiral strips were used for the experiments. Norodrenaline and Ca ~ were used as agonists. The vasoconstricting effect of the stimulants was determined by adding incremental concentrations of the compounds to the bath. Various concentrations of the antagonists were used and the shift of the concentration-response curves of the agonists were calculated. The method of Aruhnlakshana and Schild has been used for the calculation of the

Increase in cytosolic Ca ~+ is a crucial step for the stimulation of insulin secretion. Depolarising initiators of hormone release are known to elevate Ca s÷ influx via opening of voltage dependent Ca 2÷ channels. We have studied the characteristics of Ca z÷ antagonist binding to a high affinity binding site in the particulate fraction of insulin secretory RINmSF cells. In intact RINmSF cells the dihydropyridine (DHP)-type Ca z÷ antagonist [3H]-(+)-PN 200-110: (+)-[5-methyl-aH]-[isopropyl-4-(2,1,3-benzoxa diazol-4-yl)1,4-dihydro- 2,6-dimethyl-5-methoxy-carbonyl-pyridine-3-carboxylate] concentration dependently inhibited 25 m M KCl-induced insulin release with an IC~o of 0.01 laM. In the particulate fraction [aH]-(+)-PN 200110 bound in a stereoselective manner to a high affinity site, with a I~ of 7.0 9:4.8 n M and a Bmax of 858 9:556 fmo]/mg protein (means 9: SD, n=6). Benzothiazepine-type Ca z÷ antagonist d-cis-diltiazem stimulated binding of [aH]-(+)-PN 200-110 in a temperature dependent manner, whereas the calmodulin antagonists (amiodarone and CGS 9343B: 1,3-dihydro- 1-[1- ((4-methyl-4H,6H-pyrrolo[1,2-a] -[4,1] benzoxazepin-4-yl)~methyl)- 4-piperidinyl]- 2H-benz imidazol-2-one maleate) inhibited binding of [~H]-(+)-PN 200-110. CGS 9343B was roughly 50 times less potent than amiodarone. These results demonstrate the presence of a Ca 2~ entry blocking site in the insulin secretory cells (RINm5F) with analogous properties to the DHP binding subunit of the L-type Ca ~÷ channel from skeletal muscle.

INTRODUCTION:

PA2-vaines. RESULTS: Substance

Prazosln

Nicardipine

Urapidil

Naftopidil

(mS)

(R)

(S)

pA 2 NA

8.85

> 5.00

6.25

7.10

6.92

6.48

pA 2 Ca ~

> 5.00

10.11

> 4.0

5.90

6.12

5.92

Ratio > 7080 < 0.000008 > 178 16 6.3 3.6 The drugs investigated are presented in the table. Obviously, from the pA 2values and the ratios of calcium channel to nocadrenaline antagonistic activity, prazosin may be characterized as a typical c~-blocking agent and nicardipine as a typical Ca~-channel blocking agent. Urapidil showed a c~-blockadeo Interestingly, naftapidil and its enantiomers do not show any marked discrimination between its activity on the response to noradrenaliue or Ca b. ions. The ratio of 16 should not be interpreted as a marked predominance of c~blockade. Both the enantiomers show a narrower ratio between ct- and Ca ~blocking properties, which may be explained by biological variation. CONCLUSION: These results suggest that the hypotensive effect of naftopidil is not only the result of the ct-blockade but also of the Ca++-channel blocking activity. l)ep. of Pharrnacol.,BoehringerMannhehnGmbH, P.O. Box 31 01 20, D-6800 Mannheim

Ca z÷ ANTAGONIST BINDING TO TI~ FRACTION O F INSULIN SECRETORY CELLS.

PARTICULATE M. R@rd~e]dt

Pharmazeutisches Institut, Lehrstuhl f~ir PharmakologJe, Auf der Morgenstelle 8, D-7400 Tl~bingen, FRG

R 53 211

209 CALMODULIN INHIBITION BY DIPHENYLALKYLAMINES R . M a n n h o l d a n d W. V o i g t

HIGH-AFFINITY INTERACTION OF POLYMYXIN B,WITH CALMODULIN B.H. Schmidt, J. Traber, and L. Hegemann

P o t e n c y and s e l e c t i v i t y of c u r r e n t l y a v a i l a b l e c a l m o d u l i n ( C a M ) - a n t a g o n i s t s is r a t h e r l i m i t e d . We a t t e m p t e d to i m p r o v e t h e s e p r o p e r t i e s b y o p t i m i z i n g the C a M - a n t a g o n i s t p r e n y l a m i n e . T e s t system used was the CaM-stimulated phosphodiesterase. I n s e r t i o n of a d o u b l e b o n d or s u l p h u r in t h e benzh y d r y l p a r t of t h e m o l e c u l e i n c r e a s e s , i n s e r t i o n of o x y g e n d e c r e a s e s p o t e n c y . C h a i n l e n g t h is o p timal w i t h 4 a t o m s - n i t r o g e n - 3 atoms. R e g a r d i n g benzhydryl substitution halogenation surmounts m e t h y l a t i o n ; p a r a - s u b s t i t u t i o n is s u p e r i o r to os u b s t i t u t i o n . S e c o n d a r y n i t r o g e n s e e m s to be adv a n t a g e o u s as c o m p a r e d to t e r t i a r y a m i n e . L i p o p h i l i c i t y d e p e n d e n c e of C a M - a n t a g o n i s t i c p o t e n c y d e c r e a s e s in t h e f o l l o w i n g m a n n e r : saturated chain >double bond-> sulphur-> oxygen derivatives. A m o n g s t the c o m p o u n d s d e s c r i b e d n e w C a M - a n t a g o nists with remarkably high potency have been detected.

The cyclic peptide antibiotic polymyxin B is considered a rather selective inhibitor of protein kinasn C and is thnrefore widely used as a pharmacological tool to assess the involvement of this enzyme in physiological processes. We however found that polymyxin B inhibits calmodulin-dependent cyclic 3':5'-nucleotide phosphodiesterase from bovine heart in vitro at nanomolar concentrations 2+ (IC -values 80 nM at 500 ~M Ca ), that is at concentra50 tions about 200 fold lower than those required for inhibition of protein kinase C. This effect appears to be mediated by a direct interaction of polymyxin B with the z+ Ca -receptor protein calmodulin. Inhibition of phosphodiesterase was selective for the calmodu~in-dependent isoenzyme and competitive with respect to Ca z+ and calmodulin. The electrophoretic migration velocity of calmodulin in non-denaturating polyacrylamide gels was slowed down in the presence of the antibiotic. Finally, polymyxin B, covalently bound to an agarose matrix, could be used as an affinity label for calmodulin. Thus, polymyxin B may be regarded as one of the most poten~ and selective calmodulin antagonists presently available. Institute for Neurobiology, Troponwerke GmbH & Co. KG, Berliner Straee 156, D-5000 K61n 80, FRG. * Institute of Molecular Biology and Medical Biotechnofogy, University of Utrecht, The Netherlands.

C--X

R~

/ R4

I n s t i t u t e of L a s e r m e d i c i n e , H e i n r i c h - H e i n e - U n i v e r s i t y of D N s s e l d o r f , U n i v e r s i t ~ t s s t r a s s e 7, D-4OOO D~sseldorf / FRG

210

212

INFLUENCE OF DOPANINE RECEPTOR AGON~STB AND ANTAGONISTS ON CALNODULIN T~ANSLOCATION IN DIFFERENT BRAIN REGIONS. N.S. Popov

MUSCARINIC ACETYLCHOLINE RECEPTORS REGULA'VE THE RELEASE OF GTP[rS] FROM G PROTEINS IN PORCINE ATILIAL MEMBRANES G. Hilf

Acute and c h r o n i c e x p e r i m e n t s were p e r f o r m e d to s t u d y the e f f e c t s of i n t r a p e r i t o n e a l l y adm i n i s t e r e d dopamine a g o n i s t s and a n t a g o n i s t s on the t r a n s l o c a t i o n of c y t o s o l i c and membrane-bound c a l m o d u l i n i n the s t r i a t u m and h±ppocampus of the r a t b r e ± n . S i n g l e doses o f apomorphine and a l o w - d o s e amphetamine (1.25 mg/kg) r e s u l t e d i n a t r a n s l o c a t i o n of c a l m o d u l i n , as measured by a decrease i n membrane-bound and i n c r e a s e i n c y t o s o l i c calmod u l i n i n the s t r i a t u m , whereas b r o m o c r y p t i n e was i n e f f e c t i v e . A~phetamine e x e r t e d a s i m i l a r e f f e c t i n the h±ppocampus as w e l l . Howe v e r , a h i g h - d o s e amphetamine CSmg/kg) had an o p p o s i t e e f f e c t on t r a n s l o c a t i o n , £n t h a t t h e r e was an i n c r e a s e i n membrane-bound c a l m o d u l i n . C h r o n i c a l l y a p p l i e d amphetamine (5 mg/kg) and h a l o p e r i d o l (1 m g / k g ) , i . e . under c o n d i t i o n s of dopamine r e c e p t o r s u p e r sensitivity, tended to decrease c y t o s o l i c c a l m o d u l i n i n the s t r ± a t u m and hippocampus, and to i n c r e a s e membrane-bound c a l m o d u l i n . The f i n d i n g e and, e s p e c i a l l y , the c h r o n i c e f f e c t s o f h a l o p e r ± d o l and the h i g h dose o f amphetam±ne are i n t e r p r e t e d i n the l i g h t of the c u r r e n t c o n c e p t which s u g g e s t s t h a t t r a n s m i t t e r s and i n t r a n e u r o n e l s i g n a l l i n g systems c o n v e r g e , t h e r e b y i n f l u e n c i n g the more comp l e x processes of neuronal p l a s t i c i t y , rather than r e c e p t o r s e n s ± t i v ± t y o n l y . Institute o f Pharmacology and T o x i c o l o g y , N e d i c a l Academy Nagdeburg, L e i p z i g e r s t r . 44 0-3090 Nagdeburg, S a c h s e n - A n h a l t , F~G

Under appropriate conditions, i.e. in the presence of GDP, muscarinic acetylcboline receptors (mAcChR) stimulate the binding of the GTP analog GTP[rS] to G proteins in porcine atrial membranes. On the other hand, mAcChR can also regulate the dissociation of bound [35S]GTP[rS]

from prelabelled

atrial membranes.

The release of bound

[35S]GTP[rS] ffom the membranes induced by carbacbol (CCh) was rapid and was halfm~imal at about 0.3,~M CCh. Atropine competitively antagonized the stimulatory effect of CCh. In the absence of CCh, the mAcChR antagonists atropine and pirenzepine reduced the basal release of [35S]GTP[rS] by maximally about 25 %, with half-maximal effecs at 5 and 500 nM, respectively. The regulation of [35S]GTP[rS] release by mAcCbR agonists and antagonists was strictly dependent on the presence of a second guanine nucleotide in the release assay. At maximally effective concentrations, GTP[~-S], GTP, GppNHp, GDP and G.DP[I~S] promoted the stimulatory effect of CCh on [35S]GTP[~-S] release to a similar extent. GMP and ATP were ineffective up to 1 raM. In addition to guanine nucleotides, the CCh-stimulated release of bound GTP[rS] was dependent on Mg2+, being maximally effective at about 10 ,~M. Prelabelling of the membranes with increasing concentrations of GTP[~-S] largely decreased the CCh-stimulated release of bound GTP[~-S]. The data indicate that mAcChR can interact with GTP[~'S]-liganded G proteins in porcine atrial membranes, resulting in release of bound nucleoside triphosphate. This release may be caused by a direct recoptor-G protein interaction or by a receptor- and guanine nucleotide-dependent G protein-G protein interaction. Finally, the antagonist data support the hypothesis that even agonist-free mAcChR can productively interact with G proteins. Pharmakologisehes Institut tier Universi~t Heidelberg, Im Neuenbeimer Feld 366, D.-6900 Heidelberg, FRG

R 54

213

215

TIME C O U R S E OF T H E E F F E C T OF ISOPRENALINE ON T H E INHIBITORY G - P R O T E I N ~ - S U B U N I T IN T H E HEART. U. Mende, D. Geertz, H. Scholz, J. Schulte am Esch and R. Sempell

QUANTIFICATION OF GIC ( _ BY A NOVEL RADIOIMMUNOASSAY IN CARDIOMYOPATHIC HUMAN HEARTS M.B0hm, K.Larisch and E.Erdmann

In h u m a n end-stage heart failure due to idiopathic dilated cardiomyopathy the increase of the inhibitory G-protein ~-subunit (¢Gi) is thought to be due to elevated catecholamine levels. In order to clarify in vivo the pathophysiological role of catecholamines we investigated in a rat model the time course of the effect of chronic isoprenaline (ISO)-infusion on myocardial ~ G i content determined b y pertussis toxincatalyzed ADP-ribosy]ation. In addition we studied in left papillary muscles the influence of a 4 d a y ISOinfusion on the effect of carbachol (Carb) on force of contraction. Rats (n=7-8) were treated by a subcutaneous infusion with osmotic mlnipumps of ISO (2.4 m g / k g - d), propranolol (PROP, 9.9 m g / k g • d), ISO+ P R O P or 0.9% NaC] (CTR) for i, 2, 3, 4 or 8 days. ISOtreated rats revealed a constant increase in ventricular/body weight ratio of about 35% (p<0.05 vs. CTR). ISO induced an increase of ~Gi of about 15-50~ becoming significant at d a y 2 (p<0.05 vs. CTR). The potency for the negative inotropic effect of C A R D in the presence of ISO (0.i #M) was increased (mean ECs0: 0.035 )/M vs. 0.283 ~/M, p<0.05). P R O P - t r e a t m e n t alone h a d no effect but antagonized the ISO-induced effects. - In conclusion, ~-adrenergic stimulation in vivo leads to a sustained increase in ~Gi which is accompanied b y an increased responsiveness for CARD. T h e results support our hypothesis that in h u m a n heart failure the increase in ~Gi might be due to elevated catecholamine levels. (Supported b y the DFG.) Abfeilung Allgemeine Pharmakologie, Universit~tsKrankenhaus Eppendorf, Universitat Hamburg, Martinistra~e 52, D-2000 H a m b u r g 20, F R G

In dilated cardiomyopathy, an increase by 40-50% of pertussis toxin substrates (PTS) has been reported. Since posttranslational modifications affect the amounts of PTS detected with this technique, we developed a radioimmunoassay using the iodinated synthetic peptide 1251_KENLKDCGLF' the antiserum DS4 raised against the same peptlde and retinal transducin (TO) as standard in order to quantify the amount of Gic~-proteins in atrial and ventricular myocardium of patients with dilated (DCM) or ischemic (ICM) myocardium and in nonfailing myocardium (NF). B-Adrenoceptors (&-AR; 1251_cyanopindolol_binding) were studied for comparison. The results are summarized in the Table. Gi= (#g TD = equivatent/mg protein)

B-AR density (fn~t 1251-Cypbound/mg protein)

RA LA RV LV NF 4.6*0.2 5.1,0.1 2.5*0.2 1.9.0.1

RA

LV

ION 4.8±0.3 4.8*0.2 2.4±0.3 2.8±0.1"61.2±6.4" 69.7*4.3* 37.3*2.3* 49.5±2.0" 4.4±0.4 4.5*0.2* 2.3±0.1 4.1±0.2"62.8±2.4* 72±4.2* ~5.3.2.3" 49.3±2.0*

RA: right atrium; LA: left atrium; RV: right ventricle; LV: left ventricle; n = 5-17; * p < 0 . 0 5 vs NF It is concluded that the amount of immunodetectable Gi=-proteins is increased in LV of ICM and DCM. Since previous studies reported unchanged PTS in ICM, the results indirectly provide evidence for posttranslational modification of Gio~ affecting PTS-labeling. Although the decline in I~-AR was similar in all tissues, Gio( is indicating that the susceptability of LV to increase Gi(~ levels might be increased in heart failure. This new radioimmunoassay may become an important tool to study the expression of "true" Gi0( and changes thereof in pathological conditions. Medizinische Klinik I der Universit~tt, Klinikum GrolShadem, O-8000 M~nchen 70, Germany

216

TIME C O U R S E OF I S O P R E N A L I N E - I N D U C E D U P R E G U L A T I O N OF INHIBITORY G - P R O T E I N c - S U B U N I T m R N A IN T H E H E A R T T. Eschenhagen, M. Nose, W. Schmitz, A. Warnholtz and J.-M. Wdstel

CARDIAC IN VITRO PHARMACOLOGY PHOSPHODIESTERASE INHIBITOR

Abteilung Allgemeine Pharmakologle, Universit~tsKrankenhaus Eppendorf, UniversitY/ Hamburg, Martinistra~e 52, D-2000 H a m b u r g 20, F R G

RV

DCM

214

The i n h i b i t o r y G-protein a-subunit a n d i t s mRNA a r e i n c r e a s e d i n f a i l i n g h u m a n h e a r t s . We h a v e o b s e r v e d similar alterations i n aG-mRNA e x p r e s s i o n in rats treated b y a 4 d a y i n f u s i o n o f i s o p r e n a l i n e (IS0). I n t h e p r e s e n t s t u d y we r e p o r t t h e time course of t h i s e f f e c t . R a t s (n=7-8) w e r e t r e a t e d with a subcutaneous i n f u s i o n w i t h o s m o t i c m i n i p u m p s o f NaC1 0.9% (CTR), IS0 (2.4 m g / k g . d ) , p r o p r a n o l o l (PROP, 9.9 m g / k g . d ) , a c o m b i nation thereof o r £ r i i o d o £ h y r o n i n e (T3, 0.5 m g / k g - d ) f o r i, 2, 3, 4 a n d 8 d a y s . IS0 i n d u c e d a r a p i d a n d constant increase in heart/body weight ratio from day 1-8 (134-142% o f CTR). A d d i t i o n a l l y , t o t a l RNA c o n c e n t r a t i o n i n c r e a s e d r a p i d l y f r o m d a y 1 (125% o f CTR) t o d a y 3 (145% o f CTR) a n d r e t u r n e d t o CTR v a l u e s on d a y 8. Myocardial a G - m R N A concentration was determined with 32P-labeled ~ G - c D N A probes using an in vitro transcribed c R N A standard. ISO induced a gradual increase in m R N A concentrations (pg/pg total RNA) of ~Gi2 and ~Gi3 which was significant on day 3 and amounted maximally to 185% (16.7 vs. 9.0) and 136% (3.3 vs. 4.5) respectively (p<0.05 vs. CTR) on day 8. ~Gsm R N A levels (35 pg/gg) remained unchanged. P R O P alone had no effect but antagonized all ISO-effects. The T3induced cardiac h y p e r t r o p h y (115% of CTR) was not accompanied b y changes in ~G-mRNA. - It is concluded that ~-adrenergic stimulation leads to a sustained increase in myocardial ~ G i - m R N A with relative slow onset. This supports our hypothesis that the increase in ~Gi-mRNA in chronic heart failure might be due to elevated catecholamine stimulation. (Supported b y the DFG.)

LA

99±7.8" 111.5.8 65.8*4.0 83.6*4.0

OF

R 80122,

A

NOVEL

D. Hilhelm, D. de Chaffoy de Courcelle*, A. Leidig, H. Ebbert, and C. M~uter The present investigations were performed to compare the action of the phosphodiesterase inhibitors R 80122 ((E)-N-cyclohexy1-N-methyl-2-[[[phenyl (l,2,3,5-tetrahydro-2 oxoimidazo [2,l-b]-quinazolin-7-yl) methylene] amino]oxy] acetamide) and milrinone on guinea-pig isolated cardiac tissue. R B0122 inhibited human phosphodiesterase type Ill (PDE I I I ) at lower concentrations t h a n milrinone and showed a higher selectivity for PDE I I I . Force of contraction of l e f t atria paced at a frequency of 1.3 Hz was augmented by R 80122 (I-3 pM). A comparable rise in contractile force as produced by I pM R 80122 was achieved with lO ~H milrlnone. The rate of spontaneously beating atria was not altered by R 80122 in the concentration range of 0.01-0.3 pM. Higher concentrations (I-3 pM) led to a small but not significant increase of 12%. Milrinone's effect on frequency was more pronounced and amounted to 21~ at 10 pM. In the papillary muscle, stimulated at 0.2 Hz, the positive inotropic effects of both R 80122 and milrinone were inhibited by carbachol indicating the involvement of intracellular cAMP. Further support of a cAMPdependent action of R 80122 and milrinone was obtained by the leftward shift of the concentratlon-response curve of Isoprenaline in atria and papillary muscles, paced at 0.2 Hz. The action potential duration of papillary muscles was not significantly prolonged by R B0122 whereas i t was reduced by milrinone. In conclusion, R 80122 was very selective for POE I I I and increased cardiac contractility at lower concentrations than milrinone. In contrast to milrlnone, R 80122 did not change cardiac frequency and electrophysiological parameters which may be advantageous for the treatment of heart failure. JANSSEN RESEARCH FOUNDATION, D-4040 Neuss,*B-2340 Beerse

R 55 217

219

CARDIAC EFFECTS OF R 80122, A NEH AND POTENT CARDIOTONIC AGENT, IN AN ACUTEHEART FAILUREMODEL J. Schneider and E. Beck

ONLY THE COMBINED ACTION OF PIMOBENDAN AND ISOPRENALINE MARKEDLY INCREASES FORCE OF CONTRACTION AND cAMP CONTENT IN FAILING H U M A N H E A R T PREPARATIONS. Th. Bethke, C. Kohl, H. yon der Leyen, W. Meyer, H. Mehl, C. Schmidt-Schumacher, J. Starbatty and B. Stein

R B0122 ((E)-N-cyclohexyl-N-methy]-2-[[[phenyl (1,2,3, 5-tetrahydro-2 oxoimidazo [2,1-b] -quinazolin-7-yl) methylene] amino] oxy] acetamide) is a novel nonsympathomimetic and nonglycoside cardiotonic drug with a selective inhibitory a c t i v i t y on PDE I I I . The aim of this study was to examine the cardiotonic properties of R 80122 as compared to milrinone, enoximone, adibendan and digitoxin. The cardiac effects of R 80122 were investigated in the hexobarbital-depressed heart-lung-preparatlon of the guinea-pig. After induction of heart failure (reduction of cardiac output to 30% of the i n i t i a l value) cumulative addition of R 80122 (in a range of 3 x lO-8 to l x lO-6 molll) dose-dependently reversed the cardiac depressant effects of hexobarbital. Hith regard to reconstitution of c o n t r a c t i l i t y and cardiac function R 80122 appeared to be lO0 times more potent than enoximone and mIIrlnone and 10 t i m e s more potent t h a n digitoxin. Adibendan, which showed cardiotonic effects in the same dose range as R B0122, was about 50% less effective than R B0122 with respect to the maximally induceable positive inotropic effect. The cardiotonic effects of R 80122 were not accompanied by an increase in heart rate as found with milrinone in the same model. The potent cardlotonic properties and the absence of positive chronotropy revealed by this study suggest that R B0122 might exert a beneficial effect in the treatment of congestive heart failure. JANSSEN RESEARCHFOUNDATION, Raiffeisenstr. 8, D-4040 Neuss 21

In congestive heart failure (CHF) positive inotropic effects (PIE) of ~-adrenoceptor agonists and phosphodiesterase inhibitors (PDEI) are greatly reduced. We studied the effects of isoprenaline (ISO) and pimobendan (PIMO) on force of contraction (FC) and cAMP content (pmol/mg ww) in eleclrically driven (0.5 Hz) ventricular trabeculae isolated from explants of 5 nonfailing (NF) and 6 failing (F, NYHA IV, idiopathic dilated cardiomyopathy) human hearts. In NF, ISO (0.2 ~mol/l) and PIMO (i00 ~mol/1) alone increased FC by 355% and 185%, and cAMP contenf from 1.13 ± 0.06 to 1.83 + 0.23 and to 1.91 + 0.26 (n=6-13) respectlvely. In contrast, in F the PIE of ISO and PIMO were reduced amounting only to an increase in contractile force of 116% and 25% respectively. In F 'the basal cAMP contenl was only 62% of that in NF. ISO or PIMO alone increased cAMP only from 0.?0 + 0.08 to 0.98 + 0.15 or to 1.18 + 0 . 0 7 (n=15-26). However, the combination of ISO and PIMO elevated FC and cAMP in F to similar values as with ISO and PIMO alone in NF. Force of contraction was increased by about 265% and cAMP content was elevated to 2.14 ± 0.37 (n=8). -In conclusion the PDEI PIMO restores maximal effects of ~-adrenergic agonists on cAMP content and FC in F. This may explain the increase in cardiac output afier acute administration of PDEI in patients with severe CHF. (Supported by the DFG.) Abteilung Allgemeine Pharmakologie, UniversitatsKrankenhaus Eppendorf, Universit~t Hamburg, Martinistra~e 52, D-2000 Hamburg 20, FRG.

218

220

COMPERATIVE STUDIES ON THE CARDIAC ACTIONS OF AMRINONE

ANTIAGGREGATORY EFFECTS OF THE PHOSPHODIESTERASE INHIBITOR PIROXIMONE IN VIVO AND IN VITRO

AND MILRINONE IN ISOLATED GUINEA PIG HEARTS BEING STIM-

M.Buerke,

H.Darius,

T.Cyrus,

R.Erbel,

J.Meyer

ULATED WITH EPINEPHRINE R. Podehl, J. Zylka, and G. Joseph Using isolated guinea pig hems perfused according to the Langendorff technique experiments were performed to compare the functional profiles of the bipyridine PDE inhibitors amrinone (AM) and milrinone (MIL). Both compounds were examined in a concentration range of 10-7 - 10-3 mol/l without and with a threshold concentration (10-8 mol/l) of epinephrine (EP) respectively. By simultmeous registration of the appropriate parmaeters dose response curves were established for the inotropic, chronotropic, dromotropic and coronary dilating effects of these bipyfidines. Both compounds increased the heart rate (HR), AM by 30 %, MIL by 75 %, shortened the AV conduction time (AV), AM by 20 %, MIL by 30 %, and increased the coronary flow (CFI), AM by 90 %, MILby 120 % but only MIL was effective in increasing contractile force (CF) by 45 %, while AM showed an inotropic action less then 10 %. In combination with EP AM showed a strong inotropic effect (max. 70 %) even in the lowest concentration examined while MIL showed further increase in CF only in high concentrations. With respect to AV, HR and CFI the addition of EP produced no significant difference in the effects observed by the bipyfidines alone. From these data it might be concluded that those effects of AM and MIL not potentiated by the addition of EP are not solely due to PDE inhibition but may be caused by additional mechanisms. Institut ffir Pharmakologie der Universit~it zu KSln, Gleueler Str. 24, D-5000 K61n 41, FRG

Piroximone (PIR) 4-ethyl-l,3-dihydro-5-(4piridinylcarbonyl)-2H-imidazol-2-one - is a n e w agent which increases cardiac contractile force and relaxation of vascular smooth muscle t h r o u g h a n i n h i b i t i o n of t h e p h o s p h o d i e s t e r a s e t y p III. T h e s e e f f e c t s are d u e t o an e l e v a t i o n of c A M P in v a s c u l a r s m o o t h m u s c l e c e l l s a n d cardiac muscle cells. Additionally platelet a c t i v i t y is a l s o r e g u l a t e d t h r o u g h i n t r a c e l l u a r l e v e l s of t h e s e c o n d m e s s a n g e r cAMP. T h e r e f o r e we studied the effects of P I R on p l a t e l e t a c t i v i t y in h u m a n p l a t e l e s in v i t r o a n d in r a t s in vivo. In platelet rich plasma adenosindiphosphat induced platelet aggregation w a s i n h i b i t e d b y P I R w i t h a n IC50 of 8 0 ± 1 0 ~M. The inhibitory effects were time and dose dependent. PIR 50~M reduced platelet aggregation by 40, 63, 75 and 98% after i n c u b a t i o n f o r i, 15, 60 a n d 120 min. T h e P I R effects in vivo were investigated in anaesthetized rats. Injection of collagen 1 0 0 ~ g / k g b w r e s u l t e d in a s i g n i f i c a n t d r o p in p l a t e l e t c o u n t b y 49~4%. P r e t r e a t m e n t of the rats with PIR 2mg/kg bw significantly reduced t h e f a l l in p l a t e l e t c o u n t to 2 2 ± 7 % 30 m i n after drug administration. This inhibitory effect was persistent 60 m i n following PIR injection (24±4%). In w a s h e d h u m a n p l a t e l e t s cAMP levels rose dose dependently following i n c u b a t i o n w i t h P I R (50~M-500~M). P I R e x e r t e d an e f f e c t i v e i n h i b i t i o n of p l a t e l e t a g g r e g a t i o n in v i v o and in vitro, which might be of clinical importance. P r e s e n t A d d r e s s : II. M e d i z i n i s c h e Klinik, U n i v e r s i t ~ t Mainz, L a n g e n b e c k s t r . , D - 6 5 0 0 M a i n z

R 56 221

223

STRUCTURE-ACTIVITY RELATIONSHIP AND BIOAVAILABILITY OF DIFFERENTLY SUBSTITUTED XANTHINES

IN VlVO EVIDENCE THAT EMD 57 033, A NOVEL CARDIOTONIC DRUG, INCREASES LEFT VENTRICULAR CONTRACTILITY VIA SENSITIZATION OF MYOFIBRILS TO CALCIUM P.Scheiling, E.H.Becker, I.Lues, H.J.Schliep, H.Weygandt

R. Wilke,

J. G ~ r i n g and H. M a s c h l e r

A b s t r a c t w i t h d r a w n J a n u a r y 9, 1991.

EMD 57 033, (+)-5(]-(3,4-Dimethoxybenzoyl)-l,2,3,4-tetrahydro-6-quinolyl)-6-methyl-3,6-dihydro-2 H-l,3,4-thiadiazin -2-one, is a potent Ca2+-sensitizer in skinned cardiac muscle fibres, which also inhibits the c~hqP-hydrolysis induced by purified PDE-III. The contribution of each mechanism to the positive inotropio action of EMD 57 033 (E) was studied in anaesthetized open chest mongrel dogs in comparison with the pure PDE-III inhibitor Milrinone (M) and with Pimobendan (P), which also shares both PDE-III inhibiting and Ca2+-sensitizing activities. Acetylcholine (ACh), known to functionally antagonize the rise in contractility caused by an increase of c~/~P, was infused

(10-20 ~g/min) into the l e f t a n t e r i o r descending coronary a r t e r y (LAD-CA) during a p p l i c a t i o n of the cardiotonic drug, Myocardial segment length shortening (SLS) was measured in the ACh-infused LAD-region and was compared to the SLS in the untreated area (circumflex coronary a r t e r y region; CXregion). Left v e n t r i c u l a r dP/dtma × was also recorded, Rise in c o n t r a c t i l i t y (4%) during ACh-inf. into LAD-CA E (n=5) 3.0 mg/kg iv P (n=4) 0.5 mg/kg iv M (n=4) 0.1 mg/kg iv

Research Laboratories, D-3212 Gronau

SmithKline Beecham,

LV dP/dtma × 60± 7 76±13 96± 7

SLS (LAD) diast/syst -7/-7 -2/-5 0/0

SLS (CX) diast/syst -8/-8 -7/-9 -9/-9

The r i s e in myocardial c o n t r a c t i l i t y induced by the pure PDE-III blocker M is l o c a l l y abolished during ACh-application, but is restored a f t e r withdrawal of ACh. The major part of the positive inotropic effect of P i s also due to PDE-III inhibition. The in vlvo r i s e in c o n t r a c t i l i t y induced by E, on the other hand, appears to be e n t i r e l y r e l a ted to a sensitization of myofibrils to calcium. Biological Research, E. Merck, D-6100 Darmstadt, Germany

222

224

EVIDENCE FOR THE EXISTENCE OF INOSITOL PENTAK1SAND HEXAKISPHOSPHATE IN THE HEART J. Scholz. U. Troll. P. Sandig*

KINETIC ANALYSIS OF INTERVAL-FORCE RELATIONSHIP IN ISOLATED GUINEA-PIG LEFT ATRIA H.J. Mensing and R. Englert

Inosito) 1,4,5-trisphosphate (1,4,5-IP3) has been shown to mobilize intracellular calcium in several tissues. It can be phosphorylated to inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4) which has been assumed to initiate calcium entry from the exterior (Berridge & Irvine, Nature 341:197, 1989). We have recently shown that in the heart 1,4,5-IP3 may initiate and 1,3,4,5-IP4 maintain the positive inotropic effect of al-adrenoceptor agonists (Kohl et al., Circ Res 66:580, 1990). Most data published are based on radioisotopic techniques, which allow only relative estimates of fluxes. Absolute concentration data are still missing. Therefore, in this study a recently published non-radiometric HPLC-metal dye detection technique (Mayr, Biochem J, 254: 585, 1988) was used. Force of contraction was measured on isolated electrically stimulated perfused rat hearts (Langendorff technique). The hearts were incubated with the cq-adrenoceptor agonist phenylephrine (10/~M) in the presence of propranolol (1 /~M). Thereafter they were frozen and subjected to HPLC analysis. The following IP isomers could be detected: 1,4,5-IP3, 1,5,6-IP3, 4,5,6-IP3, 1,3,4,6-IP4, 1,3,4,5-IP4, 2,4,5,6-IP4, 1,2,4,5,6-IP5, 1,3,4,5,6IP5, IP6. Looking at the time course, 1,4,5-IP3 preceded, 1,3,4,5IP4 coincided and all other IP isomers followed increase in force of contraction. In conclusion, for the first time the existence of inositol pentakisphosphate and inositol hexakisphosphate is observed in the heart. 1,4,5-IP3 and 1,3,4,5-IP4 may function as second messengers. However, the physiological role of all other IP isomers in the heart remains to be elucidated. Abteilungen ffir An~isthesiologie trod *Allgemeine Pharmakologie, Universit~its-Krankenhans Eppendoff, Martinistrasse 52, D-2000 Hamburg 20

The processes t h a t regulate cycling o f myocardial a c t i v a t o r Ca are incompletely understood (e.g. the c o n t r i b u t i o n of mitochondrial Ca exchange). Among several methods used to i n v e s t i g a t e these processes k i n e t i c analysis of i n t e r v a l force data is s u i t a b l e to provide a basis f o r compartmental modeling (Mensing & Hilgemann, Trends Pharmacol Sci 2:303, 1981; DiStefano & Landaw, Am J Physiol 246:R651, 1984; Schouten et a l . , Biophys J 51:13, 1987). Here we report a new computer-aided analysis of guinea-pig a t r i a l contract i l e responses to step changes o f frequency. L e f t a t r i a were stimulated at 0.5 Hz basal frequency in mod i f i e d Krebs-Henseleit s o l u t i o n (1.2 or 1.8 mM Ca) at 28°C. Frequency inotropy was induced at 1.5 Hz f o r i min; the accumulated " p o s i t i v e i n o t r o p i c e f f e c t o f a c t i v a t i o n " (PIEA) subsequently decayed at 0.5 Hz over several minutes. Transients of peak developed force were stored in a modified Atari ST computer f o r multiexponential regression analysis: Optimal least-squares approximation (Gauss-Newton procedure) with 3 exponential terms allowed d e f i n i t i o n of 3 PIEA components I , I I , and I I I with time constants 0 . 0 1 - 0 . 0 2 , ~ 0 . 1 , and ~O.5/s. PIEA I I I k i n e t i c s are strongly beat-dependent. In order to trace PIEA-changes in unstimulated a t r i a , a var i a b l e pause of ~2 s was introduced a f t e r the 1.5 Hz period and the estimated PIEA-component values at the f i r s t 0.5 Hz beat were p l o t t e d over pause duration: Most of the PIEA I I decayed within a few seconds during a pause, while simultaneously the PIEA I I I rose steeply (before s t a r t i n g i t s well-known very slow r e s t decay). Since the PIEA I I I r e f l e c t s Ca releasable from the sarcoplasmic reticulum/SR (e.g. Mensing & Hilgemann 1981, Schouten et a l . 1987) the i n i t i a l opposite changes of the PIEA I I and I I I suggest a rapid t r a n s f e r of Ca from a " b u f f e r compartment" (mitochondria?) to the SR. Our method of k i n e t i c analysis may provide a new approach to study mechanisms of drug action on i n t a c t heart muscle. Pharmakologisches I n s t i t u t ,

Wilhelmstr. 56, D-7400 TUbingen

In vivo Hinweis, dab die neue positiv inotrope Substanz, EMD 57 033. die ]inksventrlku]~re Kontraktl]it~t ilher die

R 57 225 LONG LASTING PRETREATMENT WITH AMINOGLYCOSIDE ANTIBIOTICS INCREASES STEADY STATE AND POST-REST POTENTIATED FORCE OF CONTRACTION OF GUINEA-PIG ISOLATED ATRIA

M- Duling~ M. Pfaffendorf1 Aminoglycoside antibiotics in a millimolar concentration range acutely impair the force of contraction of mammalian cardiac muscle as a result of the displacement of calcium ions from the outer surface of the cell membrane (Liilhnann & Schwarz, Br. J. Pharmacol. 86:799, 1985). We investigated the effect of various aminoglycusides on the steady state (CFs,) and the post-rest potentiated force of contraction (CFp~) of guinea-pig cardiac muscle. Isolated left atria were paced at a frequency of 3 Hz with interspersed rest periods of 4 see duration every 15 minutes. This protocol was applied for at least 6 hours. All experiments were performed at a [Ca+÷]o of 1.8 mM. Significant differences were found concerning the negative inotropic potency of the various agents. The following EC~ values for the depression of CF,, were detected: gentamicin 1.11 _+ 0.09 raM, tobramycin 1.06 -+ 0.19 mM, sisomicin 0.28 +- 0.03 mM, netilmicin 0.23 -+ 0.01 and amikacin 0.15 -+ 0.004 mM. Under control conditions both CF, and CFpr decreased slowly over several hours. In the presence of aminoglycoside-antiblotics, after an acute reduction of both parameters, CFpr increased slowly, while CF,, remained depressed during the course of the experiment. Compared to other positive or negative inotropic drugs this effect appears to be unique. The efficacy in inducing this effect was different for the aminoglycosides in the following rank order: gentamicin> sisomicin> tobramycin > netilmicin > amikacln. After 6 hours the aminoglycosides were washed out. Both CF,, and CFp, increased markedly and remained stable at this level which amounted to 156 -+ 5.7% for CF~ and to 118 ± 2.1% for CFv~ after gentamicin (lmM) pretreatment, compared to the untreated control. This effect was not altered by the presence of gallopamil (0.5 gM) during the pretreatment with gentamicin. Furthermore, there was no difference in the acute effect of gallopamil and a reduction of [Ca÷+]o to 0.45 mM before and after the long lasting pretreatment with gentamicin. We conclude that aminoglycoside-antibioticsnot oaly induce negative inotropy by displacing calcium from the outer surface of the plasma-membran but simultaneously cause a slow calcium-accumulation in the cells which becomes only visable after a long lasting pretreatment with these drugs. However, the dependency of CF~ and CF~ on [Ca+*]o is not altered under these circumstances. Department of Pharmacology, Christian-Albrechts-University, Hospitalstr.4, D2300 Kiel, FRG and t Department of Pharmacotherapy, Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

226 PROTECTIVE EFFECT OF NIFEDIPINE ON CALCIUM-PARADOX IN GUINEA-PIG LANGENDORFF HEAR'IS. J. G6dicke, G. Mtllder Perfusion for 50 seconds of isolated guinea-pig hearts with Ca++-free Tyrode solution containing 3xl0qM EGTA produced the typical Ca++paradox: increase in tension and reduced contractile fome. Concomitantly the heart muscles lost creatine-kJnase. These changes were preceeded by an accumulation of cellular Na ÷ during the Ca+÷-free period. Perfusion for 50 seconds with nominal Ca+÷-free solution without EGTA did not produce the Ca++-paradox although the Na + content of the muscle increased (s. Table). Pretreatment of the heart muscles with nifedipine (3xl0-SM), reducing the contractile force by about 50%, mitigated the extent of the Ca ++paradox: the contracture and the loss of creatine-kinase were reduced by about 50%, the recovery of contractile force doubled. controls

nominal

1.8 mMCa ++ Ca++-free Na + content pmoles/g cells Ca++-paradox present

Ca++-free + EGTA ~

rtifedipine

8.2 +3.0

31.2 +2.5

43.9 -+2.1

36.3 -+2.7

~

~

4-t-

+

227 THE USE OF MONOCLONALANTIBODIES (mAb) TO STUDY POSSIBLE EPITOPE SHARING BETWEEN THE NA-CA EXCHANGEPROTEINS OF BOVINE HEART AND RETINA H. Porzig and R. Gutknecht The Na-Ca exchange proteins in cardiac muscle and in r e t i nal rods d i f f e r in t h e i r apparent molecular weights (160 versus 210 kDa) as well as in t h e i r t r a n s p o r t k i n e t i c s and cation s p e c i f i c i t i e s . The cardiac t r a n s p o r t e r has been cloned recently (Nicoll et al. Science, 250, 562, 1990). We have prepared mAb's directed against the r e t i n a l exchanger by using homogenized n i t r o c e l l u l o s e blots of the protein as antigen. Using the immunoblotting technique we i d e n t i f i e d 8 clones producing an IgG t h a t recognized the r e t i n a l protein. Only one of these mAb's immunoprecipitated the i n t a c t s o l u b i l i z e d protein. The epitopes recognized by the other mAb's are probably not exposed when the prot e i n is embedded in mixed l i p i d / d e t e r g e n t micelles. In an immunoblot of t o t a l myocardial membrane protein, 3 of the 8 a n t i r e t i n a l mAb's labeled one or more protein bands in the 200 and/or 100 kDa range. The pattern was somewhat d i f f e r e n t f o r each of the 3 mAb's. Since none of these mAb's was able to immunoprecipitate the func'tional deterg e n t - s o l u b i l i z e d cardiac exchanger, t h e i r s p e c i f i c i t y had to be determined by i n d i r e c t methods. We followed the immunoreactive protein through a p u r i f i c a t i o n scheme using d i f f e r e n t chromatographic methods. Immunolabeled protein bands c o - p u r i f i e d with the exchange a c t i v i t y , but a s h i f t of the l a b e l i n g pattern was observed. The 200 kDa band vanished while some l a b e l i n g was retained in the 100 kDa region. In addition several smaller immunoreactive proteins appeared. This process could not be suppressed by the usual protease i n h i b i t o r s . We concluded from these r e s u l t s that the cardiac Na-Ca exchanger shares epitopes with the r e t i n a l protein. The cardiac protein seems prone to rapid p r o t e o l y t i c degradation during the p u r i f i c a t i o n procedure. The ion exchange function seems to reside in a protein fragment that is r e l a t i v e l y r e s i s t a n t to degradation. Pharmakologisches I n s t i t u t der U n i v e r s i t ~ t , FriedbUhlstrasse 49, CH-3010 Bern

228 INCREASE IN THE POST-ISCHAEMIC RELEASE OF PURINE COMPOUNDS IN ISOLATED RAT HEARTS BY THE APPLICATION OF ADENOSINE DEAMINASE. W. B e r n a u e r In i s o l a t e d r a t h e a r t s r e p e r f u s i o n a f t e r 2, 5, o r 15 min l i g a t i o n of the left coronary artery was a s s o c i a t e d w i t h t h e r e l e a s e o f a d e n o s i n e and its degradation products inosine, hypoxanthine, x a n t h i n e and u r i c a c i d , and t h e a p p e a r a n c e o f ventricular a r r h y t h m i a s . When v e n t r i c u l a r fibrillation occurred, the release of the purine compounds was s i g n i f i c a n t l y i n c r e a s e d . For i n s t ance, t h e t o t a l r e l e a s e d u r i n g 15 min o f r e p e r f u s i o n a f t e r 2 min o f c o r o n a r y l i g a t i o n was 3272 + 374 n m o l / g d r . w t , o f t h e i s c h a e m i c a r e a in ~ibrillating h e a r t s , and 1026 + 109 in n o n - f i b rillating h e a r t s ( n = 1 2 , n=13; ~ < 0 , 0 0 1 ) . I n f u s i o n o f a d e n o s i n e d e a m i n a s e (ADA) i n t o t h e c o r o n a r y i n f l o w ( I 0 U/min) a l m o s t c o m p l e t e l y a b o l i s h e d the release of adenosine, whereas the total pos~ i s c h a e m i c r e l e a s e o f p u r i n e compounds was s i g n i ficantly i n c r e a s e d (4701 + 448 n m o l / g d r . w t . , v s . 2104 + 294 in e x p e r i m e n t s w i t h o u t ADA; n=14, n= 25; P~O.O01). The i n c i d e n c e o f f i b r i l l a t i o n was i n c r e a s e d by ADA (86 % i n s t e a d o f 48 %; P
The Table summarizes the changes of the cellular Na+ content of the hearts immediately at the end of the Ca++-free incubation period. It is evident that no simple correlation exists between the cellular Na + content at the end of the Ca÷+-free exposition period and the subsequent mechanical reaction of the heart muscles. An increase of Na + content from 8.2 to 31.2 pmoles/g cells was without any mechanical consequence whereas on the other hand an increase to 43.9 was accompanied by a marked Ca++-paradox. A N a + content of 36.3 lmaoles/g cells resulted in an intermediate place. This suggests either a definite threshold ++ between 32 and 36 tamoles Na+/g cells exists for triggering the Ca -paradox or other mechanisms than a rise of + cellular Na concentration are involved. Dept. of Pharmacology, Universifiit Kiel, Hospitalstr. 4, D-2300 Kiel

Department of Pharmacology, University of Freib u r g , H e r m a n n - H e r d e r - S t r . 5, D-7800 F r e i b u r g , F e d e r a l R e p u b l i c o f Germany.

R 58 229

231

USE OF INDIRECT STIMULATORS OF THE SLOW INWARD CALCIUM AND FAST INWARD SODIUM CURRENTS OF THE HEART IN THE PRECLINICAL TESTING OF ANTIARRHYTHNIC DRUGS K. Kelemen and R. Mark6

ELECTROPHYSIOLOGICAL EFFECTS OF THE TWO IC ANTIARRHYTHMIC COMPOUNDS FLECAINIDE AND PROPAFENONE EVALUATED IN LANGENDORFF PERFUSED GUINEA-PIG HEARTS U. Stark, S. Nagl, H. Markc~ and H.A. Tritthart

It has been previously shown that deprenyl, an indirectly acting sympathomimetic agent, mimicks the activating effect of adrenaline on the slow inward Ca ++ current in the heart (Kelemen and Mark6: in:"Neuropharmacology '85" Akad. KiadG, Budapest,pp.277-282,1985) and paracetamol, a prostaglandin releaser, causes a pros±acyclinZike increase in the a m p l i t u d e o f t h e f a s t Na + current (Kelemen and M a r k d , Pharmacol. Res. Comm.,20:£7-98,1988). In o u r present study these indirect stimulators of the cardiac depolarization mechanisms were used t o differentiate calcium and sodium blocking properties of antiarrhythmics in preclinical trials. E x p e r i m e n t s were p e r f o r m e d i n the sinoauricular fibers ot the frog with a v o l t a g e clamp technique. Verapamil (2 umol/1) and l i d o c a i n e (6 u g / m l ) were used as r e s p e c t i v e models o f c a l c i u m and sodium b l o c k e r s . D e p r e n i l induced slow inward current was p r e v e n t e d by v e r a p a m i l and n o t i n f l u e n c e d by l i d o c a i n e while paracetamol-induced increase of the amplitude of the fast inward current was selectively a n t a g o n i z e d by l i d o c a i n e and n o t aftected by verapamil. Deprenyl and paracetamol, as experimental tools, have the advantage of being stable compounds and possessing constant effects, independent ot seasonal variations, in contrast to adrenaline and especially to prostacycline.

Preliminary results of the Cardiac Arrhythmia Suppression Trial (CAST) created loss of confidence in the clinical use of class Ic antiarrhythmic agents. To study, whether different compounds of the class Ic differently influence cardiac conduction and refractoriness, we evaluated the effects of flecainide and propafenone on Langendorff perfused guinea pig hearts, using a new surface ECG recording and stimulation technique (Stark et al., J Pharm Meth 21, 195-209, 1989). Equimolar concentrations (3uM) of either drug caused a similar reduction of the. spontaneous sinus rate, whereas all conduction intervals were significantly (p < 0.01) more prolonged by propafenone than by flecainide. During programed stimulation with premature beats, flecainide and propafenone induced a similar prolongation of the AV-nodal (+21+6%, n=6; +34+9%, n=8; respectively, both p<0.01) and ventricular myocardial refractoriness (+28___5%, n=6; +49+12, n=8; respectively, both p<0.01)whereas the sine-atrial, His-bundle and atrial myocardial refractoriness were significantly more prolonged by propafenone. All rate dependent refractory periods were significantly more influenced by propafenone than by flecainide. These results indicate, that flecainide and propafenone although they belong to the same class of antiarrhythmic drugs and are described to have a similar association and dissociation behaviour to the Na+-channels, differently influence cardiac electrical activity.

Dept.of Pharmacology,Semmelweis Univ.ot Nedicine Nagyv~rad t@r ~, H-i089 8udapest, Hungary

Dept. of Med. Physics and Biophysics, University of Graz, Austria Supported by the Austrian Research Foundation (P 7141)

230

232

ARRHYTHMOGENIC ACTION OF NEUROLEPTIC DRUGS IN PURKINJE FIBRES OF GUINEA-PIG HEARTS C. Studenik and P. Haistracher

COHPARATI r E

The aim

Some neuroleptic d r u g s have been considered to be r e sponsible for the r a r e occurrence of "Torsade de pointes" in patients (Krikler DM, C u r r y PVL (1976) Br Heart J 38: 117-120; Kemper AJ, Dunlap R, Pierre DA (1983) J Am Med A s s e t 249:2931-2934). T h e r e f o r e , the effects of t h r e e neuroleptic d r u g s (chlorpromazine h y d r o c h l o r i d e , trifluoperazine h y d r o c h l o r i d e , thioridazine hydrochloride) on the action potential of isolated spontaneously b e a t i n g Purkinje fibres have been studied b y the intracellular microelectrode technique. At a potassium concentration of the b a t h i n g solution of 1.35 mmol]l, early afterdepolarizations a n d ] o r early e x t r a s y s t o l e s only o c c u r r e d in the p r e s e n c e of chlorpromazinc hydrochloride (2 and 5 Bmol/1), in 1 out of 4, and in 2 out of 5 e x p e r i m e n t s , and of thioridazine h y d r o c h l o ride (2 and 5 pmaol]l), in 2 out of 4, and in 4 out of 10 e x p e r i m e n t s , r e s p e c t i v e l y . These effects were r e v e r s i b l e on readmission of d r u g - f r e e b a t h i n g solution. At a potassium concentration of 2.7 retool/l, n e i t h e r d r u g caused early afterdepolarizations a n d ] o r early e x t r a s y s t o l e s (at 2 and 10 Ixmol]l; n=3-5). Trifluoperazine h y d r o c h l o r i d e , at the same concentrations (n=4-5), did not elicit early a f t e r d e p o l a r i z a t i o n s / e x t r a s y s t o l e s in b a t h i n g solutions of either potassium concentration. Tetrodotoxin (0.2 pmol]l) abolished early e x t r a s y s t o l e s induced b y thioridazine h y drochloride (5 limol/1) and d e c r e a s e d early afterdepolarizations (n=3). The p r e s e n t r e s u l t s are compatible with the assumption that early afterdepolarizations and early e x t r a s y s t o l e s u n d e r l y the o c c u r r e n c e of "Torsade de pointes" in h y p o kalaemic patients t r e a t e d with certain neuroleptic d r u g s . Institut fiir Pharmakodynamik u n d Toxikologie d e r Universit,it Wien, W~ihringer S t r a s s e 17, A-1090 Wien, Austria

STUDY

ON

PgOARRItYTHM~ C

THE

ANTIARRHYTHMIC AGENTS. of this

arrhythmic isolated

s t u d y was t o a n a l y s e

drugs

in a quantitative

perfused

dorff-technique, Tyrode's at

the

right

trations

aide;

class

In order

Ia:

II:

kHz/channel, mV). For

lmm

From

tion

were

ring

point

velocity

ditions

BTP were

time

well as

dispersion

lidocaine less

)

sotalol

of origin

of =

tom-purer

of

>

4 0.04

electrode

were

deflec-

of the epicardial

activa-

giving

direction

and

Single heart

and under treatment of vectors and

the

percentage total

duration

were analysed.

agents

(flecainide

propranolol), epicardial

with

(YEt) w i t h tm-

velocities,

potential

duration

the

(256

resolution:

wave f r o n t .

t h a n 5° )

and local

verpamil

and sotalol

a

more t h a n lmm) c o m p a r e d t o c o n t r o l

potential

Under quinidine

flecal-

intrinsic

calculated

conditions

antiarrhythmic

disturbation

binding

Ic:

recording

at each

Moreover, epicardial

pronouved

concen-

BTP) a n d a t e a c h m e a s u -

excitation

less

given region

were reduced under all

with

ggCl-electrodes;

negative

The p e r c e n t a g e

not

determined. of a

H20

IV: v e r a p a m i l ) .

process

amplitude

timepoints

under control

class

epicardial

was

class

multichannel

of the fastest

vector

(deviation

Langen-

cm

plasma protein

sotalol;

distance,

epicardial

(deviation

70

a low, m i d d l e a n d h i g h

activation

256

the points

mentioned above.

of

lidocaine;

("breakthroughpoints',

the local analysed

III:

activation

data

of anti-

Therefore,

with increasing

to the

Ib:

unipolar

of

the

direction

identical vation

for

an activation

of

altered

class

the timepoint

these

were

class

the epicardial

deternmined

the drugs

gz) were treated

interelctrode

analysis

determined as

pressure

with regard

simultaneously,

risk to the

w i t h 95~ 02 a n d 5~ C02 a t 37 ° C~ p a c e d

quinidine;

system

according

drugs corresponding

propranolol;

mapping

channels

beats

with 3

to measure

assisted

tion.

equilibrated

concentration

class

(prepared

at constant

antiarrhythmic

therapeutic

the arrhyth~ogenio

and comparative ~anner.

hearts

perfused atrium

of

(drugs:

rabbit

solution

EFFECTS

S,Dhei~ and A,H~ller

of con-

acti-

(ARI) a s

BTP a n d VEC > quinidine

>

indicating

a more o r

activation

process.

ARI was m a r k e d l y p r o l o n g e d

indicating

a

p r o l o n g a t i o n o£ r e f r a c t o r y p e r i o d . From t h e s e r e s u l t s i t i s c o n c l u d e d that the arrhythmogenic risk decreases in the following rank order: flecainlde lol.

>

quinidine

> lidocaine

> soltalol

> verapamiI

> proprano-

( S u p p o r t e d b y t h e DFG)

Inst.f.Pharmakol.,

U n i v . zu K r l n ,

Cleuelerstr.24,

5000 K r l n 41,

FgQ

R 59 233

235

INFLUENCE OF POTASSIUM ON THE ACTION OF AMIODARONE IN GUINEA PIG MYOCARDIAL Na*-K*-ATPASE PREPARATIONS

CARDIAC EFFECTS OF THE NOVEL K+-CHANNEL OPENER HOE 234, COMPARISON WITH CROMAKALIM AND FORSKOLIN

N. Dzlmlrl ~ and A. A. Almotrefl 2 The inhibitoryaction of amiodamne on myocardial Na+-K*-ATPase activity is probably responsible for some of its cardiac actions. In order to study further the mode of Interaction between amlodamne and this enzyme system, we investigated the influence of varying the incubation IC concentration on its enzyme inhibition in guinea pig heart preparations. Freshly isolated guinea pig hearts were homogenized, centrifuged at 14000 g, the Na* -tC -ATPase fraction separated on TSK Toyopead HW-55F gel column and dialyzed using 100 mM imidazole buffer. Concentrationresponse relationships of the amiodamne effects on the Na*-W-ATPese were examined at 2.5, 5.0 and 10.0 mM K* respectively. At these concentrations, amiodarone exerted concentration-dependent inhibitory actions, with IC.,sovalues of 10.4 + 3.2, 28.3 -+ 7.6 and 35.3 + 9.2/~M (mean + s.d.), respectively. Thus, reducing IC from 5.0 to 2.5 rnM enhanced the inhibitory potency, accompanied by a significant right-to-left shift in inhibitory effective concentration ranges, particularly at low concentrations. Increasing IC concentrationfrom 5 to 10 mM on the other hand produced opposite but less pronounced effects. The results show that the In vi~o Inhibition of myocardial Na+-IC-ATPase activity by amiodarone depends on [W]. This effect Is probably pertinent to the mechanism by which amiodarone interacts with the electmgenic pump activity of the Na*-I~-ATPase and may therefore contribute to the biochemical basis for its proarrhythmicor arrhythmogenicactions.

J. Kaiser, H. G6gelein Hoe 234, (3S,4R)-3-hydroxy-2,2-dimethyl-4-(2-oxo-l-pyrrolidinyl)6-phenylsulfonylchroman hemihydrate, is a novel K+-channel opener with relaxant properties in vascular and airway smooth muscle. In guinea pig papillary muscle paced at 2 Hz Hoe 234 (2,4.10-7 - 2,4.10-6) concentration dependently decreases the duration of the fast action potential ~APDg~) and of the refractory period. Cromakalim (3,5.10-6 - 3,5.10-o tool/l) shows similar effects but is about 10 times less potent. Glibenclamide (10 ~M) antagonizes these effects. Forskolin (1-10 ~M), a well known stimulator of adenyl cyclase, however, also shortens APD_. ~ and refractory period to a similar extent as Hoe 234 and Cromakahm. In contrast to the effects induced by Hoe 234 and Cromakalim the effects of forskolin are frequently associated with the occurrence of spontaneous action potentials. Also in this case Glibenclamide (I0 /zM) is able to reverse the shortening of APD and even reduces the occurrence of spontaneous APs. Using patch clamp technique a direct effect of Hoe 234 (1-10 ~M),on ATP dependent K+-channels of rat ventricular myocytes can be demonstrated whereas Forskolin has no influence on outward K + conductance in whole cell experiments as well as on single ATP dependent K+-channels. In conclusion Hoe 234 potently shortens the cardiac action potential probably by a direct influence on ATP dependent K+-channels. In papillary muscle of the guinea pig this shortening of the action potential does not trigger automaticity. Automaticity, however, may be accompanied by a shortened action potential as demonstrated for the adenyl cyclase activator forskolin. Dept. Pharmacol., Hoechst AG, W-6230 Frankfurt(Main)80, Germany

Biological & Medical Reseamh Department, King Faisal Specialist Hospital and Research Centre~ & Department of Pharmacology, King Saud University2, Riyadh, Saudi Arabia.

234

236

ELECTROPHYSIOLOGICAL EFFECTS OF AMIODARONE AND ITS METABOLITE DESETHYLAMIODARONE G. Stark, M. Windisch, E.M. Hailer, U.Eggenreich, M. Vincenzi, A. Lueger and Ernst Pilger

C H R O N O T R O P I C R E S P O N S E S OF C A R D I A C M Y O C Y T E S D I F F E R E N T I A T I N G IN V I T R O F R O M M O U S E P L U R I P O T E N T E M B R Y O N I C STEM CELLS A. M. Wobus, G. W a l l u k a t +) and R. Vogel ++ )

Some effects described during long term treatment with amiodarone (AM) are missing during acute application, suggesting that they were induced by desethylamiodarone (DAM), a metabolite of AM. We evaluated the effects of AM and DAM on cardiac conduction and refractoriness (ERP) in isolated Langendorff perfused guinea pig hearts using a new surface and stimulation ECG method (Stark et al.,J Pharm Meth 21, 195-209, 1989). Also the rate dependent refractoriness of the sino-atdal, AV-nodal, His-bundle conduction and of the atrial and ventricular myocardium were determined. A concentration of 10uM of either drug was used for one hour to reach adequat tissue concentrations. During one hour of perfusion, both drugs significantly prolonged AV-nodal (to 142__+12% AM; 200_+13% DAM; both n=12, p<0.01), His-bundle (to 162_+11 AM; 205_+27% DAM; both n=12, p<0.01) and intraventficular conduction (to 142+8% AM; 193-+20% DAM; both n=12, p<0.01) intervals and significantly reduced sinus rate (to 48-+5% AM; 50_+8% DAM; respectively both, n=12, p<0.01). DAM induced a significantly stronger influence on cardiac conduction and refractoriness than AM, only on the spontaneous sinus rate the effects were similarily expressed. The rate dependent QT-interval was only prolonged by DAM. During programed stimulation following 60rain, all ERP's were significantly more prolonged by AM than by DAM, except the ventdcular myocardial refractoriness and the rate dependent ERP of the AV-node. The present results show, that only DAM has class III electrophysiological effects (prolongation of the QT-interval) and stronger affects cardiac conduction.

P l u r i p o t e n t m o u s e e m b r y o n i c s t e m cells (ESC) d e r i v e d by in v i t r o c u l t u r e of the inner cell m a s s of m o u s e b l a s t o c y s t s are a b l e to d i f f e r e n tiate into spontaneously beating cardiac muscle c e l l s in vitro. D i f f e r e n t a d r e n e r g i c and c h o l i n e r g i c a g e n t s w e r e i n v e s t i g a t e d w i t h r e s p e c t to t h e i r c h r o n o t r o p i c e f f e c t s on m y o c y t e s w h i c h d i f f e r e n t i a t e d on the o u t g r o w t h s of 7 d a y old e m b r y o i d bodies. P h e n y l e p h r i n e , (-)isoprenaline, isobutylmethylxanthine a n d f o r s k o l i n increased, w h e r e a s the c h o l i n e r g i c a g o n i s t c a r b a c h o l dec r e a s e d the b a s a l p u l s a t i o n rate. The B - a g o n i s t c l e n b u t e r o l and the h e a r t g l y c o 2 . • slde digltoxine induced a positive chronotropic r e s p o n s e o n l y in c a r d i a c m y o c y t e s w h i c h d i f f e r e n t i a t e d for m o r e t h a n 5 d a y s a f t e r p l a t i n g of e m b r y o i d bodies. T h e r e s u l t s d e m o n s t r a t e d the f o r m a t i o n of h e a r t m u s c l e c e l l - s p e c i f i c r e c e p t o r s and s i g n a l t r a n s d u c t i o n m e c h a n i s m s in c a r d i a c m y o c y t e s a f t e r t h e i r in v i t r o d i f f e r e n t i a t i o n f r o m und i f f e r e n t i a t e d p l u r i p o t e n t e m b r y o n i c s t e m cells, and are d i s c u s s e d w i t h r e s p e c t to in v i t r o m e t h o d s for the i n v e s t i g a t i o n of h e a r t - a c t i v e d r u g s d u r i n g a p r e s c r e e n i n g of p h a r m a c o l o g i c a l agents.

Department of Internal Medicine, University of Graz - Austria Supported by the Austrian Development Fund, ZI 3/7098/513

I n s t i t u t f~r G e n e t i k und K u l t u r p f l a n z e n f o r schung, 0 - 4325 G a t e r s l e b e n , +) Z e n t r a l i n s t i t u t f~r H e r z - K r e i s l a u f f o r s c h u n g 0 - 1115 B e r l i n - B u c h , ++) Max yon P e t t e n k o f e r - I n s t i t u t , Bundesgesundh e i t s a m t , W - I000 B e r l i n 33

R 60 237 DEVELOPNENTOF ff-ADRENOCEPTOR NUNBER IN THE TRANSPLANTED HUNAN HEART O.-E.Brodde and M.Khamssi

The transplanted human heart is a denervated organ and hence may exhibit B-adrenoceptor (AR) supersensitivity. To monitor possible B-AR changes in the transplanted human heart we have determined in right endomyocardial biopsy samples (mlnimum size: 2 mg w.w.) of 8 heart transplant recipients B-AR number and adenylate cyclase (AC) a c t i v i t y in monthly intervals for 6-22 months after transplantation. B-AR number1~£s assessed by binding with I-2 concentrations of ( - ) - [ :~I]-iodocyanopindolol (ICYP) and total B-AR number was calculated according to the law of mass action under the assumption of a constant Kn-value of ICYP (20 pM). The Validity of this method was demonstrated by th~ fact that in patients undergoing coronary artery bypass grafting right a t r i a l ~-AR number, assessed by conventional Scatchard-analysis at 6 ICYP concentrations, was highly significantly correlated with B-AR number assessed at a single ICYP concentration in biopsy materials taken from the same atria. In t h e ~ h e a r t s of the 8 heart transplant recipients ventricular B-AR number (15-25 fmol ICYP bound/mg protein) and activation of AC by 10 ~M isoprenaline (ISO) were markedly reduced. In the ~ samples from the transplanted hearts of these patients B-AR number (40-60 fmol/mg protein) and the AC-response to 10 gM ISO were significantly higher. Over the whole period investigated in all patients B-AR number and AC-response to 10 ~M ISO were quite stable; both parameters showed no significant increases or decreases. I t is concluded that, ~n the transplanted human heart, in the f i r s t I-2 years after transplantation B-AR number and function do not increase. B~ochem. Forschungslabor, Med. Klinik & P o l i k l i n i k , Universit~tsklinikum Essen, D-4300 Essen I, F.R.G.

239 CARDIOVASCULAR ~-ADPJ~NOCEPTORS IN HYPERTHYROID DOGS A. Hoey, C. Marchant, L. Brown, R. Atwell* and C. Sernia Clinically, hyperthyroidism resembles sympathetic overactivity; ~-adrenoceptor antagonists are widely used as adjunctive symptomatic management. The aim of this study was to compare cardiac ~-adrenoceptor density and responses in hyperthyroid dogs (triiodothyronine (T3) Img/kg/day s.c. for 14 days; n=10) and in control dogs (n=10). Plasma T3 levels increased from 0.8±0.03 to 62±7.2 nmol/l. Heart rate increased from 88±4.4 to 153±12.8 beats/min but intrinsic heart rate was unchanged. Isoprenaline (0.05 ~g/kg i.v.) increased heart rate by 69±8.4 beats/min (control) and 125±13.6 beats/min (T3). Hyperthyroid dogs showed marked T wave enlargement and inversion on the ECG; echocardiography showed no change in stroke volume or ventricular wall thicknesses. In contrast, circumflex coronary arteries from T3-treated dogs showed a significantly greater will thickness (0.28±0.01 ~m) than controls (0.22±0.01 ram). Isoprenaline was a significantly less potent relaxant in isolated circumflex artery rings from hyperthyroid dogs (neg log EC~, 6.60±0.12; control, 7.23±0.06); relaxant responses to forskolin and theophylline were unchanged. ~I-Cyanopindolol binding was used to characterize ~-adrenoceptors in left ventricular homogenates. The affinities of both ~ - and ~-adrenoceptors were unchanged in T3 dogs; however, there was a selective increase in ~-adrenoceptor density from 1.4±0.2 pmol/mg protein (control) to 2.3±0.4 pmol/mg protein (T3). Maximal activation of adenylate cyclase by isoprenaline and forskolin in ventricular homogenates was unchanged after T3 administration. Thus, hyperthyroid dogs showed marked tachycardia and an increased response to isoprenaline in vivo; hypertrophy of coronary arteries but not of the ventricles; a selective reduction in potency of isoprenaline in isolated coronary arteries; a selective increase in ventricular ~-adrenoceptors but no difference in maximal production of cAMP by adenylate cyclase. Departments of Physiology and Pharmacology, and *Companion Animal Medicine and Surgery, The University of Queensland, 4072, Australia.

238

240

REGIONAL DISTRIBUTION OF ~-ADRENOCEPTORS IN NONFAILING A N D FAILING H U M A N LEFT VENT~ICULAR MYOCARDIUM. M. Steinfath, J. LavickyS, S. Rosswag, V. D~ring$, P. Kalmar$ and H.J. Krebber$

C H A R A C T E R I Z A T I O N OF T H E B - A D R E N O C E P T O R S U B T Y P E MEDIATING THE POSITIVE INOTROPIC EFFECTS OF DOBUTAMINE, DENOPAMINE AND XAMOTEROL S. Bals, S. M o t o m u r a a n d H.-R. Z e r k o w s k i

Total ~-adrenoceptor density (B,ax in fmol/mg pro±ein) and ~1/~2-adrenoceptor ratio ~i/~] in %) were determined in different regions of the human lef£ ventricufar myocardium. Portions of the papillary muscle (PM), free wall (FW), ventricular apex (VA), and interventricular septum (IS) were obtained from nonfailing hearts (NF) and patienhs with end-s±age idiopathic dilated cardiomyopathy (IDC) or ischemlc cardlomyopathy (ICM). Bmax was assessed b y (-)-(l~51)-iodocyanopindoloL ~1/O2ratio was calculated by nonlinear regression analysis of competition curves of ±he selective ~t-adrenoceptor an%agqnist CGP 20712A (1-(2-(3-carbamoyl-4-hydroxy)

We i n v e s t i g a t e d t h e p o s i t i v e i n o t r o p i c e f f e c t s (PIE) of d o b u t a m i n e (DOB), d e n o p a m i n e (DEN) and x a m o t e r o l (XAM) in i s o l a t e d r i g h t a t r i a l s t r i p s obtained from patients undergoing coronary bypass g r a f t i n g b y a s s e s s i n g its a g o n i s t i c e f f i c a c y in r e l a t i o n to the P I E of Ca ++ a n d i s o p r e n a l i n e (IPN) a n d the B - a d r e n o c e p t o r (BAR) s u b t y p e i n v o l v e d u s i n g t h e B 1 - s e l e c t i v e a n t a g o n i s t C G P 2 0 7 1 2 A (l[2-((3-carbamoyl-4-hydroxy)phenoxy)ethyl-amino]3-[4-(l-methyl-4-trifluoromethyl-2-imidazolyl)p h e n o x y ] - 2 - p r o p a n o l m e t h a n e s u l f o n a t e ) a n d the 82s e l e c t i v e a n t a g o n i s t ICI 1 1 8 , 5 5 1 ( e r y t h r o - D , L - l (7-methylindan-4-yloxy)-3-isopropylaminobutan-2ol HCl). X a m o t e r o l d i d n o t h a v e any PIE. D E N a n d D O B h a d an i n o t r o p i c e f f i c a c y s i m i l a r to t h a t of IPN, i.e. > 9 0 % of t h a t of Ca ++. pD2 v a l u e s w e r e for D E N (n=6): 5 . 8 5 ± 0 . 1 3 ; +30 n M ICI: 5 . 9 2 ± 0 . 2 2 ; +i0 n M CGP: 5 . 3 3 ± 0 . 1 0 ; + I C I + C G P : 5 . 3 0 ± 0 . 1 5 ; for DOB (n=5): 6 . 2 6 ± 0 . 1 3 ; +30 n M ICI: 5 . 7 6 ± 0 . 1 3 ; + 3 0 0 n M CGP: 4 . 9 4 ± 0 . 0 7 ; +ICI+CGP: 4.63±0.12. For both a g o n i s t s a n e a r l y i:i r a t i o b e t w e e n B A R o c c u p a n c y and contractile response was observed indicating t h e l a c k of s p a r e r e c e p t o r s for the PIE of DEN a n d D O B in h u m a n r i g h t atrium. W e c o n c l u d e t h a t D E N is a B ~ A R - s e l e c t i v e a g o n i s t ; D O B c a u s e s its P I E m a i n l y v i a B~AR w i t h a s m a l l b u t s i g n i f i c a n t c o n t r i b u t i o n b y B~AR; X A M h a s no P I E in h u m a n r i g h t atrium.

phenoxyethylamino)-3-(4-(l-me±hyl-4-±rifluorome%hyl-2imidazolyl)phenoxy)-2-propanol methanesulfonate). Results: PM: Bmax ~*/~2 FW: Bmax ~I/~2 VA: Bmax ~i/~2 IS: Bmax

NF IDC ICM 70.~±6.2 25.8±2.8* 26.2±3.4* 78.9/21.1 60.2/39.8+ 77.8/22.2 68.9±7.2 24.2±3.2* 25.1±2.9" 83.1/16.9 62.8/37.2+ 80.2/19.8 63.1±6.9 23.8±3.4* 24.4±2.8* 82.2/17.8 64.3/35.7 76.6/23.2 66.7±6.2 22.1±3.1" 25.0±2.7* ~1/~2 76.8/23.2 62.3/37.7+ 76.6/22.1 *p<0.05 vs. NF; +p<0.05 vs. NF, ICM; n=4-6 In conclusion, Bmax and ~i/@2-ratio do not vary within the different regions studied. Downregula%ion of ~adrenoceptors in end-s£age hear% failure is homogeneously dis±ributed in the lef± ventricular myocardlum. (Supported b y £he DFG and the Jung-Stiftung.)

Abteilung Allgemeine Pharmakologie and

SAb%eilung f~r

Herzund Gef~chirurgie, Universit~ts-Krankenhaus Eppendorf, Universltat Hamburg, Martlnistra~e 52, 2000 HamburE 20, FRG and SFacul%y of Fediatrics, Charles University, V uvalu 84, I5006 Prague 5, CSFR

Depts. M e d i c i n e a n d T h o r a c i c S u r g e r y , U n i v e r s i t y of Essen, H u f e l a n d s t r . 55, D - 4 3 0 0 Essen, F R G

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EFFECTS OF UNOXIDIZED AND OXIDIZED SYMPATHOMIMETIC AMINES ON THE ELECTRICAL AND MECHANICAL ACTIVITIES OF ISOLATED GUINEA PIG PAPILLARY MUSCLES UNDER HYPOXIC CONDITIONS. K. Gdttler and N. Scheuren It is well recognized that catecholamines readily undergo oxidation in solution. But little is known about the effects of these oxidized compounds in hypoxic hearts. Therefore, the aim of this study was to compare electrophysiological and functional effects of unoxidized with oxidized isoprenaline (ISO) resp. dobutamine (DOB) in hypoxia. In order to test unoxidized compounds, the drugs were freshly dissolved in i0 -~ M NaEDTA and I0 -~ M ascorbate. Spontaneous oxidation was induced by preparing drug solutions without stabilizers 24 h prior to use. Experiments were carried out on guinea-pig papillary muscles with conventional microelectrode technique. The normoxic control values of action potential duration (APD-90) of 187 ± 5 ms and of contractile force (CF) of 2.3 ± 0.2 mN were reduced continuously by hypoxia (60 min): APD-90 = 105 ± ii ms and CF = 0.5 ± 0.I mN, 120 min: APD-90 = 79 ± 5 ms and CF = 0.4 ± 0.I mN. Drugs were added during 60. - 120. min of hypoxia, At equieffective concentrations with regard to the inotropic effect of unoxidized drugs, 5 * i0 -s M ISO and i0 -~ M DOB showed similar actions on CF (+iii ± 20 % resp. +93 ± 28 %) and APD (+19 ~ 7 % resp. +13 ± 1 % ) . The comparison with the oxidized products of ISO resp. DOB revealed distinct differences. Whilst oxidation of ISO induced a loss of efficacy on all parameters tested, oxidized DOE showed a clear increase of CE = +247 ± 3 1 % and APD = +55 ± ii %. Corresponding experiments with an isomer compound of ISO orciprenaline (I0 -~ M) induced initially an increase of CE (+240 ± 62 %) and APD (+13 ± I0 %), but 30 min later cardiotoxic signs of contracture. These results suggest that under the selected experimental conditions unoxidized and oxidized sympathomimetic amines are markedly distinguished by their myocardial actions.

THE NEW"CARDIOTONIC AGENT DP120 l-106 ENHANCES CARDIAC OUABAIN TOXICITY AND ELEVATES [sH]OUABAIN BINDING S. Herzia and E. Lillenthal The piperazinylindole derivative DPI 201-106 (4--(3-(4-benzhydryl-1piperazinyl)-2-hydroxypropoxy)-1H-indole-2-carbonltrile) is the prototype of a new class of positive inotropic agents, which act mainly by increasing the cellular Na + load via Na + channels. By this mechanism, the transport burden of cardiac (Na++K+)ATPase molecules should be enhanced. Therefore, digitalis sensitivity is thonght to be Increased and digitalis toxicity to be promoted. We tested this idea studying DPI 201-106, its new derivative BDF 9148 (4-(3-(l-benzhydrylacetidine-3-oxy)-2-hydroxypropoxy)-lH-indole-2-carbonitrile), and the known Na + channel activator veratridine. In guinea pig left atria paced at 0.3 Hz, the interaction of these test compounds with the effects and binding of ouabain was investigated. In the presence of positive inotropic concentrations of DPI 201-106 (3xl0-¢M), BDF 9148 (10"TM) and veratridine (10~M), the dose response curve of ouabain (single application per experiment, 3h incubation) was shifted to the left. Toxicity (extrasystoles and/or contraeture) occurred already at two- to threefold lower concentrations of ouabaln. Additionally, in non-intoxicated muscles, the ouabain concentration exerting the highest positive inotropism was also lowered by twofold. In intact left atria, the binding of [SH]ouabain revealed the unusual, cooperative concentration-dependence reported previously (Herzig et ai. 1988, J. Physiol. 386:105-120). All test compounds apDroximately doubled equilibrium ouabain binding in the non-toxic ouabain concentration range. The binding isotherms displayed a typical inflection point, which marks the threshold concentration for ouabain toxicity. This threshold ag~n was lowered in the presence of the test compounds by a factor of two to three. The effects on ouabain binding are likely indirect (mediated by the elevated Na + l~ad), since [~H]ouabain binding to isolated cardiac membranes remained unaffected, or it was even depressed at high concentrations of the test compounds. In conclusion, it was consistently found that ouabain is two- to threefold more toxic in the presence of DPI 201-106, BDF 9148, or veratridin8. The increment of ouabain binding to intact muscles is likely responsible for this interaction. The extent to which ouabain toxicity is enhanced seems significant, due to the narrow safety range of digitalis glycosides. Dept. Pharmacology, University Kiel, Hospitalstr. 4, 23 Kiel, Germany.

Institut f~r Pharmakologie der Gleuelerstr. 24, 5000 Ktln 41

Universitat

zu

Ktln,

242 Microscopic reaction dynamics of an indol-2-carbonitrile-type sodium channel activator (BDF 9145) P. Honerj~iger, X.-G. Zong, B.I. Armah', and P. Pachaly'"

Positive inotropic indol-2-carbonitrile-type sodium channel activators such as DPI 201-106, BDF 9148 or BDF 9145"" are known to delay sodium channel inactivation, but the temporal relation between the drug, s residency time on the sodium channel and the electrophysiological effect is not known. We show here that comodification of single cardiac sodium channels with an allosteric activator, grayanotoxin-I, reveals individual BDF 9145 association and dissociation events. Outside-out patches were obtained from dissociated and cultured ventricular myocytes of late-fetal rats. BDF 9145 was added to the extracellular Na(140 mM)-cuntaining medium, grayanotoxin-I (10 or 100/aM) to the intracellular Cs(148 mM)-containing solution (20 °C). Prolonged step depolarizations to -30 mV from holding potentials between -150 and -100 mV were used to elicit single channel current. Grayanotoxin-I reduced normal open-channel current (-1.2 pA) to -0.3 pA and induced a bursting channel activity lasting between 12 s and 3 rain. Bursts occurred at intervals of about 0.1 to 1 s and were characterized by exponentially distributed open times (~'= 15 ms) and closed times (%'= 5 ms). BDF 9145 periodically altered the kinetics of this bursting activity by increasing the open time nearly 20-fold (%"= 262 ms) with little effect on closed time ( r = 7 ms). The BDF 9145-induced predominantly open state lasted 30 s on average and consumed increasing fractions of the total time of bursting activity (0.26, 0.47, 0.79) when BDF 9145 concentration was raised from 1 to 3 or 10 ~M. These results suggest that BDF 9145 binds to the open conformation of the sodium channel, and that its dissociation rate constant is 0.03 s~ trader comodification conditions. (Supported by D.F.G., Sandoz-Stiftung, and F.C.I..) "'4-(3-(4-((4-cyanomethoxyphenyl)phenylmethyl)- 1-piperazinyl)-2hydr oxypropoxy)-1H-indol-2-carbonitrile "Beiersdorf AG, Hamburg "Pharmazeutisches Institut der Universit~it Bonn Institut fiir Pharmakologie und Toxikologie der T.U. Mfinchen, Biedersteiner Str. 29, W-8000 Miinchen 40, Germany

244 ~NVESTIGATION OF POSITIVE INOTROPIC COMPOUNDS IN THE ISOIATED,WORK-PERFOIIMING GUINEA-PIG HEART ~xel Mescheder, Uta Reifenstein The positive inotropic actions of adrenaline and ouabain were compared to those Of two naphthyridine-derivatives (HV la = 4-hydroxy-ethyl-amine-7-methyl-l,8-naphthyridine-3carbonic acid-ethyl-ester; HV li = 4-methoxy- ...). These 'compounds i~creased the contractile force of isolated guinea-pig left atria. Using the "work-performing heart", pressure and hemodynamic parameters could be detel-mined. In addition, effects on hearts with myocardial depression were studied. Isolated hearts were paced at 3 Hz and perfused with Tyrode's solution (Ca2+:l,4mM, K+:5,4~LM) containing 10% 'po%ygelatine (H~maccel R) to reduce edema. The table shows relative effects of the compounds (pro-drug level = 100%). ~omDound ouabain adrenaline HV la ~D7 li

l,SxlO-IM 3XI0-SM 3XlO-SM 3XlO-SM

pla 77(16) 39(8) 99(7) 92(21)

Dlv i12~5) 125(3) 104(4) 102(6)

dp/dt 112(7) 213(18) 92(6) 98(14)

co 114(8) 154(11) 101(9) 99(9)

Parameters: pla= mean atrial pressure; plv= systolic ventricular pressure; dp/dt= first derivative of plv; co= cardiac output. Mean values (SEM) of n=4-6 hearts. Data were obtained with the highest drug concentrations not causing any rhythm disturbances. Under these conditions, no marked effects of HV la, HV li, and ouabain could be observed. In order to accentuate their effects, the influence of these substances on hexobarbitalinduced myocardial depression was studied. Hexobarbital (8xl0-SM) reduced dp/dt and co by 30% and 40%, respectively. All compounds caused restitution to initial values, but ~"~ la and HV li only temporarily elevated the reduced cardiac parameters, followed by rhythm disturbances after 25min (HV li), and reduction to levels of sole hexobarbital application after 40min (HV la). After application of ouabain, all values exceeded those of hexobarbital-treated hearts for 60 min, pressures equalled levels of non-depressed hearts. All compounds revealed positive inotropic action. Compared to adrenaline, ouabain, HV In, and HV li had smaller effects, which were accentuated in depressed hearts. Here, ouabain revealed a more stable improvement of cardiac performance than the naphthyridines. Abt. Pharmakologi~.~Uni~--_Kiel~_ Hospitals~r._~4.~ D-2300 Kiel

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THE RATE OF ASSOCIATION WITH RECEPTORS CONTROLS THE Na+,K*-ATPase-tNHIBITORY POTENCY OF A SERIES OF CARDIOACTtVE STEROIDS F.Ebner The regulation of Na*,K÷-ATPase-inhibitory potency of a series of fourteen cardioactive steroids was studied in the guinea-pig heart enzyme by quantifying the inhibition rates with pseudofirst-order rate constants at 35 oC. From the slope and the intercept with ordinate of their relation to steroid concentration the rate constants of association and dissociation were determined and compared with half-maximally inhibitory concentrations (]C~0). The correlation between the lCs0 values of the various steroids and their rate constants of association (P
A STABLE ISOTOPE TECHNIQUE FO~ INVESTIGATING CARDIAC M E T A B O L I S M USING M A S S S P E C T R O M E T R Y F.U. MHller*, D.H. Hunneman+, R. Kahles+ and G. Hel]ige+ Methods of blood gas mass spectrometry were applied to investigate cardiac metabolism with 13C-labeled substrafes. To ~alidate this technique for further studies isolated working rat hearts were per%used with 13Clabeled subs%rates in a bicarbonate-free perfusion fluid. Partial pressures of 12CO2 and 13CO 2 in the coronary effluate were measured continuously using computer assisted mass spectrometry. The fraction of CO 2 produced b y oxidation of 13C-labeled subs%rate was calculated by the f3CO2/CO2 ratio in the coronary perfusate. T h e oxidation of 13C-acetate showed a linear correlation with 13C-acetate concentrations between 0.015 and 0.16 mmol/l. A n inhibitor of acylcarnitlne translocase, 2-(3-methylcinnamylhydrazono)-propionate (BM42.30~), decreased C02 production from 13C-palmitate from 4 8 % ± 4 % to 31~+3% (n=ll, SEM). The time course of the I~C02/CO~ ratio with lSC-palmitate was markedly delayed compared with ~3C-acetate which was exp]ained b y disfributlon within intracellular palmitate stores. The rapidly accessible intracellu]ar palmitate stores were estimated b y considerations of tracer kinetic theory to be less than 900 nmol/g ww. This technique allows further specific investigations of the oxidation of labeled subs%rates in the heart and m a y be useful for basic research and~or clinical diagnosis avoiding the hazards of radiolabeled subs%rates. (Supported b y the DFG, SFB 330-Or~anprotektion.)

Institut f6r Pharmakologie und Toxikologie der Technischen Universit~t M6nchen, Biedersteiner Str.29, D-8000 M(Jnchen 40

*Present address: Abteilung Allgemeine Pharmakologie, Universitats-Krankenhaus Eppendorf, UniversitY% Hamburg, Mar£inlstra~e 52, 2000 H a m b u r g 20, FRG. +Abteilun~ f~r experimentelle Kardiologie, Zentrum Physiologic und Pathophysiologie, UniversitY% G6ttingen, Humboldtallee 23, 3400 Ggttingen, FRG.

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NA+ PUMP DENSITY AND INTRACELLULAR NA+ ACTIVITY IN GUINEA-PIG ATRIAL AND PAPILLARY MUSCLE G.X. Wang, R. Schmied and M. Korth

A NEW METHOD OF FLOW DISTRIBUTION MEASUREMENT IN LANGENDORFF HEART PREPARATIONS USING FLUORESCENT MICROSPHERES H, Weihs, and A. Walland Radioactive microspheres are used for assessing the influence of ischaemia and drugs on local myocardial blood flow. However, as a precise distinction of myocardial zones is almost impossible in rat hearts with the usual dissection techniques we aimed at measuring transmural flow distribution in rat ventricles by using transverse slices, fluorescent microspheres and a planimetric technique. Langendorff preparations of the rat, 350 g body weight, were per%used with Tyrode solution (37 ° C) at a pressure of 80 cm H20 and paced electrically at 5 Hz. Left ventricular pressure was measured with a latex balloon catheter and coronary flow by collection of perfusate over periods of 5 min. Microspheres (Fluoresbrite Carboxylate, Polysciences, St. Goar, FRG) of i0 #m diameter stained with yellow green (exalt = 458 nm, emiss = 540 nm) were injected into the aortic cannula (40000 beads/g heart). They neither affected coronary flow nor contractility. The hearts were flushed with 15 mM EGTA for relaxation followed by I0 % formaldehyde for fixation and cut into 52 #m slices with a freezing microtome. Photographs were taken from 3 slices/heart with a photomacroscope with fluorescence equipment. The left ventricular wall was subdivided on the enlarged print into equally wide zones (EPI i, EPI 2, END0 l, END0 2), which were evaluated planimetrically. The number of beads within was also counted and the density calculated. The validity of the method was tested by changing the perfusion pressure for 30 min in several groups, each of 6 - 9 hearts. Coronary flow and contractility decreased at 30 and 50 cm H20. Evaluation of the density gradients ENDO 2/EPI i revealed values of 1.81 at 120, 1.88 at 80, 0.85 at 50 and 0.52 at 30 cm H20. This indicates a particularly pronounced hypoperfusion of the endocardium at low pressure perfusion. Department of Pharmacology, Boehringer Ingelheim KG, D-6507.

Supported by the Deutsche Forschungsgemeinschaft

Intracellular Na* activity (al-Na) and membrane resting potential were measured in quiescent guinea-pig atrial and papillary muscles by means of Na* sensitive- and conventional microelectrodes. In 30 atria and 22 papillary muscles, a*Na amounted on the average to 8.0+0.2 and 4.7+0.3 mM, respectively. When both tissues were obtained from the same animal and impaled with the same ion-sensitive electrode, mean al~= values of 7.9+0.2 and 5.1+0.5 mM (n=8) were obtained from atrial and papillary muscles, respectively. Membrane resting potentials were significantly (p<0.O01) more negative in the papillary muscle (-83.5+O.7mV; n=8) than in the atrium (-77.6+0.9mV; n=8). Hydrolytic activity of sarcolemmal Na+,K+-ATPase isolated from atria amounted to only one third of that detected in ventricles (0.07+0.01;n=10 vs 0.2+0.01 gM phosphate released per rain and g tissue;n=18). Inhibition of the Na + pump by the cardioactive steroid dihydroouabain (DHO) increased atNa of the atrium within 10 rain by 0.6+0.1 (n=7), 1.3_+0.1 (n=5) and 3.2+0.2 mM (n=5) at 5, 10 and 30 ~M, respectively. In the papillary muscle, DHO up to 10 gM was without effect while a*,e rose by 1.0-+0.1 (n=5) and 3.0-+0.2 mM (n=6) at 30 and 120 ]aM DHO. Consistent with this observation, the potency of DHO to increase force of isometric contraction was three times higher in atrium than in papillary muscle (stimulation frequency 0.2 Hz). The inhibitory potencies of DHO on sarcolemmal Na*,K*-ATPase preparations were found to be identical in the enzymes from either tissue. It is concluded that a lower Na + pump density is responsible for the higher al~a and for the lower membrane resting potential in atrial as compared to ventricular cells. The regulation of cellular Na* homeostasis in atrial muscle appears to be closer at its limit than in ventricle, explaining the higher sensitivity of the atrium to interventions which impede Na ÷ pump activity. Instttut f~r Pharmakologle und Toxikologie tier Techn. Univ. M6nchen, Biedersteinerstr.29, D-8000 M6nchen 40, F.R.G.

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ACTIONS OF SPIN TRAPS ON THE ISOLATEDHEART I . E . B l a s i g and K. S c h S n h e i t

ACTION OF ~ECLOFENOXATE, T H E I R N E T A B O L [ T E S , AND P~RACETAN ON THE ISOPROTERENOL-INDUCED PROCESSES IN THE RAT HEART

A.Grisk,A.Baumann,C.Hoffmann,M.Rohde,KU.M6ritz,U.Hoffmann

As a measure of routine, free radicals generated in the f a l l i n g heart are determined by perfusion of spin traps (nitrones) and electron spin resonance (ESR) spectroscopy. However, little is known about direct cardiac effects elicited by the nitrones. Therefore, the rat isolated heart was perfused according to Langendorff with the spin trap agent a-phenyl-N-tert-butylnitrone (PBN) at a concentration of 3 mM which is commonly employed in such studies. A positive inotropic effect was recorded within 1 min of perfusion with PBN (n=10). LVDP, LVdp/dt~ax and cardiac work were increased from 13.4 ± 2.4 kPa, 580 ± 120 kPa/s and 4111 ± 927 kPa/min to 24.9 ± 3.1 kPa, 1093± 134 kPa/s and 6701 ± 1229 kPa/min, respectively,P < 0.001. Concomitantly, heart rate decreased from 305 ± 26 to 269 ± 39 min -I , P < 0.05, and coronary flow increased from 11.2 ± 1.7 to 14.3 ± 1.9 ml/min. In the effluent of PBN perfused hearts small amounts of a nitroxyl radical adduct of PBN were detected by ESR spectroscopy (aN=l.41 mT). Therefore, a contribution to the cardiac actions of PBN by the nitroxyl derivative cannot be excluded. Generally speaking, the results of the study indicate that pharmacological interactions of the spin traps may occur when the ESR-spin trap method is employed. Abteilung Herzkreislauf-Pharmakologie, I n s t i t u t ~irkstofforschung, A.-Kowalke-Str. 4, O-1136 Berlin

ffir

Using nootropics the following questions are of interest: 1. Show t h e s u b s t a n c e s any cardioprotective effect? 2. Is there a remission of the isoproterenol-induced heart hypertrophy 3. Are the effects observed retated to the intact substances or to their metabotites? Rats were pretreated with 5 mg/kg of isoproterenol given daily for ten successive days to induce heart cell necrosis and hypertrophy. Meclofenoxate, their metabolites and piracetam were given daiZy in doses from 50-100 mg/kg for ten consecutive days to prevent isoproterenol-induced effects on t h e h e a r t . T h e mass d e v e l o p m e n t , the activity of dehydrogenases and of the ornithine decarboxylase, the myocardial content o f RNA, t h e e n e r g y state, and t h e myosin isoenzyme v-l,v-2 , and v-3 were ~nvestigated, A~l drugs tested in this model are unable to prevent ~he heart hypertrophy induced by isoproterenol. Under the inftuence of the isoproterenol the v-3 myosin increases at the expense of the other two, espec£ally the v-I which disappeared completely. I~ combined application there is a shift to the redistribution mediated by the nootropics. Meclofenoxate, their metabolizes p-chlorophenoxyacetic acid, dimethylaminoethanot, and piracetam show characteristic cardioprotective effects on t h e m y o c a r d i ~ m . This was i n d i c a t e d by a reduced mortality of the animals and s reduced loss of the activity of the mitochondrial NADH-nitro-BT-reductase. The decreased content of the RNA a n d that of the reduced energy state produced by isoproterenot were gradually normalized by the nootropics. Therefore we c o n c l u d e , that the nootropics piracetam and meclofenoxate as w e l l as t h e m e t a b o l i t e s of the latter have a cardioprotective action. institute of Pharmacology EMA-University Greifswald, Friedrich-Loefflerstr. 23

and T o x i c o l o g y 0-2200 d

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R 59494 POSTPONES ISCHEMIC CONTRACIIJRE AND POTENTLY INHIBITS POSTISCHEMIC REPERFUSIOR-INDUCED FIBRILLATIONS E. Scheufler and A. Mozes R 59494 (N-methyl-N-[l-(4-phenoxybutyl)-3-pyrrolidinyl] -2-benzothlazolamine) was investigated with respect to antiischemic and a n t i f i b r i l l a t o r y properties of the compound in guinea-plg Langendorff preparations perfused at constant flow. I t was of particular interest, whether the compound is active when given only during reperfusion. Left ventricular pressure (LVP) and perfusion pressure (PP) were monitored. R 59494 was given either 45 min before ischemia or upon onset of reperfusion. In the l a t t e r case an i n i t i a l loading dose was applied to achieve fast equilibration o f the drug with the tissue. Ischemia (60 min) was e l i c i t e d by reducing the perfusion flow from about 8 ml/mln to O.i ml/min. During ischemia the onset of the ischemic contracture was defined as the time, at which the diastolic pressure was raised by 2 mm Hg. At the end of 60 min reperfusion the occurrence o f f i b r i l l a t i o n s was monitored. Ischemic contracture: lO-7 M R 59494 s i g n i f i c a n t l y delayed the onset o f ischemic contracture from 27 ± 5 to 36 ± ? min (x ± s.d.) in low flow ischemia when the compound was given before ischemia. A n t l f i b r i ] ] a t o r y properties: lO-7 M R 59494 given before ischemia s l i g h t l y reduced LVP from 83 ± 8% (controls) to 67 ± 5%. PP was not affected up to lO-7 M. The incidence of f i b r i l l a t i o n s at the end of 60 mln o f reperfusion (B3% in controls) was dose-dependently reduced to O~ in hearts pretreated with the compound in the range of ]0 -8 M to lO-7 M. Nhen given with the onset o f reperfusion R 59494 could also reduce the incidence of f i b r i l l a t i o n s (36% at lO-7 M). In conclusion, R 59494 delays the ischemic contracture at lO-? M. However, the compound was active between ]0-8 M and ]0-7 M in inhibiting the occurrence of postischemic f i b r i l l a t i o n s even when given only during reperfusion. 3ANSSEN RESEARCH FOUNDATION, D-4040 Neuss 21.

CARDIOVASCULAR EFFECTS OF THE NEW CHANNEL OPENER HOE 234. E. Klaus, H. Englert, and D. Mania

POTASSIUM

Potassium channel openers represent a new class of smooth muscle relaxing agents. Their activity is mediated by an increased permeability of the cell membrane to potassium. The aim of this study was the characterization of the acute blood pressure lowering effect of HOE 234, (3S,4R)-3Hydroxy - 2,2 - dimethyl - 4 - (2 - oxo - 1 - pyrrolidinyl) - 6 phenylsulfonylchroman hemihydrate, in animal models in comparison to cromakalim (CRO) and other compounds of this type. In normotensive anaesthetized rats HOE 234, lemakalim, and CRO given i.d. caused a dose-dependent decrease of blood pressure with ED25 values of 5.6, 31.1, and 9 7 . 4 # g / k g . In all cases the duration of the effect was longer than one hour. The activity of HOE 234 (ED25 = 9.4 #g/kg i.v.) was attenuated (ED25 = 77.5 #g/kg i.v.) by glibenclamide (20 mg/kg i.v.). HOE 234 and CRO were both more potent but shorter acting in conscious SH rats (EDb~ = 18.8 and 44.7 #g/kg, respectively) than in conscious normotensive rats (EDbs = 43.6 and 75.6 /~g/kg). In anaesthetized dogs HOE 234 and CRO (both in a dose of 10 #g/kg) decreased the mean arterial blood pressure and the peripheral resistance, whereas heart frequency, cardiac output and contractility increased. The duration of blood pressure lowering activity was 90 min for HOE 234 and 20 rain for CRO at EDbs values of 11 and 27 #g/kg, respectively, In anaesthetized rhesus monkeys HOE 234 in a dose of 5 #g/kg i.v. lowered MAP by 17% and after a dose of 30 #g/kg i.d. by 15% whithout any increase in heart frequency. In conclusion, HOE 234 is a potent blood pressure lowering agent with prolonged duration of action. Its hemodynamic profile is that of a peripheral vasodilator. Its mechanism of action is based on the increased conductivity of K + ions through smooth muscle cell membranes. Hoechst AG, POB 800320, D-6230 Frankfurt/M. 80

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P H A R M A C O L O G I C A L P R O F I L E O F MCF-1084. M. Leitold, H. Laufen, T. Zimmermann, R.-A. Yeates (+)-5- [ 1 , 4 - d i h y d r o - 2 , 6 - d i m e t h y l - 3 - m e t h o x y c a r b o n y l - 4 - ( 2 nitrophenyl) -5-pyrldylcarbonyl] -isosorbide-2-nitrate, MCF-1084, i s , l i k e n i f e d i p i n e , a d i h y d r o p y r i d i n e calcium channel blocker, but also contains the isosorbide-2nitrate moiety (IS-2-N)(1). This minor structural change results in substantial differences in pharmacological properties between MCF-1084 and nifedipine. In in-vitro experiments MCF-1084 was more potent in relaxing phenylephrine-induced contractions than those induced by potassium-chloride (KCl). Moreover, the MCF-1084-induced relaxations of the phenylephrine- and KCl-contractions of rabbit aortic rings were antagonized by the soluble guanylate cyclase inhibitor (6-anilino-5,8-quinolinedione), L Y 83583. The relaxation of KCl-induced contractions by nifedipine was not antagonized by LY-83583. MCF-1084 inhibited the adenosine diphosphate-induced h u m a n platelet aggregation and elevated the activity of the soluble guanylate cyclase in rabbit aorta-homogenates. MCF-1084 produced a potent relaxation of isolated dog coronary arterial strips contracted with KCI. In contrast to nifedipine, this drug-induced relaxation was not overcome by the cumulative addition of extra calcium to the bathsolution. MCF-1084 relaxed the KCl-induced contractions in dogs' saphenous venous strips, also dose-dependently. In the isolated perfused Langendorff-heart of guinea pigs MCF-1084 caused an increase in coronary flow, depending on concentration. After oral administration to the anesthetized rat or to the conscious dog MCF-1084 exhibited antianginal and hypotensive activity for 6 hours. Also after oral administration, MCF-1084 increased the survival rate of rats after an acute coronary ligation, in distinctly lower dosage levels than with the organic nitrates and nicorandil. Orally administered M C F 1084 did not reduce the systolic blood pressure in spontanously hypertensive rats in comparison to nifedipine. (i) Stoss P., M. Leitold: EP 0114270 B1 01.04.1987. Dept. of Pharmacology, Heinrich M a c k Nachfolger, Chem.pharm. Fabrik, Postfach 2064, D-7918 Illertissen]Bayern.

LU 49938: A COMBINI~D CA~+- AND 5HT~-ANTAGONIST WITH POTENT ACTION IN HYPERTENSION AND ON CORONARY CIRCLTLATION M. Kirchengast, J. Grles, H.D. Lelmaann The optically pure compound LU 49938 (LU; (2S)-5-[N-methyl-N-(n-hexyl)] amino2-(3.4.5- trimethoxyphanyl) -2-isopropyl-valeronitril-hydrochioride) is almost equipotent as a blocker of Cai*-charmels and 5I-IT~-receptors. A chronic experiment with SHRSP fed on 8 % salt diet without and with LU (21.5; 46.4 mg/kg/d) in the feed from the 10th to the 16th week of life was carried out. At the end of the 6-week treatment period 46.4 mg/kg/d LU had reduced systolic blood pressure by 27 %, ventricular mass by 14 %, and albuminuria by 95 % compared to untreated controls. The coronary reserve, determined in isolated hearts under constant perfusion pressure was as high in LU-treated SHRSP as in age-matched WKY. In untreated control SHRSP it was reduced to 4 1 % of WKY's values. In anaesthetized mongrel dogs a fixed coronary stanosis was made after mechanically damaging the endotheliura in that part of the LAD (Folts et al. (1976), Circulation 54: 365-370). After positioning of the constrictor coronary flow reductions up to 0 occurred which were spontaneously reversible or had to be shaken loose. This decrease in flow was due to thrombus formation. The cyclic flow reductions (CFRs) occurred with a frequency of 6 to 8 per 30 mln and were stable over 2 h in control dogs. In the treated groups 1 h after induction of CFRs the test compound was given i.v. over 2 rain and measurements were' carried out for 1 h. LU 49938 (0.05 mg/kg) totally and ketanserin (0.01 mg/kg) almost totally abolished CFRs in the first 30 rain after application without significantly influencing hemodynamics. The highest hemodynamieally applicable doses of verapamll (0.5 mg/kg), gallopamil (0.2 mg/kg) and diltiazem (0.5 mg/kg) were only about 50 % effective, with nifedlpine (0.01 mg/kg) no significant effect on CFRs could be seen. Intraduodcnnlapplicationof4.64mg/kgL U toanaesthetizeddogsincreasedblood flow in the coronary sinus by 60 % without significantly altering femoral artery flow, thus indicating potent coronary dilatation. We conclude that LU 49938 is a highly effective agent in lowering blood pressure as well as in increasing coronary flow and in preventing intraarterial platelet deposition. Knoll AG, cardiovascular Pharmacology, D-6700 Ludwigshafen

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SPASMOLYTIC EFFECTS OF THE NEN CA~+-ARTAGONIST L~ 49.938 ON BOVINE CORONARY ARTERIES IN VITRO M. Braun

REDHCED ENDOTHELIAL INJHRY CHOLESTEROL-FED RABBITS. P. Kienbaum

Evidence is accumulating that enhanced platelet activation and secretion of vasoactive mediators (i.e. thromboxane (TX) A2 and serotonin (5-HT)) are involved in the thromboembolic complications of ischemic heart disease. This study investigates the effects of LU 49.938 ((2S)-5[N-methyl-N-(n-hexyl)]amino-2-(3.4.5-trimethoxyphenyl)-2isopropyl-valeronitril-hydrochloride) on contractions of isolated bovine coronary arteries induced by exogenous 5HT and a stable TXAi/PGH2 mimetic as well as arachidonic acid-stimulated (30 pM) washed human p1atelets. LU 49.938 was equipotent in inhibiting maximum 5-HT (I0 pM)- and KCI (40 mM)-induced contractions (ICs0: = 0.2 ~M; n = 5 - 7). Maximum contractions induced by the thromboxane mimetic were reduced to 54 ± 3% of control in the presence of LU 49.938 (1 ~M; n = 5). The biphasic contraction induced by platelet-derived TXAz and 5-HT [1] was concentration-dependently inhibited by pretreatment of the arteries with LU 49.938 (10 nM - 1 ~M). The inhibitory effect on 5-HT mediated contraction was much more pronounced:

Naftidrofuryl (NAF, Dusodrils) is clinically used for treatment of ischaemic vascular diseases. This study investigates the morphological and functional endothelial impairments in cholesterol-fed rabbits (I %, 3 months) and the effects of chronic oral administration of NAF (60 mg/kg x d). The extent of aortic-plaque formation was quantified by a subjective score (0-5) and was significantly reduced by NAF treatment: 4.8 ± 0.i vs. 3.6 ± 0.3 (p < 0.05). Rings of the thoracic (A. thor.) and abdominal (A. abdom.) aorta were equilibrated in an organ bath containing oxygenated Erebs-Henseleit-buffer and indomethacin (3 ~M) at 37°C. Maximum effective acetylcholine concentrations (ACH, 1 ~M - 10 pM) and substance P (SP, 30 nM) induced endothelium-dependent relaxations of PGFa= precontracted vessels were markedly suppressed by cholesterol-feeding and partially restored by NAF.

Contractile response [% control] COH LU 49.938 lit/l] 0.01 0.i TXA~ 5-HT

100 100

84 ± 12 61 ± 6*

68 ± 8* 40 ± 10"

1 56 ± 8* 6 ± 3*

*)p ~ 0.05 vs. control (CON); x ± sem, n = 6 - 9 At these concentrations LU 49.938 did not affect basal vessel tone. These data suggest LU 49.938 as a potent inhibitor of coronary vasopasm induced by hyperreactive platelets. ill Verheggen R, Schr~r K (1986) J Cardiovasc Pharmacol 8:483-490 Institut fur Pharmakologie, Heinrich-Heine-Universit~t, Moorenstr. 5, W-4000 DUsseldorf 1 Deutscher Titel: Pharmakologisches Profil yon MCF-1084

BY

ORAL

NAFTIDROFUHYL

IN

[% relax, of PGFia precontracted vessels]: CON ACH: A. ACH: A. SP: A. SP: A.

thor. abdom. thor. abdom.

84 91 45 40

NAF ± ± ± ±

6 3 4 6

77 85 46 49

CHOL ± ± ± ±

5 5 3 6

19 37 12 20

± 6+ ± 10+ ± 4+ ± 6÷

CHOL+NAF 33 68 24 28

± ± ± ±

5 9* 4* 6

*p < 0.05 vs. CHOL, ÷p < 0.05 vs. CON, n = 6-8 Contractile responses to KCI and PGFi, as well as relaxing effect to SIN-I were not different between the 4 groups. These data demonstrate improved preservation of endothelial function by NAF. Institut fur Pharmakologie, Heinrich-Heine-Universit~t DUsseldorf, Moorenstr. 5, 4000 D~sseldorf i

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NEUTROPHIL HYPERREACTIVITY IN CHOLESTEROL FED RABBITS I8 REDUCED BY ORAL TREATMENT WITH NAFTIDROFURYL A. Weber and Th. Hohlfeld

STRUCTUREACTIVI~ RELATIONSHIPOF SONETHEOPHYLLINEDERIVATIVES

Leukocytes accumulate in atherosclerotic plaques and may contribute to vasospasm of cholesterol fed primates (Lopez JA, Circ Res 65: 1078, 1989). This study investigates the effect of naftidrofuryl (Dusodril R, NAF) on cholesterol-induced neutrophil hyperreactivity. New Zealand White rabbits (3.5-5 kg) were fed a cholesterol-rich diet (i % for 3 months, CHOL), resulting in severe aortic plaque formation as compared with a control group fed a standard diet (CON). Total plasma cholesterol was not changed by NAF. Ex vivo measured neutrophil-dependent, zymosan-induced chemiluminescence (NDC) in whole blood was significantly increased by CHOL. Oral treatment with NAF (60 mg/kg-d) during CHOL feeding reduced NDC only in CHOL, but not in CON rabbits (table). The severity score of aortic plaque (AP) formation (table) was significantly correlated with neutrophil hyperreactivity (r = 0.82, p<0.001) and reduced by NAF treatment. Group

n

NDC [AU]

AP [score 0-5]

CON NAF CHOL CHOL+NAF

9 7 6 8

14 13 60 29

0.2 0 4.8 3.6

± 4 ± 2 ± 10 *) ± 2 +)

± ± ± ±

0.I 0 0.1 *) 0.3 +)

*) p<0.01 vs CON, +) p<0.05 vs CHOL, AU: arbitrary units Additional in vitro experiments on isolated human neutrophils showed that NAF inhibited NDC only at high concentration (ICBo = 4.7.10-5 M). The data demonstrate a significant cholesterol-induced neutrophil hyperreactivity and a marked reduction by oral NAF treatment, which was accompanied by a considerable reduction of aortic plaque formation.

(DIAIXFff'DROIDIDNITRATES) - C(]M)ARISONOF THEIR IN VI'IRO AND IN VIVO

PHAF@~ACOLOGICAL #£TIVITY R. Schlegelmilch Startingfrom Teoprani%ol(KC 046) a seriesof derivativescarrying a substitutionat the N-atcmof the propylamino-chainwere synthetized. The aim was to studythe structureactivityrelationof these N-alkylatdd derivatives, and eventuall~to obtain a compoundwith bettertherapeutic potential. ~he sobstituentsconsistedof alkyl residuesas well as carbonic acid esters, alcohols,ethersand other structures. Pharmacologicalactivi%ieswere comparedusing several isolatedorgan models like precontractedrat aorta spiral s%rips,dog coronaryartery

segments, and the Langendorff heart. In ¥~vo experiments c~prised mainly the detection of blood pressure lowering and heart rate influencing activity in the conscious dog. - ISDNserved as a positive control in all models. In isolated organ preparations structures carrying a long chain lipophilic substituent had an excelIc~Tt spasnolytic effect on precontracted s~Doth m~scle preparations and most strikingly increased coronary flow in the Langendorff heart. These ccn~oounds,however, also showeddose dependent negative inotropic effects and a remarkable ihcrease in heart rate. In contrast, derivatives carrying only a short alkyl residue (up to C4) at the propylamino-N atom showeddearly less influence on these parameters, lhe cc~ounds carrying more hydrobhilic cc~onents, like piperazinyl- or acetylglycyl-residues were even less effective. Contrary to the findings mentioned above, in experiments with conscious dogs after oral a~inistration, effects comparableto KC 046 (i.e. considerable decrease in systolic blood pressure, weak influence on heart rate, and long duration of action s t i l l in low doses) occured only with those derivatives carrying short alkyl residues (up to C4). ~ explar~tion may be provided by distri~tion phenc~nenaor by the lower e~teral absorption of derivatives with long lipophilic substituents. Present address: Departmentof Pharmacology, Dr. Willmar Schwabe~ H , Willmar-Schwabe-Str. 4, 7500 Karlsruhe 41, FRG

Institut fur Pharmakologie, Heinrich-Heine-Universit~t D~sseldorf, Moorenstr. 5, D-4000 DUsseldorf, FRG

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260

SPASMOLYTIC AND ANTIPLATELET EFFECTS OF THE NOVEL TXA2 ANTAGONIST BAY U 3405 IN AN IN VITRO MODEL OF CEREBRAL ARTERIAL SPASM L. Bruch

STIMULATION OF t-PA C O M P O U N D S IN V I V O

BAY u 3405 ((3R)-3-(4-Fluorophenylsulfonamido)-l,2,3,4tetrahydro-9-carbazolepropanoic acid) is a potent PGH2/TXA2 receptor antagonist. This study investigates the spasmolytic effects of BAY u 3405 on isolated precontracted rings (1-2 mm OD) of bovine brain arteries (BBA) and on release of platelet derived vasoactive substances (TXA2, 5-HT). BBA segments were placed in an organ bath containing oxygenated Krebs-Henseleit buffer under a resting tension of 1 g at 37°C. In BBA rings precontracted by PGF2, (I0 ~M) or U46.619 (9-11-epoxy-methano-PGH2, 0.I ~M) BAY u 3405 caused a dose-dependent and finally complete relaxation (ICBo: 18 nM and 32 nM respectively) but did not affect 5-HT (i ~M) induced contraction. Thrombin (0.6 IU/ml) stimulated washed human platelets (109 cells per assay) caused a hiphasic contractile response of the BBA, consisting of an initial, TXA2 dependent component and a second 5-HT-mediated component. Pretreatment of the platelets by a maximum effective concentration of BAY u 3405 (i ~M) reduced the TXA2-component to 59±6% (mean ± SEM, n = 8) and the 5-HT component to 49 ± 11% of control. This was paralleled By a reduction of the TXB2 content of the Bath medium to 49 ± 9% and an inhibition of 14C-5-HT secretion to 42 ± 10% (n = 4) of control. These data demonstrate a potent spasmolytic effect of BAY u 3405 on precontracted isolated cerebral arteries, involving both direct antagonism of TXA2-mediated vasoconstriction and inhibition of TXA2-dependent release Of platelet-derived vasocontractile mediators. Heinrich-Heine-Universit~t DUsseldorf, Moorenstr. 5, W4000 DUsseldorf 1 Present address: Klinik fur Innate Medizin, Charit4, Schumannstr. 20/21, O-1040 Berlin

RELEASE BY POLYANIONIC H.-P. K l S c k i n g

The acute tissue-type plasminogen activator (t-PA} r e l e a s i n g e f f e c t of s e v e r a l p o l y a n i o n i c c o m p o u n d s w i t h d i f f e r e n t in v i t r o e f f e c t i v e n e s s was s t u d i e d in rats a f t e r i.v. a d m i n i s t r a t i o n . The r e s u l t s are e x p r e s s e d as m a x i m u m i n c r e a s e in IU of t - P A / m l plasma. C o m p a r e d to the c o n t r o l (saline), the following polyanionic compounds caused a statistically significant increase in a c u t e t - P A r e l e a s e a f t e r i.v. a p p l i c a t i o n of i0 mg p o l y a n i o n i c c o m p o u n d / k g : heparin: 16.8 + 5.6 IU/ml; s o d i u m humate: 21.6 + 3.1 IU/ml; a m m o n i u m humate: 12.9 + 5.0 IU/ml; p o l y m e r i c o x i d a t i o n p r o d u c t f r o m e a f f e i c acid: ii.3 + 1.7 IU/ml; hypersulfated bis-lactobionic acid amides LW 10121: 10.8 + 4.1 IU/ml; LW 10086: 6.0 + 2.8 IU/ml; enoxaparin sodium: 4.8 + 1.4 IU/ml; p e n t o s a n p o l y s u l p h a t e : 18.4 + 5.8 IU/ml. T e m p o r a r i l y i n s u f f i c i e n t f i b r i n o l y s i s may h e n c e be normalized by acute stimulation of t-PA release. The d e c r e a s e of t - P A activity, e.g. in the p r e s e n c e of i n h a l a t i o n a n a e s t h e t i c s , could be p r e v e n t e d by p r e v i o u s administration of a t-PA releasing substance. Partial thrombolysis w a s a c h i e v e d in a n i m a l e x p e r i m e n t s a f t e r a c u t e s t i m u l a t i o n of t - P A r e l e a s e w i t h a p o l y a n i o n i c compound. Institute of Pharmacology and Toxicology, Medical Academy Erfurt, N o r d h i u s e r Str. 74, O - 5 0 1 0 Erfurt, FRG

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MODE OF ACTION OF POTASSIUM CHANNEL OPENERS IN THE GUINEA-PIG ILEUM LONGITUDINAL MUSCLE PREPARATION H.-J. Pfannkuche

T E D I S A M I L B L O C K S 86Rb E F F L U X A N D S I N G L E K + C H A N N E L C U R R E N T S IN V A S C U L A R S M O O T H MUSCLE. V.A.W. Kreye, D. P f r ~ n d e r and U. Theiss

Cromakalim, pinacidil and P-I075 (N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridinylguanidine) are smooth muscle relaxants, believed to produce their effects via the opening of K+-channels in the plasma membrane. However, it is unclear whether those drugs function solely to open muscular plasmalemmal K + channels or whether their effects may be due, in part, to neuronal activity (H. Schwdrer and H. Kilbinger, Naunyn-Schmiedeberg's Arch Pharmacol (1989) 339 : 706 - 708). We have examined the effects of three K + channel activators in the guinea-pig ileum longitudinal muscle preparation (GPI-LM) to address this question. Cromakalim, pinacidfl and P-I075 produced a concentration-dependent inhibition of electrically stimulated (0.1 Hz, 2 ms, submaximal voltages) twitch contractions of the GPI-LM (pICso values of 6.4, 6.2 and 7.6, respectively). These values correlated well with ICs0 values obtained for their inhibitory actions in the mt portal vein. However, the effects of the K + channel-openers in bethanechol (pDz= 5.9) or carbachol (pDz= 7.2) stimulated GPI-LM were significantly weaker (factor > 10) than in the field-stimulated preparation. In contrast to the K + channel openers, atropine (used as a reference inhibitory agent), was a morn potent antagonist of exogenously- induced, as compared to field-stimulation- induced contractions. Therefore, it is suggested that in the GPI-LM, K + channel openers act primarily at the pre-junctional level to inhibit smooth muscle contraction. This may indicate a presynaptic site of action for K + channel openers, in addition to their post-synaptic action in smooth muscle.

T e d i s a m i l , a new b r a d y c a r d i c agent w i t h an inhibit o r y a c t i o n on K + c u r r e n t s in h e a r t muscler was inv e s t i g a t e d w i t h r e s p e c t to its p o s s i b l e a c t i o n s on v a s c u l a r smooth muscle. - W i t h i n a range of I - 700 ~M, t e d i s a m i l was found to c o n t r a c t h e l i c a l strips of r a b b i t aorta p r e s u m a b l y b e c a u s e of K + channel b l o c k a d e und s u b s e q u e n t d e p o l a r i s a t i o n . - In ion flux studies u s i n g B6Rb+ as a m a r k e r for K + ions, t e d i s a m i l i n h i b i t e d the C a 2 + - d e p e n d e n t R b + e f f l u x w h i c h can be i n d u c e d by n o r a d r e n a l i n e or by e l e v a t ed [KCI] e. Also, the R b + e f f l u x induced by the K + c h a n n e l o p e n e r c r o m a k a l i m was i n h i b i t e d by tedisamil, w h i l e w h e n g i v e n alone, the d r u g had a slight s t i m u l a t i n g e f f e c t on R b + efflux. - U s i n g the p a t c h c l a m p technique, we i n v e s t i g a t e d the e f f e c t of tedisamil on C a 2 + - d e p e n d e n t large-conductance K+ c h a n n e l s (BKCa) in i n s i d e - o u t m e m b r a n e p a t c h e s of e n z y m a t i c a l l y d i s p e r s e d smooth m u s c l e cells from g u i n e a pig p o r t a l vein. A p p l i e d at the c y t o s o l i c side of the m e m b r a n e patches, t e d i s a m i l c o n c e n t r a t i o n - d e p e n d e n t l y i n c r e a s e d the f r e q u e n c y of c h a n n e l c l o s u r e s , led to a c o n s i d e r a b l e s h o r t e n i n g of the m e a n d u r a t i o n of o p e n i n g s (from 8 ms to <1 ms), and r e d u c e d the o p e n - p r o b a b i l i t y ; the IC50 for the latter e f f e c t was 74 ~M (n=5). The m e a n time of resid e n c y of the d r u g m o l e c u l e at the c h a n n e l was estim a t e d to be I-2 ms. - In c o n c l u s i o n , tedisamil seems to b l o c k d i f f e r e n t types of K + c h a n n e l s in v a s c u l a r s m o o t h muscle. II. P h y s i o l o g i s c h e s I n s t i t u t der U n i v e r s i t ~ t Heid e l b e r g , Im N e u e n h e i m e r Feld 326, D - 6 9 0 0 H e i d e l berg, Fed. Rep. of G e r m a n y

SANDOZ PHARMA Ltd., Preclinical Research, CH-4002 Basel

262

264

DO MINOXIDIL SULPHATE AND CROMAKALIM OPEN P H A R M A C O L O G I C A L L Y S I M I L A R K + - C H A N N E L S IN H A T ISOLATED AORTA? Ulrich Quast and Katharine Bray

CALCIUM-DEPENDENT EFFECTS OF ERYTHROPOIETIN ON RESISTANCE VESSELS AND THROMBOCYTE FUNCTION. S. Heidenreich, J. Kienast, M. Tepel, W. Zidek, and K.-H. Rahn Development of hypertension and thrombotic shunt occlusions are the main side effects of treatment of hemodialysis patients with erythrepoietin (rhEPO). To detect a possible direct vasopressor effect of rhEPO we determined the contractile responses of microdissected renal resistance vessels of normotensive Wistar-Kyoto rats to rhEPO by a small vessel myogragh. We found a direct dose-dependent vessel contraction to rhEPO in a range between 10 U/ml and 200 U/ml rhEPO, with a median active force of 0.86 _+ 0.09 mN (n=10) for 20 U/ml rhEPO. The presser effect was abolished by incubation of vessel segments in calcium-free buffer. The blockers verapamil, phentolamine and saralasin did not attenuate active force. Enzymatic digestion of endothelium revealed a direct vasal effect of rhEPO on smooth muscle. On a cellular level we could show that rhEPO exerted a rise of intracellular free [Ca] in isolated smooth muscle cells with the Fura-2 probe. To elucidate a direct effect of rhEPO on platelet function, we performed aggregation studies using platelet rich plasma. RhEPO alone did not induce thrombocyte aggregation up to doses of 100 U/ml. However, under minute, sub-effective doses of adrenalin (1 tiM) rhEPO exerted an irreversible platelet aggregation under concentrations down to O.1 U/ml. This effect was mediated by a rise of intracellular [Ca] of thrombocytes. These results indicate that rhEPO directly acts on smooth muscle cells and as a cofactor on thrombocytes via Ca-fluxes. Both effects might contribute to alterations of microcirculation and vascular problems of dialysis patients under hormone therapy.

Minoxidil sulphate (MxS), the active metabolite of minoxidil, is a smooth muscle relaxant with K+-channel opening properties (Meisheri et al., J. Pharmacol. Exp. Ther.~ 245: 751, 1988). The present study compares the 42K+/86p~+ effluxes induced by MxS with those induced by the K+-channel opener cromakalim (CRK), in rat aortic rings using methods previously described (Quast and Baumlin, N. S. Arch. Pharmacol. 338: 319, 1988). Both MxS and CRK increased the rate constants (k) of 42K+ and 86Bb+ efflux from rat aorta concentration-dependently (EC50 values for 42K+ in the ~M rangeq maximum increases of 4.4 ± 0.2 and 0.5 ± 0.05 (10-2.min-l), for CRK and MxS respectively). The ratio of 86pdD+:42K+ efflux was concentration-dependently increased by CRK (from 0.15 at 0.1~M to = 0.5 at 100~M), whereas it remained approximately constant (= 0.35) after treatment with MxS. In crossdesensitization experiments, exposure to MxS (I and 30~M) for 20min inhibited subsequent CRK (l~M)-induced peak increases (/kk) in 42K+/S6Rb+ efflux by up to 50 % of control values; similarly, preincubation with CRK (0.3~M) reduced MxS (10~M)-induced tracer efflux. Glibenclamide (30-500nM) inhibited the 42K+/86Rb+ effluxes stimulated by MxS (10~M) and CRK (I~M) in a concentrationtion-dependent manner with IC50 values between 0.05 - 0.1 ~M. In rat aorta, MXS and CRK open K + channels which are Rb + permeable and show a similar sensitivity to blockade by glibenclamide. The cross-desensitisation experiments suggest a considerable overlap between the K + channels opened by these drugs. Cardiovascular Department, Preclinical Pharma Ltd., 4002 Basel, Switzerland.

Research~

Sandoz

Dep. of Medicine, Univ. of Muenster, W-4400 Muenster, Germany.

R 67 267

265 RAMIPRILAT INDUCED CYCLIC GMP AND PROSTACYCLIN PRODUCTION IN BOVINE AORTIC ENDOTHELIAL CELLS IS MEDIATED BY BRADYKININ G. WIEMI~RAND R.H.A. BECKER

The stimulation of endothelial bradykinin [Be] receptors by bradykinin (BK) increases the cytosolic Ca~+ concentration which in turn promotes synthesis of prostacyclin (PGI~) as well as endothelium derived releasing factor (EDRF) as asssessed by 6-keto-prostaglandin F~ (6-KPGF~) and endothelial cyclic GMP contents, respectively. The present experiments were performed to elucidate this mechanism considering a possible interaction of converting enzyme (CE) inhibition, kinins and prostanoid metabolism. Primary endothelial cells grown to confluence were incubated for 1 rain with BK and for 10 min with the CE inhibitor ramiprilat (both lxl0-S-lxl0 -4 mol/1) after a 15 min preincubation period in KREBS-buffer with IBMX (lxl0 -4 mol/l) with and without the B~ antagonist Hoe 140 (H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-TicO~c-Arg-OH) which was preincubated for 5 rain. Cellular cyclic GMP as well as 6-KPGF~ in the supernatant were determined by radioimmunoassays. There was a dose dependent stimulation of cyclic GMP and 6KPGFle' synthesis by RT as well as BK. This was completely suppressed by the B~ antagonist Hoe 140 (lxl0 -7 mol/l). These findings suggest that inhibition of CE, localized on the luminal side of the vascular endothelium, results in increased synthesis of cyclic GMP and prostacyclin by enhancement of kinin activity. PGU HEARTCIRCULATION, DPT. PHAPJvIACOLOGY, HOECHST AG, W-6230 FRANKFURT AM MAIN 80, GEP,2vlANY

DETECTION OF NO FROM L-ARGININ IN ENDOTHELIALCELLS BY ELECTRON SPIN RESONANCE.A. M~lsch, P. Mordvintcev*, A. Vanin* and R. Bt~$~ Despite indirect evidence that the endothelium-derived relaxing factor may be identical with nitric oxide (NO), its nature is still controversial. We used the characteristic triplet electron spin resonance (EPR) signal at g = 2.04 and g = 2.02 of NO-Fe2+diethyldithiocarbamate (DETC)-complexes to detect NO-formation in isolated cytosol and intact endothelial cells from porcine aorta (PAEC). Endothelial cytosol (0.1 mg protein) incub~ated(37 °C) with L-arginine (1 mM), NADPH (0.2 mM), free Caz+ (2 pM), calmodulin (1 pM), 6R-tetrahydrobiopterin (0.1 ,uM), and heatkilled yeast loaded with DETC generated 12 nmol NO/60 rain, as calculated from the comparison of the EPR signal in frozen (77 K) incubates and of standards containing dinitrosyl-Fe2+-thiosulfatecomplexes. The generation of NO depended on each of the above additives (except yeast), was not observed with nitrite and hydroxylamine, and was stereospecifically inhibited by NG-nitro-Larginine. The rate of NO formation in the incubates was followed at 37 °C in an EPR quartz flat cell and correlated with the activation of a purified soluble guanylyl cyclase, the physiological NO-effector enzyme. In cultured PAEC loaded with DETC and stimulated with thimerosal NO-Fe2+-DETC-complexes were detected. In conclusion, we identified NO as the immediate product of the NOsynthase reaction in cytosol and intact cells by a new method which allows the realtime measurement of the kinetics of NOformation at 37 °C. *Present address: Department of Chemical Physics, Academy of Sciences, Moscow, SU 117977. Dept. of Applied Physiology, University of Freiburg, HermannHerder-Str. 7, D 7800 Freiburg, Germany.

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THE PH~P&{ACOLO~ICAL ROLE OF SYNERGISTIC R E C E P T O R SUBTYPES INVOLVED IN PHASIC AND ~ONIC S~OOTH ~,~USCLE CONtRACtIONS J. B e r g m a n n

CHARACTERIZATION AND PURIFICATION OF PARTICULATE EDRF-SYNTHASE FROM BOVINE AORTIC ENDOTHELIAL CELLS U. F6rstermann**, J.S. Pollock*, J.A. Mitchell* and M. Heller**

Adrenergic and muscarinsrgic agenists as well as substance P analogs elicit phasic and tonic contractions in " isolated s~nooth muscles ~ which are , ~ ~ ~ ~ ~ +~ ~" ~ relates to ~nt~ace~lul~r Ca ~elease arts Ca' infl~, respectively. }tudies of this complex contraction mechanism with agonists and antagonists suggest the involvement of different synergistic subreceptors. Activation of both subreoep~qrs produces the same second messenger (Ca~), but ~rom different sources, and the same response (contraction), but w i t h a different time course. Synergistic subreeeptors with different a ~ i m i t i e s ~sr agonists and anta~onists and tkeir Dresence in different ratios in va~ rious smooth muscle organs could be the cause fo~ such pharmacological phenomena as imt~insic activity amd receptor reserve. Central Institute of ~ o l e c u l a r Biology Robert-R~ssle-...tr~ 10, 0-i115 }~er~in

The stimulating effect of niu-ic oxide-containing endothelium-derived relaxing factor (EDRF/NO) on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL-6 cells) was used as an assay of EDRF/NO synthesis by bovine aortic endothelial cells (BAE cells) or subcellular fractions thereof. The EDRFfNO release of intact BAE cells could be stimulated with bradykinin, thrombin or adenosine 5'-diphosphate (ADP) and was abolished in Ca2+-free medium. When subcellular fractions were analyzed, less than 10% of the total EDRF/NO-synthesizing activity was found in the cytosolic fraction of BAE ceils, more than 90% of the activity was associated with the particulate fraction. Cytosolie and particulate enzyme activities required L-arginine as a substrate and reduced B-nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor;, both were inhibited by N°-nitro-L-arginine and N°-methyl L-arginine, and hemoglobin or methylene blue abolished the effect of the EDRF/NO produced by both enzymes. Both enzymes were highly sensitive to Ca 2+, the major increase in activity occurring between 100 nM and 500 nM free Ca2+. The calmodulin (CAM) antagonists calmidazolium and trifluoperazine, as well as the CaM-binding protein calcineurin, inhibited the EDRF/NO synthesis by both the cytosolic and the particulate enzymes. Washing the crude particulate enzyme with 1M KC1 reduced total activity by over 40%, but the activity could be restored by adding exogenous CaM. The KCl-washed particulate enzyme could be solubilized with the detergent CHAPS (3-[{3-cholamidopropyl} dimethylammonio]-l-propanesulfonate). Purification of the solubilized particulate enzyme preparation by affinity chromatography on 2'5' ADP Sepharose followed by Superose 6 gel permeation chromatography (Pharmacia) resulted in a single protein band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (Mr -135 kDa). The purified enzyme produced EDRF/NO in an L-arginine-, NADPH- and Ca2+/calmodulin dependent fashion and also converted L-arginine to L-citrulline. We conclude that the particulate enzyme isolated from BAE ceils represents a new isoform of EDRF/NO synthase. *Abbott Laboratories, Abbott Park, 1L 60064, USA, and *Department of Pharmacology, Northwestern University, Chicago, IL 60611, USA

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Soluble S y n t h a s e s of G u a n y l y l Cyclase-Activating F a c t o r s

ENDOTHELIAL CYCLIC NUCLEOTIDES DO NOT REGULATE EDRF/NO RELEASE IN BOVINE AORTIC ENDOTHELIAL CELLS M. Kuhn and A. Otten

In Rat Brain (Type I) And Mouse Macrophages (Type II) Harald KKW. Schmidt, Timothy, D" warner, Masaki Nakane & Fetid Murad. The oxidative L-arginine signal transduction pathway regulates the intracellular cGMP content. The key enzyme of this pathway is the L-arginineconverting guanylyl cyclase-activating-factor (GAF) synthase. This enzyme exists in at least three isoforms: soluble Ca2÷/calmodulin-regalated (I), inducible soluble Ca2+/calmodulin-independent (II), and particulate (III). All isoforms have in common the ability to catalyze the NADPH-dependent oxidation of a terminal guanidino-nitrogen of L-arginine yielding different NO-containing forms of GAF and L-citrulline as a co-product. ---Type I GAF synthase was purified from rat cerebellum to homogeneity by sequential affinity chromatographic steps on 2'5' ADP Sepharose and calmodulin agarose. Enzyme actvity, bit-assayed by both the formation of a plasma membrane-permeable factor that stimulated soluble guanylyl cyclase in RFL-6 cells and the conversion of L-arginine into L-citrulline, depended on the presence of NADPH, Ca2*/calmodulln and was about four-fold stimulated by high thiol concentrations (GSH > 10mM) and tetrahydrobiopterin (1 gM). The physical characterization revealed: isoelectric point 6.1 5:0.3 (Mono P), Stokes radius Rs 7.9 + 0.3 nm (Superose 6), sedimentation coefficient S~o, 7.8 5:0.2 S. From Rs and sm, and assuming a partial specific volume of 0.7~5 cm~ .g'~ the calculated M~for the native ezyme was 279 + 25 kDa. SDS/PAGE revealed a single protein band with Mr 155 + 3 kDa, suggesting that Type I GAF synthase is a homodimer of two 155 kDa subunits. --Macrophages contain type II GAF synthase (Stuehr et al. 1989) with GAF being identical to NO. NO release (148 h) by RAW 264 macrophages (RAW) was determined spectrophotometrically by measuring total ,Jtrite and nitrate (NOr) accumulation after reduction of nitrate with nitrate reductase. Lipopolysaccharide (LPS) or interferon-~ (INI0 induced the release of NO with similar efficiciency and were not additive, reaching a maximum after 12-24 h. The ionophore A 23187, a potent stimulator in intact cells of the CaZ+-regulated type I GAF synthase, was inactive in INF/LPS-treated RAW. However A 23187 caused a small but significant release of NO in non-induced RAW. Pretrealment of RAW with actinomycin D (EC5o=35 ng/ml) or cyclobeximide (ECho=50 riM) completely inhibited the apparent induction of NO release. Thus, RAW contain both soluble forms of GAF synthase. The induction of type II requires transcription and intact de nero-protein biosynthesis, suggesting that type II GAF synthase itself is induced and type I is inactivated or that the synthesis of a protein which converts type I into type II is induced. Department of Pharmacology, Northwestern University, Chicago, Illinois 60611, USA; Abbott Laboratories D-47R AP9A, Abbott Park, Illinois 60064, USA.

It has recently been suggested that endothelium-derived relaxing factor/ nitric oxide (EDRF/NO) might inhibit its own release by increasing the concentration of cyclic GMP in endothelial cells. This study was designed to investigate this possibility. As most stimulants of E D R F / N O production lead to the simultaneous formation of prostacyclin (PGI2) , we also investigated whether PGI 2 and increased levels of endothelial cyclic AMP have a regulatory effect on EDRF/NO production. Conditioned medium from cultured bovine aortic endothelial cells (BAE cells) was transferred onto rat fetal lung fibroblasts (RFL-6 cells) and the increase in cyclic GMP in these 'detector cells' was used as a bioassay for EDRF/NO. BAE cells released EDRF/NO and the synthesis could be stimulated with bradykinin (10 nM) or Ca 2+ i0nophore A23187 (1 ~tM). The EDRF/NO release in response to bradykinin and A23187 was accompanied by an about 2-fold increase in the cyclic GMP content in the producing BAE cells in the presence of the cyclic GMP phosphodiesterase inhibitor M&B 22,948 (2-o-propoxyphenyl-8-azapurin-6-one) (0.1 mM). Incubation of BAE cells with atrial natriuretic peptide (0.1 ~tM) or sodium nitroprusside (10 btM) enhanced cyclic GMP content of BAE cells 6.5-fold and 4.1-fold, respectively (in the presence of M&B 22,948). These increases in the cyclic GMP levels in BAE cells had no effect on basal or bradykinin- and A23187-stimulated release of EDRF/NO. Bradykinin (10 nM) and A23187 (1 ~tM) also stimulated PGI2 production in BAE cells 2.4-fold and 5.6-fold, respectively. Neither this endogenous PGIz nor exogenous PGIz (3 ~tM) raised cyclic AMP in BAE cells, but A H 21-132 ([+]-cis-6-[p-acetamidophenyl]1,2,3,4,4a, 10b-hexahydro- 8,9-dimethoxy-2-methylbenzo- [c] [1,6] naphthyridine) (0.1 raM), a selective inhibitor of cyclic AMP phosphodiesterase, increased the cyclic AMP content of BAE cells 3.7-fold. However, basal and stimulated release of EDRF/NO by BAE cells remained unchanged in the presence of AH 21-132 or PGIg. We conclude that cyclic nucleotides do not influence EDRF/NO formation or release in BAE cells. Abteilung Klinische Pharmakologie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Str. 8, D-3000 Hannover 61

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A Ca2+-REGULATED NITRIC OXIDE SYNTHASE PURIFIED FROM PORCINE CEREBELLUM REQUIRES TETRAHYDROBIOPTER/N AS A COFACTOR M. John, B. Mayer and E. B6hme.

C Y T O C H R O M E P-450 MAY MEDIATE CYCLIC GMP STIMULA-

Nitric oxide ('NO) enzymatically formed from L-arginine as precursor acts as an inter- and intracellular signal molecule with eytosolic gnanylyl cydase as the effectur system. Two NO syuthase isoanzymes with NADPH as an essential cofaetur are postulated: a cytokine-inducible enzyme in maerophages and a Ca2+regulated constitutive enzyme in various other ceils. The Ca2+-regulated NO synthase was isolated from porcine cerebellum by ammonium sulfate precipitation and affinity chromatography on 2',5'-ADP-Sepharose. The enzyme was purified 4,500-fold from a crude superantant with 5 % yield. The purified enzyme showed one stained protein band corresponding to 160 kDa on SDSPAGE and exhibited a molecular mass of about 200 kDa in gel filtration expcrlments. Specific enzyme activity, as determined by measurement of the formation of the stable byproduct L-eltrulline, was about 0.8 Izmol x rain"1 x mg"1. The purified enzyme formed NO from L-arginine in a Ca2+/calmodulin-depundent manner as demonstrated with a specific chemiluminescence method. Enzyme activity was largely dependent on tetrahydrobiopterin, which stimulated the formation of NO and L-eltrulline 4-fold with an ECs0 value of about 0.5 ~¢I, whereas dihydrobiopterin (up to 10 ~M) was without effect on enzyme activity. The same dependency on tetrahydrobiopterin was found for Ca2+-dependent increases of eGMP formation which were observed when purified cytosolic guanylyl eyelase was incubated in the presence of the isolated NO synthase and L-arglnine. Thus, both isoanzymes, Ca2+-regulated and cytoklne-indueible NO synthase, appear to require NADPH as well as tetrahydrobiopterin as cofaetors for the conversion of L-arginine into NO. In addition, reeousfitufion of isolated NO synthase with purified cytesollc gnanylyl eyelase enables to mlmlek in vitro the Ca2 + -mediated increases of intracellular cGMP levels observed in various intact cell systems. Instimt ftir Pharmakologie, Freie Universittit Berlin T~ela~ce67-73,D-1000Berl~.F.R.G.

TION BY GLYCERYL TRINITRATE IN CULTURED CELLS H. Schr6der LLC-PK 1 pig kidney epithelial cells were used to investigate the mechanism of cyclic GMP stimulation by glyceryl trinitrate. The inhibitor of cytochrome P-450 cimetidine (0.i mM) decreased cyclic GMP stimulation by glyceryl trinitrate (0.03-1 ~M) by up to 66%. However, glyceryl trinitrate-induced cyclic GMP stimulation remained unaltered by ranitidine (0.1 mM) which has a much lower affinity for the cytochrome P-450 enzyme system. Another inhibitor of cytochrome P450, miconazole (0.1 mmol/l) also attenuated glyceryl trinitrate-induced cyclic GMP stimulation. In contrast, cimetidine and miconazole did not affect cyclic GMP stimulation by sodium nitroprusside that spontaneously releases nitric oxide. A 24-h pretreatment with the inducer of cytochrome P-450 3-methylcholanthrene (10-30 ~M) increased glyceryl trinitrate-induced cyclic GMP stimulation by up to 100%. Similar results were obtained in cultured rat lung fibroblasts, another established cell line that has previously been reported to show a cyclic GMP response to organic nitrates (i). These findings provide evidence that in intact cells, glyceryl trinitrate-induced cyclic GMP stimulation involves cytochrome P-450-dependent pathways leading to nitric oxide formation from organic nitrates. (1) Schr6der et al. 311

(1987) J Appl Cardiol 2:301-

Institut f~r Pharmakologie der Heinrich-HeineUniversit~t, W-4000 D~sseldorf, Moorenstr. 5

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273 N I T R O X I D E FORMATION FROM G L Y L C E R Y L T R I N I T R A T E PERIPHERAL ARTERIES AND VENES. R.R6sen, G.Jordt, E.K6nig, and A . F . E . R u m p

BY

In therapeutic c o n c e n t r a t i o n organic nitrates like g l ¥ c e r y l t r i n i t r a t e (GTN) decrease cardiac p r e l o a d by a specific dilation of peripheral venes, whereas the dilation of arteries is o b s e r v e d at higher concentrations only. A l t h o u g h these effects are v e r i f i e d 'in vivo' as well as 'in vitro', there is still no e x p l a i n a t i o n for this different behaviour of arteries and venes. The active Drinciple of GTN is nitroxide (NO) which is thought to be generated by the v a s c u l a r smooth muscles. We, therefore, estimated the NO formation rate from GTN by preparations of denuded A.fem. and V.fem. of rabbits at different c o n c e n t r a t i o n s . NO was trapped by Hb-Oz and m e a s u r e d by the formation rate of M e t - H b difference spectrophotometrically. The NO-formation rate could be e n h a n c e d with increasing concentrations of GTN. At all c o n c e n t r a t i o n s t e s t e d of GTN the venous preparations were more potent than the arterial to generate NO (see table). GTN mol/l i0-~ i0-~ i0-~ 10-s

N O - f o r m a t i o n rate A.fem. (n) 4.6Z0.6 (6) 4.5ZI,2 (7) 6.6~i.2 (ii) 6.3±1.3 (12)

(nmol/lxminxmg ww) ¥.fem. (n) i0.9 Z i.i (6) 20.8± 2.7 (7) 16.6~ 3.3 (9) 26.8±10.6 (9)

Moreover, the NO formation rate correlated very well with the v a s c u l a r dilation of V.fem. but not of A.fem.. In A.fem. the r e l a x a t i o n was higher than the c o r r e s p o n d i n g N O - f o r m a t i o n rate, supporting the idea that in arteries G T N - i n d u c e d relaxation is m e d i a t e d by additional mechanisms.

STIMULATED BUT NOT BASAL EDRF RELEASE FROM CULTURED ENDOTHELIAL CELLS IS INHIBITED BY SULFHYDRYL REAGENTS M. Hacker, I. Siegle, W.C. Sessa and J.R. Vane Endothelium-dedved relaxing factor (EDRF) may be nilric oxide or a nitrosorhiol such as S-nitrose-L-cysteine (Myers et al. (1990) Nature 345, 161-163). However, the depletion of L-Cysteine(L-Cys) and the m~lification of intracellular sulfhy&3,1 (SH) groups inhibits the basal (/e flow-induced) but not the stimulated release of EDRF from cultured endothelial cells (Minor et al. (1989) Circulation 80, 281). This apparent discrepancy prompted us to investigate in more detail the effect of SH reagents on the release of EDRF from bovine aortic endothelial cells grown on microearder beads. EDRF release was detected with a superfused, de-endothellalized rabbit aortic ring prec~nstric~edwith U46619 (9a,1 la-methancepoxy-prestaglandin F2~. Basal EDRF release was defined as the degree of relaxation upon infusion of superoxide dismutsse (10 units/ml) through the column of endothelial cells. L-Cys and glutathione (GSH) levels were measored by HPLC analysis of their monobromobimane derivatives. Ethacrynic acid (infused through the column of endothelial cells at 10 or 100 gM for 15 rain; n=3), diamido (100 and 250 I.tM;n=3) or 1-chloro-2,4-diullrobenzene(CDNB; 100 tIM; n=3) had no effect on either basal or stimulated EDRF release, whereas N-ethylmaleimide (NEM; 25 gM for 15 rain) significantly inhibited the stimulated release of EDRF induced by ADP (3 gg; from 36.1 to 9.8% relaxation; n=6) or ionomycin (20 ag; from 40.2 to 8.9% relaxation; n=5). In contrast, NEM had no effect on basal EDRF release (26.9 and 30.8% relaxation respectively) or the rclease of EDRF induced by the non-receptor stimuhis poly-L-lysine (10 gg; 20.7 and 16.6% relaxation respectively; n=4). The reactive disulfide 2,2-dithiodipyridine (25 I.tM for 15 rain) exhibited the same pattern of inhibitory activity as NEM (n=3), whereas N°Lnitro-L-arginineOMe (100 I.tM) inhibited to the same extent both basal and stimulated (ADP; 3 ~tg) EDRF release (from 28.6 to 4.1% and from 27.0 to 4.6% relaxation respectively; n=3). The inWacellular concentrations of L-Cys and GSH were 0.125 and 1.8 mM (n=5) respectively. Like diamide or CDNB, NEM caused a significant decrease in both inWacellularL-Cys and GSH (58.8 and 70.8% respectively; n=5). We conclude that NEM selectively inhibits the stimulated release of EDRF from cultured endothelial cells, suggesting a fundamental difference between basal and stimulated EDRF release. As the level of L-Cys was similarly affected by diamide, CDNB or NEM, Snitroso-L-eysteine is unlikely to be identical with EDRF. (Supported lay a graDl from Glaxo Group Research Ltd.)

The William Harvey Research institute, St. Bartholomew's Hospital Medical College, Charterhouse Square, London EC IM 6BQ, United Kingdom

Institut fur Pharmakologie, U n i v e r s i t & t zu K61n

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RELEASE OF INTACT EDRF DEPENDS ON INTRINSIC SUPEROXIDE DISMUTASE: IMPLICATIONS FOR ATHEROSCLEROSIS A. Mfigge*, J.H. Elwell, and D.G. Harrison

METABOLISMOF GLYCERYLTRINITRATE: SUICIDE INHIBITION OF THE MICROSOMALGLUTATHIONE S-TRANSFERASE E,. L~ibold and J. Werrin~loe~

Endothelium-derived relaxing factor (EDRF) is rapidly inactivated by radicals. It is not known which of the endothelial antioxidant defense mechanisms preserve the release of intact EDRF. Antioxidant defense was impaired in isolated rabbit aorta by inhibiting catalase activity with 3-amino-l,2,4-triazole (AT, 5 raM), or by inhibiting superoxide dismutase (SOD) with diethyldithiocarbamate (DETC, 5 raM). Pretreatment with DETC markedly reduced acetylcholine and A23187induced relaxation, and, to a lesser extent, endothelium (EC)independent relaxation with nitroprusside, Pretreatment of cultured bovine endothelial cells (BAEC) with DETC did not alter the release of nitrogen oxides (measured by chemiluminescence), but, the effluent of treated cells showed m a r k e d depression in vasodilator activity (measured by bioassay). Pretreatment of rabbit aorta with AT did not alter EC-dependent and independent relaxations. Pretreatment of B A E C with buthionine (S,R)-sulfoximine (20 hours, I mM) resulted in depletion of glutathione content by about 85%, but did not alter the release of nitrogen oxides or vasodilator activity. Thus, imbalance of intrinsic SOD activity and production of superoxide anions or related radicals may predispose to impaired EDRF-mediated relaxations. This imbalance may contribute to impaired EDRF-mediated relaxation in atherosclerosis. To test this hypothesis, we increased vascular SOD activity in normal and atherosclerotic (AS) rabbits (1% cholesterol-fed for 4 months) by pretreating these animals with polyethylene-glycolated (PEG) SOD (41,000 U/kg/d i.m.) for 7 days. PEG-SOD pretreatment increased vascular SOD activity two-fold in normal and AS rabbits. This pretrealment partially restored to normal the impaired acetylcholine and A23187-induced relaxation in the isolated aorta of AS rabbits, PEG-SOD treatment did not alter ECdependent and independent relaxation in the isolated aorta of normal rabbits. Thus, impaired EDRF-mediated relaxation in AS may be due to augmented E D R F degradation by radical species, rather than reduced EDRF-production.

The enzymatic degradation of organic nitrates is of part i c u l a r interest regarding the generation of n i t r i c oxide ('NO) which mediates the activation of guanylate cyclase. Studies were carried out, therefore, to investigate the role of the microsomal glutathione S-transferase (GST) in the metabolism of glyceryl t r i n i t r a t e (GTN). Ion chromatographic methods were employed f o r the determination of n i t r i t e and nitrate and spectrophotometric procedures f o r the measurement of "NO via analysis of i t s co-oxidation with oxyhemoglobin (Wolf & Werringloer, Biol. Chem. HoppeSeyler 368:1142,1987). Using rat l i v e r microsomes the f o l lowing results were obtained: I. The degradation of GTN by the membrane-bound GST resulted in the formation of "NO and n i t r i t e (Vmax = 1 resp. 50 nmol min- I mg p r o t e i n - l ) ; only very low levels of nitrate were detected. The substrate a f f i n i t y of the GST in this novel pathway f o r the generation of "NO (Km < 200 ~M GTN) was found to exceed the a f f i n i t y of the corresponding cytosolic enzyme(s) by more than one order of magnitude, 2. The formation of "NO and n i t r i t e declined progressively during the metabolism of GTN. This decline of product formation was due neither to substrate or cofactor depletion nor to the presence of molecular oxygen, 3. Incubation of microsomes with GTN in the presence of GSH resulted in a concentration dependent inactivation of the GST. This was reflected in an attenuation of the formation of "NO and n i t r i t e from GTN and of the conjugation of l-chloro-2,4-dinitrobenzene (CDNB) in re-isolated microsomes. Further, in microsomal fractions prepared from luminal tissue portions of porcine aortae a GSH-dependent conjugation of CDNBwas detected. This a c t i v i t y was also observed to be susceptible to inhibition by pre-incubation with GTN in the presence of GSH, suggesting a possible role of the membrane-bound GST in the vascular generation of'NO and the developement of tolerance to GTN.

*Present address: Kardiologie, Medizinische Hochschule Hannover, D-3000 Hannover 61, Germany; Cardiovascular Division, University of Iowa, Iowa City, IA 52242, USA

Institut f~r Toxikolo~ie der Eberhard-Karls-Universit~t, Wilhelmstr. 56, D-7~O0 T~binEen I, F.R.G.

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A P P L I C A T I O N OF THE POLYMERASE CHAIN REACTION FOR THE m R N A M E A S U R E M E N T OF THE C O M P O N E N T S OF THE RENIN A N G I O T E N S I N SYSTEM IN THE H E A R T M. Paul, J. Wagner, Y. Liu, N. Niedermaier

EFFECTS OF DES-ARG-BRADYKININ ON STIMULATED SYMPATHETIC OUTFLOW AND BLOOD PRESSURE OF PITHED SHR AFTER ACUTE AND CHRONIC INHIBITION OF THE CONVERTING ENZYME. M~artin Simon and Peter Dominiak Converting enzyme inhibition (CEI) not only inhibits the metabolism of Ang I to Ang II but also the degradation of bradykinin (BK) to biologically inactive fragments. Therefore the antihypertensive effects of CEI are in part dued to accumulated BK. During CEI bradykinin is metabolized, for a less degree, to des-Arg-BK by the enzyme kininase I. Since BK is capable of releasing catecholamines from the sympathetic tissues it was of interest to investigate the actions of des-Arg-BK on stimulated sympathetic outflow and blood pressure of SHR treated with a CE inhibitor.- Ramipril (1 mg/kg x d for 14 days or 0.1 mg/kg i.v.) was administered to male SHR (250 g) for CEI. Animals were pithed by a standard method and the sympathetic outflow was induced by stimulating the thoracolumbar spinal cord (50 mA, 0.5 Hz, 1 ms, 3 min). Blood pressure was measured in a carotid artery and the blood samples for the determination of stimulated circulating catecholamines, as a parameter for the sympathetic outflow, were collected from the contralateral carotid artery at the end of each stimulation period. DesArg-BK was infused during the stimulations at doses of 0.9 up to 27 pg/kg x rain. Circulating stimulation dependent catecholamines were assayed by HPLC and ELCD.- In control and ramipril treated animals (acutely and chronically) blood pressure was dose dependently increased when des-Arg-BK was administered. However, when compared to the control group blood pressure of the ramipril treated SHR increased to a less degree. Stimulation dependent noradrenaline overflow remained unchange during the acute control and CEI experiments. After chronic ramipril administration, however, a weak but dose dependent increase of noradrenaline release could be observed when des-Arg-BK was infused. Concerning adrenaline, a distinct dose dependent increase was existent in the chronically rarnipril treated group during des-Arg-BK.It is suggested that des-Arg-BK is only formed by kininase I if CE is chronically inhibited since the sympathetic outflow was dose dependently enhanced after chronic ramipril treatment and additionally infused desArg-BK. The effects of the latter on blood pressure and catecholamine release are somewhat comparable to BK-antagonistic actions, Institut for Pharmakologie, Medizinische Universit~t zu L0beck, Ratzeburger Alice 160, 2400 L0beck

The presence of the components of the renin angiotensin system (RAS) has been demonstrated in multiple organs. The detection and quantitation of the mRNAs for these components in the heart has been cumbersome, caused by the low mRNA abundance and by insufficient yields of total RNA needed for analysis. To overcome these difficulties we employed the polymerase chain reaction (PCR) as a highly sensitive method for the measurement of cardiac mRNAs. In initial experiments we wanted to detect the mRNA levels of angiotensin converting enzyme (CE) in humans by this method. For this purpose double stranded cDNA was synthesized from human cardiac RNA, which was then amplified by PCR using two primers spanning a region of 380 basepairs of the CE cDNA sequence. Amplified products were visualized after gel electrophoresis, blotting and hybridization to a specific CE probe. Results showed high levels of CE mRNA in atria and ventricles. The regulation of CE mRNA in the heart was then tested in a rat model. Measurements were performed in cardiac tissues of animals with chronic heart failure, induced by ligation of the right coronary artery. Results showed a significant increase in t~E mRNA in the right ventricles of the heart failure animals. We conclude that CE expression is upregulated in the chronic phase of heart failure induced in this model. Furthermore, we were also able to detect renin and angiotensinogen mRNAs in human and rat hearts by PCR analysis. Prelimimary experiments in our laboratory demonstrated increased angiotensinogen mRNA expression in cardiac hypertrophy. Taken together, these experiments provide important information on the role of the tissue RAS in cardiovascular disease. German Institute for High Blood Pressure Research and Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D6900 Heidelberg, FRG

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CHRONIC INHIBITION OF THE CONVERTING ENZYME INCREASES THE EFFECTS OF ANGI AND ANG II ON STIMULATED SYMPATHETIC OUTFLOW IN BROWN-NORWAY-RATS. Paolo Brenner and Andrea BI6chl

Relation between inhibition of angiotensin-converting enzyme (ACE) in vivo and in perfused hindlimbs of the rat H.J.M.G. Nelissen, P.J.A. Leenders, G.P.J. Best, and J.F.M. Smits

Ang I and Ang II, both are capable of releasing adrenaline (A) from the adrenal medulla and noradrenaline (NA) from the sympathetic nerves (Peach 1977, Physiol Rev 57:313). Investigating the probable participation of bradykinin in converting enzyme inhibition (CEI) we examined Brown-Norway-Rats (BNR) because they are lacking of high and low molecular weight kininogen. Since nothing is known about the actions of Ang I and Ang II on the sympathetic system in those rats we studied the role of those peptides during chronic CEI.- Control and defect BNR (250 g) were chronically treated with ramipril (1 mg/kg x d) by gavage for 14 days. Rats were pithed by a standard method and the sympathetic outflow was induced by stimulating the thoracolumbar spinat cord (50mA, 0.5Hz, 1 ms, 3min). Ang~ (33ng up to 3.3 pg/kg x min) or Ang II (10 ng up to 1 pg/kg x rain) were ad.ditionally infused during the stimulations. Blood pressure was measured in a carotid artery and blood samples for the determination of stimulated circulating catecholamines were obtained from the contralateral carotid artery at the end of each stimulation period. Circulating NA and A as parameters for the sympathetic outflow were assayed by using HPLC and ELCD.- Not surprisingly, the pressure responses to Ang I were blunted in both groups of rats when ramipril was administered. However, Ang II exerted more distinct effects on blood pressure increases in the ramipril treated group. In both groups of BNR Ang I increased dose dependently A but not NA, the effects were more pronounced after CEI. Ang II, however, enhanced dose dependently NA but not A in both groups of rats. Chronic CEI Caused a significant enhanced NA release by 100 % in control and defect BNR. A release remained unaffected.Concerning BNR it is concluded that Angl preferentially st!mulates the adrenal medulla whereas Ang II affected the sympathoneuronal system. CEI increased the Angl effects on A-release possibly by accumulation of Ang I and the NA responses to Ang II by sensitization of the presynaptic Ang II receptors. Institut for Pharmakologie, Medizinische Universit~it zu Lfibeck, Ratzeburger Alice 160, 2400 Li3beck

Local renin angiotensin systems (RAS) may play an important role in regional blood flow regulation and may thus also be important in the therapeutic effects of ACE inhibitors. To investigate influences of local RAS without interference of the circulating RAS, we used the perfused hindlimb model. In this model we wanted to determine whether local ACE is active and how several ACE inhibitors inhibit local ACE in relation to in-rive inhibition. The hindlimbs of spontaneously hypertensive rats were perfused with a modified iso-oncotic Krebs-Ringer solution (37°C, gassed with 95% 02 and 5% 02) after carmulation of abdominal aorta and vena cava. Flow in both common lilac arteries and perfusion pressure were measured. Baseline values of pressure and total flow were 43.5-1-0.2 mmHg and 7.8-t-0.2 ml/min (mean+SEM). The effects of injections of [Val~]-angiotensin I (AI) and II (AII) on resistance with and without continuous infusion of enalaprilate (E), lisinepril (L), zabiciprilate (Z) or captopril (C) were determined. The doses for E, L and Z were 25 and 250 rig/rain and for C 0.75 and 7.5 /~g/min. The ratio of the doses corresponded with the ratio of the doses in rive (E, L, Z: 0.1 mg/kg; C: 3 mg/kg). In the hindlimb perfusion model, both AI and AII increased resistance (AI: EDs0 1.64+0.39 #g; AII: ED~o 0.52+0.08 #g; n=8-10). The ACE inhibitors increased EDge for AI dose-dependently and significantly without a significant effect on the dose-response curve of All. The effects of ACE inhibition in the hindlimb perfusion model and in rive are summarized below (n=6-12; ED~0: /zg AI; ACE activity plasma at t=15 min after injection: nmol/min/ml). control E L Z C EDgehindlimb 1.64+0.39 3.71+0.58 3.69+0.78 2.655:0.21 9.095:1.1 EDgein rive 0.21+0.03 1.455:0.26 4.235:0.96 1.935:0.42 2.385:0.73 ACE activity 136.85:6.8 1.39+0.66 1.85+0.70 1.515:0.58 7.655:2.3 In conclusion, local ACE is active in this hindlimb perfusion model and can be inhibited dose-dependently by ACE inhibitors. Based upon results in vivo and in the perfused hindlimbs, C seems to have the most pro-nounced local action of the ACE inhibitors tested. Dept. of Ph~macology, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands

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ANGIOTENSINOGEN AND THE ACUTE PHASE REACTION C.Klett, M. Soden, E.Hackenthal Since during experimental inflammations (e.g. injection of lipopolysaccharides or partial hepatectomy) increased plasma concentrations of both angiotensinogen (Ao) and of acute phase proteins (APP) are observed, Ao is often considered as an acute phase reactant. We have shown, however, that injection of turpentine, a classical inflammation model, significantly decreased plasma concentrations and hepatic secretion of Ao, although two typical APP (~lacid glycoprotein,~2-macroglobulin) were stimulated. To further examine the relationship of Ao to APP we have studied the effects of interleukin 6 (IL6) and interleukln 1 (ILl), two eytokines involved in the induction of APP, on plasma concentrations, hepatic secretion rate, and mRNA concentrations of Ao in the rat, in comparison to the behaviour of the two APP mentioned above. Injection of IL6 (i00 000 U, i.p.) increased plasma Ao from 847 ~ 53 to 1109 ~ 49 pmol/ml at 12 h, a n d ~ l - a c i d glycoprotein from 34 ~ 3 to 268 J ii pmol/ml. Isolated perfused livers taken from 15 h IL6-treated animals showed enhanced secretion for both proteins (Ao: 2.6 fold, control: 6.5 ~ i.i fmol/mg ww/h, ~l-acid glycoprotein: 6.7 fold, control: 3.0 ~ 0.5 pmol/mg ww/h). In isolated hepatocytes, IL6 (500 U/ml) stimulates Ao secretion from 99 J 17 to 167 ! 18 fmol/mg ww/h, and Ao mRNA from 6.0 ~ 0.3 to 8.9 ~ 0.8 pg/Dg total RNA, as well as the secretion of ~lacid glysoprotein from 52 ~ 5 to 306 ~ 27 fmol/mg ww/h. These direct effects of IL6 could be amplified by subacute doses of 5 nM dexamethasone. In contrast, ILl decreased Ao secretion to 56 % of control, whereas ~i-acid glycoprotein secretion was stimulated 2.7 fold. In conclusion, Ao cannot be considered as a typical APP since its regulation by ILl is contrary to that of other APP. Furthermore, during experimental inflammation the induction of APP occurs 12 h later than that of Ao indicating a more complex mechanism for the induction of APP.

DISTRIBUTION OF ANGIOTENSIN II RECEPTOR SUBTYPES IN RAT BRAIN. N. Obermfiller+, J. Culman+, M. de Gasparo* and S.P. Bottaa'i*.

Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 6900 Heidelberg, F.R.G.

Angiotensin II (Ang II) infusion in the brain has been shown to elicit several responses. Recently, two receptor subtypes for Ang II, AT 1 and AT2, which differ in their binding properties as well as in theh" signal transduction mechanisms have been described. In order to adress the role of these receptor subtypes in mediating the central effects of Ang II, we determined tile concentration of both receptor subtypes in fourteen areas of Wistar rat brain selected for theh putative role in Ang II action. The homogenized tissues were incubated for 90 rain. with 0.2 nM [125I]-SarllleSAng II to deterraine total binding. Bound was separated from free by filtration . Non-specific binding was measured in the presence of 1 ~tM Ang II. The proportion of AT1 and AT 2 receplors were determined in the presence of the selective AT l antagonist Dup 753, 2-butyl-4-chloro- 1 -[(2'.-( 1H-tetrazol-yl)biphenyl-4-yl)-methyl]-5(hydroxymethyl)imidazol, (l~tM) and AT 2 antagonist CGP 42112A, nicotinic acid-Tyr-(Na-benzyloxycarbonyl-Arg)-Lys-His-Pro-Ile-OH (50 nM). These concentrations correspond to approx. 100 x their Ki for the receptor for which they have the highest affinity and are approx. 100x below their Ki for the other subtype. The distribution of Ang 11 receptors appears to be very heterogenons. Whereas it is low in the cortical areas, it is high in the hy-pothalamns. Within the hypothalaraus, the Ang II receptor concentration is also unevenly distributed; e.g. it is particularly high in the periventricular nucleus, but at the limit of detection in the supraoptic and preoptic medial nuclei. Their concentration is also high in the median eminence. Almost all Ang I-I receptors present in most of the investigated areas are of the AT~ type. AT 2 receptors were only found in significant concentrations in the pefiventricular nucleus and in the median eminence. These findings suggest that the known central effects of Angioter~sin II are mediated by AT 1 receptors, whereas AT 2 receptors probably mediate as yet unknown effects of this peptide. Deutsches Institut ffir Bluthochdruckforschung, Pharmakolog. Inst. Univ. Heidelberg D-6900 (+) and Cardiovascular Research, Ciba-Geigy, CH-4002 Basel (*).

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CHARACTERIZATION OF THE ANGIOTENSIN II RECE~£OR SUBTYPE IN RAT ILEUM

ROLE OF ANGIOTENSIN II RECEPTOR ANTAGONISM IN THE PROGRESSION AND REGRF~SION OF CARDIAC HYPERTROPHY IN RATS AFTER AORTIC BANDING. W. Linz, U. Athus, R. Henning and W. Scholz

M. Schinke, H.N.Doods I and M. Entzeroth I Two different angiotensin II receptor subtypes can be distinguished by their differential affinities for the nonpeptide antagonists DuP 753 (2-n-butyl-4-chloro-5hydroxymethyl-l-[(2"-(IH-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazol) and PD 123.177 (EXP655, l-(4-amino-3methyl-benzyl)-5-diphenylacetyl-4,5,6,7-tetrahydro-imidazol[4,5-c]pyridine-6-carboxylic acid) (Chiu et al., Biochem..Biophys. Res. Commun. 165, 196-203). Specific binding sites for angiotensin II have been identified in the smooth muscle layer of the rat intestine (Cox e t a l . , Br. J. Pharmac., 87, 201-209). In the present study we characterized the angiotensin receptor subtype in rat ileal membranes using binding and functional experiments. 125I-Sarl,IleS-angiotensin II binds to rat ileal membrane preparations with a K d of 0.26 nM and a Bma x of 98 fmol/mg protein. DuP 753 could be shown to be a potent competitor with a Ki of 12.7 nM, whereas PD 123.177 did not displace the radioligand in concentrations lower than i0 ~M. Furthermore, angiotensin II and III contracted ileal smooth muscle preparations with -log EC50 values of 8.19 and 7.69, respectively. These responses were potently inhibited by DuP 753 with pA 2 values of 7,53 and 7.68. Schild regresssion revealed a slope not different from unity. PD 123.177 was almost inactive with pA 2 values lower than 5.0. It is concluded that angiotensin II and III evoke a potent contractile response in ileal smooth muscle by interaction with angiotensin II receptors which are of the AT 1 subtype. Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, F.R.G iDept. Biochemical Research, Dr. Karl Thomae GmbH, POB 1755, D-7950 Biberach, F.R.G.

Angiotensin II (ANG II) is a putative growth hormon affecting vascular smooth muscle and cardiac cells. To evaluate its role in the progression and regression of hypertrophy of the myocardium rats were subjected to banding of the abdominal aorta. For studying the effect of ANG II antagonism on progression of hypertrophy the animals were treated with the ANG II receptor antagonist 2-n-butyl-4-chloro-5-hydroxymethyl- 1-[2'-(1H-tetrazol-5yl)biphenyl-4-yl)mthyl]imidazole, potassium salt (DuP 753) for 6 weeks starting immediatly after surgery. In another experiment where the influence of the antagonist on regression was investigated the animals received the compound six weeks after surgery when cardiac hypertrophy was expected to have developed. Sham operated (SHAM) as well as untreated animals with aortic banding (CON) were used for comparison. In CON mean arterial blood pressure and heart weight was increased 6 weeks after surgery in comparison to SHAM. However, in the group which had received the ANG II antagonist (3mg/kg/day) the increase of blood pressure was significantly lower whereas heart weight was slightly but not significantly reduced. In the regression study blood pressure and heart weight of CON remained on a high level during the following 6 weeks of the experiment, whereas a significant decrease of both parameters was seen in the ANG II antagonist treated group. These results suggest, that the presence of increased concentrations of ANG II at the receptor level is not only important for the development of cardiac hypertrophy in pressure overlaod, but it is also necessary to maintain the elevated mass of the cardiac tissue. Hoechst AG, W-6230 FrankfurdM. 80, Germany

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A n g i o t e n s h l I does not contribute to striated muscle arteriolar tone in conscious spontaneously hypertensive rats M.W.J. Messing, H. van Essen, H.A.I. Struyker Boudier

VASCULAR BUT NOT PLASMA RENIN ACTIVITY IS DECREASED AFTER CHRONIC TREATMENT OF RATS WITH LY$O$OMOTROPIC DRUO$ Ruth Coonen and Lntz Hein

Vasenlar tone is determined by different structural, metabolic, neural and endocrine influences on the vessel wall. To evaluate the contribution of several of these factors on striated muscle arteriolar tone, male, 5 weeks old spontaneously hypertensive rats (SHR), were implanted with a dorsal microcirculatory chamber. Four weeks later experiments were performed. Mean arterial pressure (MAP) was measured from an intra-arterial catheter, drugs were injected through an intravenous catheter. Arterioles, ranging from A I (60-100 #m) to A3 ( < 3 0 #m), were recoreded on video tape for off-line analysis of diameter changes, using an image shearing device. Bolus injections of the following drugs were given: dthydropyridine calcium antagonists (DHP's) nifedipine and felo~ipine (3-30 #g/kg), ,-adrenoceptor antagonists prazosin ("1:0.1 mg/kg) and yohimbine (-2:1 mg/kg), potassium channel opener lemakalim !30-300 #g/kg) and angiotensin-converting enzyme inhibitors (ACEI s) captopril (3-30 mg/kg) and enalaprilat (30-300 pg/kg). Solvent was used for control experiments. All drugs lowered MAP by 10-20%. D H P ' s and lemakalim dilated arterioles at lower doses, but higher doses were needed for the larger ones. Prazosin dilated only large arterioles, while yohimbine dilated all ty.p.e.s. ACEI's, on the other hand, had no significant dilatory effect on either vessel type. From these data we conclude that angiotensin II does not contribute to the in vivo striated muscle arteriolar tone in conscious SHR. University of Limburg, Dept. of Pharmacology, P.O. Box 616, 6200 MD Maastricht, The Netherlands.

In recent years biochemical and functional evidence has been obtained that the blood vessel wall contains ~ components of the renin-angiotensinsystem, thereby contributing to local regulation of vascular tone. The cellular localization of the different components is unclear at present. During biosynthesis, renal renin aqulres phosphomannosyl residues that enable it to bind to the mannose 6-phosphate receptor and to be transported to lysosomes (Faust et al., J. Cell Biol. 105, 1947~ 1987). The aim of the present study was to investigate in rat portal vein whether lysosomes contribute to renin-like activity of isolated vessels. As an experimental model for disturbance of lysosomal catabolism the induction of a generalized lysosomal storage disorder by chronic administration of di-cationic amphiphilic drugs was used (Hein et al., Xenobiotica 20, 1259, 1990). Wistar rats were chronically treated with the lysosomotropic compounds chloroquine and tllorone (3-6 weeks, 65-90 mg/kg • day p.o.), respectively. Using a small vessel myograph, no differences of the contractile responses to K ÷, norepinephrine, angiotansin II and angiotensin I were observed between portal veins from rats treated with chloroquine or tilorone and control rats. Upon addition of the renin substrate tetradecapeptide, the vessels of untreated rats developed a vasoconstriction. The responses to the renin substrate were considerably smaller with portal veins from drugtreated rats in comparison with vessels from control rats. Administration of a low-sodium diet to the rats had no influence on the responses of portal veins. However, the low-sodium diet caused an increase of the plasma renin activity from 22 _* 3 to 65 +_ 7 ng angiotensin I/ml • h. No effect of the drug treatment on the plasma renin activity was observed. Upon electron microscopical examination both, iuxtaglomerular ceils of the kidney and vascular smooth muscle cells contained inclusion bodies typical for lysosomal storage of polar lipids after chloroquine or tllorone treatment. Thus treatment of rats with di-cationie lysosomotropic compounds inhibits the conversion of renin substrate to angiotansins in isolated vessels but has no measurable influence on the ability of the kidney to release renin. Dept. Pharmacolo~', University of Kiel, Hospitalstr. 4, D-2300 Kiel I

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METABOLISM AND DISTRIBUTION OF ANGIOTENSIN I (ANG I) IN THE BLOOD VESSE~ WALL P. Goblke, P. Biinning

CHARACTERIZATION OF TRANSGENIC RATS TGR(mRen2d)27 WITH SEVERE HYPERTENSION

A local generation of ANG II in different tissues has gained increasing interest in view of possible local actions of converting enzyme iultibitors (CEI). In this study the metabolism and distribution of ANG I as well as the distribution of the CEI ramiprilat in the blood vessel wall was investigated using an in vitro system, the isolated thoracic rabbit aorta.

Henln le one of the candidate-genes for hypertension. We have generated transgenlo rat4, harboring the ren-2d gane of the mouse (Nature 344: 541,1990). These animals exhibit exlremely high blood pressure despite low plasma renln. Northern blotting anelyels revealed transgene mRNA In various tissues among which the adrenal gland was outstanding. The DNA elements of the ren-2d gene responsible for preferenclel expresslon in the adrenal gland were examined by Investigating the 5'flanking region of the ren-2d gene linked to a chlorsmphenlcol eeetyltransforase {CAI~ reporter gene. These studlee allowed the definition of cls- elements responsible for adrenal expression of the ron-2d gene. In addition to the adrenal gland, ran-2 ~'ansgene mRNA was detected In various tissues, e.g. brain, fat, skin, muscles, spleen, gastrointestinal 1tact and thymus. Charactarlstic changes were observed at different times of development. In the kidney the t;'anegene was expressed at high levels in the embryonic stage and than decreased. In other tissuee levels of t~ansgene expression were low in the embryo and then increased In the newborn and were high throughout the ilfespen (brain, small intestine). Adrenal renin was high in fetus and remained high from embryonic ~tuga till 12 weeks. The data obtained with RNase protection as~.~ were largely confirmed by histological studies and in sltu-hybridization, using a S-labelled mouse renin-RNA probe as well as by immunonyteehemical studies. At an age of 6 months mete trendgenie rats developed dramatic pathological ohaogee in kidney morphology (mssanglel proliferation, dilatation of Bowman's sepeule and endstage giomeruloselereeis). Aoda and coronary arteries of the heart revealed ee increased media thickness; cardiac hypertrophy was aiee observed. These date show that TGR(mHen2)27 ~'eesgenle rate display eli typical characteristics of hypertensive pathology, but at a much a~elieretud rate. The fact, that these changes occur In animals with a single gene difference to normoteeelve controls makes them particulerly suitable for pethophyeleloglsel and pharmacological studies.

The incubation of ANG I (3x10"9M) in the aortic lumen resulted in a partial generation of ANG II-immtmereactivity (14% after 60 min.). Most of the luminal ANG I was degradated to peptid-fragments or diffused as intact ANG I /or as peptid-fragments into the vessel wall. Incubation studies with -ANG I revealed that ANG I or ANG I-fragments were mainly distributed in the medial layer of the aorta and to a lesser extent in the adventitia and the endothefium.

W

Denudation of the aorta or the addition of the CEI rumiprilat (10-7M) to the incubation medium lead to a complete blockade of the ANG II generation. Under these conditions there seemed to be an increased catabolism of ANG I by other enzymatic processes. The time dep~endent distribution of the CEI ramiprllat was investigated by incubation of 3H-rumiprilat (10"7M) in the aortic lumen. The ieafibitor qulcldy diffused into the vessel wall, reaching a distribution equilibrium in endothelium, media and adventitia within a few minutes. After incubation of a lower dose of 3H-rumipriint (10"9M) an accumulation of the CEI in the endothelinm and to a lesser extent in the advanfitia could be detected. Our reseats provide no evidence for an extraendothefial generation of ANG II in the blood vessel wall and point to the endothelium as the major site of ANG II generation and major target for the action of CEI. Present address: Department of Ph~trmacology, University of Heidelberg, FRG * Hoechst AG Franlff~rt/Main, FRG

D. GAHTEH, M. BADER, J. PETERS, S. BACHMANN, YI ZHAO, M. LEE, J. MULUNS

German institute for High Blood Pressure Research and Department of Pharmacology, Univers;ty o! Heidelberg, Im Neuenhelmer Feld 366, D-6900 Heidelberg, FHG (Supported by DFG/SFB3|7; NIH R01 HL45468-01; and EEC Trens-Gen-Eur).

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THE TRANSGENICmRen2 RAT: CARDIOVASCULARCHARACTERIZATION . Oliver Chung, Thomas Schips , The transgenic mRen2 rat (TGR) is a hypertensive animal model with an additional mouse Ren2 renin gene in Its geoome, that has been developed using transgenic techniques. We compared the cardiovascular responses of TGR to intravenous bolus applications of phenylephrine (PHE), angiotensin II (ANG II), sodium nitroprusside (NNP), hexamethonium (HEX) and 2- n- butyl4- chloro- 5- hydroxymethyl- 1- [(2'- (1H- tetrazol- 5- yl)biphenyl- 4- yl)methyl] imidazole (DUP), a selective ANG II AT1- receptor antagonist, with those of spontaneously hypertensive rots (SHR) and normotensive Spmgue Dawley rats (SD). In addition, we exposed the animals to a defined cold stress (4°C/30min) and examined the plasma catecholamine levels. Mean arterial blood pressure (MAP) was 100+__6mmHg in SD, 146_+2.4mmHg in TGR and 144_+2.8 mmHg in SHR. PHE (0.1-10 ug) and ANG II (1-100 ng) led to equal increases in MAP, but the bradycardia was attenuated in TGR and SHR compared with SD. NNP (0.1-10 ug) caused a comparable decrease of MAP and increase of heart rate (HR) among all animals. HEX (20 mg/kg) led to a greater decrease of MAP in TGR (-72+4.2 mmHg) and SHR (-70+__4.6mmHg) than in SD (-47+5 mmHg). DUP (10 mg/kg) significantly lowered MAP in TGR (-15+3.5 mmHg) and SHR (-13_+2.3 mmHg) but not in SD (-2+__0.9mmHg). The increase in plasma norepinephrine, epinephrine and dopamine under cold stress did not differ significantly among groups. Our data show similar cardiovascular responses betweenTGR and SHR. Both

CHARACTERIZATION OF NEUTRAL ENDOPEPTIDASE 3 . 4 . 2 4 . 1 1 (NEP) IN THE K I D N E Y : C O M P A R I S O N B E T W E E N NORMOTENSI~/E, G E N E T I C A L L Y H Y P E R T E N S I V E ~ EXPERIMENTALLY HYPERTENSIVE RATS R. S C H U L Z r Y. SAK/~NE r C. BERRYr R.D. G H A I N E P is t h e m a j o r ~ d e g r a d i n g e n z y m e in t h e kidney, t h e r e f o r e s u g g e s t i n g a p o s s i b l e r o l e for t h i s e n z y m e in v o l u m e a n d b l o o d p r e s s u r e r e g u l a t i o n . T h e o b j e c t i v e of t h i s s t u d y w a s t o d e t e r m i n e if d i f f e r e n t i s o f o r m s of N E P e x i s t in D O C A - s a l t a n d S H R r a t s a n d t h e i r n o r m o t e n s i v e c o n t r o l s . The p r e s e n c e o f d i f f e r e n t i s o f o r m s of N E P in t h e v a r i o u s h y p e r t e n s i v e rat m o d e l s w o u l d h a v e r e l e v a n c e w h e n s e a r c h i n g for n o v e l N E P i n h i b i t o r s . W e t e s t e d for p o s s i b l e d i f f e r e n c e s in e n z y m e p r o p e r t i e s of N E P f r o m k i d n e y c o r t e x h o m o g e n a t e s in t h e f o l l o w i n g rat m o d e l s : S H R vs WKY and Sprague Dawley DOCA-salt h y p e r t e n s i v e rats v s c o n t r o l . N o r e l e v a n t differences were found when comparing the f o l l o w i n g p a r a m e t e r s for NEP: (I) s p e c i f i c a c t i v i t y (mean: 204 U / m g p r o t e i n ) , (2) M i c h a e l i s c o n s t a n t (mean Kin: 280 ~M), (3) ICs0 o f t h i o r p h a n (mean: 6.5 riM) a n d p h o s p h o r a m i d o n (mean: 54 nM), (4) p H p r o f i l e s ( o p t i m u m at p H 8.0), (5) h e a t i n a c t i v a t i o n p r o f i l e s ( h a l f - l i f e 20 m i n at 65 °C), (6) i m m u n o t i t r a t i o n of k i d n e y c o r t e x h o m o g e n a t e s , (7) m o l e c u l a r w e i g h t as d e t e r m i n e d b y gel f i l t r a t i o n (92000) a n d (8) a f f i n i t y c h r o m a t o g r a p h y w i t h c o n c a n a v a l i n A. W i t h o u t e v i d e n c e for t h e p r e s e n c e of d i f f e r e n t N E P i s o f o r m s in d i f f e r e n t h y p e r t e n s i v e rat m o d e l s , it is u n l i k e l y t h a t d i v e r g e n t f i n d i n g s in D O C A - s a l t rats a n d S H R u s i n g a g i v e n N E P i n h i b i t o r are d u e t o IqEP i s o f o r m s . Research Department, Pharmaceuticals Division, C I B A - G E I G Y C o r p o r a t i o n , Summit, N.J., U S A

strains exhibit an attenuated

HR bamreceptor reflex to pressor stimuli

compared with normotensive SD. Similar to SHR TGR exhibit partial dependence of the increased basal blood pressure on ANG II and the sympathetic nervous system. Our results display no evidence for specific hemodynamic differences between the genetic hypertension of adult SHR and TGR. *Present address: Department of Pharmacology, University of Heidelberg, INF 366, W-6900 Heidelberg, FRG Deutscher Titel: Die transgene mRen2 Ratte: kardiovaskul&reCharakterisierung 290 C H R O N I C S A L T R E T E N T I O N IN R E C I P I E N T S OF GRAFTS FROM SPONTANEOUSLY HYPERTENSIVE (SHR). C. G r a f a n d R. R e t t i g

292 RENAL RATS

R e n a l s a l t r e t e n t i o n is o f t e n a c c u s e d of b e i n g a p r i m a r y c a u s e for a r t e r i a l h y p e r t e n s i o n . A l t h o u g h it has b e e n s h o w n p r e v i o u s l y t h a t h y p e r tension in S H R can b e t r a n s m i t t e d b y r e n a l t r a n s p l a n t a t i o n to n o r m o t e n s i v e r e c i p i e n t s , t h e u n d e r l y i n g m e c h a n i s m s h a v e not b e e n e l u c i d a t e d . We subjected bilaterally nephrectomized recipie n t s (male a d u l t F1 hybrids) of k i d n e y s f r o m SHR (n=9) and normotensive Wistar-Kyoto rat (WKY) d o n o r s (n=9) to a l o w - s a l t d i e t (LSD, 0.18%) d u r i n g the s e c o n d w e e k a f t e r t r a n s p l a n tation, immediately followed by a high-salt d i e t (HSD, 1.8%) d u r i n g the t h i r d a n d f o u r t h w e e k . A t the e n d of t h e f o u r t h w e e k , d i r e c t l y measured systolic blood pressure was 195±10 m m H g in r e c i p i e n t s of SHR k i d n e y s vs 142±5 m m H g in r e c i p i e n t s of W K Y k i d n e y s (p<0.05). G l o m e r u far f i l t r a t i o n r a t e (566±44 vs 6 0 6 ± 7 8 ~ i / m i n / g ) and renal plasma flow (1.4±0.1 vs 1.5±0.2 m l / m i n / g ) w e r e s i m i l a r in b o t h groups. A f t e r the t r a n s i t i o n f r o m L S D to HSD, r e c i p i e n t s of SHR kidneys retained significantly more sodium for m o r e d a y s t h a n r e c i p i e n t s of W K Y k i d n e y s (e.g., 2 . 8 ± 0 . 4 vs 1 . 8 ± 0 . 3 m m o l / d a y o n the f i r s t d a y of HSD, p < 0 . 0 5 ) . T h i s w a s d u e to d i m i n i s h e d r e n a l s a l t e x c r e t i o n in the p r e s e n c e of s i m i l a r s a l t i n t a k e in b o t h groups. E x t r a c e l l u l a r f l u i d v o l u m e w a s s i m i l a r in b o t h g r o u p s (45±2 vs 47±2 ml/100g). We conclude that chronic renal sodium r e t e n t i o n m a y p l a y a m a j o r r o l e in the p a t h o g e nesis of p o s t t r a n s p l a n t a t i o n hypertension in r e c i p i e n t s of r e n a l g r a f t s f r o m S H R d o n o r s . Dept. of P h a r m a c o l o g y , Univ. of H e i d e l b e r g , Im N e u e n h e i m e r F e l d 366, 6900 H e i d e l b e r g , G e r m a n y

INCREASE IN BRAIN GABA LEVEL BY VIGABATRIN DELAYS THE DEVELOPMENT OF HYPERTENSION IN SPONTANEOUSLY HYPERTENSIVE RATS N. Singewald, A. Pfitscher, A. Philippu It has been reported that the GABA level in the hyp0thalamus of adult spontaneously hypertensive rats (SHR) is lower than that in normotensive rats (WKY), thus pointing to a dysfunction of the GABA system in SEN (J.W. Hambley et al. Neurochem.Int.6:813,1984). TO prove whether GABAergic neurons are involved in the development of genetic hypertension, we treated WKY and SHR with the irreversible GABA transaminase inhibitor vigabatrin and investigated the effects of this compound on blood pressure (BP) and GABA levels in various brain areas. Eight-week-old, five-week-old and one-week-old rats were treated daily with vigabatrin (150 mg/kg, s.c.) for 27, 33 and 55 days, respectively. Systolic BP was measured plethysmographically at the rat tail. One day after the last injection, the brain was removed and GABA determined in the hypothalamus, frontal cortex, brainstem and rest of the brain. The long-lasting treatment with vigabatrin did not influence the development of hypertension either in 8week-old, or in 5-week-old WKY and SHR. In l-week-old SHR, treatment with vigabatrin for 28 days led to a significant decrease in BP (controls: 145± 2 mm Hg, vigabatrin: 133 ± 2 mm Hg, n=6 each). In the following two weeks the BP of the treated SHR remained unchanged, while that of the control SHR steadily increased. Thereafter, the BP of SHR also started to elevate, although vigabatrin treatment was continued. At the end of the experiment, the BP of the control animals was 194 ± 4 mm Hg, that of the vigabatrin-treated rats 173 ± 5 mm Hg. In all animals, vigabatrin led to a decreased body weight. The results show that vigabatrin delays the development of hypertension only in l-week-old SHR. Institut f~r Pharmakodynamik und Toxikologie der Universit,t, Peter-Mayr-Str. I, A-6020 Innsbruck, Austria

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EFFECTS OF CHOLERA TOXIN ON PROTEIN AND GLYCOPROTEIN SYNTHESIS IN AND RELEASE FROM ISOLATED GASTRIC MUCOUS CELLS ~.-K. Heim, A. Oestmann and K.-Fr. Sewin~

HIGH pH-SELECTIMITYVERSUSTHE H,K-ATPase OF THE NOVEL PROTON PUMPINHIBITOR PANTOPRAZOLE W.A.Simon, E.Sturm and B.Koh]

N-acetyl-[*~C]DIncorporation of [SH]L-leucine and into acid-precipitable material was used to glucosamine analyse effects of cholera toxin (CT) on protein and glycoprotein synthesis in and release from isolated (collagenase/pronase) and enriched (>70% by counterflow centrifugation) pig gastric mucous cells, Cells were incubated with the radioactive tracers for 20 h at 37~C in Dulbecco's modified Eagle's medium in the absence (control) or presence of the test compounds. CT alone or in combination with the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) 30 ~mol/l resulted in a significant (p<0.05) stimulation of all four measured parameters.

Basal CT (ng/ml)

0.01 0.I i i0 i00

Protein synthesis release IBMX IBMX + + 100% 120% 100% 108% 132% 149% 100% 116% 152% 164% 123% 129% 167% 175% 124% 155% 169% 184% 134% 151% 176% 189% 144% 144%

Glycoprotein synthesis release IBMX IBMX + + 100% 115% 100% 78% 125% 136% 122% 115% 134% 132% 140% 136% 133% 138% 138% 164% 131% 138% 111% 141% 130% 138% 114% 152%

CT is believed to enhance intestinal secretion of mucus by stimulating AC of intestinal epithelial cells via activation of Gs. Our results argue in favor of a similar stimulatory effect of CT on mucus synthesis in and release from gastric mucous cells. Abteilung Allgemeine Pharmakologie, Medizinische Bochschule Hannover, Konstanty-Gutschow-Str. 8, D-3000 Hannover 61, FRG. Supported by BMFT grant no. 0319279B.

Acid secretion in the stomach is mediated ~y the H,KATPase acting as a proton pump in the apical membraneof the parietal c e l l . In v i t r o acid secretion can be stimulated in isolated gastric glands by dibutyry] cAMP (dbcAMP), histamine (His) or carbachol 1~Carb) and followed by measuring the accumulation of C-aminopyrine (AP). The 2-((2-pyridyl)-methy]sulphinyl)-benzimidazoles (PSBs) are known to i n h i b i t the H,K-ATPase after conversion under acidic condition to the active principle by binding covalent]y and irreversibly to SH-groups of the enzyme. Every PSB should inhibit the enzymeafter complete activation to the same extent. However, the degree of inhibition by PSBs differs remarkably depending on obviously different states of internal acidification. The three PSBs pantoprazole (PAN), omeprazole (OME) and lansoprazole (LAN) were tested in isolated gastric glands after stimulation by dbcAMP (ImM), histamine (tO#M) and carbachol (tO#M). I t was found that after dbcAMP stimulation the inhibitory potency (-log ICbO, mol/l) of PAN (5.5) is 5-6 fold lower than for OME or LAN (6.3), respectively. After stimulation by histamine, PAN and LAN are similar active (6.3} in contrast to the more potent OME (6.7). After the complete activation by Carb the three PSBs are equipotent in inhibiting acid secretion (6.6-6.8). Less a c t i v i t y of PAN in comparison the OME and LAN was also observed regarding the quenching of the fluorescent dye acridine orange in proton pumping gastric vesicles with an obviously higher internal pH than supposed to be necessary for complete activation. The results suggest that PAN is completely activated only at low pH values whereby the extent of inhibition increases with a rise in acid secretion. Forschungslaboratorien, Byk Gulden Pharmazeutika Postfach 6500, D 7750 Konstanz, Germany

294

296

THE NSAID FLUFENAMIC ACID I~CR~ASES THE INHIBITORY EFFECT OF SH-REAGENTS ON GASTRIC H-/K -ATP ASE. W. Beil

HIGH GASTRIC ACIDITY PROLONGS ANTISECRETORY ACTIVITY OF PANTOPRAZOLE, A NOVEL PROTON PUMP INHIBITOR, IN THE DOG S.Postius and U.Br~uer We recently demonstrated that pantoprazole, BY1023/ SK&F 96022, a substituted benzimidazole, displayed enhanced antisecretory efficacy, if administered under conditions of exogenously stimulated acid secretion in the gastric fistula dog (Arch. Pharmacol~_4_l, R69, 1990)~ By contrast, histamine H2-blockers lost efficacy under such conditions. We now aimed at confirming these findings by investigating the impact of pretreatment with H2-blockers on the pHelevating effect of pantoprazole. pH-metry was performed using a combined glass electrode. The data were evaluated by use of both hard- and software from Synectics, Stockholm. Gastric acid secretion was continuously stimulated by s.c. pentagastrin (6 ug x kg-I x h-l) in freely moving dogs from noon to 8 a.m., next day, using an extracorporal minipump (Chronomat, Fresenius)~ Pantoprazole (i0 ~mol/kg i.v.) was administered at 3 p.m. without (controls) or with twice i.v. administration (2 and 4 p.m.) of either cimetidine (84 #mol/kg), ranitidine (6 #mol/kg) or famotidine (0.75 or 1.5 #mol/kg). H2blockers were also administered alone (controls) at the indicated doses, causing profound inhibition of acid secretion for ca. 4 h. Intragastric pH was followed under the various treatments over a total of 18 h. Pantoprazole alone elevated intragastric pH by at least 1 pH unit for ca. 13 h. Combined treatment with pantoprazole plus H2-blocker shortened t h l s p e r i o d by ca. 5 h. The most effiresults clearly demonstrate that pantoprazole ciently elevates intragastric pH, if administered under conditions of strong acid secretion• Under conditions of reduced acid secretion, i.e. during H2-blocker treatment, its duration of pH-elevation will also be reduced. This is best explained by an acid dependent activation of substi(J.Chem. Soc. Chem. Commun., 125-127, tuted benzimidazoles 1986). This feature of the proton pump inhibitor pantoprazole may well fit the clinical requirements, i.e. in DU or Zollinger Ellison syndrome. BYK GULDEN Pharmaceuticals, D-7750 Konstanz

NSAIDs accelerate the reaction between SH-reagents and protein SH-groups in lymphocytes. This effect is attributed to their ability to unmask SH-groups which are normally hidden• inside + + the tertiary structure of proteins. The gastric H /K -ATPase, the key enzyme for proton secretion, is a thiol enzyme. In the present wsrk+we studied the effect of flufenamic acid (FA) on H /K -ATPase-mediated H + transport and on the interaction sf the enzyme with ~ifferent SH-reagents. Methods: H-/K-ATPase-mediated H uptake in intact, inside-out vesicles was monitored with the pH-sensitive dye acridine orange as described (Beil et al., Biochem. Pharmacol. 37, 4487, ~988). ~ F~ in concentrations Sp to 25 ~mol/l failed to affect H /K ATPase driven H uptake. In concentrations 50 ~mol/l FA. + . reduced H uptake and steady-state N concentration. Thls inhibitory effect was not affected by prostaglandin Ep or the dithioerythritol. In vesicles, in whicN the + mercaptane + H /K+-ATPase reaction was stoppsd at the steady-stste level of H uptake, FA enhanced the H dissipation rate. The membrane permeable SH-reage~t 2,2'-dithio-bis-5-nitropyridine (DTNP) inhibited H uptake in a concentrationdependent fashion. Preincubation of the membranes with 25 ~mol/l FA increased the inhibitory action of DTNP. FA also increased the inhibitory action of the membrane impermeable SH-reagent carboxypyridine disulphide (CPDS) b~t not that of omeprazole, which after acid activation selec$ively reacts with intravesicular SH-groups of the H-/~ -ATPase. Conclusions: High concentrations of FA reduce H uptake in intact membrane vesicles by a detergent like effect. Low concentrations of FA enhance the inhibitory action of the S~-rsagents DTNP and CPDS but not that of omeprazole at the H-/K -ATPase. These data indicate that FA unmasks ~H-$roups which are localized at the cytosolic site of the H-/K ATPase. Abteilung Allgemeine Pharmakologie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Str., D-3000 Nannover

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HN-I1203, A NEW H + / K + - A T P a s e INHIBITOR W I T H O U T EFFECTS ON SERUM G A S T R I N LEVELS A T T H E R A P E U T I C DOSES IN DOGS D Sfimmoclar, H. Fellier, H. Stmil3nig, T.P. Pruss

IS ~ SECRETION

HN-11203, 5-(4,5-Dihydro-2-oxazolyl)-2-[(4-methoxy-3,5-dimethyl-2-pyridil)-methyl sulfinyl]-lH-benzimidazole, belongs to the new class of "proton pump inhibitors" which selectively act by blocking the terminal step of gastric acid secretion. The antisecretory effects in rive were evaluated in histamine stimulated, chronic gastric fistula dogs. HN-11203 exerted the same antisecretory efiqcacy but 4-10 fold lower potency compared to omeprazole following intravenous (EDge = 2.3 mg/kg) or oral (EDeo = 9.0 mg/kg) administration, respectively. Moreover HN-11203 has a milder onset of action. Maximal antisecretory effects are not reached before the 3 rd hour after oral administration. As both compounds have the same duration of action 24 hours after administration of the oral EDs0, acid output was still inhibited by about 40 % - the mild onset of action of HN-I1203 is considered to be one reason for the lack of influence on serum gastrin levels as measured in dogs following repeated treatment (1 x daily for 5 days) with the oral EDso. In contrast, the reference compound omeprazole, led to a marked hypergastrinaemia in a similar study as shown in the following table: gastrin levels in % of basal value compound day 1 daoY52 day 3 day 4 day 5 HN-11203 100 o 80 89 124 omeprazole 100 141 610 445 821 (Gastrin control levels were in the range of 24-78 pg/ml, blood samples were taken before the daily drug administration each). One week after treatment, gastrin levels were back to control values. It is postulated that the hypergastrinemia induced by omeprazole is responsible for the gastric tumors observed in rats. Because of this, omeprazole is limited to short term treatment. Thus, if the lack of hypergastrinaemia with HN-I1203 carries over to man, it will favour HN-11203 tbr the clinical use in the treatment and maintenance therapy of peptic ulcer disease. Hafslund Nycomed Pharma AG, Department of Pharmacology St. Peter Strasse 25, A-4020 Linz, AUSTRIA

W.

STI~F~TLATION OF GASTRIC BY M 1 OR NON-M 1 RECEPTORS

ACID ?

Kromer

It is w i d e l y b e l i e v e d that vagal stimulation of gastric acid secretion, mimicked in t h e m o u s e isolated stomach by field stimulation, is m e d i a ted by muscarine M 1 receptors located on neurons and paracrine ceils. However, based on pA 9 values, Black and Shankley (Br. J. P h a r m a c o l . 8__8, 291, 1986) h a v e q u e s t i o n e d that concept and postulated non-M 1 receptors to be operative under this condition. Both atropine and pirenzeplne produced pA 2 values lower than expected. The authors explained the low pA 2 of atropine by potential loss of antagonist from the receptor compartment into the acid stream and that of pirenzeplne by the assumption that non-M receptors were operative. However, the forme~ explanation might similarly explain the low pA 2 value of pirenzepine. We therefore reevaluated the issue by comparing 10 m u s c a r i n i c antagonists . exhibiting both different receptor type selectivities and different absolute affinities. Telenzepine and pirenzepine were used as M 1 - s e l e c t i v e antagonists. The percentages inhibition of field-stimulated acid secretion obtained by 1 ~mol/l of each antagonist were plotted against the antagonist binding affinities t o e i t h e r M1, M 2 o r M ~ r e c e p tors. An affinity-inhibition ~urve was 5nly obtained when its calculation (one site receptor m o d e l ) w a s b a s e d o n M 1 (r = 0 . 7 3 ; p < 0 . 0 2 ) , b u t not M ^ (r = 0.34; p > 0.2) o r M ~ (r = 0 . 5 8 ; p > 0~05), affinities. Thus, our ~ata obtained by this novel method (Kromer, T I P S , i n press) support the concept of M~ receptors to be functionally relevant for field stimulation of acid secretion in t h e m o u s e i s o l a t e d stomach. Byk

Gulden

Pharmaceuticals,

D-7750

Konstanz.

3OO

298 INHIBITION OF H+-SECRETION AND AN OMEPRAZOLE IN THE GUINEA PIG COLON A. Mehlich Ouabain acts as an i n h i b i t o r

(FIELD) ~DIATED

of

OUABAIN-INSENSITIVE

÷ . N-secretzon

of

isolated

ATPASE

BY

mucosa from

guinea pig colon which is probably caused by the activity of a K+~H +ATPase in the brush border-membrane (I). Since i% is ~e11 known that the gastric K+-H+-ATPase is inhibitable by + omeprazole ~e tried to inhibit the H -secretion of guinea pig colon by omeprazole. For this purpose isolated colonic mucosae were mounted in flux chambers as previously described (2). Within an incubation period of one hour the luminal administration of I m~ native omeprazole in a ~eakly buffered + solution~ pH 6.6~ decreased the H -secretion by about 50 % according ~o the buffer titration curve. H+-secretion during a second incubation period ~iih omeprazole-free buffer after ~ashing the mucosae once indicated a partial reversibility of omeprazole inhibition under these conditions. Furthermore the inhibitory effects of ouabain and omeprazole on ATPase ~ere demonstrated in a. homogenate of colonocytes. I mN ouabain reduced . . + + total ATPase actzv~ty zn a Na , K -medium to 90 %. The same ~c%ivity (90 %) yielded an incubation mixture without sodium using Tris-ATP. Therefore ~e concluded that ouabain ~ilI inhibit only the kTP~se ~hich can be activated by sodium. Addition of I m~ ouabain plus 0.2 ~ acid-activated omepr~zole reduced the initial MPase-activity to 72 %. kddition of o~eprazole alone decreased the activity to 82 %. So the effects of both inhibitors are additive suggesting that each inhibitor is specific for one distinct ATPase. These results are ~n contrast %o the finding that o~eprazole as ~e11 as ouabain inhibits the H+-secretion of the isolated mucosa. It is proposed to use the ouabain-insensitive omeprazole-inhibitable kTPase as a so far missing marker enzyme for brush border-membranes of colonocytes. (I) Suzuki, Y. and Kaneko, K.; Am. J. Physiol. 25~, G 979 - G 988 (~98g). (2) L~uterbaeh, F.; Naunyn-Schmiedeberg's Arch. Pharmacol. 29~, 201 ZlZ (1g77). krbeitsgruppe 81ochemische Phar~akologie, kbteilung for Ph~rmakologie und Toxikologie~ Ruhr-Universit~t, D-%~O ~OCHU~

THE RELATIVE ROLE OF 5-HT AND CYCLIC NUCLEOTIDES IN ENTEROTOXIN,-1NDUCED INTESTINAL FLUID SECRETION.

E. Beubler, G. Horina, A. Schirgi-Degen, P. Badhri, C. Pothoulakis*, and J.T. LaMent*

It is commonly accepted that enterotoxins act on intestinal electrolyte and fluid transport via cyclic AMP-, cyclic GMP- or Ca-dependent mechanisms. Recent studies also suggest a role for 5-hydroxytryptamine (5-HT) and PGE 2 in enterotoxin-induced fluid and electrolyte secretion. We therefore investigated the involvement of all these mediators in cholera toxin (or)-, heat-stable E. cell enterotoxin (ST)- and clostridium difficile toxin A (TxA)-induced fluid secretion in the rat jejunum in rive. The changes in luminal and mucosal mediator levels following exposure to these secretagogues is summarized below. luminal

or ST TxA

5-HT + 0 0

mucosal PGE2 + n.s. +

cAMP + 0 +

cGMP n.s. +

( + ) = increase, (0) = no change, (-) = decrease, n.s. = not studied The 5-HT2 blocker ketanserin and the 5-HTa blocker tropisetron each partially and in eombinatinn totally inhibited CT- and ST-but not TxA-induced secretion. Even total inhibition of the CT- or ST-effect was accomplished without reducing the enhanced cyclic nucleotide levels. Indomethacin inhibited the effect of all toxins by about 50 %. These findings suggest the secretory effect of CT and ST is mediated largely by 5-HT release which in turn activates 5-HT2 receptors on neuronal sturctures and induces PGE2-release via 5-HT2 receptors on the enterocyte. Exactly what role the cyclic nucteotides play in enterotoxin-induced secretion remains unclear.

Dept. of exp. & clin. Pharmacology, University of Graz, Austria and *Section of Gastroenterology, University Hospital, Boston, MA.

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R E D U C T I O N OF D U O D E N A L M U C O S A P E R M E A B I L I T Y B Y M U T U A L L Y

EFFECTS

D E P E N D E N T ACTIONS O F H C O 3 - A N D C A 2+

G A L L B L A D D E R EPITHELIUM

H. J. Macherey

K.-U. Petersen~ M. Reuter, and J. M. Winterhager

Effects of HCO 3- and Ca ~÷ on permeability of guinea-pig duodenal epithelium were investigated in stripped preparations mounted in Ussing-type chambers. Tracer fluxes (No ÷, CI-, mannitolfM) and electrical conductance (Gt) were found to be straightly correlated. In the presence of HCO 3- (20 mM), a change from Ca~+-free to Ca ~+containing solutions (1.2 raM) caused significant decreases in G t (from ~, 25 to = 16 mS/cm ~) and unidirectional fluxes of Na ÷ (j~a : from 9.2 -+ 0.9 to 6.8 + 0.7 #mol/cmeh; J ~ : from 8.6 + 1.1 to 5.5 -+ 0.8 ~tmol/cm2h; n=19-20). Smaller reductions were found in the absence of HCOs- that were significant only in jNa Ca 2+ being ins" added, G t and flux rates of Na ÷, CI-, and M were lower in HCo3-containing than in HCO3--free media. In HCO3--free Ringer's, P~/Psa' P~/Pct' and PNa/Pct (P=permeability) were close or equal to ratios predictable from free solution diffusion coefficients; in HCO3-containing Ringer's, ratios became different or more different from predicted values. As compared to HCO~--free conditions, HCOssignificantly reduced P,/P~e. There was a straight correlation between conductivities of differently composed HCOs--free salines (No + and/or C1- substitutions) and G t of tissues bathed therein, that did not embrace the data point representing HCO 3- Ringer's. Pretreatment with cytochalasin D diminished the HCOs--inducible decrease in G t. We conclude that HCO 3- reduces paraeellular solute flow by an action dependent on Ca ~+ and sensitive to cytochalasin D. Without HCO 3being added, Na +, CI-, and M permeate the epithelium mainly by watery structures; HCO 3- activates or enhances some barrier able to distinguish between M and Na +, the occluding junctions being the most likely target. Institut ftir Pharmakologie, RWTH Aachen, D-5100 Aachen, FRG

Effects of secretin on electrolyte transport by guinea-pig gallbladder epithelium were studied in isolated preparations mounted in Ussingtype chambers. Tissues were continuously short-circuited to determine tissue conductance (Gt) ~ short-circuit current (Ise), and transepithelial voltage (Vms). Unidirectional fluxes of Na * and C1- were determined simultaneously by means of radioactive tracers, those of HCO 3- by pH~ stat titration (from mucosa to serosa: Jmst from serosa to mucosa: Jsm)" Serosal addition of seeretin caused concentration-dependent increases in Gt, Isc, and (lumen-negative) y~s~"At 13.1 #mol/1, seeretin raised net HCO 3- secretion by 1.5 #mol/cm h, due to inhibition of Jm (from 1.2 to 0.7 #mol/cm2h;. n=9) and stimulation of Jsm (from 3.7 to 4.7 #mol/cm2h; n=9). The associated increase in Is¢ by 3.8 btmol/cm2h (up from 0.5 #mol/cmZh) was not different from the evolving net HCO 3secretion (4 /tmol/cm~h) suggesting that the latter accounts for the Ise. Thus secretin converts HCO 3- secretion from an electroneutral into an electrogenic process. Neither No* nor C1- net fluxes did contribute to secretin-induced Ise: Na + (2.7 /~mol/cm2h) and C1- (2,5 btmol/cm~h) were absorbed at equal rates in the presence of secretin. Before exposure to secretin, respective net fluxes were 4.6 and 6.4 #mol/cm2h. The inhibition of Jnet was solely due to reduction of Jm (n=23-25). Jst, (nffil6) remained unchanged. The excess in net (electrogenie) HCO 3secretion over (electroneutral) absorption of NaC1 explains the longknown ability of secretin to transform gallbladder fluid absorption into secretion. In all cases examined, secretin effects equalled those of cAMP and PGE 1 we have previously described. Since secretin is known to stimulate gallbladder cAMP synthesis, our results are consistent with cAMPmediated inhibition of apical membrane Na/H exchange (leading to inhibition of Jm of HCO~-, No*, and C1-) and induction of eleetrodiffusive pathways for anions at the apical membrane (leading to electrogenic HCO 3- secretion). Institut f~r Pharmakologie, RWTH Aachen, D-5100 Aachen, FRG

302

304

H202-INDUCED INCREASE IN PERMEABILITY IN THE VASCULARLY PERFUSED RAT SMALL INTESTINE

ROLE

Tobias Schwarz S o m e m a j o r f o r m s of s h o c k a r e a s s o c i a t e d w i t h d r a s t i c p e r m e a b i l i t y c h a n g e s in t h e gut. I n t h i s c o n t e x t w e w e r e i n t e r e s t e d in i n v e s t i g a t i n g p h y s i o l o g i c a l l y i m p o r t a n t m e d i a t o r s , which reversibly alter microvascular permeability in small intestine, The isolated, vascularly and luminally perfused rat s m a l l i n t e s t i n e is a s u i t a b l e m o d e l s y s t e m f o r s t u d y i n g t h i s problem. W e u s e d m a l e W i s t a r r a t s ( 2 0 0 - 250 g). T h e p e r f u s i o n of t h e v a s c u l a r a n d l u m i n a l s y s t e m s w a s m a d e in a n o p e n m o d e . T h e v a s c u l a r i n f u s i o n of H 2 0 ~ r e s u l t e d i n a d o s e - a n d t i m e d e p e n d e n t r e d u c t i o n of t h e x ; a s c u l a r p e r f u s i o n f l o w a t c o n stant vascular perfusion flow and pressure. The enhancement of m i c r o v a s c u l a r f l u i d p e r m e a b i l i t y in r a t s m a l l i n t e s t i n e w a s a s s o c i a t e d w i t h a f l u i d m o v e m e n t f r o m t h e v a s c u l a r to t h e luminal side. This phenomenon was reversible. The increase in m i c r o v a s c u l a r p e r m e a b i l i t y i n d u c e d by a t w o m i n u t e s inf u s i o n of 1 0 0 / ~ M I-I202 w a s r e v e r s e d by a n a u t o r e g u l a t i o n m e c h a n i s m . T h e r e d u c t i o n of t h e v a s c u l a r p e r f u s i o n f l o w ind u c e d by a c o n t i n u o u s i n f u s i o n of 1 0 0 / ~ M H 2 0 2 w a s a n t a g o n i z e d by i n f u s i o n of 1 / ~ M s o d i u m - n i t r o p r u s s i d e . T h i s sugg e s t s t h a t e x t r a c e l l u l a r H 2 0 2 is i n v o l v e d in h o m e o s t a s i s of microvascular intestinal permeability. U n i v e r s i t y of C o n s t a n c e , F a c u l t y of B i o l o g y , B i o c h e m i c a l P h a r m a c o l o g y , P O B 5560, D - 7 7 5 0 C o n s t a n c e , G e r m a n y

OF

SECRETIN

OF K - D E P E N D E N T

ON

ELECTROLYTE

TRANSPORT

l-{ S E C R E T I O N IN H C O 3 / O H

BY

GUINEA-PIG

TRANSLOCATION BY

ISOLATED GUINEA-PIG C O L O N I C M U C O S A

G.Sprakties Guinea-pig distal colonic epithelium is able to acidify its lumen by means of an apical membrane H, K-ATPase. The corresponding alkaline equivalents are exported to the blood side. We have investigated the properties of this transport and the handling of exogenous HCO3-. Scraped mucosae were mounted in Ussing-type chambers and shortcircuited continuously. Acid and base transport were measured by a pH-stat system. When bathed in nominally HCO3--free Ringer's, tissues acidified the luminal bath at a rate of 1.8 -+ 0.1 #reel/era 2 h; contraluminal alkalinization determined simultaneously amounted to 1.2 + 0.1 #mol/cm 2 h (n=26). Both titration rates were inhibited by luminal, not contraluminal addition of ouabain (10 -5 reel/l), an inhibitor of colonic H, K-ATPase; methazolamide (10 "~' mol/l, added bilaterally); contraluminal, not luminal addition of SITS (10 -3 reel/l); luminal or contraluminal addition of NPPB (5-nitro-2-(3-phanylpropylamino benzoic acid, 3x10 "5 mol/l), suggesting that alkaline equivalents (HCO3-) cross the basolateral membrane by electroneutral exchange for CI-. There were no changes in transepithelial voltage and short-circuit current that would be expected for the alternative of HCO 3- electrodiffusion. For measurement of absorptive HCO3--fluxas (Jm), HCO 3was present in the luminal bath. The Jm was drastically reduced when luminal K ÷ concentration was set to zero. Restoring K ÷ to 5 mmol/1 prompted an increase (from 1.3 -+ 0.2 to 2.1 -+ 0.3 ltmol/cm 2 h, n=4 ), which was reverted by luminal addition of ouabain. Jm was inhibited by SITS and NPPB (same conditions as above). Rates of the opposite flux, Jsm, were close to those of inhibited J ~ and thus probably represent paracellular flow. In conclusion, apical H, K-ATPase activity gives rise to absorptive HCO 3- flux whleh crosses the basolateral membrane by the same mechanism (C1/HCO3 exchange) as do alkaline equivalents generated during H + secretion. Institut fiir Pharmakologie, RWTH Aachen, D-5100 Aachen, FRG

R 77

305

307

INHIBITION OF POTASSIUM-MEDIATED INCREASES OF INSULIN SECRETION AND CYTOSOLIC CALCIUM OF RINm5F-CELLS BY TERT-BUTYLHYDROPEROXIDE (t-BHP) H.P.T. Ammon, I. Koopmann

ADENINE NUCLEOTIDES AND GLIBENCLAMIDE BIND TO DIFFERENT SITES AT THE SULFONYLUREA RECEPTOR OF MOUSE PANCREATIC ISLETS M.Schwanstecher and S.L6ser

In the pancreatic islets the redox state of the intracellular glutathione, which depends on glucose concentration, is thought to modulate calcium entry and insulin release. RINm5F-cells do not respond to glucose with insulin release. Aim of the study was to investigate the effect of glucose on the redox status of GSH and the influence of t-BHP - a thioloxidant - on GSH/GSSG, cytosolic calcium (Quin2-method), and insulin release (RIA} of RINm5F-cells when depolarized by KCI, In contrast to pancreatic islets the RINm5F-cells even at low glucose (2.8 mM) showed a high GSH/GSSG ratio (30.7 4. 3.6 (mean + S.E.) n=6), which was independent of the glucose concentration. This ratio was decreased by t-BHP (4 mM to 15.2 4- 1.8). t-BHP also inhibited KCI (25 mM)-induced insulin release (0 raM: 6.0 ± 0.6; 0.4 raM: 5.4 + 0.6; 4 raM: 4.6 4. 0.7; 10 raM: 3.6 4- 0.3 taU IRI/laO protein x 60 rain) and the KCI-mediated increase in cytosolic calcium (control: 269 + 61 vs. 4 raM: 92 + 36 nM (n=4). In a concentration range from 1 to 10 mM t-BHP did not affect viability of RINmSF-cells. We conclude that in RINm5F-cells basal GSH/GSSG ratio is much higher than in islets, and can not be modified by a raise of the glucose concentration and therefore here posseses no regulatory function in insulin release. On the other hand in RINmSF-¢ells depolarization-induced calcium entry along the voltage sensitive calcium channels and insulin secretion also seems to depend on a certain redox state of glutathione. Pharm~zeutisches Institut, Universit~t TObingen, Auf der Morgenstelle 8, D - 7 4 0 0 T~Jbingen

Closure of the ATP-sensitive K+channel is induced by sulfonylureas and intracellular adenine nucleotides. However, for adenine nucleotides there was no correlation between the affinity of binding to the sulfonylurea binding site and the potency of blocking the ATP-sensitive K + channel. Evidence was presented recently that MgATP modulates the affinity of the snifonyturea binding site for glibenclamide and diazoxide. Therefore we investigated whether MgATP altered the affinity for binding of adenine nucleotides to the sulfonylurea binding site. In microsomes obtained from mouse pancreatic islets and incubated in the presence of 100 gmol/l MgATP the nonhydrolyzable ATP-analogue AMP-PNP (300 ~tmol/1) did not displace specifically bound 3H-glibenclamide. However, the Mg complex of AMP-PNP induced a concentration-dependent reversal of the MgATP-induced inhibition of 3H-glibenclamide binding. MgATP (100 ~tmol/1) increased the K o for binding of tolbutamide by threefold indicating that the response to MgATP is not restricted to glibenclamide. The data indicate that adenine nucleotides bind neither to the state of the binding site for sulfonylureas which is observed in the absence of MgATP nor to the state which is observed in the presence of MgATP. lnstitut fiir Pharmakologie und Toxikologie der Universit~it Robert-Koch StraBe 40, D-3400 G6ttingen

306

3O8

CHARACTERIZATION OF THE NUCLEOTIDE RECEPTOR REGULATING THE TOLBUTAMIDE-SENSITMTY OF THE ATP-DEPENDENT K + CHANNEL IN PANCREATIC B-CELLS C.Schwanstecher, C.Dickel and U.Panten

EFFECTS OF BETA-ADRENERGIC ANTAGONISTS AND CALCIUM CHANNEL BLOCKER ON CYCLIC AMP-RELEASE IN CULTURED THYROID CELLS. F.W. Jekat

Evidence was presented recently that the cytosolic MgADP complex controls the tolbutamide-sensitivity of the ATP-dependent K+charmel (K-ATP-channel) in mouse pancreatic B-cells by interaction with a receptor site not identical with the site mediating channel closure and that this control is effective in the intact Bcell. In mouse B-cells the inside-out configuration of the patchclamp technique was used to further characterize this receptor site. In the absence or presence of tolbutamide Mg2+complexes of nucleotides were applied to the cytoplasmic face of the inside-out patch. As compared to ADP, ADPgS (0.2 retool/l) or GTP (0.2 - 1 mmol/1) only slightly enhanced the tolbutamide-sensitivity of the K-ATP-channel whereas AMP and GDP were without effect. Due to its strong channel blocking effect, ATP alone could be tested only at a 20 rtmol/1 concentration and did not change the potency of tolbutarnide. However, in conjunction with ADP, which antagonized the inhibitory effect of ATP, ATP (100 ~tmol/1) enhanced the tolbutamide-sensitivity of the K-ATP-channel. AMP-PNP (a nortmetabolized analogue of ATP), too, enhanced the tolbutamide-sensitivity when tested in the simultaneous presence of ADP. The results indicate that adenine nudeotides applied to the cytoplasmic face of the B-cell are effective on the tolbutamide-sensitivity of the K-ATP-chaanel in the order ATP>AMPPNP > ADP > ADPgS > AMP and that the corresponding guanine nudeotides are much less effective. Institut fiir Pharmakologie und Toxikologie, Universit~it G~ttingen, Robert-Koch-Str. 40, D-3400 Giittingen

It is well known that catecholamines are rapid and potent stimulators of intracellular cyclic adenosine-3',5'-monophosphate (cAMP) accumulation of thyroid cells in the presence of thyrotropin (TSH). Thus the effects of betaadrenergic antagonists (propranolol, pindolol) and calcium channel blocker (nitrendipine, nicardipine) on cAMP-production were studied in cultured thyroid cells of the differentiated cell line FRTL-5 both in the presence and absence of TSH. Phosphodiesterase was inhibited to accumulate cAMP in the medium. All test substances were applied at concentrations of i00 ~g/ml, I0 ~g/ml and 1 ~g/ml. At the highest concentration of i00 Bg/ml the betaadrenergic antagonists propranolol and pindolol clearly amplified the stimulating effect of TSH. But at the lowest concentration of 1 ug/ml both substances did not influence TSH-dependent cAMP-production. At the highest concentration both calcium channel blocker counteracted the stimulating effects of TSH on cAMP-production, but such effects were not observed at the moderate and lowest concentration. In the absence of TSH none of the test substances revealed any stimulating or hindering effect on the production of cAMP. These in-vitro experiments show that at a high concentration of 100 ~g/ml all test-substances interfere (in a positive or negative manner) with TSH-dependent cAMPproduction of cultured thyroid cells. On the other hand an almost normal situation was observed at a moderate and low concentration, respectively, and in the absence of TSH. These findings are a basis for both a better understanding of drug effects on the mechanisms of hormone formation and secretion and proper in-vivo experiments to clarify the effects of antihypertensive agents on the thyroid. Institute of Pharmacology and Toxicology, University M~nster, DomagkstraBe 12, D-4400 MUnster, West-Germany

of

R 78 3O9 cAMP STIMULATES H U M A N TSH/~ G E N E EXPRESSION IN P I T U I T A R Y CELLS H.J. Steinfelder, B.D. Weintraub, and F.E. Wondisford The regulation of the expression of the/~-subunit gene of h u m a n thyrotropin (hTSH/~) by the cAMP/protein kinase A system was studied in transient expression experiments. The effect of forskotin or cAMP analogues on chloramphenicol acetyltransferase (CAT) activity was tested in two cell lines that had been transfected with chimeric plasmids containing the C A T gene, the hTSHfl promoter and variable lengths of its 5'flanking region. Forskolin had no effect on hTSH/~ promoter directed C A T expression in 293 cells, a h u m a n embryonal kidney cell line. In pituitary GHa cells transfected with plasmids containing either 1200 or only q28 base pairs (bp) o f hTSH~ 5'flanking D N A forskolin induced CAT activity approximately tenfold. Deletion of 100 bp of the 5'flanking region from -128 to -28 bp resulted in a 50-60% decrease in cAMP induction of hTSHBCAT expression. Further deletional analysis revealed that the 5'flanking region from -128 to -92 bp is essential for maximal induction by cAMP. Deletion of a portion of the first exon from 8 to only 2 bp resulted in an additional decrease in 8Br-cAMP stimulated expression. Thus, it seems likely that in addition to the 5'flanking region (-128 to -92 bp) the area covering the start site of transcription (-28 to. + 8 bp) is involved in cAMP regulation of hTSH/3 gene expression. Cotransfection with a plasmid coding for a protein kinase A inhibitor protein resulted in dose dependent decrease of cAMP stimulation of hTSH/~ gene expression. Therefore, we conclude that the 6-10fold stimulation of hTSH/~ gene expression by cAMP in pituitary cells is mediated by two separate D N A regions, and is the result of the activation of the catalytic subunit of protein kinase A. Institute of Pharmacology, D-3400 G6ttingen, FRG, National Institutes of Health, Bethesda, MD 20892, and Case Western Reserve University, Cleveland, O H 44106, USA.

311 TYLOSIN INHIBITS

THE TESTOSTERONE PRODUCTION IN

MOUSE LEYDIG CELLS IN VITRO M. L. Meisel Tylosin, an antibiotic of the macrolid group, is widely used as a promoter of growth and as a therapeutical antibiotic in food-producing animals. Former own investigations in rats yielded reduced levels of LH and FSH in serum and pituitary gland as well as a decreasead weight of seminal vesicles after 15 days t r e a t m e n t with tylosin. This results indicate an influence on the pituitary-gonadal-axis. To prove if in addition the reactivity of Leydig cells is modified by this antibiotic the following experiments were performed: 1- Mice were treated for 8 days with tylosin and the influence on Leydig cells investigated in vitro. 2-Leydig cells were taken from untreated mice and the antibiotic added in vitro. The technique of isolation of Leydig cells was performed according to Van Damme et al., Acta endocrinologica 77, 655, 1974. Ad 1- In a dose range from 10 mg to 10 pg],kg bw a clearly reduced testosterone production in presence of hLH was observed. Ad 2- Addition of tylosin to incubation medium in a dose range from 1 to 100 /zg/ml caused a dose dependent inhibition of basal testosterone production. LH addition lead to a significant reduced increase in testosterone at a concentration of 10 pg/ml and completely blocked at 100 /~g/ml. These data suggest that tylosin acts directly on testicular tissue, maybe by inhibiting early steps of steroidogenic pathway. Institut ftir Pharmakologie und Toxikologie der Westf~lischen Wilhelms-Universit~it MOnster, DomagkstraBe 12, 4400 Mtinster, FRG

310

312

ANTITHYROTROPIC A C T M T Y OF PLANT CONSTITUENTS "IN VITRO" H. Winterhoff, S. Kleemann u. M. LOcke

THE ~.'w~'~CTOF DIHYDROLISURIDE APPLIED IN VIVO AND/OR VITRO O~ ~ I C LIPOLYSIS. K. Elisova

Aqueous extracts from Lithospermum and Lycopus species showed a pronounced antithyrotropic activity in rats. The thyrotropin (TSH) caused increase in thyroid hormones was reduced or even abolished by these plant extracts. As m o d e of action a direct binding of phenolic plant constituents to the hormone and as a consequence a loss of receptor binding was discussed (Winterhoff, Sourgees and Kemper:

We have studied the effect of dihydrolisuride (DHL) - an inhibitor of prolactin secretion - on adrenergic lipolysis in epididym~l adipose tissue of male rats and parametrial adipose tissue of female rats. We have administered DHL in _v~vo to the experimental animals at a dose of 0.12 mg.kg 24 hours prior to the experiment where the lipolytic activity of noradrenaline in vitro was estimated. The criterion of lipolysis was the amount of free fatty acids released into the incubation medium. The parametrial adipose tissue of female rats was more sensitive to the effect of catecholamines after pretreatment with DHL while the effect on catecholamine induced lipolysis in male adipose tissue was not changed compared to the control groups. We have studied the effect of DHL in vitro also. Addition of different concentrations of DHL to the incubation medium did not affect the catecholamine induced lipolysis both in male and in female adipose tissue. The possible rflechanismof DHL administered in vivo on adrenergic lipolys±s in vitro will be discussed.

Horm. metab01. Res. 1~5, 503, 1983). In vitro studies should clarify, if the cAMP increase following TSH receptor binding was diminished by such active plant-extracts or plant-constituents. For these investigations FRTL-5 cells were cultured according to the methods of Ambesi-Impiombato (Proe. nat. Acad. Sci 7_/7: 3455). Extracts from Lycopus virginicus as well as oxidized rosmarinic acid or caffeic acid inhibited the TSH caused cAMP-increase in a dose dependent manner. Moreover in the same experimental model the stimulatory action of forskolin, a diterpene of Coleus forskohlii, could be antagonised too. These experiments prove that not only a reduced receptor binding but also an attack on intracellular processes must contribute to the activity of these plant constituents. Anywhere comparatively higher amounts of substrate are required to suppress TSH in vitro than in vivo. Additional effects on thyroid hormone binding and metabolism in vivo will be discussed. Institut far Pharmakologie und Toxikologie der Westffilischen Wilhelms-Universitat Mfinster, Domagkstr. 12, D-4400 Mtinster, FRG

IN

st Present address: Dept. of Pharmacology, I Medical School of Charles University in Prague, Albertov 4, 128 000 Prague 2, Czechoslovakia

R 79 313

315

THE ANTIALLERGIC D R U G CROMOLYN IS A POTENT BLOCKER OF CI- CHANNEL ACTIVITY IN RAT MUCOSAL MAST C~LLS (RBL-2H3 LINE). Chr. Romanin, M. Reinsprecht, I. Pecht , H. Schindler.

TUMOR NECROSIS FACTOR (TNF) ITSELF AND INTERLEUKIN 1 INDUCE SELECTIVE HYPORESPONSIVENESS TO CERTAIN EFFECTS OF TNF M. K6nig, B. Janzen, R. Winzen, K. Resch, *D. Wallach, and H. Holtmann. The cytokine TNF is involved in the defense of the body against pathogens. The wide spectrum of effects induced by TNF as well as their potentially lethal consequences demand that responsiveness to TNF is tightly regulated. Our previous studies have shown that TNF itself as well as interleukin i (IL-I), a cytokine which functions similarly to TNF, induce in cells a state of hyporesponsiveness which is maintained in the presence of normal levels of TNF receptors. That hyporesponsiveness was reflected in an increased resistance against the cytocidal effect of TNF (applied with protein synthesis inhibitors) in cells that had been pretreated with TNF or IL-I. It was asked whether the hyporesponsiveness induced by TNF or IL-I is selective for the cytocidal effect of TNF, which develops within several hours, or whether it also affects more rapid responses to TNF: the increase in phosphorylation of the small heat shock protein, HSP27, and the down modulation of the receptors for EGF. In cells pretreated with IL-I or TNF the response to TNF in terms of stimulation of phosphorylation of HSP27 was decreased, as was the response to the cytocidal effect of TNF. In contrast, the down modulation of EGF-receptors in response to TNF occurred in the cytokine-pretreated cells to the same extent as in non-pretreated cells. Thus TNF and IL-I can activate mechanisms which decrease the responsiveness to TNF by affecting an early event in the signalling pathways activated by TNF. That event appears to be involved in certain responses to TNF, while others (e.g. the down modulation of EGF receptors) remain unaffected and thus may be initiated by different signalling mechanisms. The selective modulation of cellular responsiveness to TNF may represent an important element in the coordination of the effects of TNF in the organism. Inst. Molecular Pharmacology, Medical School, D-3000 Hannover, FRG, *Dept. Molecular Genetics & Virology, The Weizmann Institute of Science, Rehovot 76100, Israel.

Crosslinking of IgE-carrying receptors at the surface of basophils or mast cells by the corresponding antigen initiates a cascade of cellular processes culminating in the secretion of inflammatory mediators. In RBL cells cromolyn has recently been shown to inhibit mediator release when introduced into the cells in a membrane-permeant form (Hemmerich et al., Biochemistry in press, 1991). Its antiallergic action has so far been attributed to an inhibition of the antlgen-induced Ca2+ uptake. We investigated now the effect of cromotyn on a CIchannel activity found in RBL cells by employing the patch-clamp technique in the ins/de-out patch recording configuration. Excision of previously mute membrane patches of RBL cells led to the activation of C1" channels in 45 out of 89 membrane patches studie& Channel activation occurred in a slow fashion with activation times best approximated by a single exponential fit with r =7.7 rain. The CI" channels exhibited a slope conductance of 29-+4 pS and a reversal potential close to the equilibrium potential for CI'. Application of cromolyn (1 raM) to the cytoplasmic side of the membrane of an inside-out patch resulted in a complete inhibition of this CI- channel (n=7). Blockage was fast (within 15 see) and fully reversible upon wash-out of cromolyn. A dose response relation, based on the open state probability of the CI" channel, revealed an ICsf~of about 15/xM cromolyn (n=3) and a Hill coefficient of 0.9. The parallelity of inhibition of CI- current and mediator secretion by cromolyn suggests a functional role for CI- channel activity in stimulus -secretion coupling. (Supported by Austrian Research Funds, project S 45-03) * Present address: The Weizmann Institute of Science, Rehovot, Israel Institute for Biophysics, University of Linz, Altenbergerstr. 69, A-4040 Linz, Austria

314

316

PERIPHERAL BENZODIAZEPINE BINDING SITES LYMPHOCYTES AND HUMAN LYMPHOMA CELL LINES B. E. E. Alexander, E. Roller" and U. Klotz

ON

HUMAN

Peripheral type benzodiazepine binding sites (PBR) have been identified in various mammalian tissues such as kidneys, adrenals, heart, testes and some neuronal regions.Whereas the well knownclinical effects of benzodi~zepines are mediated by the central type receptor, a physiologicalrole of the PBR ?artsnot yet been elarlfied. Since it was assumedthat PBR mightbe involvedin the regulationof ceil ~rowth and differentiation, we characterized PBR on different lymphocytepopulat~ousto study the receptor mediated effects of specific ligands. Venousblood was collected from healthyvolunteers and peripheral mononuclear ceils were prepared using Ficoll-Paque"densitygradient centrifugatiun. Pure Tand B-cell fractious were separated by adherence of monoeytesto tissue culture plates. T-cells were isolated on nylon wool colurans. Lymphoma cells were harvested from suspensioncultures. Radioligand-bindlng-studies w i t h [aH]l-(2-chlorophenyl)-N-methyl.N-(1. methylpropyl)-3-isoquinolineearboxamide([ tt]Pk 11195), a specifleligand for the PBR, characterized a single, saturable and high atFanityspecific binding site on different cell populations (values represent mean --- S.D.): cell population

I~ [mM]

Br,~ [fmol/2x 10~ cells]

T+B-lymphoeytes 4.6 -+ 0.3 1142 _+ 320 T-lymphoeytes 6.9 --. 0.9 1043 ~: 13 RAIl (B-lymphomacells) 1.8 -+ 0.5 58I -+ 8 CCRF-CEM (T-lymphomacells) 3.9 -+ 1.7 850 -+ 82 MOLT (T-lymphomacells) no sigxfificantspecificbinding K562 (myelogan.leukemia cells) 2.0 -+ 0.2 517 + 38 We found no significant difference in the maximal binding capacity (B~) between the isolated lymphoeytesbut distinct l~wer Bm~ values for the human lymphoma cell lines. The dissoeiafiun constants (Ko) for [aHlPk 11195 were sfightly lower for lymphomaceil populations. Values for b~di~g capacity and affinity of the easilyaccessible and homogenousCCRF-CEM ceil line population came closest to values for lymphocytebinding. This cell line could therefore be used as a model for further study of PBR. Current displacement studies with different llgands and thek corresponding .effects .on lymphaeyte growth and differentiation will provide additional informatma whether binding to PBR has any physiologicalmeaning. Supported by the Robert-Busch-FoundationStuttgart; Dr. Margarete FiseharBosch-InstitutfiirKlinischePharmakologie,AuerbachstraBe112,W-7000Stuttgart 50, FRG; "Abteilung fiir Hfimatologie, Onkologie and Immtmologie, RobertBnseh-Kraukeahans, Stuttgart, FRG

PHARMACOLOGICAL PROPERTIES OF A NOVEL D R U G C O U N T E R A C T I N G T N F a 1N S H O C K Marcus N i e h 6 r s t e r Bacterial lipopolysaccharides (LPS) are known to induce m.ulti.organ failure and lethal shock, w h e n p r e s e n t in circulau o n , Le. in the state of septicaemia. This activation of leukocyte p o p u l a t i o n s leads to synthesis and release of t u m o r necrosis f a c t o r a ( T N F a ) into the circulation. T N F a was shown to induce all characteristics of LPS shock. P h a r m a c o l o g i c a l interventions were carried out in an in vivo model of LPS- or T N F a - i n d u c e d lethal shock, i.e. intrav e n o u s a d m i n i s t r a t i o n of 10 m g / k g LPS ( s a l m o n e l l a a b o r t u s equi) or 400 /zg/kg r e c o m b i n a n t m u r i n e T N F a ( r m u T N F a ) to BalbC mice. The animals died within the following 72 hours. I n t r a p e r i t o n e a l a d m i n i s t r a t i o n of 100 m g / k g of the methyl xanthine derivative 1-(5-hydroxy-5-methyl)hexyl-3-methyl-7-propylxanthine, (A 802715, H o e c h s t AG, Werk Kalle-Albert, Wiesbaden, F . R . G . ) one h o u r p r i o r to LPS-challenge p r o t e c t e d against LPS-induced shock and lethality, Protection against r m u T N F a - i n d u c e d lethality was achieved by similar p r e t r e a t m e n t with A 802715 in a dose of 200 m g / k g . Protective p r e t r e a t m e n t with A 802715 p r i o r to LPS-challenge did not affect LPS-indueed increase of s e r u m T N F c o n c e n t r a t i o n s as assessed one h o u r after challenge. T h e s e findings suggest that A 802715 is a true inhibitor of TNFa-toxicity r a t h e r than a c o m p o u n d i n t e r f e r i n g with synthesis or secretion of this cytokine. Biochemical Pharmacology, Faculty of Biology, University of Konstanz, D-7750 Konstanz, F R G

R 80 317

319

CONCANAVALIN A INDUCED LIVER INJURY: AN EXPERIMENTAL AUTOIMMUNE DISEASE

THE EFFECT OF ENDOTOXIN AND LEAD ACETATE ON THE HEPATIC UPTAKE OF COLLOIDAL CARBON IN RATS $. B~hring

G i s a Tiegs

Concanavalin A (Con A) caused hepatic failure in several mouse strains, i.e. in NMRI-, Balb c- or C3H/HeJ-mice, when given i.v. in a dose greater than 1.5 mg/kg. Liver injury was assessed by transaminase release as well as histopathological evaluation 8 hrs after Con A challenge. Only the liver was affected. Administration of the immunosuppressing agents dexamethasone (0.5 mg/kg) or cyclosporine A (50 mg/kg) or fujimycin (FK 506, 50 mg/kg) protected against Con A hepatitis, whereas indomethacm did not. Experiments with genetically manipulated mice (SCID. athymic nude) identified T-lymphocytes as effector cells fo~ induction of organ injury. Further evidence was obtained using several monoclonal antibodies against T cells (Thv 1.2 or anti CD4 + plus anti CD8 + ). However, also macrop~age depletion protected the animals. The results suggest that Con A hepatitis is a useful experimental model for autoimmune disease. Biochemical Pharmacology, Faculty of Biology, University of Konstanz, D-7750 Konstanz.

In the l i v e r , K u p f f e r cells which are resident ~nacr~phages are m a i n l y responsible for the elimination of most c i r c u l a t i n g foreign particles. The p h a g o c y t o t i c function is of importance for unspecific and specific d e f e n c e mechanisms and it influences~.the k i n e t i c of d r u g carrier systems. Many reports have indicated that heavy metals can react with cells of. th e immune system and alter humoral immunity and c e l l - m e d i a t e d immunity responses. Lead salts have been shown to induce a profound s e n s i t i z a t i o n to endotoxins in rats. However, many aspects of these reports a r e conflicting. The half live of colloidal carbon elim i n a t i o n was used as a m e a s u r e of the RES activity. The effect of lead acetate, endotoxin or lead acetate and endotoxin t o g e t h e r on the c l e a r a n c e of colloidal carbon were studied in rats and in the isolated p e r f u s e d rat liver. After treatment of rats with acute toxic doses of lead acetate 15,10 mg/kg) the clearance of carbon was inhibited dose dependently. A f t e r adm i n i s t r a t i o n of endotoxin (i mg/kg) the clearance function was enhanced. Simultaneous administration of e n d o t o x i n and lead acetate increases the lethality. The clearance function of the isolated p e r f u s e d rat liver was also influenced dose d e p e n d e n t l y after application of lead acetateI5, 10,20 mg/l) in vitro. A p p l i c a t i o n of endotoxin (20Lug/ml) in vitro lead to an enhanced clearance function. P r e t r e a t m e n t of rats with lead acetate [I0 mg/kg) and a p p l i c a t i o n of endotoxin in vitro impairs the clearance of carbon. Present adress: Institute of P h a r m a c o l o g y and Toxicology, U n i v e r s i t y of Rostock, Leninallee 70, 0-2500 Rostock, G e r m a n y

318

32O

E N D O T O X I N - I N D U C I B L E C Y T O T O X I C I T Y IN LIVER CELL CULTURES

EFFECTS OF SMALL MULTIPLE DOSES OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) ON PERIPHERAL LYMPHOCYTES OF A NEW WORLD MONKEY (Ca/lithrixjacchus) Reinhard Neubert*, Rail Stahlmann, Hans Helge*

T.Hartung and A.Wendel It is known that rodents challenged with a combination of galactosamine and endotoxin develop a fulminant hepatitis. Until now, no in-vitro correlate for this organ-specific lesion was described. Here, in-vitro conditions are established which allow to examine lipopolysaccharide(endotoxin)-inducible cell injury to hepatoeytes. U n d e r these in-vitro conditions which require the presence of functionally intact Kupffer cells, a concentration-dependent lactate dehydrogenase release is inducible by different lipopolysaccharides in hepatocyte cultures from Fischer rats. It can be abrogated by polymyxin B. These coeultures secreted tumor necrosis factor-a upon an lipopolysaccharide stimulus. The presence of a tumor necrosis factor-a antiserum reduced the major part of the endotoxin-indueible eytotoxicity. Further similarities to the in-vivo situation such as the different individual cytotoxic potency of various endotoxin species and the different responsiveness of hepatocytes from two different rat strains support that this coculture system might be useful for studying endotoxin-inducible lesions in-vitro. University of Constance, Biochemical Pharmacology, POB 5560, 7750 Constance, Germany

Although information for man is still scanty and controversial, there is ample evidence that exposure to relatively high doses of TCDD (#g/kg body wtrange) is able to induce several deviations of the immunological functions in rodents. We have found the immune System of Callithrix jacchus (common marmoset) to be exceptionally sensitive to the action of this xenobiotic (R. Neubert et el. Arch Toxico164: 345-359) subsequent to a single dose of 10 ng TCDD/kg body wt. Here we present the results of studies on peripheral lymphocytes during a long-term exposure. TCDD was given once weekly (0.3 ng/kg body wt; s.c.) to mature marmosets for 6 months. This dosing regime is expected (t/2 of 8 weeks) to lead to an about lO-fold accumulation compared with the single dose. Monoclonal antibodies to human lymphocyte markers and flow cytometry (FACScan) were used to analyse lymphocyte subpopulations in venous blood about every 4 weeks. No adverse effects were observed with the dosing regime in this non-human primate. Thus, for a chronic treatment a dose corresponding to daily 43 pg TCDD/kg body wt represents a NOEL (noobserved-effect-level) under these experimental conditions. Subsequently, the weekly dose was raised to 1.5 ng/kg body wt. A significant decrease in the CD4+CDw29+ subpopuiation ("memory cells') and in the CD20 + ("BI") cells, as well as an equally significant increase in the CD8+CD56 + subpopulation could be demonstrated 6 weeks after the change in treatment. The 6 doses may have accumulated to about 5-fold of this single dose, and the total effective dose given during the study may correspond to a single dose of about 10 ng/kg body wt. in fact, the same dose was found before to be effective as after single exposure. When dosing with TCDD was stopped after a total of 34 weeks, the effect on peripheral lymphocytes persisted for another 4 weeks, but no deviation from control values was seen 12 weeks after the end of the treatment. These studies were supported by grant No. 0765002 from the Bundesministerium for Forschung und Technologie (BMFT). Institut for Toxikologie und Embtyopharmakologie, Freie Universit~t Berlin, Gatyatr, 5, D-1000 Berlin 33 und Kinderk/inik* der Freien Universit~t Berlin, Heubnerweg 6, D-1000 Berlin 19

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IN VITRO EFFECT OF FIVE NUCLEOSIDE ANALOGUES USED AS ANTIVIRAL DRUGS ON LYMPHOPOIESIS IN DEVELOPING FETAL RAT THYMUS Michael Foerster, Ulla Kastner, Hans-Joachim Merker, Diether Neubert

EFFECT OF NEUTROPHIL-ACTIVATING PEPTIDE-I (NAP-I)/INTERLEUKIN-8 (IL-8) ON NO-MEDIATED INHIBITION OF HUMAN PLATELET AGGREGATION AND RELAXATION OF RAT AORTIC STRIPS A. Dembi~ska-Kie~*, D. Pallapies and B.A. Peskar

The spreading outbreak of the "Aquired Immune Deficiency Syndrome" (AIDS) has resulted in extensive efforts to find more effective drugs in the therapy of the infections of the HIV-1- and the opportunistic viruses. For this reason the therapeutic use of nucleoside analogues is increasing. Information on toxic effects of these drugs on the perinatal development of the thymus are of special interest and the knowledge of influences on the developing immune system are one prerequisite for a sensible therapy. We have tested the effect of the five nucleoside analogues zidovudine, aciclovir, ganciclovir, 2'-3'-dideoxycytidine and vidarabine phosphate on the in vitro development of thymic lobes of 17-day-old rat fetuses. The lobes were cultivated at medium/air interphase up to 7 days. Aciclovir was added to the medium in concentrations of 30 #M, 100 #M and 300 #M. The investigations showed an influence of the nucleoside analogues on the proliferation of the lymphatic cells. Even the lowest concentration resulted in a significant reduction in the number of thymocytes as compared to untreated controls. In this respect 2'-3'-dideoxycytidine was more effective than aciclovir, ganciclovir and vidarabine phosphate. Zidovudine had the slightest effect. Results of flow cytometry show the decrease in the number of the double positive CD4+CD8 ÷ cells. Ught and electron microscopical investigations indicate effects of the drugs on the thymocytes, whereas the epithelium is only slightly affected. Supported by grant Nr. 07 VDX 019 from the Bundesministeriumfor Forschung und Technologie(BMFT). Institut for Toxikologie und Embryopharmakelogie, Freie Univerait~t Berlin, Garystr. 5, £)-1000 Berlin 33

Polymorphonuclear leukocytes (PMN) have been shown to produce n i t r i c oxide (NO), which i n h i b i t s human p l a t e l e t aggregation. NO is destroyed by superoxide anions (Gryglewski et a l . , Nature 320, 454, 1986), which can also be produced by PMN during stimulation with various chemoa t t r a c t a n t s including NAP-I/IL-8 (Baggiolini et a l . , J. Clin. Invest. 84, 1045, 1989). We have investigated the e f f e c t of NAP-I/IL-8 on PMN-mediated i n h i b i t i o n of platel e t aggregation, Rat PMN (4 - 10 x 105) harvested 12 16 hours a f t e r i . p . i n j e c t i o n of oyster glycogen i n h i b i ted thrombin (45 - 120 mU/ml)-induced aggregation of washed human p l a t e l e t s (2.5 x 108 c e l l s / m l ) . The same number of PMN preincubated with NAP-I/IL-8 (I ~g/ml) at 4oc for I min exhibited a markedly reduced antiaggregatory a c t i v i t y , while preincubation for 8 or more min did not s i g n i f i c a n t l y a f f e c t the antiaggregatory act i v i t y of rat PMN. These results correspond to the time course of the NAP-I/IL-8-induced oxygen burst. NO released from rat a o r t i c endothelial cells, in response to carbachol (50 - 300 nM) causes a concentration-dependent r e l a x a t i o n of a o r t i c strips precontracted with phenylephrine (100 riM). The carbachol-induced r e l a x a t i o n of a o r t i c strips preincubated with NAP-I/IL-8 (500 ng/ml) for I min was s i g n i f i c a n t l y reduced. Restoration of the carbachol-induced r e l a x a t i o n was observed immediately a f t e r change to NAP-I/IL-8 free Krebs-Henseleit buffer. The results suggest that NAP-I/IL-8 can diminish NO-med~ated effects by releasing superoxide anions. The r a t i o NO/superoxide anions produced by leukocytes and vascular tissue might be relevant f o r the expression of proinflammatory effects of NAP-I/IL-8. *Present address: Copernicus Academy of Medicine, Department of Pharmacology, PL-31 531 Cracow, Poland Department of Pharmacology and Toxicology, Ruhr-Universit y , D-4630 Bochum, F.R.G.

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SIMILARITIES AND DIFFERENCES OF THE NADPH-OXIDASE SYSTEM OF HUMAN GLOMERULAR MESANGIAL CELLS AND PHAGOCYTES. H.H. R a d e k e , , O.T.G. J o n e s ' , M, N a k a m u r a ~ Eec~fit~--E~e--~hs~-nt h a ~ - h ~ / h i ~ n gls~a%-{fila}- ~h-e~an~-ial ceils (HMC) c h a r a c t e r i z e d a s s m o o t h m u s c l e l i k e c e l l s release o x y g e n r a d i c a l s in r e s p o n s e to c y t o k i n e s and s u g g e s t e d t h e p r e s e n c e of a p h a g o c y t e - l i k e N A D P H - o x i d a s e . The following results support similarities: Using diphenylene i o d o n i u m (DPI) d e s c r i b e d a s a c o v a l e n t l y b i n d i n g i n h i b i t o r of a c t i v a t e d 45 kDa f l a v o p r o t e i n in n e u t r o p h i l s r a d i o l a b e l e d w i t h *~J i d e n t i f i e d a s i n g l e 45 kDa p r o t e i n b a n d in separated HMC plasma membrane fraction, Direct p o t e n t i o m e t r y of HMC m e m b r a n e s ( - 8 4 0 mV to - 1 6 0 mV) showed a low p o t e n t i a l c y t o c h r o m e (76 I)mol/mg HMC m e m b r a n e p r o t e i n ) , w h i c h so f a r c o u l d o n l y be a t t r i b u t e d to c y t o c h r o m e bama. In s l o t b l o t s t h e 7D5 MoAh s p e c i f i c for the extracellular d o m a i n of t h e a-subunit showed a p o s i t i v e r e a c t i o n w i t h HMC. Moreover, in W e s t e r n b l o t s MoAb 449 d i r e c t e d a g a i n s t t h e c y t o p l a s m a t i c e p i t o p e of t h e s s u b u n i t i d e n t i f i e d a 28 kDa p r o t e i n a n d MoAb 48 r a i s e d a g a i n s t t h e l a r g e (8) s u b u n i t of c y t o c h r o m e baaa of human neutrophils detected a smear between 75-100 kDa in d e n a t u r e d HMC m e m b r a n e p r o t e i n . H o w e v e r , on a f u n c t i o n a l l e v e l t h e r e a r e i m p o r t a n t d i f f e r e n c e s c o m p a r e d to t h e r e s p i r a t o r y b u r s t o x i d a s e : The O f - r e l e a s e of HMC w a s n o t s u i c i d a l . D e s p i t e t h e r e l a t i v e l y h i g h c o n t e n t of c y t o c h r o m e has, calculated by the potentiometrlc studies (20% of n e u t r o p h i l s ) t h e r a d i c a l r e l e a s e of a d h e r e n t HMC w a s low (3 to 10 nmol O~-/10~ HMC x h o u r ) b u t c o n t i n u e d f o r u p to 6 h o u r s . A l t h o u g h c o m p l e t e l y i n h i b i t a b l e b y SOD, DPI (up to 25 NM), so f a r , f a i l e d to i n h i b i t O~- r e l e a s e in b o t h a d h e r e n t a n d s u s p e n d e d HMC. Moreover, p o t e n t s t i m u l i of n e u t r o p h i l s a s p h o r b o l e s t e r (PMA), FMLP or LTI~ h a d o n l y m a r g i n a l or no a c t i v i t y a t all, w h e r e a s HMC r e l e a s e d h i g h a m o u n t s in r e s p o n s e to c a l c i u m i o n o p h o r e s (A~,a7 ) or cytokines. This data demonstrate that, although the p r i n c i p l e c o m p o n e n t s a r e h o m o l o g o u s in p h a g o c y t e s and HMC, t h e r e are important differences in the signal transduction mechanism and the efficiency of the NADPH:O~- o x i d o r e d u c t a s e , M o l e c u l a r P h a r m a c o l o g y , Medical School; H a n n o v e r , F.R.G.; ~ Biochemistry, University Bristol, U.K., z I n s t . Medical Science, Tokyo, Japan.

INTERFERENCE OF GLUCOCORTICOIDS WITH THE ADHERENCE OF M O N O C Y T I C CELLS M. Goppelt-Strtibe an~ A. EmmendSrffer* M0nocytes are c h a r a c t e r i z e d by their ability to adhere to p l a s t i c of other supports in vitro which c o r r e l a t e s w i t h their interactions with in vivo. To study the efffect of glucocorticoids on adherence we u s e d the m o n o c y t i c cell line U937 as modell system . Adherence of these cells is effectively induced by the phorbolester TPA. The morphological changes are accompanied by the expression of the adhesion molecules CD 54 (ICAM1) and CD18. Incubation of the cells with glucocorticoids such as prednisolone or dexamethasone m a r k e d l y inhibited cell adherence and the expression of the surface molecules. The mlneralocorticoid aldosterone or the gestagene progesterone had no effect on these processes. The T P A - d i f f e r e n t i a t e d U937 cells remain in their d i f f e r e n t i a t e d state for up to three weeks even in the absence of TPA. Incubation of 2weekold cells w i t h glucocorticoids for 3 days resulted in m a r k e d morphological changes and loss of adherence. This reorientation was accompanied by a reduced expression of proteins known to be involved in cell-cell interactions (CD54 (ICAM i) and CDI8). Preliminary experiments indicate similar effects of g l u c o c o r t i c o i d s on the in vitro differentiation of h~Iman p e r i p h e r a l blood monocytes. Zentrt~m P h a r m a k o l o g i e und Toxikologie, Abt. Molekularpharmakologie, Medizinische Hochschule Hanno~er * F r a u n h o f e r Institut f~r Toxikologie, Abt. Immunologie, W-3000 Hannover 61, ~RG

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INHIBITION BY BOSWELLIC VITRO AND IN VIVO H. S a f a y h i

and

ACIDS

OF

5-LIPOXYGI~NASE IN

T. Mack

Boswellic acids ( a - and (~-BA), l l - k e t o - 8 - B A and t h e i r acetyl d e r i v a t i v e s were i s o l a t e d from the gum r e s i n of b o s w e l l l a ~errata and c h a r a c t e r i z e d by MS, IR, UV and ~HNMR s p e c t r a and by t h e i r melting points. G l y c o g e n - e l i c i t e d r a t p e r i t o n e a l PMNL were s t i m u l a t e d by Ca~÷/A23187 to produce the 5-1ipoxygenase (5-LO) products l e u k o t r i e n e B~ (LTB~) and 5-HETE (H.Safayhi, G.Tiegs, A.Wendel, Biochem. Pharm. ~.~, 2691-2694, 1985). BA and d e r i v a t i v e s s i g n i f i c a n t l y reduced the formation of LTB~ and 5-HETE, whereas steroidal-type antiinflammatory drugs (e.g. prednisolone 10-~ 10-~ M) had no immediate effect on t h i s system. Among BA, acetyl-11-.keto-~-BA induced the most pronounced i n h i b i t i o n of 5-LO product formation in a c o n c e n t r a t i o n dependent manner with an IC~8 at 2 pM. In c o n t r a s t to BA, 18-~-glycyrrhetinic acid (also a pentacyclic t r i t e r p e n e ) did not affect 5-LO a c t i v i t y at comperable concentrations. Acute h e p a t i t i s was induced in NMRI mice by i.p. injection of galactosamine/endotoxin. Liver damage was a s s e s s e d by serum t r a n s a m i n a s e a c t i v i t i e s 8 h later. BA (1500 mg/kg, p.o.) s u b s t a n t i a l l y and s i g n i f i c a n t l y p r e v e n t e d acute h e p a t i t i s . Since i t was p r e v i o u s l y shown t h a t i n h i b i t o r s of 5-LO are effective in this system (A.Wendel and G.Tiegs, Biochem. Pharm..8.~, 2115-2118, 1986) we s u g g e s t t h a t the in vltro demonstrated suppression of leukotriene production by the BA might contribute to i t s benefits in the h e p a t i t i s model and to the reported in v l v o a n t i inflammatory q u a l i t y of the gum r e s i n crude e x t r a c t s of b o s w e l l i a s e r r a t a (G.B.Singh and C.K. Atal, Agents Actions 18, 407-412, 1986). Dept. of Pharmacology, Pharmaceutical Inst., U n i v e r s i t y of Ttibingen, Auf der Morgenstelle 8, 7400 Tfibingen, FRG

326 PROANTHOCYANIDINES AND OTHER STRUCTURES AS DUAL INHIBITORS OF ~YSO-PAF TRANS/~ETYLASE AND 5-LIPOXY~ENASE B. Quern#[ , F. Petereit , F. v. Bruchhausen , A. Nahrstedt Constituents of Cistus incanus ssp. tauricus have been attributed to antiinflammatory effects (Petereit et a l . , Planta Medica 55, 650, 1989). Now we have studied the influence of some gallocatechins isolated from this plant in v i t r o on both microsomal lyso-PAF acetyltransferase (LPTA; EC 2.3.1.67) and 5-1ipoxygenase (5-LOX; EC 1.13.11.34) preparations in comparison to some other structures. The compounds were: I. gallocatechin(4~-8)catechin ; I I . gallocatechin(4~-8)gallocatechin as proanthocyanidins; I I I . (+)gallocatechini IV. g a l l i c acid ; V. piriprost; VI. nordihydroguaiaretic acid (NDGA). The inhibitions of the enzymes revealed as reductions of their radiolabelled products PAF or oxidized arachidonic acid metabolites, respectively, on thin layer plates were expressed as the corresponding IC~ values (I~M). As shown in the table some gallocatechin ffErivatives principally inhibited both enzymes similar to gossypol and some other bicatechols (v. Bruchhausen et a l . , Arch. Pharmacol. 342, R6, 1990). A:ICSO I II Ill IV V VI LP-TA 70 140 >1000 >1000 300 100 B:IC50 5-LOX 100 100 80 80 15# 6,7 A/B 0,7 1,4 > 100 > 100 20 15 # data from literature Other lipoxygenase inhibitors like EP 10045 (2-Hydroxy4(isopentanylmercapto)phenol), L651896 (dihydro(hydroxymethyl)phenylpropenyl-benzoflurano|)0 esculetin or tiron had no i~hibitory potency on LP-TA added in concentrations of 3"10TM M. The dual inhibitory effect in i t s e l f is of interest because some diseased states like asthma bronchiale or c o l i t i s ulcerosa are discussed for their participation of both mediators, PAF and leukotrienes, ~espectively. Inst. f. Pharmakp~ogie, FU Berlin, Thielallee 67-73, D1000 Berlin 33; Inst. f. Pharmazeutische Biologie und Phytochemie, Uni MUnster, Hittorfstr. 56, 4400 MOnster

THE MODULATION NUCLEOTIDES W.Gielen

OF S I A L Y L T R A N S F E R A S E

BY C Y T I D I N E

The serum sialyltransferase deserves further interest as a m a r k e r of m a l i g n a n c y and as an indihat~r i n the e a r l y s t a g e of inflammation. The t r a n s f e r a s e a c t i v i t y d e p e n d s on the intact glycoprotein s t r u c t u r e of the e n z y m e molecule. S i a l i d a s e t r e a t m e n t r e d u c e s the e n z y m e activity significantlyand in c o r r e l a t i o n to the amoun% of s i a l i c a c i d h y d r o l y z e ~ . T h e s o l u b l e e n z y m e is not m o d u l a t e d by f a c e s i a l i c a c i d or galactose as it has b e e n s h o w n r e c e n t l y for the membrane bound sialyltransferase on the lymphocyte surface. A n o t h e r r e g u l a t i o n s y s t e m s e e m s to be r e p r e s e n t e d by the c y t i d i n e n u c l e o t i d e s CMP, C D P and CTP. T h e d e g r e e of i n t e r a c t i o n d e p e n d s on the e n z y m e origin. The h u m a n s e r u m s i a l y l t r a n s ferase a c t i v i t y is s t r o n g l y r e d u c e d e v e n at low concentration (0.25-1.0 mM) by all 'three nucleotides, w i t h C D P as the b e s t i n h i b i t o r and w i t h a r e m a i n i n g a c t i v i t y of o n l y 35%. The sialyltransferase a c t i v i t y in the rat serum is e n h a n c e d by all c y t i d i n e n u c l e o t i d e s tested, at a c o n c e n t r a t i o n of 0 . 2 5 m M C M P up to 190%, considering that C M P is" l i b e r a t e d from CMPsialic a c i d in the s i a l y l t r a n s f e r a s e reaction. An i n h i b i t i o n of the r a t s e r u m s i a l y l t r a n s f e r a s e c o u l d be o b t a i n e d at and a b o v e a concentration of 1 mM nucleotides. The differences of the modulation may probably be related to the different amount of ~ 2 , 6 - s i a l y l t r a n s f e r a s e and ~2,3-sialyltransferase present in both sera, e a c h of the i s o e n z y m e s p o s s e s s i n g an individual s e n s i t i v i t y to the c y t i d i n e n u c l e o t i d e s . Institute of Pharmacology, C o l o g n e , G l e u e l e r Str. 24, 5000

University C o l o g n e 41

of

328 SIMULTANEOUS MEASUREMENT OF WHOLE BLOOD CHEMILUMINESCENCE IN MICROTITER PLATES AS AN EFFECTIVE DRUG SCREENING SYSTEM V. Kaever and J.T. Robitzsch Human polymorphonuclear leukocytes and monocytes are able to produce large amounts of reactive oxygen species (ROS) upon appropriate stimulation. The measurement of ROS in whole blood by amplified chemiluminescence (CL) may therefore provide a simple method reflecting the activation stage of such immunocompetent cells in various disorders and just as well to determine the influence of different drugs. We have established an assay system in a microtiter plate format luminometer that permits the simultaneous handling of up to 96 samples. Using heparinized venous human blood from healthy donors optimal CL intensities were determined at a 100-fold blood dilution in a total volume of 0.25 ml of Hank's balanced salt solution containing 0.4 mM luminol as CL enhancer and either opsonized zymosan (i mg/ml) or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) (I0-6 M) as stimuli. Under these conditions maximal ROS production was found after 5 - 10 min. In order to demonstrate the usefulness of this system for the efficient screening of drugs which either change the cellular activation stage or directly interfere with the ROS synthesis the in vitro effects of nordihydroguaiaretic acid (NDGA), dipheny]ene iodonium (DPI), and dic]ofenac were tested. After preincubation of the diluted whole blood with the drugs for 15 min the zymosan-stimulated CL was diminished in all cases. The specific NADPH oxidase inhibitor DPI was most effective (IC~o = 1.5 x lO-8 M) whereas higher concentrations of the antioxidant NDGA (1.6 x 10 -6 M) or the nonsteroidal antiinflammatory drug diclofenac (1.4 x lO-~ M) were needed to achieve halfmaximal inhibition. Institute of Molecular Pharmacology, Medical School, Konstanty-Gutschow-Str. 8, D-3000 Hannover 61, FRG

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329 CHEMILUMINESCENCE GENERATION BY GUINEA-PIG ALVEOLAR MACROP~AGES J. Schmidt, R. Lindstaedt and I. Szelenyi

.,'~'.r.zt'T OF PHARMACOLOGICAL AGenTS ON THE SYNI~IESIS OF THE AflrfE-P}~SE REACI~NT ALPHAI-ACID G L Y C O ~ (AAG) IN RATS WITH CARRAGE~AN-I~VJC~D PAW O~D~4A E. Koch

A/veolar macrophages are believed to play a critical role in inflsm,mtory reactions in the lung, both by the generation of a number of specific mediators and by serving as effector cells in response to i~fls,m~tory mediators. One important proi~fl-mm-tory mediator generated by alveolar macrophages is the phospholipid

AAG is a plasma protein whose synthesis is dramatically increased during the so-called acute-phase reaction (APR) in the rat. Tne APR is an immediate host defence mechanism to infectious or inflanm~tory stimuli. It includes fever as well as changes in the plasma concentration of proteins and ions. The APR is mediated by interleukin-l, interleukin-6 and tumor necrosis factor. Pharmacological intervention in the production of these cytokines represents a major effort in the development of innovative new drugs. Detection of acute-phase proteins offers a convenient way to i r ~ e c t l y investigate the effect of agents on the production of inflaran~tory cytokines. Pat hind paw oedema is an established model for testing drugs on inflanm~tcry reactions. Tne aim of the present study was therefore to exam/ne if evaluation of antiphlogistic agents in this test system may be cc~bined with the search for c ~ which influence production or activity of mediators of the APR. l>aw oedema was induced by the subplantar injection of 0.i ml 1% carrageenan. The resultant swelling was quantified 4 h later. After 24 h a blood sample was collected and the serum concentration of A~E was measured by single radial i ~ f f u s i o n or cc~c petitive ELISA. Substances tested included corticosteroids, non-steroidal anti-inflau~atory drugs, antiarthritic agents and immuncsuppressants. The results indicate tbmt the local inflammatory response and the production of AAG are individually influenced. Thus, it seems reasonable to combine this assay for anti-inflammatory properties with the evaluation of drug effects on the APR. Such an approach could be helpful in characterizing the activity profile of potential new agents.

CHARACTERIZATION

OF

PAF-IND~CED

platelet activating factor (PAF). The aim of the present study was to investigate the effector function of the alveolar macrophages in response to PAF. Cultured guinea-pig alveolar Emcrophages stimulated by PAF showed a concentration-dependent increase in the generation of superoxid anions by the membrane enzyme NADPH-oxidase as measured by means of incigeninchemilu~tinescence (CL). Optimal stimulato~ concentrations of PAF were found in the range of I0 -M. Lyso-PAF. the inactive precursor molecule and metabolite of P~KF, was completely inactive. It is likely that the signal transduction pathway is calcium-dependent. Depleting extracellular calcium by the calcium chelator EDTA as

well as inhibiting intracellular calcium-fluxes by TMB-8 resulted in a marked inhibition of the eL-response to PAF. An inhibitory effect was also observed following the treatment with a calmodulin-antagonist W-7. In addition, inducin 8 elevated cytosolic cAMP-levels by application of PGE 2 or forskolin modulated the PAFinduced eL-response in an inhibitory manner. On the other hand, the calcium-dependent enzyme protein kinase C (PKC) seems not to be involved in the signal transduction since PKC-inhibitors staurosporine and H-7 proved to be completely inactive. ASTA

Pharma

AG,

Department

of

Pharmacology,

Weism~llerstr. 45. 6000 Frankfurt/Main, FRG

Department of Pharmacology, Dr. Willmar Postfach 410925, 7500 Karlsrlthe 41, F.R.G.

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EFFECT~ CE THE PHTHALAZINE]bE AZE]~TINE PROL IFERAT ION M. Kietzmann, D. Lu~:~ch 1 a n d M. N o l l i e r e 2

IN

EPIDERMAL

Azelastine is a phthalazinone derivate with antiallergic e99icacy. The drug exerts a histamine H I- and PAF-antagonistic activity and inhibits also the release o÷ 5lipoxygenase products. Because o~ the putative role o~ these mediators in the regulation oE epidermal matabolism, epidermal e~Gects o9 azelastine ~ere measured in ~emale NMRI-mice. The substance ~as administered orally over 7 days (i, 3, 5 mg/kg per day). ThereaEter a mechanical skin irritation was perEormed by abrasio oE super~icial epidermal layers. BeEore and aEter skin irritation, tail skin ~as sampled. The workup oE the skin specimens was ~oll~ed by the measure~ent o~ the incorporation rate o9 3H--thymidine triphosphate, 3H-isucins and 3H-histidine and the epidermal concentration o9 prostaglandins and isukotrisnss. Additionally, epidermal thickness and cell number ~ r e measured histometrically. In unirritated epidermis, azelastine induced a ~ecrease o9 the thymidine triphosphate and amino acid incorporation rate. Within 1 hour agter mechanical skin irritation, an increase o~ PGD2 and LTC4 ~as ~ound. Cc~pared to controls, the increase oG LTC4 was diminished and that oE PGD2 was enhanced in mice treated ~ith azelastine (i mg/kg) be9ore skin irritation . The increase oE the thymidins triphosphate incorporation into DNA ~as also diminished by azelastins. No signiGicant digEerences o~ the amino acid incorporation rate ~ere measured det~een azelaetine treated and control mice. Compared to controis, the epidermal thickness (maximum 3 days aGter skin irritation) was enhanced by azelastins. The results demonstrate that azelaeti~, which inhibits the release oE 5-1ipoxygenase products, induces a decrease o9 epidermal proliferation reactions. Institut 9~r Pharmakologie, Toxikologie und Pharmazie, TierArztliche Hochschule Hannover und 1Hautklinik Linden, 3000 Hannover, 2ASTA Pharma AG, 6000 FrankEurt, FRG

Schwabe GmbH,

GLYCOSAMINOGLYCAN POLYSULFATE (GAGPS} STIMULATES THE SYNTHESIS OF FIBRONECTIN (FN} BY CULTURED ARTICULAR CARTILAGE CHONDROCYTES

J. S t e i n m e y e r I, N. B u r t o n - W u r s t e r 2 a n d G. L u s t 2 Several studies disclosed that osteoarthritic c a r t i l a g e of s e v e r a l s p e c i e s c o n t a i n s u p t o 20 times more FN than disease free cartilage and t h a t d e g e n e r a t e d c a n i n e a r t i c u l a r c a r t i l a g e exp l a n t s s y n t h e s i z e 3 t o 5 t i m e s m o r e FN. T h e a i m of o u r p r e s e n t s t u d y w a s t o i n v e s t i g a t e t h e e f f e c t of a p u t a t i v e c h o n d r o p r o t e c t i v e agent, GAGPS, o n FN, t o t a l p r o t e i n a n d D N A s y n t h e s i s as w e l l as on e x t r a d o m a i n A c o n t a i n i n g F N (EDA FN) a n d k e r a t a n s u l f a t e c o n t e n t of c h o n d r o cyte cultures. - Chondrocytes were isolated from adult canine articular cartilage and c u l t u r e d in H a m F12 m e d i a s u p p l e m e n t e d w i t h 10% F N d e p l e t e d f e t a l b o v i n e serum. A t t h e init i a l s e e d i n g G A G P S w a s a d d e d t o t h e c u l t u r e s at a c o n c e n t r a t i o n of 10 -7 , 10 -6 or 10 -5 mol/l. After 3 days culture media were harvested, the c e l l s fed, t h e s a m e a m o u n t o£ d r u g a d d e d a n d c u l t u r e d for a n o t h e r 3 da~s. O n d a y 6 c h o n d r o cytes were labeled with ~H-thymidine or 3 5 S m e t h i o n i n e . T h e c o n t e n t s of t o t a l FN, E D - A FN and keratan sulfate were determined with ELISA assays. - GAGPS stimulated dose dependently incorporation of 3 5 S - m e t h i o n i n e i n t o FN a n d p r o t e i n w h e r e a s i n c o r p o r a t i o n of 3 H - t h y m i d i n e i n t o D N A w a s u n a f f e c t e d . T o t a l FN, E D - A F N a n d k e r a t a n s u l f a t e c o n t e n t w e r e e l e v a t e d in c h o n d r o c y t e c u l t u r e s t r e a t e d w i t h GAGPS. It h a s to be considered whether, in t h e l o n g run, G A G P S h a s a l s o a d e l e t e r i o u s e f f e c t on c h o n d r o c y t e m e t a b o l i s m v i a an e l e v a t e d F N s y n t h e s i s . IInst. fur P h a r m a k o l o g i e u n d T o x i k o l o g i e d e r Univ. Bonn, R e u t e r s t r . 2b, D - 5 3 0 0 Bonn, F.R.G. 2 j a m e s A. B a k e r I n s t i t u t e for A n i m a l H e a l t h , C o r n e l l U n i v e r s i t y , Ithaca, N.Y. 14853, U.S.A.

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AUTOANTIBODIES TO CHONDROCYTE MEMBRANE PROTEINS IN RAT ADJUVANS ARTHRITIS.

IS THERE A NONSPECIFIC ACTION OF ANTIINFLAMMATORY STEROIDS AFTER MEGADOSE ADMINISTRATION? I.Moldenhauer, R.Hirschelmann and J.GieBler The concentrations needed for antiinflammatory effects of glucocorticoids are low since this action is receptor-mediated. The K D value e.g. for dexamethasone is in the nanomolar range. High-dose pulse glucocorticoid therapy results in serum concentrations much higher than those for receptor saturation. Essentially necessary higher levels, e.g. higher than 10 -6 mol/l cannot produce extra glucocorticoid effects unless one assumes nonspecific steroid action. Such effects have been proposed formerly, for instance stabilization of lysosomal membranes by intercalation of the amphiphilic antiinflammatory steroids into the membrane lipids. This view has a weak theoretical basis, however, it is still referred to. After high and low dose dexamethasone administration, i.e. after i0 mg/kg and 0.i mg/kg, respectively, we determined in rats the antiinflammatory effects, the serum steroid levels and the osmotic stability of membranes. We found no notably enhanced antiinfla~Lmatory effect of dexamethasone in carrageenin paw edema after increasing the dose from 0.I mg/kg to i0 mg/kg although the dexamethasone pgak serum level after i0 mg/kg was around i0-"D mol/l, a concentration, which can produce nonspecific membrane stabilization. We found indeed an increased resistence of erythrocytes against hypotonic baemolysis. After further experiments with a glucocorticoid receptor antagonist and with RNA/protein synthesis inhibitors we conclude, that there is no convincing evidence for nonspecific antiinflammatory dexamethasone activity. FB Pharmazie, Institut fur Pharmakologie Martin-Luther-Universit~t, 0-4010 Halle (Saale).

C. Franzelius, A. Schulmeister, and J. Mollerthauer.

Rat mycobacterial adjuvans arthritis is known to be a model for rheumatoid arthritis in man. As of yet, the mechanism for the joint-specific targetting of the induced autoimmunity in the rats is not completely understood. Here we present evidence that in part the B cell response is directed against defined cartilage cell surface components. The antibody titers rise from the first week after arthritis induction and remain stable for as long as six months, correlating to the macroscopically observable joint swelling. Treatment of the animals with immune modulating drug.s (dexamethasone, cyclosporine A, leflunomide) reduces antibody titers together with the joint swelling. Indometacin and other NSAID neither influence autoantibody production nor paw swelling. By western blot analysis a 65 KD glycoprotein of cartilage membrane could be demonstrated to be a major antigen in this autoimmune reaction. The protein is constitutively expressed in cartilage tissue and absent in the extracellular matrix. The anti-65 KD antibodies rising in the diseased animals are interspezies-crossreactive. As tested by monoclonal antibodies, the protein is immunologically unrelated to 65 KD heat shock protein from mycobacteria, a protein which is known to play an essential role in the mduction of the adjuvans arthritis. This work was supportedby the BMFT, grant No. 01VM88072,and by the DFG-SFB 1392. Institut fiir Pharmakologie und Toxikologie, Universit~t Erlangen-N~rnberg, Universit~itsstr. 22, 8520 Erlangen.

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EFFECTS OF VARIOUS ANT]PHLOGISTIC DRUGS ON ~XPER]~ENTAL INDUCTION OF ECTOPIC BONE FORMATION S. Hoedt-Schmidt, D.A. Kalbhen, W. Riither*, G. Wahl**~ and M. Gebhardt*** Clinical and experimental studies have shown that non-

PHARMACODYNAMIC AND TOXICOLOGICAL DIFFERENCES BErWEEN FLURBIPROFEN ENANTIOMERS iN RATS W.S. Beck and M. P/auchithiu

steroidal anti-inflammatory drugs may exert important effects on bone metabolism. In order to elucidate the influence of various antiphlogistic drugs on bone mineralization the aim of the present study was to investigate effects of diclofenac sodium ( 2 . 5 mg/kg), ibuprofen (25 mg]kg), and indomethacin (2.5 mg]kg) on ectopic bone formation using an experimental system for bone induction according to Urist 1965 (Science 150: 893). We applied histological methods as well as a direct measurement of the Ca/P molar ratio in bone tissue after calcination at 900 °C by X-ray diffraction examination based upon the construction of a standard curve from known mixtures of hydroxyapatite and [~-tricalciumphosphate, since these are the only two crystallized mineral phases remaining in the bone ash. Six weeks after implantation of demineralized bone matrix derived from rat femora into muscle pouches created in the paravertebral muscle of rats, the ash weight of explants was determined. Administration of the drugs was started on the day of implantation and continued five times weekly over a period of 42 days. The ash weight of bone was decreased by 14% and 15% with diclofenac and indomethacin respectively, while ibuprofen caused an increase of 20% compared to control, but showing no significant differences between the treated groups and control. Mineralization of implants 42 days after implantation was not markedly affected by treatment with diclofenac, ibuprofen or indomethacin further sustaining the view that only early phases of osteogenesis may be influenced b y these drugs in this model.

Institut f~ir Pharmakologie und Toxikologie, Universit~it Bonn, Reuterstr. 2, D-5300 Bonn 1, FRG; *Orthop~Idische Klinik, **Poliklinische Abteilung fiir Chirurgische Zahn-, Mund- und Kieferheilkunde, ***Mineralogisch-Petrologisches Institut, Universit~it Bonn

Aspirin-like drugs are believed to exert their anti-inflammatory and analgesic activities via inhibition of the cyclo-oxygenase system particularly in inflamed tissue. However, there is no satisfying correlation between prostaglandin (PG) synthesis inhibition and analgesic effects. Some nonopioid analgesics (e,g. salicylic acid) do not inhibit cyclo-oxygenase at analgesic concentrations. The enantiomers of flurbiprofen, a chiml antiinflammatory and analgesic 2-arylpropionic acid derivative, are converted into each other to a neglegible extent (< 5%) in rats. Using standard experimental models we determined the anti-inflammatory, analgesic and toxic effects together with the inhibition of PG production by $- and Rflurbiprofen in cells and tissues. We could show that R-flurbiprofen (R) has only 1/500 the activity as compared to S-flurbiprofen (S) as an inhibitor of PG production in mouse macrophages. Furthermore S inhibited inflammation and nociception in rats. By contrast, R did not affect inflammation, but was almost as active as S as analgesic in two different rat models of pain. S was an effective ulcerogen in rats, damaging both the stomach of fasted animals as well as the small intestine of fed animals. R was devoid of both damaging effects at analgesic doses. An investigation of the PG synthesis inhibition in inflamed tissue, as well as the mucosa of the stomach and the small intestine shows that S inhibited PG production almost completely. R at identical doses had only marginal effects on PG production in the gastric mucosa, and was devoid of any measurable effects in the mucosa of the small intestine. Both enantiomers had approximately equal access to plasma and the inflammatory fluid, and there was very little conversion of R into S (2-5%) in these body compartments. Taken together, we obtained evidence that PG synthesis inhibition is of dominant importance as a mechanism of action of aspirin-like drugs with regard to inflammation and GI-tract toxicity, but that PG synthesis independent mechanisms contribute to the analgesic effects. These results indicate additional molecular mechanisms of analgesia and suggest the use of Rarylpropionic acids as analgesics.

Department of Pharmacology and Toxico/ogy, University of ErlangenNuernberg, UniversitaetastraBe22, 8520 Erlangen, F.R,G.

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337 CYCLIC N U C L E O T I D E PHOSPHODIESTERASE CYTOSOL OF HUMAN NEUTROPHILS S.Winder, and Chr. Schudt*

339 ISOZYMES

IN

~ive different families of cyclic nucleotide phosphodiesterases (PDE) have been defined. Several of these isozymes may exist in parallel in one cell type. In order to analyse the composition of PDE isozymes in human neutrophils, PDE activity from PMN cytosol was separated by FPLC - Mono Q - ion exchange chromatography. Elution with sodium acetate (0.05 - 1.2 M) resolved three separate peaks of PDE activity: a c G M P - s p e c i f i c peak (PDE V) at 0.2 M and two cAMP-specific peaks (PDE IV) at 0.4 and 0.7 M. Activities of PDE I (calmodulin stimulated), ~DE II (cGMP stimulated) and PDE I I I (cGMP inhibited) could not be detected. The predominant cAMP hydrolysing activity (0.7 M) revealed a K value of 1.2 ~M. By inhibition studies with th~ selective PDE inhibitors rolipram, motapizone, and zardaverine, IC~o-values of 0.26, 46 and 0.16 ~ M were o b t a i n ~ . Thus, this isozyme is characterized as PDE IV. Hill coefficients deviate from unity and especially for rollpram nH = 0.51 ± 0.04 was found, suggesting two different b i n d i n g sites or two states of PDE IV * Present address: Byk Gulden Pharmaceuticals, Postfach 10 03 10, D-7750 Konstanz, University of Konstanz, Dept. of Biochemical Pharmacology, Postfach 55 60, D-7750 Konstanz

THE EFFECTS OF THE 4-QUINOLONES CIPROFLOXACIN AND OFLOXACIN ON THE HUMERAL IMMUNE RESPONSE OF HEALTHY AND IMMUNE SUPPRESSED NHCE R. Brmmengr~ibcr und K.D. Friedberg The influence of the 4-quinolones clprofloxaein and ofloxacin on immunoglobulin secretion (IgM- and IgG-response) was determined in healthy mice and the regeneration phase in animals immuae suppressed with cyelophosphamide with the aid of an enzyme linked immtmosorbent assay (ELISA). In our in rive experiments we administered ciprofloxacin (50 and 100 mg/kg b.w.) and otloxaein (25 and 50 mg/kg b.w.) to female Balb/e mice for various periods (4, 7,10, 14,18, 2~. and 28 days). We determined the antibody level (IgM and IgG titer) before and after the chemotherapeutic treatment. The IgM level had increased slightly dose-dependently from day 4 after both eiprofloxacia and ofloxaein administration, but not statistically significantly. In contrast we detected a statistically significant dose-dependent increase in IgG content of the serum already after 4 days administration of the two 4-qulnolones ciprotloxacin and ofloxacin. The regenerationphase of the immune suppressedmice was not influencedby the additionaladmlnlstradonof the qainoloncs.Afterrecoveryof the animals (from about day 14) therewas again a duse-dependentincreasein both the ciprofloxadn-and ofloxac'm-treatedanimals. The resultsof our study show that both 4-qulnolonesinvestigatedin the treatmontregimenthatwe selectedhavea positiveimmunomodulatlngeffecton thespecifichumoraiimmunityofBalb/cmice. Imtitut flit Pharmakologie and Toxikologie der Fakult~it air Kliulsche Medizin Mamaheim der Universit[it Heidelberg, MaybachstraBe 14-16, D-6800 Manaheim 1, FRG

338

34O

Idiopathic lung eosinophilia in guinea pigs as a model for testing antiinflammatory drugs. G.Hanauer, R.Beume, H.Wolf, U.Kilian Induction of eosinophilia by antigens like ovalbumin in guinea pig (g.p.) lungs, m e a s u r e d in bronchoalveolar lavage (BAL), is a well established model for testing antiinflammatory/antiallergic drug effects. While p e r f o r m i n g such experiments, we found that our Dunkin Hartley g.p. strain exhibits a spontaneous=idiopathic eosinophilia without ovalbumin sensitisation. We validated this observation p e r f o r m i n g cell number measurements in BAL from the day of arrival up to 35 days in 4 groups (n=4xl0 g.p.). The percentage of eosinophils out of total cell number decreased from =40% at day 1 to =25% 7 days later and reached a plateau of =15% on day 14 and day 35. For pharmacological tests in this situation, 2x10 male g.p./group received oral doses of 12 m g / k g / d dexamethasone(D) or the newly developed phosphodiesterase III/IV inhibitor zardaverine(Z) or placebo for 4 consecutive days. BAL was p e r f o r m e d on day 5 (day 7 after arrival). Compared to placebo, the absolute cell number decreased; the relative part of eosinophils in BAL decreased significantly and reproducibly after D to =40% and after Z to =70%. Thus, idiopathic lung eosinophilia in g.p. seems to be a suitable test model for effects of antiinflammatory drugs. In contrast to tests w i t h lung eosinophilia in ovalbumin sensitized g.p., no long lasting sensitisation p e r i o d w i t h great housing capacities is required. The reasons why g.p. develop idiopathic eosinophilia are not clearly identified and need further investigations. Possible explanations are transport, handling stress or housing conditions. Departments of Pharmacology and Biochemistry, Byk Gulden Pharmaceutics, D-7750 Konstanz

A POSSIBLE MECHANISM FOR THE INHIBITION OF OXYGEN METABOLISM IN PHAGOCYTESBY MERCURY S. Magour The phagocytic leukocytes undergo, upon stimulation, a complex series of events which include phagocytosis, degranulation, and an abrupt increase in oxygen uptake and metabolism. Accordingly, they produce large quantities of superoxide radicals (O2"), hydrogen peroxide (H202), and metabolize large amounts of glucose via the hexose monophosphate shunt to supply the cells with high concentration of NADPH. Concomitantly, the membrane bound NADPH-oxidase catalyse the oneelectron reduction of molecular oxygen to 02" using NADPH as an electron donor. The data show that 1/uM of either mercury chloride or methylmercury chloride irthibited 09- production by 50 % within 10 min. Under these experimentaFconditions both mercurials neither enter the phagocytes, nor inhibit the production of NADPH. However, NADPH oxidase was inhibited by both mercurials similar to that observed with the intact phagocytes. Pretreatment with either sulfhydryl-containing substances or with blood plasma protected the phagocytes against the inhibition caused by mercury. The presented data indicate that the inhibition of 09" production is due to an irreversible binding of both mercurial~ to the sulfhydryl groups of NADPH oxidase.

Inst. of Toxicology, GSF, D-8042 Neuherberg, FRG

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ENHANCEMENT OF TRIS OXIDATION BY STIMULATION OF HUMAN GRANULOCYTES M.R. Steinwachs, S. Zaki, R. Kahl

B i o a s s a y of a tracheal c o n s t r i c t i n g factor released by respiratory epithelial cells: Is endothelin involved ? A. Becker, J.H. Wilkensl, K. Plein, D. Tsikas, F.M. Gutzki, J.C. Fr61ich

The formation of oxidants by granulocytesis pathophysiologicallyimportant with regard to the activation and maintanance of many inflammatory processes.Since the granulocytes are subject to low pH value in inflamed tissue we have investigated the effect of pH 6.0 vs. pH 7.4 on the formationof oxidizingagents in stimulated and unstimulated human granulocytes by means of the detector substance TRIS [tris(hydroxymethyl)-aminomethane].14C-TRIS is oxidized in a Fenton type reaction to 14C-formaldehyde which is then intracellularty metabolized to 14CO2. The granulocytes used in these investigations were isolated from pooled human blood. After addition of 14C-TRIS (1.25 Ci/0.227 tool) and opsonized zymosan,the cells (7.5 x 106/ml) were incubated in Hank's buffer for one hour at 37~C.The 14CO2 formed was equilibrated with a hyamine solution in a second reaction vessel.The sample thus obtained was then analyzed in a liquid scintillation counter. Human granulocytes release 14CO2 into the medium trader all conditions tested. At pH 7.4 stimulated granulocytesproduced approximataly 1.5 times more 3-4CO2 than unstimulated cells. In contrast, at pH 6.0 a 2-fold increasewas found. The difference in the 14CO2 formationat the two pH levels was statistically significant for stimulated ceils (P<.06). In control experimentswe did not find evidence for pH dependent formationof 14CO2 from 14C-formaldehyde indicating that the primary oxidation of TRIS is the pHsensitive step. In conclusion, the formation of the TRIS oxidizing agent in stimulated human granulocytes is dependent on pH indicating increased production of oxidantsby zymosanstimulationat low pH. Universit/it G6ttingen, Abteilung fiJr Klinische Pharmakologie, Robert-KochStage 40, 3400 GOttingen

Epithelium-derived factors of unknown identity have been proposed to modulate airway smooth muscle tone. Guinea pig trachea (GPT) responses were assessed as diameter changes by computerized videomicroscopy (resolution 5 gm). A permanent hamster lung epithelial cell line was grown on microcarrier beads and peffused in a cell colutrm. GPTs from which the epithelium had been removed mechanically were cannulated from both sides and mounted in an organ bath. When the outflow tubing from the epithelial cell column was connected to the inflow cannula, the detector GPT contracted reaching 28 + 6 % of the maximum methacholine (100 I.tM)-induced contraction (n=12, p<0.001). Perfusion of the cell column with diclofenac (10 p-M) abolished the GPT contraction, whereas perfusion of the detector GPT only with diclofenac (10 gM) did not block the contraction. Analysis of the effluent of the epithelial cell column by gas-chromatography/massspectrometry demonstrated a basal release of prostanoids (PGFzcc 2,6 + 0,6; PGE2:4,8_+0,6 ; 6-keto-PGFla: 2,7_+0,2 pmol/ml; TxB2:35+8 fmol/ml ). Superfusion of the GPT with prostanoids in these concentrations caused contraction. Diclofefiac markedly (>90%) suppressed prostanold release. In order to assess the possible role of endothelin a dose response curve (1 pM to 10 nM endothelin) was performed. Endothelin dose dependently contracted the GPT with a diameter change of 54 -+ 8 % (n=6 p<0.001) at 10nM. The effect of the concentration of endothelin which caused approximately 30% constriction of the GPT could be blocked by diclofenac (10gM). It is concluded that lung epithelial cells can modulate GPT tone by releasing a transferable factor. This epithelial factor is most likely a cyclooxygenase product and not endothelin because in contrast to the epithelial factor endothelin contraction of the GPT was blocked by diclofenac given to GPT, Abteilung f/Jr Klinische Pharmakologie, Medizinische Hochschule Hannover, D-3000 Hannover 61 1Abteilung Innere Medizin V, Universit/~tskliniken des Saarlandes, D6650 Homburg/Saar.

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THE EFFECT OF ECHINACEA ANQUSTIFOLIA ON THE PHAGOCYTIC ACTIVITY OF PERITONEAL MACROPHAGES FROM MICE

ENDOTHELIN GENERATION DURING OLEIC-ACID-INDUCED LUNG INJURY IN RATS S. P r i t z e , K. Thelen and Th. Simmet

A. Schumacher

.Oleic acid (OA)-induced lung i n j u r y is an experimental model f o r the acute r e s p i r a t o r y d i s t r e s s syndrom (ARDS). A pathophysiological key f e a t u r e in t h i s model is the rapid and severe increase in pulmonary a r t e r i a l pressure which might at least p a r t i a l l y be due to pulmonary vasoc o n s t r i c t i o n . Only r e c e n t l y a new family of highly potent v a s o c o n s t r i c t o r peptides called endothelin (ET) I, 2 and 3 with s l i g h t l y d i f f e r e n t amino acid compositions has been i d e n t i f i e d (Yanagisawa et a l . , Nature 332: 411, 1988). We have now investigated whether ET may be formed in rats injected i . v . with OA suspended in saline containing BSA 0 . 1 % ( w t / v o l ) . For q u a n t i t a t i v e analysis a radioimmunoassay has been established. Anti-ET-1 antiplasma was raised in r a b b i t s immunized with a conjugate of ET-I and BSA coupled by a water-soluble carbodiimide. The antiplasma obtained recognizes mainly ET-I but has a r e l a t i v e cross-reaction of 17.6 % and 1.5 % with ET-2 and ET-3, r e s p e c t i v e l y . OA i n j e c t i o n (250 ~i suspension containing 42 ~I OA) was followed by an increase in plasma levels of immunoreactive ( i r ) ET-I which peaked at 45 min with 114 + 19 pg/ml but which was s i g n i f i c a n t l y elevated as e a r l y - a s 15 min a f t e r i n j e c t ion. In bronchoalveolar lavage (BAL) f l u i d (5 x 10 ml s a l i n e ) r a t h e r large amounts of ir-ET-1 were detected. A steady increase was observed up to 120 min (2939 + 344 pg/BAL). In both plasma and BAL f l u i d ir-ET-1 w a s - f u r ther analysed by reversed phase HPLC using a continuous gradient from 25-60 % a c e t o n i t r i l e / w a t e r (v/v) c o n t a i ning t r i f l u o r o a c e t i c acid 0 . 1 % ( v / v ) . In e i t h e r case, ir-ET-1 was found to consist mainly of ET-I, with small amounts of ET-2 and even smaller amounts of ET-3. Our results suggest t h a t ET might be of pathophysiological s i g n i f i c a n c e f o r the OA-induced lung i n j u r y . Their possible relevance to ARDS remains to be established. Department of Pharmacology and Toxicology, Ruhr-Universit y , U n i v e r s i t ~ t s s t r . 150, W-4630 Bochum

Vegetable immunostimulants~ especially preparations which contain Echinacea-extracts are among the most frequently administered drugs. We used a new special model of phagocytosis to investigate the action on nonspecific humeral immunity. Opsonized sheep red blood cells (SRBC) were presented to isolated peritoneal macrophages from female C57B16 mice. After lysis of the macrophages and the SRBC they have ingested we measured the amount of haemoglobin released using a sensitive colour reaction with trimethy]benzidine. The intensity of the co/our is directly proportional to the number of phagocytosed 5RBC. Endotoxin was used as the reference substance because of its ability to increase phagocytosis to about 50% - 70% after a singte dose of 1 ug/kg BW i.p. 2/4 hours before ceil extraction. Eehinacea angustifolia was investigated as aqueous extract and as a commercial product, both at doses of 25mg/kg and 250mg/kg BW, after intraperitonea! and oral administration. After administration of the aqueous extract the rate of phagocytosis decreased markedly in comparison with the control group. This effect was more intense after i.p. than p.o. administration. The duration of treatment (1, 5, 7, 28 days) did not influence the result. The administration of the commercia| product also decreased phagocytosis. We could not therefore confirm the claim that Echinacea angustifolia is a drug which stimulates the immun e response. lnstitut felt Pharmakologie und Toxiko]ogie tier Faku]t~t fiJr K]JnJsche MedJzin MannheJm, Universit~t Heidelberg, Maybachstr. 14-16, 13-6800 Mannheim 1, FRG

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PERFUSION OF ENDOTOXIN INTO THE ISOLATED P E R F U S E D R A T L U N G AS A M O D E L F O R T H E ACUTE RESPIARTORY DISTRESS SYNDROM (ARDS).

SIMPLE DIRECT HPLC METHOD FOR DETERMINATION OF ACETYLCHOLINE AND CHOLINE IN TISSUES OF RATS P.-T. Nguyen and J. Remien

Stefan Uhlig I n t r o d u c t i o n . Adult respiratory distress syndrom in association with multiple o r g a n failure is a major cause of death [I ]. Sepsis accounts for most of these cases. E x p e r i m e n t a l app r o a c h e s to sepsis-induced A R D S r e q u i r e a model in small in small l a b o r a t o r y animals. The m a j o r aim of this study was to r e p r o d u c e pathophysiological changes seen in A R D S , such as a decrease of compliance and lung edema, in the isolated rfused rat lung. e t h o d s and Results. Lungs f r o m f e m a l e Wistar rats (200250g) were p e r f u s e d in a recirculating system with KrebsH e n s e l e i t buffer s u p p l e m e n t e d with 2 % albumin. 40 m i n u t e s after starting the perfusion, a bolus of Salmonella m i n n e s o t a endotoxin was injected into the p e r f u s a t e . A b o u t 30 m i n u t e s after the a d m i n i s t r a t i o n of endotoxin a massive d e c r e a s e of tidal volume, lung compliance and lung conductance took place and the lung b e c a m e e d e m a t o u s . Perfusate flow was not affected. The loss of lung f u n c t i o n due to endotoxin was a c c o m p a n i e d by the release of t u m o r necrosis factor into the perfusate. A n t i - i n f l a m m a t o r y drugs, like d e x a m e t h a s o n a m e l i o r a t e d the loss of endotoxin-induced lung function. Conclusions. 1. I n f u s i o n of endotoxin into islolated p e r f u s e d rat lungs leads to reactions also observed in A R D S in man. T h e s e s y m p t o m s are b r o u g h t a b o u t in the absence of white blood cells. 2. The isolated p e r f u s e d rat lung is capable of p r o d u c i n g t u m o r necrosis factor, u p o n an LPS stimulus.

~v~

Biochemical Pharmacology, Faculty of Biology, University of Konstanz, D-7750 Konstanz, F R G 1. R.J.A. Goris. I n t e n s e Care News 1 (1987) 1-7

Tissue concentrations of acetylcholine (ACh) and Choline (Ch) are very sensitive to postmortem changes. The method of tissue extraction however may contribute to the observed variations as well. Therefore a new technique was developed avoiding any solvent extraction or precolumn purification steps. The method is based on an enzymatic on-line postcolumn derivatization of ACh and Ch. Rats were killed by decapitation and the heart and brain excised within less than 2 min. The organs were either homogenized in 6% perchloric acid or heated in a microwave oven and subsequently homogenized in buffer solution. After centrifugation an aliquot of the diluted supernatant was injected onto a Cla column. ACh and Ch were eluted in separate HPLC runs using a phosphate buffered EDTA-solution (pH 7.4) with either 0.015 or 4 mM tetramethylammoniumperchlorate. ACh was enzymatically hydrolysed in the postcolumn with immobilized enzymes (Acetylcholin-Biosensor, Biometra) and the resulting Ch oxidized to give HeOe for the electrochemical detection. Preparation of the tissues by microwave heating resulted in 1.5 to 2 fold increase of the ACh concentrations in heart (4.3+_0.8 vs. 6.9+_1.2 nmol/g) and brain (13.5+-4 vs. 22.6+-5 nmol/g) as compared to the direct homogenization in perchloric acid. This increase of ACh was accompanied by a decrease of Ch concentrations of 43% (heart) and 670/0(brain), respectively. ACh was rather stable after the excision of the tissues. Leaving the organs at room temperature for two hours resulted only in a decrease of about 30% of the initial concentrations. In contrast, Ch concentrations increased rapidly, indicating other sources for its production besides ACh, e.g. phospholipids. Short term pretreatment with physostigmine resulted in an 1.5 fold increase of ACh in the brain but not in the heart. Ch concentrations were not changed. The results show, that microwave killing of animals and microwave heating of organs in vitro give equivalent results of ACh concentrations but not of Ch. Obviously a degradation of ACh and other substances to Ch rapidly occurs during homogenization with perchloric acid. It is highly probable that mainly intracellular ACh is measured under the normal condition of fully active ACh-esterase. In contrast, Ch seems to accumulate already in intact cells during the ischemic postmortem phase. Walther Straub-Institut ffir Pharmakologie und Toxikologie, Nussbaumstrasse 26, 8000 MiJnchen 2

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DOXOFYLLINE/AMOXYCILLIN: INVESTIGATIONS ON PMARMACOKINETICS AND MUTUAL INTERACTION IN HEALTHY VOLUNTEERS A.H.Staib*, Fuhr,U.*, Raab,E.**, Braun,W.**, Hauck,C. ~, Chojnowski,M.* Pharmacokinetics of doxofylline (D), an antiasthmatic methylxanthine derivative, and amoxycillin (A), were determined alone and during coadministration following 7 days bid of D (400 mg) and/or 4 days bid of A (750 mg). Additionally, kinetics of D were measured after the ist dose. D and A concentrations in serum were determined by reversed phase HPLC. Subjects were 18 healthy male volunteers (age: 26.8t2.1 ys, weight: 74.9±6.1 kg). TABLE: Pharmacokinetic parameters of doxofylline (D) and amoxycillin (A), means ! SD, n = I$ substance and Cma x tma x tl/2e ] AUC0_oo application (mg/l) (h) (h) (mg/l*h) ~ ist dose alone 0.9Z0.6 0.8~0.4 0.9~0.3 1.5 i 0.9 D 13th dose alone 6.7±3.5 1.0±0.4 3.0±1.5 35.4±30.6 D 13th dose + A 6.3±3.4 0.8!0.4 2.7~1.7 33.7±34.0 A 7th dose alone 6.4ZI.7 1.7Z0.6 1.2!0.3 19.0± 5.0 A 7th dose + D 7.7±2.1 1.7±0.6 1.1±0.2 23.6± 9.1 A prononnced increase of D concentrations was observed within the application period, with Cmax, tl/2el, and AUC0_>o o significantly (p<0.05) higher following the last dose. Since accumulation in the serum was not observed (trough values: 0.74Z0.96 mg/l), the existence of a deep compartment is presumed. Concentrations of D were lower than described in literature. Thus, estimation of D bioavailahi]ity was carried out by additional i.v. applications in two volunteers resulting in a mean f value of about 0.15 which is considerably lower than that of 0.6 reported. Coadministration of A caused no significant changes in D kinetics, but the Cma X and AUC0_oo of A increased slightly, but significantly (p<0.05) on concomitant application of D. * Dept.Clin. Pharmacology, University Hospital Frankfurt, Theodor-Stern-Kai 7, D-6000 Frankfurt/Main 70 **Minden Pharma GmbH, P.O.Box llg0, D-4950 Minden

I D E N T I F I C A T I O N A N D R E G U L A T I O N OF H I G H A F F I N I T Y T R A N S P O R T E R FOR C H O L I N E IN S Y N A P T O S O M E S M . K n i p p e r * , C.Kahle, H . B r e e r T b e r e is c o n s i d e r a b l e e v i d e n c e i n d i c a t i n g t h a t t h e a c c u m u l a t i o n of c h o l i n e v i a a s p e c i f i c h i g h a f f i n i t y u p t a k e s y s t e m is the r a t e - l i m i t i n g step for the s y n t h e s i s of a c e t y l c h o l i n e . It is one of the most interesting properties of this t r a n s p o r t s y s t e m that its c a p a c i t y a p p e a r s to be coupled to neuronal activity. Some recent experiments s u g g e s t that this c o u p l i n g m a y b e b r o u g h t about v i a i n t r a s y n a p t o s o m a l m e s s e n g e r s . M o d y f i n g the c o n c e n t r a t i o n of c y c l i c n u c l e o t i d e s in s y n a p t o s o m e s s i g n i f i c a n t l y i n c r e a s e d the rate of c h o l i n e a c c u m u l a t i o n ; a similiar effect was i n d u c e d by p h o r b o l esters, k n o w n to a c t i v a t e the p r o t e i n k i n a s e C. The o b s e r v e d c h a n g e s in the r a t e of h i g h - a f f i n i t y c h o l i n e a c c u m u l a t i o n c o u l d e i t h e r r e s u l t from a l t e r a t i o n s in t h e i n t r i n s i c a c t i v i t y of the t r a n s p o r t e r or via c h a n g e s in the actual n u m b e r of f u n c t i o n a l carriers. In experiments using tritiated hemicholinium-3 as s p e c i f i c p r o b e for c h o l i n e t r a n s p o r t e r w e h a v e found that the number of binding sites significantly increased upon kinase activation. In a p p r o a c h e s to i d e n t i f y the c a r r i e r protein, monoclonal antibodies were produced which s p e c i f i c a l l y b l o c k the h i g h a f f i n i t y t r a n s p o r t of choline. These antibodies were found to recognize a single polypeptide band (Mrs.= 90.000) on W e s t e r n b l o t s and to label s p e c l ~ l C areas in the nervous system. By FPLC-ion e x c h a n g e - and i m m u n o a f f i n i t y - c h r o m a t o g r a p h y the 90 kd p o l y p e p t i d e has b e e n p u r i f i e d to h o m o g e n e i t y and was s u b s e q u e n t l y reconstituted in liposomes catalyzing the accumulation of e x o g e n o u s choline. * P r e s e n t address: D e p a r t m e n t of Z o o p h y s i o l o g y , U n i v e r s i t y of S t u t t g a r t - H o h e n h e i m , Garbenstr.30 7000 S t u t t g a r t 70

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EFFECTS OF GALANTHAMINE (GAL) AND TETRAHYDROAMINOACRIDINE (THA) O N C O L I N E S T E R A S E ACTIVITY, AND CONCENTRATION AND TURNOVER OF A C E T Y L C H O L I N E (ACH) I N R A T B R A I N IN-VIVO. O. P l e u l GAL and THA are c o n s i d e r e d as c a n d i d a t e s for clinical t r e a t m e n t of the c h o l i n e r g i c d e f i c i t in senile dementia of A l z h e i m e r t y p e (SDAT). However, satisfactory therapeutic success was not available as yet. Additional knowledge regarding the m o d e of action is therefore requested. We estimated time course and dose response r e l a t i o n of 3 cholinergic parameters in p a r a l l e l a f t e r a d m i n i s t r a t i o n of G A L or THA, r e s p e c t i v e l y , in rat brain. The a c t i v i t y of A C h esterase (ACHE) w a s i n h i b i t e d in a time and dose dependent manner b y b o t h the i n h i b i t o r s . Maximal effects were higher after THA than after GAL treatment. ACh levels were increased by 25-30 % maximally in animals sacrified by microwave radiation. The i n c r e a s e was not exactly related in time to the inhibition of ACHE. The turnover rate of ACh, measured with the a i d of i n c o r p o r a t i o n of r a d i o l a b e l l e d choline into ACh, was reduced by 40-50 % maximally by THA and to a minor extent by GAL. The a l t e r a t i o n was time r e l a t e d to the inhibition of ACHE. It is concluded that ACh turnover rate may be a more marked parameter for effects on cholinergic mechanisms than ACh l e v e l only. The t u r n o v e r rate of A C h w a s increased by 2.5 mg/kg scopolamine i.p. in untreated rats a n d in rats p r e t r e a t e d w i t h G A L as well.

RELEASE OF 3H-ACETYLCHOLINE FROM THE ISOLATED T R A C H E A EVOKED BY PREGANGLIONIC NERVE STIMULATION; COMPARISON WITH T R A N S M U R A L STIMULATION I. Wessler, A. Klein and D. Pohan We have recently reported that the airway epithelium strongly inhibits the release of acetyIcholine (ACh) from the gulnea-pig trachea and that transmural stimulation causes, in addition to the release of ACh, the liberation of phosphorylcholine (PCh) from the epithelium (this journal 1990, 342:387 - 393). In the present experiments a local stimulation was applied to the peripheral ends of the cut left recurrent laryngeal nerve and of the right vagal nerve to induce th~ release of H-ACh. Isolated rat tracheae were incubated with H-choline and after a 70 mln washout period tritium efflux was measured in 3 min fractions. Radioactive compounds (choline, ACh, PCh) present in the tissue or liberated into the incubation medium were separated by reverse phase HPLC. 3HPCh (335 000 +- 38 000 dpm/trachea, n=4) was the dominant radioactive ~ompound in the acid-soluble supernatant of homogenized tissue and H - A C h was a minor fraction (35 000 + 3 000 dpm/trachea). Electrical stimulation (15 Hz, 1 200 pulses) of the innervating parasympathetic nerves caused a 2fold increase in tritium efflux that was abolished by 1 I~M tetrodotoxin or by the removal of extracellular calcium and was substantially reduced (80%) by 300 BM hexamethonlum or tubocurarine. Separation of the radioactive compounds by HPLC showed 3H-PCh to contribute to about 50% to the spontaneous t[itinm efflux. Preganglionic stimulation did not a~ffect the outflow of "H-PCh, but produced an exclusive release of OH-ACh. Similar results were obtained with the guinea-pig trachea. Epithelium-dependent inhibition of 3H-ACh release was operating with both preganglion~c and transmural stimulation. Potassium channel opener did not reduce ~H-ACh release. Scopolamine (0.3 BM) produced a substantially increased 3H-ACh release (121 + 13%, n=6). The present experiments show the release of 3 H - A C h evoked by pregangllonic stimulation of the parasympathetic nerves innervating the trachea. Preganglionic stimulation offers considerable advantages in comparison to transmural stimulation; partlcularly, it simulates the physlological mode of excitation and does not cause an artificial outflow of PCh.

I n s t i t u t fGr K l i n i s c h e P h a r m a k o l o G i e d e r F r e i e n U n i v e r s i t & t Berlin, H i n d e n b u r ~ d a m m 30, D-1000 B e r l i n 45 W e s t

Pharmakologlsches Institut der Universit~tMains, Obere Zahlbacher Str. 67, 6500Mainz.

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EFFECTS OF T U B O C U R A R I N E AND a - B U N G A R O T O X I N ON P R E T E R M I N A L NICOTINE RECEPTORS (BACKFIRING) OF THE RAT PHRENIC NERVE R. Besser and T. Vogt Two neuronal nicotine receptors are present at motor nerves. Ereterminal nicotine receptors localized beyond the diffusion radius of released acetylcholine mediate repetitive discharges of the axon (backfiring) and presynaptic nicotine receptors localized near by the active release zones mediate a positive nicotinic feedback mechanism. So far, the preterminal nicotine receptors are less well characterized; therefore, we have attempted to investigate their pharmacological properties. Isolated rat phrenic nerve-hemidiaphragm preparations were superfused in an organ bath with the phrenic nerve extended outside and incubated with paraffin. The phrenic nerve was placed in two stimulating and two recording electrodes. Depolarizations of the muscle fibres were recorded by two steel needles placed into the hemidiaphragm and contraction was recorded by a force displacement transducer. Stimulation and recording unit was a Medelec 92A. Partial blockade of cholinesterase (2.5 min exposure to 3 BM neostigmine) was performed, to stimulate the preterminal nicotine receptors by released acetylcholine. After exposure to neostigmine a single stimulus was foIlowed by an increased contraction and by repetitive discharges in both the muscle and the phrenic nerve (backfiring). Tubocurarine (10 - 100 riM) blocked backfiring with concentrations that affected neither repetitive discharges of the muscle fibres nor skeletal muscle contraction nor nicotinic autofacilitation, a-Bungarotoxin (2 and 10 Bg/ml) abolished backfiring and muscle contraction. However, with lower concentrations (0.15 - 0.6 Bg/ml), a-bungarotoxln reduced or prevented repetitive muscular discharges and the neostigmineinduced increase in contraction without affecting backfiring. The present experiments show a different potency for tubocurarine and a-bungarotoxin to preferentially block preterminal nicotine receptors suggesting a difference between muscular and preterminal nicotine receptors.

PULSE-TO-PULSE DEPENDENT EFFECT OF SYMPATHETIC CARDIAC NERVE STIMULATION ON ACETYLCHOLINE OVERFLOW EVOKED BY VAGUS STI~ULATION E. Muscholl and Alice Habermeier-Muth In a rabbit perfused atria preparation electrical stimulation of the vagus nerve (VNS) inhibits noradrenaline (NA) overflow elicited by sympathetic nerve stimulation (SNS) when each VNS impulse precedes each SNS impulse by a fixed time interval, Cog. 3-10 ms or 200-283 ms, respectively [Habermeier-Muth & ~uscholl, J.Physiol. 401: 277,198~]. VNS applied 100 ms before SNS has no significant effect on NA overflow in the absence of an MI muscarine receptor blocker but inhibits release when pirenzepine is added, and enhances release after AF-DX 116 [Habermeier-Muth et al,, NSAP 342:483,1990]. Thus, an individual VNS pulse exerts an ~2 mediated inhibition of NA overflow for a brief time period of 3 to 283 ms. In the present study the effect of SNS on acetylcholine (ACh) overflow evoked by VNS was studied using the above preparation. We determined the fractional rate of overflow (FRO) of [14C]ACh after labelling the cholinergic nerves with [14C]choline, and NA overflow by HPLC/ECD. 3 VNS periods (2 Hz, 3 min) were carried out at 10 min intervals. SNS (2 Hz, 3 min) was applied simultaneously with VNS 2, but each impulse was placed either 7, 19, 50, 132, 350, or 500 ms before the VNS impulse. There was a facilitation of ACh FRO which peaked at an impulse interval of 19 ms (+45 ± 4.2 % vs. controls, mean ± SEM, n=6) and dissipated at ~ 132 ms. This facilitation was unaltered in the presence of propranolol 100 nM (+63 ± 19.8 % n=5), idazoxan 100-300 n~ (+33 ± 7.1%, n=4), idazoxan 1.0-3.0 I~4 (+33 ± 10.8 %, n=7), or rauwolscine 1.0 ~M (+64 ± 11.0 %, n=5). However, there was a further enhancement of FRO after prazosln 20 nM (+87 ± 19.8 %, n=5, p<0.05 vs. +45 ± 4.2), indicating an ul-receptor mediated inhibition by SNS of ACh release. The mechanism of the predominant sympathetic £ a c ~ ] ~ o n of ACh release remains to be established. Pharmakologisches Institut der UniversitRt, Obere Zahlbaoher Strasse 6 % D-6500 Mainz

Clinic for Neurology, University of Mainz, Langenbeckstr. 1, D-6500 Mains

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FORMATION AND RELEASE OF [3H]ACETYLCHOLINE (ACH) IN A CIRCULAR MUSCLE PREPARATION OF THE GUINEA PIG ILEUM C. Dietrich and H. Kilbinger In contrast to most other isolated tissues from which ACh release is usually studied, the circular muscle of the guinea-pig ileum is free of neuronal cell bodies and contains only axon terminals (Wilson et al., Ceil Tissue Res 247:497, 1987). Despite this advantage release of ACh from the circular muscle preparation (CMP) has not been studied so far. - The CMP was prepared from a segment of distal ileum (Johnson et al., this Journal 338: 397,1988), incubated with 10 ~.Ci [3H]eholine (60 rain, field stimulation at 20 Hz 1 s at intervals of 30 s) and then superfused with Tyrode solution containing 10 ].tmol/1 hemieholinium-3. Incubation with [3H]choline led to the formation of [3H]ACh (51 +- 6% of the total [3H]eontent in the tissue, N=4). The electrically evoked [3H]outflow was ealeium-dependent and prevented by 300 nmol/1 tetrodotoxin. The evoked outflow as measured in the presence of 10 l.tmol/l neostigmine eonsisted to 93 -+ 8 % (N=3) of [3H]ACh. The agonists DMPP (300 lxmol/1) and 5-hydroxytryptamine (5-HT, 10 ]~tmol/l) which increase basal ACh release from the myenterie plexus longitudinal muscle preparation through stimulation of ganglionic nicotine and 5 - H T 3 receptors, respectively (el. Kilblnger & Pfeuffer-Friederich, Br. J. Pharmac. 85:529, 1985), did not affect the basal [3H]outflow from the CMP. The muscarine receptor antagonist A F - D X 116 (0.1 and 1 ].tmol/l) did not significantly increase the electrieally evoked [3H]outflow (10 Hz 3 s at intervals of 30 s, 10 min), whereas pirenzepine and hexahydrosiladifenidol (HHSiD) concentration-dependently enhanced the evoked outflow, Pirenzepine (-log ECs0: 7.1) was slightly more potent than HHSiD (-log ECs0: 6.7) which may indicate that the muscarlne autoreeeptors in the CMP are of the M 1 subtype. In conclusion, the CMP provides a useful cell body-free preparation in which to study the receptor mediated modulation of depolarizationevoked ACh release.

SDZ ENS 163 IS A S E L E C T I V E M 1 A G O N I S T A N D I N D U C E S ~ R L R A S E O F A C E T Y L C H O L I N E H . W . G . M . B o d d e k e , A. Enz. T h e n e w m u s c a r i n i c a g o n i s t SDZ ENS 163; (+)-(3S, cis)-3-Ethyldihydro-4-[(l-methyl-iH-imidazol-5-yl) methyl-2(3H)-thiopentane dihydrogenphosphate] was investigated in in v i t r o m o d e l s for M 1 (rat s u p e r i o r c e r v i c a l g a n g l i o n ; RSG), M2 (rat atria) a n d M3 m u s c a r i n i c r e c e p t o r s ( g u i n e a - p i g ileum). In addition, the effect of SDZ ENS 163 upon a c e t y l c h o l i n e r e l e a s e in r a t h i p p o c a m p a l s l i c e s a n d its e f f e c t u p o n r a t h i p p o c a m p a l EEG was investigated. In RSG SDZ ENS 163 induced concentration-dependent depolarizations; a pD 2 v a l u e of 6.5 + / - 0.3 (s.e.m.) a n d e f f i c a c y of 128 +/- 4.2 % ( c a l c u l a t e d as p e r c e n t a g e of t h e m a x i m u m of m u s c a r i n e ) w e r e found. In r a t a t r i a SDZ ENS 163 w a s a p a r t i a l a g o n i s t w i t h r e s p e c t t o d e c r e a s e in c o n t r a c t i l e f o r c e ( e f f i c a c y = 14 +/- 2.9 % ; pD2 = 5.8 +/- 0.2). In g u i n e a - p i g i l e u m SDZ ENS 163 w a s a p a r t i a l a g o n i s t w i t h r e s p e c t t o f o r c e of c o n t r a c t i o n (pD 2 = 5.3 +/- 0.I, e f f i c a c y = 72 +/4.2 %). T h e o x o t r e m o r i n e - i n d u c e d i n h i b i t i o n of t h e e l e c t r i c a l l y s t i m u l a t e d r e l e a s e of a c e t y l c h o l i n e in r a t h i p p o c a m p a l s l i c e s is i n h i b i t e d b y SDZ ENS 163 (pA2 = 5.5 + / - 0.i). SDZ ENS 163 (0.3 to 30 uM/kg) i n d u c e d a n i n c r e a s e in l o w f r e q u e n c y e n e r g y (2-5 Hz) in r a t h i p p o c a m p a l rEG. D u e t o t h e d i s t i n c t a f f i n i t y b u t v e r y low e f f i c a c y of SDZ ENS 163 a t M2 r e c e p t o r s t h i s c o m p o u n d m a y a c t as a n M2 a n t a g o n i s t in r a t h i p p o c a m p u s . T h i s m a y e x p l a i n the acetylcholine releasing activity of the c o m p o u n d . W e c o n c l u d e t h a t SDZ ENS 163 is a selective M1 agonist in vitro. The central m u s c a r i n i c a c t i v i t y of SDZ ENS 163 in v i v o m a y r e s u l t b o t h f r o m its p o s t s y n a p t i c M 1 e f f e c t b u t also from presynaptically m e d i a t e d r e l e a s e of acetylcholine.

Pharmakologisehes Institut tier Unlversit~t, Obere Zahlbaeher Str. 67, D-6500 Mainz

Sandoz CH4002

P h a r m a Ltd, Switzerland

Preclinical

Research,

354

356

A F F I N I T Y C O N S T A N T S OF A N T A G O N I S T S ON THE M U S C A R I N I C RECEPTOR OF T H E R A B B I T IRIS AND O N M I , M2, A N D M 3 RECEPTORS.

ANTAGONIST BINDING STUDIES ON FIVE CLONED HUMAN MUSCAI~INIC RECE,PTOR SUBTYP,ES (Hml-Hm5) .~ F. DSrje '#, J. Wess . M. R. Brann and E. Mutschler ~

U. Altes, I. Kessler, and H. Fuder muscarinic receptor of the rabbit iris mediating sphincter contraction is neither of the M I nor M 2 type and cannot unequivocally be characterized as M 3 according to p A 2 values of M 3 selective antagonists (this journal 340:597-604, 1989). To further classify the receptor we determined -log K B or pA 2 values for the M 3 selective pfluor-hexahydrosiladifenidol (pFHHSiD) and for vale~ham~te bromide (VAL) against methacholine aD~ compared them with those on the rabbit vas deferens (M1-mediated inhibition of twitch response), on the rat iris (M2-mediated inhibition of stimulation-evoked [3H]-noradrenaline overflow), guinea-pig ileum (M3-mediated contraction) and guinea-pig uterus (methacholine-induced contraction). The affinity (-log KB or P&2 with confidence limits} of p F H H S i D for the prejunctional M 2 receptor in the rat iris (6.18±0.06, n=5), for the postjunctional receptor in the guinea-pig uterus (6.47, 6.29-6.69, n=8) and in the rabbit iris sphincter (6.17, 6.02-6.38, n=8) did not differ from that reported for the rabbit vas deferens (6.68, Lambrecht et al., Eur J Pharmacol 168:71-80, t989), but was lower than that for the M 3 receptor in guinea-pig ileum (7.84, ibidem). This confirms the idea that the Postjunctional iris receptor is not an M 3 type despite high pA2 values found for hexahydrosiladifenidol and 4-DAMP (see ou~ paper quoted above). The affinity of VAL was similar at the pre9unctional M I (rabbit vas deferens, 8.57, 8.43-8.73, n= 13), M 2 (rat iris, 8.52±0.03, n=7), and the Postjunctional M 3 receptor (guinea-pig ileum, 8.49, 8.32-8.71, n=6) indicating a lack of selectivity for these receptors. The pA 2 of VAL in the uterus (8.10, 7.84-8.45, n=7) tended to be, and that in the rabbit iris (7.7, 7.56-7.83, n=7) was lower than those at the M I , M 2 or M 3 receptors. The affinity constants extend the unusual spectrum found in the rabbit iris which argues against a heterogenecns receptor Population and for the presence of a novel muscarinic receptor. Pharmakologlsches Institut der Universit~t Mainz Obere zahlhacher Sir. 67, D-6500 Mainz Supported by DFG

Basle

The

The development of selective antagonists has led to the pharmacological subclassification of muscarinic receptors into four subtypes (M1-M4). Recently. molecular cloning studies have revealed the existence of five distinct muscadntc receptor proteins (ml-m5). To further characterize the antagonist binding properties of the cloned receptor proteins and to compare their binding characteristics with those of the pharmacologically defined subtypes, we determined the binding profiles of a series of selective muscarinic antagonists for the five cloned human receptor subtypes, individually expressed in CHO-K1 cells. The affinity estimates (pKi = -log Ki) were derived from displacement binding experiments employing [3H]NMS (N-methylscopolamine) as radioligand. Pirenzepine 4-DAMP* Himbacine AQ-RA 741"*

Hml 8.2 9.2 7.0 7.5

Hm2 6.7 8.4 8.0 8.4

Hm3 6.9 9.3 7.0 7.2

HM4 7.4 8.9 8.0 8.2

Hm5 7.1 9,0 6,3 6,1

*4-Diphenylacetoxy-N-methylpiperidine methobromide; **11-[[4-[4-(diethylo amino)butyl]-1 -piperidinyl] acetyl] -5,11-dihydro-6H-pyrido[2,3-b] [1,4]benzodiazepine-6-one. The Ml-selective antagonist pirenzepine bound with high affinfty to ml, Intermediate affinity to m4, and low affinity to m2, m3 and m5 receptors. 4-DAMP exhibited similarly high affinities for ml, m3, m4 and mS but up to 7-fold lower affinity for m2 receptors. Himbacine and AQ-RA 741 displayed high affinities for m2 and m4, intermediate affinities for ml and m3 and substantially lower affinities for mS receptors. Although, with the exception of pirenzepine, none of the tested antagonists showed more than 2-fold selectivity for one subtype over all other subtypes, each receptor displayed a unique antagonist binding profile. In general, the antagonist binding properties of Hml-Hm4 correlated well with those of the pharmacologically defined subtypes, M1-M4, respectively. The approach described here should facilitate the future development of novel subtype-selective muscarinic antagonists. * Lab. of Molecular Biology, NINDS, Bethesda, MD 20892, USA and # Dept. of Pharmacology, Univ. of D-6000 Frankfurt/M, FRG.

R 90 357 ANTIMUSCARINIC PROPERTIES OF PRIDINOL AND RELATED ANTAGONISTS AT THREE MUSCARINIC RECEPTOR SUBTYPES: CONSTITUTIONAL AND STEREOCHEMICAL ASPECTS G.Lambrecht, T.P.Friebe, H.Egerer, A.J.Aasen*, P.Sjt*, A.Tafel** and R. Tacke**

In order to assess the structural requirements for antimuscarinic potency and subtaroe selectivity of pndinol (El=C; R1,R2=phenyl; R3,R4=H; n=5) "(pA~: M1=8.4; M2=7.7; M3=7.8) and its methiodide (PA.2: M1=9.5; M2=8.7; M3=8.3) a series of analogues were invesugated for their functional antagonistic properties at muscarinic M1 (rabbit vas deferens), M2 (guinea-pig left atria) and M3 receptors (guinea-pig ileum). ~ The affinity profiles are controlled by • structural parameters: the [ R1 R~\ /OH the followang I ~£(~ c~ ~ nature of "El" (carbon or silicon), the R/'-\~H - ~ , - ~ ~CHo~_ str~cture of the ring systems "R1'' and " T'.' T~' '( ~,~,w, ,,RZ,,(ohenvl or cvclohexyl), the nature R~ R~ ~ of "R3" anal "R4"(H or CH3), the ring ~R~,R~=PhenyI,Cyclohexyl; size of the cyclic amino group (n=4 or Ra,R4=H,CH~; n=4,5; EI=C,Si 5) and the absolute configuration at "El". Furthermore, the antimuscarinic potency and receptor selectivity are affected by N-methylation. High stereoselectivity (up to 1600-fold) was observed for the (R)and (S)-enantiomers tested, the MI-M3 receptors making different stereochemical demands for these stereoisomers. Thus, muscarinic receptors can be differentiated on the basis of their enantioselectivity. Favourable results were obtained by C/Si-exchange in case of compounds with R~=R2=phenyl or Rl=R2=cyclohexyl. For instance, sila-dicyclidine methiodide (EI=Si; R~,R2=cyclohexyl; R3,R4=H; n=4) (pA~: M1 =9.2; M2=7.7; M3=8.2) displayed a 13(M1), 6- (M2) and-16-fold (M3) higher affinity for muscarinic receptors than its carbon analogue dicydidine methiodide (El = C).

359 CHARACTERIZATION OF THE MUSCARINIC RECEPTORS IN THE MESENTER1C VASCULAR BED PREPARATION OF SHR AND WKY M.G.C. Hendriks and P.A. v~r~Z w i ~ n , To characterize the muscarinic receptors on the endothefium of rat mesenteric vascular bed we determined the pA2-values of several specific antagonists using a perfused isolated mesenteric vascular bed, versus the muscarinic agonist (acetyl-8-) metacholine chloride (MCh). The preparations were either obtained from spontaneously hypertensive rats (SHR) or Wistar-Kyoto rats (VCKY). The isolated vascular beds were perfused with oxygenated Tyrode solution at a constant flow of 5 ml/mln. The resulting perfusion pressure was 17.5 -+ 7.8 mm Hg and 18.2 _+7.5 mmHg for SHR and WKY, respectively. In order to detect the relaxing effect of MCh equieffecfive concentrations of the a~-adrenoceptor agonist methoxamine (WKY: 5 gM and SHR: 0.7 #M) were added as a stimulus to the perfusion medium. SHR proved more sensitive to methoxamine stimulation, whereas no differences were found for the Em~ (89.6% -+ 4.3% relaxation) and EC~0 (44 nM MCh) of the relaxing effect of MCh between the two groups. As specific antagonists we used pirenzepine (MI) , AF-DX I16 (M~) and 4-DAMP (Me). The pA~-values obtained are listed in the table.

atropine

WKY

SHR

9.9

10.7

pirenzepine

7.2

6.7

AF-DX 116

5.8

5.8

4-DAMP

9.6

10.6

The pA2-vahies were in the expected range established for other arterial vessels (Eglen R.M. and Whiting R.L., J. Auton. Pharmacol., 19: 233-245, 1990) and clearly indicated the presence of muscarinic M3 receptors. In this respect no significant difference was found between the SHR and WKY groups. Department of Pharmacotherapy, Academical Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

Dept. of Pharmacology, Univ. of D-6000 Frankfurt/M, FRG. • Dept. of Pharmacy, Univ. of N-0316 Oslo 3, Norway. • * Dept. of Chemistry, Univ. of D-7500 Karlsruhe, FRG. 358

36O

INFLUENCE OF 4 MUSCARINIC ANTAGONISTS ON ENDOTHELIAL MEDIATED RELAXATION IN RAT MESENTERIC ARTERY Si~rid Dohrmann Acetylcholine causes relaxation of vascular smooth muscle mediated via a stimulation of muscarinic receptors on the endothelial cell. The aim of our study was to characterize the nature of the receptors involved in the endothelial response. We compared the potency of 4 different muscarinie antagonists - Atropine, Hrenzepine, AF-DX 116 ~ 11[ [2[(Diethylamino)- methyl- 1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido [2,3-b] [1,4] benzodiazepine-6-one, and 4-DAMP = 4-Diphenylaeetoxy-Nmethylpiperidine since these substances are thought to possess different receptor affinities. The experiments were carded out in the isolated perfused mesanteric artery of Wistar rats (perfusion pressure), in isolated electrically driven guinea-pig atria (contractile force) and in isolated taenia coli preparations (shortening of the muscle). In all these experimental preparations Acetyl-8-methylcholine was used as the antagonist and the dose response curves were estabfished as usual. Parallel shifts of the dose response curves were obtained in guinea-pig atria and in guinea-pig taeuia coli and in the presence of Hexamethonium (10 pM) also in the mesenterie artery of the rat. The pA~-values obtained are summarized in the Table. They are based on 5 to 8 andividual experiments and the mean dose ratios were used for the estimation of the pAz-vaiues.

BISPYRIDINIUM COMPOUNDS AS ALLOSTER/C MODULATORS OF M2-CHOLINOCEFrORS. STRUCTURE-ACTMTY-CONSIDERATIONS. D. Heber. K. Mohr. and H. Ohnesor~e The bispyridinium oxime Uno3 (trimethylene-bis-[4-hydroxyiminomethylpyridinium]dibromide mono-2,6-dichlorobenzyletber) is an ailosteric modulator of mnscarinic eholinoceptors. Previously, it has been shown that the 2,6-diehlorobenzyl-substituent is important for the ailostedc activity. Derivatives of Uno3 were synthesized which contained the dichlorobenzyl-group at both ends of the molecule. In these symmetrically built Duo-substances the central alkyl-chaln connecting the pyfidinium rings was elongated from trimethylenn (Duol.3) over tetramethylene (Duol.4) to haxamethylene (Duol.6). In suspensions of guinea pig cardiac membranes (3raM MgHPO4, 50mM Tris, pH 7.3, 37"C) the effect of the compounds was measured with respect to the equilibrium-binding of [SH]N-methylscopolamine ([aH]NMS, 0.5nM), and to the rate of [aH]NMSdissociation revealed by addition of 104M atropine. The IC~o-COncentrations reducing [SH]NMS-blnding to 50% of the control amounted to: Uno3 3)aM, Duol.3 4)aM, Duol.4 6,uM_, Duol.6 5kLM. All compounds retarded the dissociation of [SH]NMS eoncontrationdependently, which reflected the ailosteric activity. Concentrationeffect-curves for the drug-lnduced diminution of k_ 1 (control-value: 0.14 min 4) were evaluated. The ECho-concentrations, at which [aH]NMSdissociation was retarded by a factor of two, served as a measure of the allosteric activity. The ECru-values amounted to: Uno3 ll}.tM, Duol.3 6kL,M, Duol.4 3)~vl, Duol.6 3).flVl. In conclusion, "introduction bf the dichlorobenzyl-group at both ends of the bispyridinium-center almost doubled the allosteric activity (Duol.3 vs. Uno3). Elongation of the alkyl-chain connecting the pyridinium rings from trirnethylene (Duol.3) to tetramethylene (Duol.4) again increased the allosteric activity by a factor of two; however, the elongation to hexamethylene (Duol.6) had no further effect. The dependence of the allosteric activity on the length of the central methylane-ehain suggests that more than just one half of the symmetrical bispyridinlum-molecules is involved in the interaction with the putative allosteric site of the cholinoceptor.

.

antagonist Atropine

AF-DX 116 4-DAMP Hrenzepine

A.mesenteriea 9.9 8.6 9.7 8.2

g.-p. atrium 9.1 7.6 9.1 6.8

g.-p. taenia coli 9.7 7.8 9.0 6.9

All four antagonists were found to be most effective in rat mesetuerie artery. Pirenzepine proved to be 25 times, AF-DX 116 8 times,4-DAMP 5 times and Atropine 3 times more effective in mesenteric artery of the rat than in the other tissues. The differences in atfinity between Pirenzepine and the other antagonists are thought to be too small to support a conclusion euneerning the prevailing receptor type on the endothelium of the mesenteric artery of the rat. Abt. Pharmakologie, Universit~ K.iel, Hospitalstrasse 4, D-2300 Kiel

Abt. Pharmakolngie, Universifftt Kiel, Hospitalstr. 4, 2300 K i d *Pharmazeufiscbes lnstitut, Universit~t Kid, Gutenbergstr. 76-78

R91 361

363

COMPARISON OF THE ALLOSTERIC EFFECTS OF W84 AND TETRAHYDROAMINOACRIDINE ON [sH]PIRENZEPINE-DISSOCIATION FROM MC~h-RECEPTORSIN RAT CEREBRAL CORTEX ristian Tr~nkle Tetlahydreaminoacridine (THA) has recently been described to be a very potent allosteric modulator of Mt-receptors (Potter et at., Mol. Pharmacol. 35:652-660 (1989). Also the haxamethonium-derivative W84 = hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)-ammoniumbromide] is known to have marked allosteric effects on muscarinic cholinoreceptors. Aim of the present study was to compare the potency of W84 with that of THA to retard [SH]pirenzepine-dissociation from cerebral cortex M~receptors. Binding studies were performed using membrane suspensions of rat cerebral cortex in an incubation buffer consisting of 3 rrlM MgHPOa and 50 mM Tris, pH 7.3, at 23"C. After an incubation period of 90 rrffnutes membrane bound radioactivity was separated by rapid tiltration and determined by liquid scintillation counting. Presented data are mean values of at least 2 experiments. [ZH]pirenzepine-binding took place at a single population of binding sites with a K D of 5 nM and a B of 1100 fmol/mg membrane protein. W84 and THA i n h i ~ d [SH]pirenzepine-binding concentration-dependently with ICso-values of 1.3 uM and 2.8 uM, respectively. Dissociation of [ZH~irenzepine was visualized by addition of 10-~M atropine. It proceeded monophasically with t~,~ , =0.030 to . ! =23 ~ rain" . ) k ~x . .min . -~. Both ~ W84 and . THA started . retard [ H~renzepme-dissoc~atton at concentrations exceeding thetr ICso-Values. A plot of K 1 versus W84/THA-concentrations yielded doseresponse curves for tffd retarding action on the dissociation. The concentrations reducing the rate of [ZH]pirenzepine-dissociation by 50% amounted to EDr,o= 1.9xl0-~M for W84 and to 5.2x10-SM for THA, respectively. The slopes of the concentration response-curves (nrr W84 = 1.1; nu THA = 2.2) differed remarkably suggesting a ditYerent mechanism of action. Both compounds completely inhibited [SH]pirenzepine-dissociadon at high concentrations. With respect to the allosteric action on complexes of [SH~)irenzepine with M~-cholinoreceptors, the bisquarternary compound W84 is more potent tfian THA over a wide range of concentrations but rather by a different mechanism.

MEMBRANE SOLUBILITY AND AFFINITY TO THE GRANULE CARRIER OF VARIOUS AGENTS AFFECTING UPTAKE OF AMINE INTO CHROMAFFIN GRANULES. V. Schmitt, W. Stengel, H. Striffler & A. Burger

Dept. of Pharmacology, University Kiel, Hospitalstr. 4, D-2300 KJel

For various monoamines, transported into chromsffin granules, the affinity to the carrier increases with increasing lipophilicity. Moreover, for seven derivatives of the uptake inhibitor tetrahenazine, Scherman et al. 1988 (Molec Pharmacol 33:72-77) showed a strict inverse correlation over a wide range hetween the affinity to the carrier and the apparent octanol/buffer partition coefficient (P o/b), which reflects the membrane solubility of the inhibitor. Therefore, for various drugs affecting the uptake of catecholamine (CA) the ICso of uptake inhibition was compared with P o/b. The unidirectional carrier-mediated uptake of CA into resealed membrane vesicles ("ghosts") of bovine adrenal chromaffin granules was determined by membrane filtration after 3 min of incubation (30°C; pH 7.4) with 8 ~mol/l (= i Km) oH-CA (= 70/30 % adrenaline/noradrenaline). P o/b was calculated without correction fon the protonated/unprotonated form of the agent sfter spectrophotometric determination of the concentration in the aqueous phase (60 mmol/l Na2H/KNz phosphate; pH 7.4). Several groups of drugs were examined. For 12 ~-adrenoceptor antagonists the ICso (14 - 612 ~mol/l) was inversely correlated with P ~/b (30 - 0.002), if l-isopropylamino analogues were considered. Lahetalol, however, with a different structure, showed a very high affinity to the carrier (IC~ 0.5 ~mol/l) without the corresponding high P ~/~. Surprisingly, among C~a÷ entry blockers, opioids, MAO inhibitors~ aadrenoceptor antagonists, and dipyridamole analogues, substances were found with high affinity to the carrier (IC~ ~ 12 ~mol/l) sharing a relatively high P ozh. For these drugs a close correlation between affinity and membrane soluhility was found for dipyridamole and its analogues only, whereas small changes of the structure of the other drugs were followed by the loss of correlation. Institut f~r Pharmakologie u. Toxikologie der Universit~t, Versbacherstr. 9, D-8700 W0rzburg (FRG)

362

364

THE ALLOSTERIC ACTION OF W84 ON MUSCARINIC RECEPTORS IS WEAKLY TEMPERATURE DEPENDENT. C.-M. Staschen The bisquarternary ammonium compound W84 (hexamethylene-bis-[dimethyl= (3-phthalimidop~ropyl)-ammoniumbromide]) ~strongly retards the dissociation of [°H]N-methylscopolamine ([°H]NMS) from muscarinic acetylcholine receptors in guinea pig myocardium (Jepsen et al., Pharmacol. & Toxicol. 63:163-168, 1988). This effect is indicative of an allosteric action. In the present study the atte,m~t was made to evaluate the temperaturedependence of this effect. [ H]NMS dissociation was determined in membrane suspensions of guinea pigs hearts and brains, respectively, at 0°C, 23"C and 37"C with an incubation buffer medium of 3 mlvl MgPO~, 50 mM Tris, pH 7.3. After the binding has reached equilibrium, tile dissociation was made visible by addition of an excess of atropine in combination with increasing concentration of W84. In both membrane suspensions and at all three investigated temperatures the dissociation of [SH]NMS was found to be monophasic and the rate constant (k t, listed below) strongly temperaturedependent. Under all conditio-des W84 retarded the [SH]NMS-dissociatiun concentration-dependently. In order to obtain a measure for its potency the rate constant of [~I-qNMS-dissociation versus the concentration of W84 was plotted, using computer aided nonlinear regression analysis. From these dose-response curves the ECru-values (W84-concentration retarding the [SH]NMS-dissociation by a f~ctor of 2) are listed below. Hill coefficients (n)i) are indicated to characterize the slope of the dose-response curves. Temp. ventricle • rain ,, [°C] / ~ - ~ ECru**84nr~ NI~§ ECho W84n)I

EXPRESSION T~SPORTER

203 ~

1.3 t 0.4 35.0 0.3 180.0 1.2

-0.65 -0.65 -0.63

0.4 8.6 46.4

0.3 1.0 1.9

-0.67 -0.76 . -0.68

•: [10-S/mini; **: [tiM] The ECm-valuas showed, that with increasing temperature the dissociatio-5-retarding effect of W84 is somewhat lowered. In conclusion: in both membrane suspensions the allostaric effect of W84 is only weak temperature dependent in contrast to the dissociation of NMS. Abt. Pharmakologie, Universit~it Kiel, HospitalStr. 4, D-2300 Kiel 1. Abt. Pharmakologie/Physiologie, Schiffahrtmedizinisches Institut tier Marine, Posffach 6161, D-2300 Kiel 14

OF THE IN XENOPUS

D. C o D D e n e u r ,

NEURONAL NOI~ADRENAL~NE LAEV~S OOCYTES

B. L i n q e n a n d H. B 6 n i s c h

The ' neuronal, sodium-dependent and nisoxetinesensitive noradrenaline (NA) transporter of a d r e n e r g i c nez-~e t e r ~ i n a l s is a l s o a n i n t e g r a l m e ~ b r a n e p r o t e i n of rat p h a e o c h r o m o c y t o m a (PC12) c e l l s a n d o f t h e b o v i n e a d r e n a l m e d u l l a (B~24). As a first step towards cloning and sequencing the NA-transporter gene, P~A from PC12 c e l l s a n d B A M w a s isolated, size fract i o n a t e d a n d p o l y ( A +) ~u~NA w a s s e p a r a t e d . To recognize the NA-transporter P~NA t h e sizeselected RNA fractions were microinjected in X e n o p u s l a e v e s oocytes, a n d a f t e r 3 d a y s t h e expression of the NA-transporter was monitored b y m e a s u r i n g t h e n i s o x e t i n e - s e n s i t i v e u p t a k e of 3 H - N A (iO0 nmol/1, 2 h). 3 H - N A u p t a k e w a s f o u n d in o o c y t e s i n j e c t e d w i t h an P~NA c h a r a c t e r i z e d b y a s i z e of a b o u t 2 k i l o b a s e s (~JD) o b t a i n e d f r o m b o t h PC12 c e l l s a n d BAM. T h e P~NA-induced 3 H - N A u p t a k e (about 1,5 f m o l / h p e r oocyte) w a s s o d i u m dependent, sensitive to desipramine, stereos e l e c t i v e l y i n h i b i t e d b y t h e (+) e n a n t i o m e r of o x a p r o t i l i n e , b u t n o t i n h i b i t e d b y iprindol, a p u t a t i v e a d r e n a l i n e t r a n s p o r t i n h i b i t o r . Thus, a f t e r s u c c e s s f u l e x p r e s s i o n of t h e n e u r o n a l N A t r a n s p o r t e r b y a 2 k b P~NA fraction, t h i s f r a c t i o n m i g h t b e u s e d t o o b t a i n t h e s t r u c t u r e of the NA-transporter gene by means of expression cloning using Xenopus laeves oocytes. I n s t i t u t f~r P h a r m a k o l o g i e u n d T o x i k o l o g i e Univ. Bonn, R e u t e r s t r a B e 2 b, D - 5 3 0 0 B o n n 1 S u p p o r t e d b y t h e DFG.

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THE CONCENTRATION GRADIENT FOR EXTRACELLULAR 3H-NORADRENALINE GENERATED BY UPTAKE~ IN THE INCUBATED RAT VAS DEFERENS. U. Trendelenburg, 1. Azevedo and D. Moura. Rat vasa deferentia were incubated with 0.2 I~mol/I 3H-noradrenaline for 60 min and then washed out with amine-free solution for 100 min. Autoradiographs (Azevedo et al., Naunyn-Sehmiedeberg's Arch Pharmacol 342: 245-248, 1990) revealed a preferential storage of the exogenous amine in the varicosities close to the surface of the tissue. Autoradiographie images were digitized and the silver grain area was quantified as fraction of total area. The density of silver grains was expressed as a function of distance from surface; it is assumed to reflect the steady-state concentration gradient of 3H-noradrenaline in the extraeellular space as it existed during the incubation. Grain density fell by 1.7 (95 % confidence limits: 1.2 and 2.3) % per ~m depth of entry (n = 7). From the endogenous noradrenaline content of the tissues (88 nmot/g) and the study of Dahlstr~Sm et al. (Acta Physiol Seand 67: 271-277, 1966), the cube of tissue surrounding each varicosity was calculated; the length of each side was 7.4 I~m. Hence during inward diffusion each • . ~' 3 • varicosity lowers the extracellular concentration of H-noradrenahne by 12.4 %. This value somewhat overestimates the avidity of uptak% since uptakez was not inhibited in these experiments. During spontaneous efflux of 3H-noradrenaline, neuronal uptake occurs not only into that varicosity from which the 3H-amine leaked, but also ('en passant') into the varieosities encountered during diffusion to the medium. Calculations reveal that "neuronal uptake en passant" a) accounts for 50 % of the ~H-noradrenaline leaking from varicosities and b) is independent of the slope of the concentration gradient. However recent experiments indicated ~ • are subject • that 90 0~/0 of the spontaneous efflux of 3H-noradrenabne to neuronal re-uptake (Seh~Smig et al., Naunyn-Sehmiedeberg's Arch Pharmacol 340: 502-508, 1989). Hence, re-uptake into each "leaking varicosity" must have amounted to 80 %. It is concluded that the steady-state concentration gradient generated by uptake~ in the extraeellular space (during incubation with the 3H-amine) can be quantified. During spontaneous efflux there is not only a corresponding "neuronal uptake en passant', but also about 80 % "re-uptake into each leaking varicosity". Dept. of Pharmacology, Versbaeher Str. 9, D-8700 Wiirzburg. (supported by the Deutsche Forsehungsgemeinschaft - SFB 176)

INHIBITION OF THE RENAL TRANSPORT SYSTEM FOR ORGANIC CATIONS BY STEROIDS 1 Babin-Ebell~ H. Russ, M. Gliese and E. Sch~mi~ The corticosterone-sensitive extraneuronal transport system for noradrenaline (uptakes) and the renal transport system for organic c~tions (RTOC) may well be identical or at l~st closely related (Schtimig and SchiSnfeld 1990; Naunyn-Sehmiedeberg's Arch Pharmaeol 341:404-410). Hence, it was of interest to investigate the effect of eorticosterone and other steroids on RTOC. Confluent monolayers of elonal LLC-PK~ cells (pi~ kidney, proximal tubule) were incubated for 120 s at 37° C with-~4C-tetraethylammonium (TEA) in order to measure initial rates of organic cation transport through the apical plasma membrane. (1) The specific t4C-TEA transport was saturable, with a Km of 44 lamol/l (95% confidence interval: 28, 66) and a V~x of 230 + 30 pmol/(min mg protein) (n=8). (2) Various steroids were found to inhibit RTOC. Progesterone, cortieosterone, testosterone and testosterone acetate potently inhibited 1~C-TEA uptake into LLC-PK~ cells, the Ki's being 0.14 (0.09, 0.19), 0.24 (0.12, 0.45), 0.55(0.12, 2.46) and 0.61 (0.37, 0.99) lamol/1, respectively (n=4-6). In fact, these compounds belong to the most potent inhibitors of RTOC known so far. Hydrocortisone and dexamethasone were less potent inhibitors, the Ki's being 2.9 (1.4, 6.0) and 24 (13, 44) lamol/l, respectively (n=4). The Ki of aldosterone was above 10 lamol/1. (3) The mechanism of inhibition seems to involve a direct interaction at the carrier. Corticosterone inhibited ~C-TEA uptake competitively and the inhibitory potency was not affected when preineubation with corticosterone was reduced from 30 rain to 1 min. (4) The results support the likelyhood of a very close relationship between RTOC and uptake2. Supported by the Deutsche Forschungsgemeinsehaft (SFB 176 and Scho 373) Dept. Pharmacol., Univ. Wiirzburg, Versbacher Str. 9, 8700 Wiirzburg, Germany

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368

EVIDENCE THAT UPTAKE~ OF ISOP~ENALINE IS ENHANCED HYPERPOLARIZATION OF GUINEA-PIG THACHEALIS MUSCLE L.J. Eryan-Lluka and H.E. Vuocole

BY

CATECHOLAMINERGIC NEURONS MODULATE THE IN VIV0 RELEASE OF HISTAMINE IN THE HYPOTHALAMUS

H. Prast and M. Heistracher

There is evidence that depolarization inhibits uptake 2 (U2) of catecholamines into various cell types (Trendelenburg U, Naunyn-Schmiedeberg's Arch. Pharmacol. 336:33, 1987). The aim of this study was to investigate whether U~ is affected by hyperpolarlzation of cells. The effects of the ~-adrenoceptor antagonists, propranolol (PROP) and ICI 118,551 (erythro-dl-l-[7-methylindan4-yloxy]-3-isopropylaminobutan-2-ol; ICI), and the K÷-channel opening drug, lemakalim (LEM), on U2 of isoprenaline (ISO) in guinea-pig trachealis muscle (GPT) were examined. U2 was measured as the rate of corticosterone (CORT)-sensitive 3-O-methylisoprenaline (OMI) formation in segments of GPT incubated in 1 n~ol/l (which does not hyperpolarize GPT) or 25 nmol/l (which hyperpolarizes GPT; Allen SL et al., Br. J. Pharmacol. 86:843, 1985) [~H]-(±)-ISO • (PROP or ICl) ± LEM ± I00 ~mol/l CORT at 37°C for 60 min. At 1 nmol/l ISO, the rate of CORT-sensitive OMI formation was not affected by 10 ~mol/l PROP or ICI, but was increased by 27% by i0 Bmol/l LEM. At 25 nmol/l ISO, the rate of CORTsensitive OMI formation was decreased by 10 ~mol/l PROP (24% inhibition) or ICI (29% inhibition) and was increased by I0 ~mol/l LEM in the absence (35% increase) or presence (55% increase) of I0 ~mol/l PROP. Neither PROP nor ICI (10 ~mol/l) affected U 2 of ISO (25 nmol/l) in rat perfused hearts, in which IS0 does not cause hyperpolarization. Hence, it appears that (i). PROP and ICI inhibit U2 of ISO in GPT by preventing ISO-induced hyperpolarization of the cells (Allen et al., 1985) and (ii) LEM enhances U 2 of ISO due to hyperpolarization of GPT by LEM (Allen SL et al., Br. J. Pharmacol. 89:395, 1986). These results support the hypothesis that hyperpolarization enhances U~ of catecholamines.

In vitro experiments have shown that u2-heteroreceptors are. located on histaminergic neurons of rat cortex. HOWever, these receptors do not seem to modulate the release of histamine under in vivo conditions (C. Gulat-Marnay et al.,J.Neurochem.53:519, 1989). We now investigated the presence of u2-heteroreceptors in the rat hypothalamus and their importance for the modulation of the histamine release. The posterior hypothalamus of conscious, freely moving rats was superfused with artificial CSF and the superfusate collected continuously in time periods of 10 minutes. Released histamine was determined radioenzymatically (A. Philippu et.al.,Naunyn-Schmiedeberg's Arch.Pharmacol. 308:137,1979). Hypothalamic adrenoceptors were stimulated or blocked by superfusion with their agonists or antagonists for 10 minutes. In some experiments, the effect of potassium-rich (120m~ K +) CSF on histamine release was investigated. Superfusion with potassium-rich CSF increased the release rate of histamine significantly. Treatment of rats with u-fluoromethylhistidine abolished this effect. Bi-Agonists (isoprenaline, salbutamol) or antagonists (propranolol) were ineffective. Histamine release was significantly increased by yohimbine and idazoxan (I0-4M), while it was decreased by the agonists clonidine (5x105M) and noradrenaline (I0-4M). Thus, in the rat hypothalamus noradrenaline released from its neurons inhibits the release of histamine. At least in the hypothalamus, histaminergic u2-heterorecepto~s seem to fullfill this concrete function.

Department Of Physiology and Pharmacology, The University of Queensland, Brisbane 4072, Australia

Institut f~r Pharmakodynamik und Toxikologie der Universit,t, Peter-Mayr-Str.1, A-6020 Innsbruck, Austria

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SUPERFUSION AND ELECTRICAL FIELD STIMULATION OF CULTURED CHICK SYMPATHETIC NEURONS: MODULATION OF NORADRENALINE RELEASE VIA PRESYNAPTIC ALPHA-2 ADRENOCEPTORS. S.Boehm*, S.Huck*, H.Drobny and E.A.Singer.

R E L E A S E OF N O R A D R E N A L I N E A N D A T P F R O M G U I N E A - P I G V A S D E F E R E N S E L I C I T E D BY E L E C T R I C A L S T I M U L A T I O N AND NICOTINE I. y o n K ~ g e l g e n

The lumbar halves of paravertebral sympathetic chains wore dissected from twelve day old chick embryos, dissociated and plated onto collagen-coated tissueculture type polystyrol plates (5 mm diameter) at a density of 40,000 cells/plate. The cells were kept in a culture medium containing 5% fetal Calf s~rum for 5 days. After prelabelling the cells with H-noradrenaline (40Ci/mmol; 0.05~moi/i, 37°C, 30 min), the plates were transferred to superfusion chambers, where they were placed between two platinum wire electrodes 5 ~ apart, and superfused with physiological salt solution. After a washout period of 30 min the collection of 4 min fractions was started. Electrical stimulation was performed with trains of 36 monophasic rectangular pulses delivered at 3 Hz (0.5 msec, 20V/cm, 60mA) at 12 min (Sl), 32 min (S2) and 52 min (S3) after the beginning of sample collection. Tritium overflow evoked by $I, expressed as percentage of total radioactivity on the plates, amounted to 0.78% (corresponding to 0.41 nCi above a baseline e~flux of 0.68 nCi). It was abolished in the absence of Caz+ or the presence of tetrodotoxin (I ~mol/l). The addition of the alpha-2 adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK 14304) at increasing concentrations (0.01-100 ~mol/l) 16 min before $2 and $3 caused a concentration-dependent decrease in stimulation-evoked overflow of tritium. The maximum reduction amounted to about 30% of control values. The alpha-2 adrenoceptor antagonist yohimbine (0.3 pmol/l) antagonized the effects of UK 14304. The antagonist alone had no effect on electrically evoked overflow of tritium indicating the absence of autoinhibitory feedback under the chosen experimental conditions. *Institute of Neuropharmacology and Institute of Pharmacology, University of Vienna, w~hringer StraBe 13a, A-1090 Vienna, Austria.

N o r a d r e n a l i n e and A T P are t h o u g h t to be p o s t g a n g l i o n i c sympathetic c o t r a n s m i t t e r s in g u i n e a - p i g vas deferens. Is A T P r e l e a s e d t o g e t h e r w i t h n o r a d r e n a l i n e by a c t i v a t i o n of, p r e s y n a p t i c n i c o t i n e receptors, and if so, h o w does this r e l e a s e c o m p a r e w i t h e l e c t r i c a l l y e v o k e d release? - ~ a s a d e f e r e n t i a w e r e p r e i n c u b a t e d w i t h H - n o r a d r e n a l i n e and t h e n s u p e r f u s e d and stimulated either e l e c t r i c a l l y (60 p u l s e s at i, 2, 5 or i0 HZ) or by l-min s u p e r f u s i o n w i t h n i c o t i n e (56, i00, 180 or 320 ~M). ATP in the s u p e r f u s a t e was d e t e r m i n e d by the l u c i f e r i n - l u c i f e r a s e method. - I n c r e a s i n g f r e q u e n c i e s of electrical s t i m u l a t i o n and i n c r e a s i n g c o n c e n t r a t i o n s of n i c o t i n e e l i c i t e d i n c r e a s i n g contraction, o v e r f l o w of t r i t i u m and o v e r f l o w of ATP. The ratio "evoked ATP o v e r f l o w / e v o k e d t r i t i u m overflow" was similar t h r o u g h o u t the range of n i c o t i n e c o n c e n t r a t i o n s t e s t e d and for electrical s t i m u l a t i o n at 1 and 2 Hz, but was about 4 times h i g h e r for s t i m u l a t i o n at 5 and i0 Hz. T e t r o d o t o x i n 0.5 ~ M b l o c k e d all e f f e c t s of electrical s t i m u l a t i o n but did not c h a n g e t h o s e of nicotine, w h e r e a s the r e v e r s e w a s true for h e x a m e t h o n i u m I00 ~M. - The results d e m o n s t r a t e for the first t i m e a r e l e a s e of ATP f o l l o w i n g a c t i v a t i o n of p r e s y n a p t i c n i c o t i n e receptors. T h e y also d e m o n s t r a t e by d i r e c t m e a s u r e m e n t of A T P o v e r f l o w that ATP is p r e f e r e n t i a l l y r e l e a s e d by h i g h frequencies of e l e c t r i c a l stimulation. P h a r m a k o l o g i s c h e s Institut, H e r m a n n - H e r d e r S t r a s s e 5, D-7800 F r e i b u r g i.Br.

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EPITHELIUM-DEPENDENT MODULATION OF ENDOGENOUS NORADRENALINE RELEASE FROM RAT ISOLATED TRACHEA. K. Rack~, G. Brunn, M. Eisner and C. Langer. The airway epithelium (EP) plays an important role in the regulation of the airway smooth muscle tone. Recently, a strong inhibition by the airway EP of acetyleholine release was demonstrated (Wessler et al. this journal, 342:387, 1990). In the present study, possible effects of the airway EP on the sympathetle neurotransmisslon were tested. Rat isolated tracheae were incubated in 1.7 ml Krebs-HEPE$ solution and the release of endogenous noradrenaline (NA) was determined. The tissue was stimulated twlee (S1, $2), either by electrical field stimulation (3 times for 1 mln with 1 min intervals, at 3 Hz or 15 Hz) or by exposure to high potassium (K ÷, 60 mmol/1 for 10 min). In some experiments the EP was removed prior to the start of the incubation. Tissue NA determined at the end of the experiments amounted to about 1.6 nmol/g and was reduced by about 35 % in EP-denuded tracheae. Electrical stimulation of intact tracheae at 3 Hz caused the release of 52 pmol/g NA (3.3 % of tissue NA). After removal of the EP the evoked NA release was reduced to 12 pmol/g (0.96 % of tissue NA). Stimulation at 15 Hz evoked the release of 5.4 % and 3.4 % of tissue NA from EP-eontaining and EP-denuded tracheae, respectively. K + evoked the release of 45 % of tissue NA from EP-eontaieing and EP-denuded tracheae. Scopolamine did not affect the release of NA, neither from EP-containing nor EP-denuded tracheae, but oxotremorine inhibited almost completely the release of NA evoked by 3 Hz from EP-containing and EP-denuded tracheae. Indometacin (3 v.mol/l, 40 min before $2) increased the release of NA from EP-cantaining as well as EP-denuded tracheae by about 100 %. In conclusion, removal of the airway EP caused a marked inhibition of NA release from the trachea which was surmountable by increasing the stimulus intensity. This inhibition appears not to be mediated by endogenous aeetylcholine nor by eyelooxygenase products of arachidonie acid, although endogenous eyelooxygenase products or exogenous activation of musearine receptors inhibited NA release in the EP-eontaining and EP-denuded tracheae. Supported by the Deutsche Forsehungsgemeinsehaft (Ra 400/3-1)

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Pat vena cava segments were preincubated with 3H-noradr~naline and superfused with l~hysiological salt solution contain/rig desipramine 0.6 ~mol/l, c o r t i o ~ r o n s 40 ~m~i/l plus rauwolscine 1 ~m~'I/l. ~he electrically (0.66 Hz; 6 min) evoked t r i t i ~ overflow was not affected by histamine 0.1 - i0 )tool/l, but inhibi~d by GABA ( m x i m ~ degree of inhibition by 70 %; pIC35 5.83). ~ne inhibitory effect of GABA was hardly affected by emission of rauwolscine but was reduced when electrical stimulation was carried cut at 2 Hz (2 mJ_n). The effect of GABA on the electrically (0.66 Hz; 6 min) evoked tritit~ overflow was mimicked by the GABA B receptor agonist R-(-)-baclofen and its S-(+)-enanticmer (pIC35 6.08 and 3.28, respectively); the GABAA reoeptor agonist muscimol showed a very weak potency (pIC35 < 4.0). The inhibitory effect of GABA was not affected by the GABAA receptor antagonist (-)bicuculline methiodide I00 )/mol/l, whereas the effects both of GABA a~d R-(-)-baclofen were a ~ t e d by the GABAB receptor antagonist CGP 35348 i00 )anol/l (P-(3amincpropyl)-P-diethoxymethyl-l~hos~h/nic acid). The effect of GABA was not altered in preparations frcm rats pretreated with RS-baclofen I0 mg/b~ i.p. acutely (one injection 24 h prior to decapitation) o r chronically (daily injections for 14 days; last injection 24 h prior to decapitation), cc~pared to tissues from animals injected with the vehicle only. - T n e results suggest that the postganglionic sympathetic nerve fibres innervating the rat vena cava are endowed with presynaptic GABAB receptors. An effect of histamine could not be detected despite the presence of rauwolscine (in the rat brain cortex and the pig retina, blockade of c~2-adrencceptors strongly increases, or even is the prerequisite for, the inhibitory effect of histamine on noradrenaline release; Sc/llicker et al., this j ~ 340, 633, 1989 ar~ 342, 497, 1990). Institut fdr Pharmakolcgie und Toxikologie der Universit&t Bo~n, Reuterstr. 2 b, D-5300 Bonn 1

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It is well known that the release of noradrenaline from sympathetic nerve terminals can be increased by blocking prejunctional e2-adrenoceptors and by stimulating protein kinase C (PKC). The present study was done to find out whether these two regulatory pathways are independent from each other or whether they are linked in some way. Guinea-pig left atria were incubated at 32 °C in Krebs-Henseleit solution (2.5 mmol/I Ca2+), stimulated at a rate of 0.5 Hz, loaded for 1 h with 10 pCi 7-3H-noradrenaline and washed repeatedly during the next hour. Noradrenaline release was then induced by refractory-period field stimulation; one field pulse (30 V, 0.2 ms) was applied during each refractory period. The first 5 min stimulation period ($1) served as control, while drugs were applied 15 rain before S2. In control experiments the $2/S 1 release ratio was close to 1. Phorbol-12myristate-13-acetate (PMA) concentration-dependently increased the noradrenaline release, the mean $2/S 1 ratio being 2.39 + 0.34 with 10-7 mol/I. Phorbol-myristateacetate-0-methylether which d o e s not stimulate PKC was ineffective. The effect of PMA was greatly attenuated in muscles pretreated with 7 x 10-5 mol/I polymyxin B. Clonidine 3 x 10"7 mol/I halved the noradrenaline release and this effect was completely abolished by 10"7 mol/I PMA. Idazoxan 3 x 10"7 mol/I increased the release ratio to 2.26 ± 0.30 and, in the presence of this a2-adrenoceptor antagonist, PMA caused no further increase. When prejunctional autoinhibition was eliminated by a slightly modified fieldstimulation protocol (4 pulses of 0.05 ms duration during each refractory period; 8rasch, Eur. J. Pharmacol. 171, 49 ff. 1989), neither idazoxan nor PMA had a significant influence on the release ratio. These results suggest that PKC is fully activated when the prejunctional a2-adrenoceptors are blocked while a-adrenoceptor stimulation decreases the activity of the enzyme. Thus regulation of noradrenaline release by ~adrenoceptors and PKC seems to be closely linked. Institut for Pharmakologie der Medizinischen Universitfit zu L0beck, Ratzeburger Allee 160, D-2400 L0beck

376

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REGULATION OF NORADRENALINE RELEASE FROM GUINEA-PIG ATRIA BY PREJUNCTIONAL e-ADRENOCEPTORS AND PROTEIN KINASE C. H. Brasch

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ROLE OF SECOND MESSENGER P A T H W A Y S IN THE PREJUNCTIONAL c~s-ADRENOCEPTOR M O D U L A T I O N OF NORADRENALINE RELEASE F R O M THE RAT TAIL ARTERY tB. Bucher and =J. Neuburger

.

~iti~ ~ i ~ ~o~ ~ ~ of 16 ~ ~ 2 ~ at 3 ~ ~ ~ i ~ ~ ~ i0 ~ i0 ~ 34 % ~ ~ e ~ ~ p ~ of ~ i ~ 0.32 ~ i / i (P), ~ively. ~e ~iti~ ~ ~ 12"~ 43 % ~ ~ p ~ of p ~ z ~ 0.i~i/i ~ ~ i ~ i0 ~i/i, ~iv~y. (2) S l i ~ ~ ~ ~e p ~ of P ~ ~ of 24 ~ ~ 2 ~ ~ a~s~ at 3 ~ . ~ e ~ i ~ eff~ of ~ s ~ i0 ~ 28 % ~ ~e ~2+ ~ t i o n ~ 1.3 ~ i / i ( ~ ~tion ~ i ~ ~ly); it ~ ~ 46, 39, 24 ~ 18 % at ~ 2 + ~ t i ~ of 0.65, 0.98, 2.6 ~ 5.2 ~i/i. ~, at a ~ 2 + ~ t i ~ of 1.3 ~ i / i , ~e ~ati~ ~ ~ 0.i, 0.3, 1 ~ i0 ~ , ~ ~~iti~ ~ ~ 48, 47, 37 ~ 22 %. (3) S l i ~ ~ ~ ~ ~e p ~ of P ~ ~ of 24 ~ ~ 2 ~ ~ ~li~ at 0.3 ~ . ~ s l i ~ ~ ~ N~yl~e~de (~) 32 ~ i / i for 45 ~ prior ~ ~ t i ~ wi~ ~ H - ~ ~ " , ~ 0.I, 1 ~ i0 ~ i ~ ~e ~ ~ i ~ ~ 17, 35 ~ 39 %; ~ ~ i v e ~u~ ~ sli~ ~ ~ ~ ~ ~ 32, 42 ~ 50 %. (4) ~ e f o ~ l ~ (i0 ~ I / l ) - s t ~ a ~ a~ation of ~ ~ ~ f~ ~ ~t b ~ ~ ~ ~ aff~ ~ ~ ~ H3 ~ r ago~ R-(-)-~-~y~s~ 1 ~ ~0 ~ / ~ . - ~ ~ i ~ , (~) ~ p ~ i c ~2a u t o ~ ~ ~ ~t b ~ ~ ~ ~ ~ H3 h ~ ~ , (2) ~ H3 ~ ~ ~ ~ition of ~ ~ ~ d ~ ~ ~ ~ ~ ~2+ ~ t i o n ~ (3) ~ e H 3 ~ ~y ~ i ~ ~ a G p ~ , ~ do ~ a ~ ~ ~ ~ i ~ ~ ~ ~yla~ ~cl~. ~. ~ i . U~v. ~, ~ . 2 b, ~ 5 3 0 0 ~ 1

Activation of a~-adrenoceptors depresses nerve stimulation-induced noradrenallne release. The biochemical mechanisms underlying this modulatory process are still unclear. One of the possibilities is that the prejunctional receptor is coupled either to adenylate cyclase and/or to phospholipase C. We investigated whether these second messenger pathways are involved in the inhibitory function of a~-adrenoceptors situated at postganglionic nerve terminals innervating the rat tail artery. Forskolin (0.3-10 pmol/l) and 8-Br-cAMP (10-800 l~mol/l) increased, while BHT 983 (8-80 l~mol/l) inhibited the stimulation-evoked release of [aH]-noradrenallne. The effect of B-HT 988 80 pmol/l was unaltered in the presence of forskolin 10 ~mol/l and was slightly decreased in the presence of 8-Br-cAMP 100 pmol/l. When the stimulation conditions were changed in order to obtain the same release both before and after the application of 8-Br-cAMP 100 l~mol/l, the effect of B-HT 938 30 l~mol/l was not modified by the cyclic nucleotide. Phorbol 12-myrlstate 18-acetate (PMA; 1 llmol/l), a protein kinase C activating phorbol ester, increased [aHl-noradrenaline release, while phorbol 13-acetate (1 ]~mol/l) which does not affect protein kinase C was inactive. Although P M A i l~mol/l almost doubled the release, it did not alter t h e e f f e c t of B-HT 933 10 limol/1. As a whole, t h e p r e s e n t r e s u l t s do n o t s u p p o r t t h e h y p o t h e s i s t h a t a d e n y l a t e c y c l a s e a n d / o r p r o t e i n k i n a s e C a r e i n v o l v e d in t h e u=a d r e n o c e p t o r m o d u l a t i o n of n o r a d r e n a l i n e r e l e a s e . 1 L a b o r a t o i r e de P h a r m a c o l o g i e C e l l u l a i r e e t Moleculaire, C.N.R.S. URA 600, B.P. 24, 67401 I l l k i r c h Cedex, F r a n c e a n d ~Pharmakologisches Institut, Universit~t Freiburg. H e r m a n n Herder-Strasse 5, D - 7 8 0 0 Freiburg, F.R.G.

R95 379

377 PERTUSSIS

TOXIN

SENSITIVE

~i- AND NEM

SENSITIVE

u2-ADRENOCEPTOR MEDIATED INHIBITION OF NORADRENALINE R E L E A S E I N R A B B I T R E N A L A R T E R I E S . V. Wolk,

P. S c h o l l m e y e r u n d L.C. R u m p

PHYSIOLOGICAL u2-AUTOINHIBITION: EFFECTS OF YOHIMBINE ON RENAL SYMPATHETIC NERVE ACTIVITY AND RENAL NORADRENALINE SPILLOVER IN ANAESTHETIZED RABBITS B. Szabo, A. Schramm and K. Starke

Noradrenaline (HA) release from sympathetic n e r v e s in t h e r a b b i t r e n a l a r t e r y is m o d u l a t e d t h r o u g h ~ I - a n d ~ 2 - a d r e n o c e p t o r a g o n i s t s (Rump e t al. 1990, J. A u t o n o m . P h a r m a c . I0, 32-33) w h i c h s u g g e s t s t h e e x i s t e n c e of p r e j u n c t i o n a l U l - a n d u 2 - a d r e n o c e p t o r s . It w a s t e s t e d w h e t h e r Ul- a n d u 2 - a d r e n o c e p t o r m e d i a t e d i n h i b i t i o n o f N A r e l e a s e is c o u p l e d t o p e r t u s s i s t o x i n (PTX) or N-ethylmaleimide (HEM) s e n s i t i v e p a t h w a y s . Rabbit renal arteries were either incubated for 14 h a t 37 ° C u n d e r 95% 0 2 / 5 % C O 2 w i t ~ P T X (I a n d 5 Mg/ml) a n d t ~ n incubated with ~H-NA or preincubated with H-NA and then pretreated w i t h N E M (i ~M) for 30 min. T h e a r t e r i e s w e r e e l e c t r i c a l l y s t i m u l a t e d a t 2 Hz for 9 0 s a n d t h e s t i m u l a t i o n i n d u c e d (S-I) o u t f l o w of r a d i o a c t i v i t y w a s t a k e n as an i n d e x o f N A r e l e a s e . T h e u 2 - a d r e n o c e p t o r a g o n i s t U K 14304 (0.I ~M) i n h i b i t e d S-I o u t f l o w o f r a d i o a c t i v i t y a n d t h i s inh i b i t o r y e f f e c t w a s n o t a l t e r e d b y P T X (I a n d 5 ~g/ml) pretreatment. The ul-adrenoceptor agonist methoxamine (i0 ~M) ~iso inhibited S-I o u t f l o w of r a d i o a c t i v i t y and this inhibitory e f f e c t w a s n o t a l t e r e d b y P T X (i ~g/ml) b u t attenuated by PTX (5 ~g/ml) p r e t r e a t m e n t . In c o n t r a s t t h e i n h i b i t o r y e f f e c t of U K 1 4 3 0 4 b u t not that of methoxamine was markedly attenuated by NEM (i ~M). These results suggest that prejunctional ul-adrenoceptors may be linked to a PTX and prejunctional ~2-adrenoceptors to a H E M s e n s i t i v e p o o l of i n h i b i t o r y G - p r o t e i n s .

We studied the effect of intravenous yohimbine on blood pressure, heart rate, renal sympathetic nerve aetivity (RSNA), the renal noradrenaline spillover rate (RNSR; calculated from renal blood flow, the arterial and renal venous concentrations of endogenous noradrenaline and the fractional renal extraction of 3H-noradrenaline) and the total body noradrenaline spillover rate (TBNSR; calculated from the arterial concentration of endogenous noradrenaline and the total body 3H-noradrenaline clearance) in anaesthetized rabbits (alfadolone + alfaxalone). The parameters were measured before and at the end of 5-min infusions of sodium nitroprusside, which was given in order to modulate efferent sympathetic nerve activity through the baroreflex. In control experiments, sodium nitroprusside produced hypotension and simultaneously increased RSNA, RNSR and TBNSR. TBNSR increased much more than RNSR. Yohimbine (I mg kg -I + 0.2 mg kg -I h -l) had no major central effect on sympathetic nerve activity since it did not change the blood pressure-RSNA relationship. However, yohimbine shifted the RSNA-RNSR and the RSNA-TBNSR relationships in a manner indicating an increased release of noradrenaline per action potential in the kidney as well as in the whole body. This is the first simultaneous measurement of RSNA and RNSR in the rabbit. The results demonstrate that yohimbine enhances the renal release of noradrenaline via a peripheral site of action, and indicate that the a 2adrenergic autoinhibition of noradrenaline release operates physiologically in the kidney with intact sympathetic innervation.

Univ. K l i n i k F r e i b u r g , I n n e r e Med. t e r Str. 55, 7 8 0 0 F r e i b u r g , FRG.

Pharmakologisches Institut der Universit~t Hermann-Herder-Strasse 5, D-7800 Freiburg i. Br., FRG

IV,

Hugstet-

378

380

~2-ADRENOCEPTOR MEDIATED INHIBITION OF EXOCYTOTIC NORADRENALINE RELEASE IN T H E A B S E N C E OF EXTRACELLULAR CALCIUM R. J a c k i s c h , D. L a u t h and H.Y. H u a n g

EFFECTS OF INHIBITION OF EDRF F O R M A T I O N ON H A E M O DYNAMICS AND NORADRENALINE RELEASE T. H a l b r i i g g e a n d K. Liitsch

Recently, we have shown that 3,4-diaminopyridine (DAP) -evoked [3H]overflow from hippocampal s l i c e s p r e l a b e l e d w i t h [3H]NA m a y be a u s e f u l m o d e l for a c t i o n p o t e n t i a l - i n d u c e d NA release (Huang et al., Eur J P h a r m a c o l 169:115-123, 1989). T h e p r e s e n t s t u d y f u r t h e r i n v e s t i g a t e s t h e m e c h a n i s m of D A P - e v o k @ d N A r e l e a s e a n d t h e r o l e of e x t r a c e l l u l a r Ca 2 + in the i n h i b i t o r y e f f e c t of p r e s y n a p t i c a 2 - a u t o r e c e p t o r s . It w a s p e r f o r m e d on rat h i p p o c a m p a l s l i c e s in t h e p r e s e n c e of t h e N A r e u p t a k e i n h i b i t o r d e s i p r a m i n e a n d shows, t h a t D A P is a b l e to i n d u c e N A r e l e a s e a l s o in t h e a b s e n c e of e x t r a c e l l u l a r Ca 2+ (+imM EGTA throughout). This release was enhanced by Li + a n d b y an i n h i b i t o r of m i t o c h o n d r i a l Ca 2+u p t a k e , r u t h e n i u m red, b u t a b o l i s h e d b y t h e N a +c h a n n e l b l o c k e r t e t r o d o t o x i n a n d in t h e a b s e n c e of N a +. T h e s e f i n d i n g s i n d i c a t e an i n v o l v e m e n t of N a + - i n f l u x , f o l l o w e d by a c t i v a t i o n of i n t r a c e l l u l a r Ca 2+ - s t o r e s a n d l i b e r a t i o n of Ca 2+ for t h e e x o c y t o t i c r e l e a s e m e c h a n i s m . In t h e a b s e n c e of e x t r a c e l l u l a r Ca 2+ t h e D A P - e v o k e d N A r e l e a s e was also strongly inhibited by ~ -conotoxin, suggesting that under these conditions Na +influx occurs via voltage-sensitive Ca 2+c h a n n e l s of the N-type. Since the ~2-agonist clonidine potently inhibited the DAP-evoked NA r e ~ e a s e e v e n in t h e a b s e n c e of e x t r a c e l l u l a r Ca z+ (+ EGTA) w e conclude, t h a t a 2 - a u t o r e c e p t 0 r m e d i a t e d r e d u c t i o n of N A r e l e a s e i n v o l v e s e i t h e r i n h i b i t i o n of p r e s y n a D t i c N - t y p e C a 2 + - c h a n n e l s or of N a + - i n d u c e d Ca 2 4 r e l e a s e f r o m i n t r a c e l l u lar stores. Inst. P h a r m a c o l . , D - 7 8 0 0 F r e i b u r g , H . H e r d e r s t r . 5

L - N G - m o n o m e t h y l - a r g i n i n e (L-NMMA), a specific i n h i b i t o r of EDRF s y n t h e s i s , w a s u s e d in a n a e s t h e t i z e d r a b b i t s ( a l f a d o l o n e ÷ a l f a x a l o n e ) to a s s e s s the r o l e of E D R F - i n d u c e d v a s o r e l a x a t i o n in the regulation of haemodynamics and noradrenaline release into p l a s m a (RNA). To t h i s end, L - N M M A w a s given i.v. a t 10-min i n t e r v a l s in 3 c u m u l a t i v e d o s e s o f 3, 10, a n d 30 (n=S) o r 10, 30, a n d 100 m g k g -1 (n=9). F o r c o m p a r i s o n , t h r e e s u c c e s s i v e p h e n y l e p h r i n e (PHE) i n f u s i o n s (2.5, S, a n d 10 [xg k g - l m i n - ~ ; n=9) w e r e given i.v. f o r 10 min e a c h to a s s e s s b a r o r e f l e x - m e d i a t e d r e s p o n s e s t o d i r e c t v a s o c o n s t r i c t i o n . Mean b a s e l i n e v a l u e s : m e a n a r t e r i a l p r e s s u r e (MAP, m m H g ) 64-+2, h e a r t r a t e (HR, min -1) 285 _+ 6, c a r d i a c o u t p u t (CO, ml min-:) 336 £ 12, t o t a l p e r i p h e r a l r e s i s t a n c e (TPR, Pa s m1-1) 1457 _+56, a n d RNA (pmol k g -1 min -1) 181 _+ 21. The o b s e r v e d c h a n g e s in h a e m o d y n a m i c s ( a b s o l u t e values) a n d RNA (%) i n d u c e d b y L - N M M A a n d PHE w e r e as f o l l o w s : L - N M M A (mg k g -~) 3 10 30 MAP HR CO TPR RNA

5_+2 -13£2 -27-+9 303_+83 -28£3

9,+1 -29£3 -42_+5 454-+52 -48£3

13,+1 -40£3 -60-+7 739_+80 -57£3

100 16,+2 -53_+6 -69-+9 915,+113 -66-+2

PHE (gg k g -1 min -~) 2.5 5 10 7£1 -15_+2 -28£4 281_+22 -30_+3

11_+1 -31,+3 -60£8 581,+47 -52£3

23£3 -61,+13 -59£8 930-+98 -73£3

L - N M M A d o s e - d e p e n d e n t l y i n c r e a s e d MAP a n d TPR while d e c r e a s ing HR, CO, a n d RNA. The inverse r e l a t i o n s h i p b e t w e e n TPR a n d RNA w a s v i r t u a l l y i d e n t i c a l f o r b o t h d r u g s , i n d i c a t i n g t h a t d e c r e a s e s in RNA i n d u c e d b y inhibition of EDRF f o r m a t i o n are solely b a r o r e f l e x - m e d i a t e d . H o w e v e r , the d o s e - r e s p o n s e c u r v e f o r L - N M M A a p p r o a c h e d a m a x i m u m inhibition o f RNA by 69~, w h i l e t h a t f o r PHI~ a p p r o a c h e d 1OOZ. Hence, t h e c o u n t e r r e g u l a t i o n a g a i n s t p e r i p h e r a l v a s o r e l a x a t i o n by EDRF a c c o u n t s f o r 69% o f r e s t i n g RNA. ( S u p p o r t e d by t h e DFG; Gr 490/5) P h a r m a k o l o g i s c h e s I n s t i t u t d e r Llniversit~it W t i r z b u r g , V e r s b a c h e r Str. 9, W - 8 7 0 0 W i i r z b u r g , FRG

R 96 383

381 IWIDI~NCI~. FOR BLOOD Cl~I.L~ CONTItI~tITING TO ~ C L R ~ J L N C ] ~ Ol~ C A T ~ C H O L A M ] I ~ S Bernd Friedgen

PIJ~MA

A n a e s t h e t i z e d ( a l f a d o l o n e p l u s a l f a x a l o n e ) r a b b i t s w e r e given s i multaneous infusions of trace amounts of aM-labelled noradrenaline (NA), a d r e n a l i n e (A), i s o p r e n a l i n e (ISO) a n d d o p a m i n e (DA) e i t h e r i n t o t h e f e m o r a l vein (i.v.; n= 22) or i n t o t h e a s c e n d i n g a o r t a (i.a.; n= 20). C e n t r a l v e n o u s (right ventricle; Cv) a n d a r t e r i a l ( f e m o r a l a r t e r y ; Ca) p l a s m a ~ H - a m i n e c o n c e n t r a t i o n s w e r e d e t e r m i n e d a t 60 mie d u r i n g t h e i n f u s i o n . P l a s m a c l e a r a n c e s (CI= inf u s i o n r a t e / C v } a n d p u l m o n a r y e x t r a c t i o n s (ERp = l - C a / C v ) w e r e o b t a i n e d f r o m i.v., a n d s y s t e m i c e x t r a c t i o n s ( E R s = 1 - Cv/Ca) f r o m i.a. i n f u s i o n s . In lO a n i m a l s i n f u s e d w i t h vehicle, t h e c a r d i a c o u t p u t o f p l a s m a (CO; t h e r m o d i l u t i o n ) w a s 92.6 ± 11.3 m l k g - * m i n -~. The m e a n g r o u p r e s u l t s w e r e u s e d to c a l c u l a t e t o t a l - b o d y e x t r a c t i o n s []~Rtot = ERp + EI{$ (1 -ERp)] a n d t h e o r e t i c a l p l a s m a c l e a r a n c e s (Clcalc = CO x ERror). The r e s u l t s (means w i t h 95% c o n f i d e n c e limits) w e r e as f o l l o w s (Cl values in m l k g -~ min-~; ER v a l u e s in %):

NA A ISO DA

Cl

ERp

ERs

E R t o t Clcalc C L / C l c a l c

7S.9 (70.7;81.5) 83.2 (76.3; 90.7) 80.1 (72.7;88.2) 150.6 (138.3; 164.0)

9.2 (5.1;13.1) 3.5 (-0.4; 7.2) 4.4 (-0.1;8.9) 25.0 (19.4; 30.2)

69.4 72.2 (64.0;74.1) 74.4 75.3 (68.7; 79.1) 66.3 67.8 (58.5;72.7) 78.0 83.5 (72.1; 82.7)

66.9

1.13

69.7

1.19

62.8

1.28

77.3

1.95

C1 w a s h i g h e r t h a n Clcal¢, w i t h t h e r a t i o of C 1 / C l c a l c d e p e n d i n g on t h e amine. Hence, t h e amine e x t r a c t i o n a c r o s s the p u l m o n a r y and systemic circulation does not fully account for the observed Cl, i n d i c a t i n g a m i n e r e m o v a l b y b l o o d cells. This w a s c o n f i r m e d in i n - v i t r o e x p e r i m e n t s w i t h r a b b i t b l o o d (39°C) in w h i c h p l a s m a levels o f NA, A, ISO a n d DA declined w i t h t e r m i n a l h a l f - l i v e s o f 24.5, 19.5, 11.3 a n d 9.3 min, respectively; this decline w a s p a r t l y due to C O M T activity~ ( S u p p o r t e d by t h e DFG~ Gr 490/S)

O N SUBTYPE SELECTIVITY OF ~I-ADRENOCEPTOR MEDIATED REDUCTION IN CARDIAC TRANSIENT OUTWARD CURRENTS. Xo-L. Wang, E. Wettwer, G. GroB and Uo Ravens

A STUDY

Alphal-adrenoceptors of rat heart consist of about 20% of UlA- and 80% of UlB-Subtypes as distinguished with several subtype-selective antagonists. We have investigated the contribution of either subtype to the depressant effect of ~l-adrenoceptor stimulation on transient outward currents (Ito) in rat heart myocytes. Single-electrode voltage clamp studies were performed at room temperature (20-22~C). Propranolol (i ~M) was used to block S-adrenoceptors and Cd 2+ (0.i mM) to block Ca2+-currents. Peak Ito and the late outward current (Ii) at the end of clamp steps from -40 mV to 0 mV (duration, 250 ms) decreased with phenylephrine (PE, 30 ~M) to 64.4 ± 2.5 % and 45.0 ± 5 . 1 % (n=4), respectively. The effects on peak Ito wer8 markedly reduced in the presence of prazosin (PRAZ, 0.3 ~M, n=4), after pretreatment with the irreversible UlB-Subtype selective antagonist chlorethylclonidin (CEC, i00 ~M, 30 min at 37~C, n=5) or in the presence of the UlA-Subtype selective antagonist niguldipine (NIG, 0.i ~M, n=4) or both antagonists (n=5). I 1 was not affected by PE in the presence of PRAZ or CEC plus NIG, but was reduced in cells treated with either CEC or NIG. The results demonstrate that the PE effects on peak Ito and Ii are clearly ul-adrenoceptormediated. Both Ul-Subtypes seem to contribute to the reduction of outward currents.

P h a r m a k o l o g i s c h e s l n s t i t u t d e r Hntversit~it W t i r z b u r g , V e r s b a c h e r Stral]e 9, W - 8 7 0 0 W i i r z b u r g , Federal Republic o f G e r m a n y

Pharmakologisches Institut, Universit~t -GHSEssen, Hufelandstr. 55, 4300 Essen i, Germany

382

384

CIRCADIAN AND SEASONAL RHYTHM OF CATECHOLAMINE NETABOLITE E L I M I N A T I O N I N RAT AND MAN DURING INFLAMMATORY STATES E . I < r a u s e - , B. H a u f f

INOTROPIC RESPONSIVENESS TO ADRENOCEPTOR STIMULATION AND RECEPTOR DENSITY IN HEARTS FROM DIABETIC AND AGEMATCHED CONTROL RATS. J.B. Heijnis, M-J. Mathy.

We i n v e s t ± g a t e d the quest±on ±f the chronobiolog±cally determined elimination of catecholamine metabolites (vanillylmandelic a c ± d and homovan±llic acid) is altered by i n f l a m m a t o r y processes. We u s e d r a t paw edema i n d u c e d by carrageen±n, dextran or yeast and an UV-erythema in man. The r e n a l e x c r e t i o n of catecholamine metabolites i n 24 h o u r s ±n r a t and i n man s h o w e d c ± r c a d ± a n and s e a s o n a l v a r i a t i o n s , w h i c h c o u l d be p a r t l y quant±fied b y ue±ng COSINOR a n a l y s ± S o The max±mum o f t h e c i r c a d ± a n var±ations was a t t h e end o f t h e a c t i v ± t y phase, i.e. in rats at 5 h i n t h e m o r n ± r i g . En r a t w i t h c a r r a g e e n i n edema o r i n man w i t h U V - e r y t h e m a no s ± g n i f ± c a n t c h a n g e s o f t h e a c r o p h a s e s c o u l d be f o u n d b u t t h e m e s o r was ± n c r e a s e d by 34 % ±n c a r r a g e e n ± n paw e d e m a . The a m p l i t u d e s were pract±cally not altered: 5.1 vs. 7.1 nmol/6 h in healthy and edema rats, respectively. In a n i m a l s w i t h y e a s t paw edema t h e s e a s o n a l variation changed froa 4-month oscillat±on to a 6 - m o n t h one and t h e m e s o r d e c r e a s e d by a b o u t 30 ~ . I n r a t s w i t h t h e s h o r t - l a s t i n g dextran paw e d e m a , h o w e v e r , t h e s e a s o n a l r h y t h m was no longer significant it only resambled the variations in healthy animals. T h e r e was a l s o no alteration of the mesor values.

The inotropic effects of the ¢l-adrenoceptor (AR) agonists methoxamine (MET), cirazoline (CIR), phenylephrine (PE), the 8-AR agonist isoprenaline (leO) and the binding characteristics of the corresponding AR were investigated in hearts from diabetic rats (DR, 6 weeks) and age-matched control rats (CR). Diabetes was induced by iv injection of streptozotocin (50 mg/kg). Hearts from both groups were peffused according to Langendorff and stimulated at 5 Hz. The left ventricular pressure (LVP) was measured via an intraventricular balloon. Binding studies were performed with aH-prazosin and (-)J=%iodocyanopindolol as Iigands for ¢cAR and 8-AR, respectively. The sensitivity of LVP to MET, CIR and PE (PE in the presence of 10~ M propranolol) as well as the maximal effect of these drugs was significantly enhanced in hearts from DR compared with those from CR. However, despite an increased responsivenessa small but significant reduction of the number of ¢~-AR was found without a change in affinity in hearts from DR, compared with these from CR. A small decrease in sensitivity of LVP to leo was observed in hearts from DR compared with those from CR. The same maximal increase of LVP was reached in both types of hearts. Concomitantly, a small, marginally significant reduction of the number of BAR was found without any change in affinity in hearts from DR compared with those from CR. Our results indicate that there is no direct relationship between the inotropic responsiveness to AR agonists and alterations in number of the corresponding AR in hearts from DR in comparison with those from CR. It is conceivable, that post-receptor events may be subject to important changes in hearts from DR.

~resent address: Wissenschaftsbereich Pharmakologie, Sektion Pharmazie, Martin-Luther-Universitar Halle-Wittenberg, Weinbergweg 15, 0-4010 Halle (Saale)

Department of Pharmacotherapy, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands.

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ALPHA-ADRENOCEPTORS IN THE NORMOXIC AND ISCHAEMIC MYOCARDIUM OF THE RAT. R. van den Ende

THE INFLUENCE OF "FULL" AND "PARTIAL" a.ADRENOCEPTOR AGONISTS ON SPONTANEOUS MYOGENIC ACTIVITY IN RAT PORTAL VEIN. H.R. Schwietert The isolated rat portal vein is a convenient in vitro model to study the effects of drugs on myogenic mechanisms in vascular smooth muscle, The aim of the present study was to investigate the ability of "full" and "partial" ~adrenoceptor agnnists to elicit phasic and tonic types of responses in the rat portal vein. The ~-adrenoceptor agonists cirazoline, adrenaline, noradrenaline, phenylephrine, St 587 (2-[2-chlor o-5-trifluoromethylphenyl-imino] imidazolidine), indanidine, B-HT 920 (6-allyl-2-amino-5,6,7,8-tetrahydro-4-Hthiazolo-[4,5-d] -azepine) and UK-14,304 (2-[8-bromoq ulnoxalyl-7-imino] imidazolidine) all enhanced phasic myogenic activity in the rat portal vein. In addition to an increase in phasic myogenic activity, the agonists cirazoline, adrenaline, noradrenaline, and phenylephrine were also able (in higher concentrations) to raise the basal tone of the rat portal vein preparation. Since St 587 maximally stimulated phasic myogenic activity without causing an increase in basal tone (even in high concentrations) the interaction of this imidazolidine with a~-adrenoceptors was investigated in more detail with the competitive antagonists prazosin (pA~=8.74) and 5-anethyl-urapidil (pA~=8.37). This finding indicated that the contractile responses to St 587 and phenylephrine were mediated by the same a~-adrenoceptor subtype. The irreversible a -adrenoceptor antagonists phenoxybenzamine and chlorethyldonidine both attenuated the contractile response to St 587. The actions of chlorethylclonidine could be solely explained on the basis of an effect on receptor reserve. Chlorethylclonidine behaved as a partial agonist in the rat portal vein. In conclusion, our observations indicate that agents which have been characterised as "partial" agonists in other vascular preparations can elicit only phasic types of responses in the rat portal vein, whereas agents which have been designated "full" agonists can induce both phasic and tonic types of responses. The extent to which the phasic and tonic types of contraction are stimulated by agonists is governed independently by the affinity and intrinsic efficacy for each of the receptor-coupled pathways.

Myocardial ~-ad/'enoceptor mediated responses become increasingly important under certain pathological conditions, including ischaemia (I; II). It was the aim of the present study to investigate the influence of 30 rain of global ischaemia on the ~-adrenoceptor binding characteristics and on the functional responses to the selective ~-agonist cirazoline in rat isolated hearts and homogenates. We established the [ZH]-prazosin binding activity of membranes isolated from aerobically perfused and ischaemic rat Langendorff hearts. Global ischaemia (30 min) did not cause any change in either affinity or density of these binding sites, In functional studies the selective ~-agonist cirazoline increased the contractile force by maximally 13.0 _+ 4.6 % after ischaemia, whereas under normoxic conditions the maximal inotropic effect was 110.2 _+ 13.5 % (n=5-7; p<0.05). In conclusion, global ischaemia did not change the density or affinity of cardiac ~-adrenoceptors. However, the contractile response to cirazoline was reduced as a result of ischaemia. This reduction in inotropic action could not be attributed to changes in ~-adrenoceptor density or affinity and might therefore be caused by post receptor events. (I) J Clin Invest 72, 802-818 (1983) (ll) J Mol Cell Cardiol 17, 399-409 (1985) Division of Pharmacotherapy, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam~ The Netherlands.

Department of Pharmacotherapy, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands.

386

388

Changes in al-adrenoceptor density in homogenates from rat myocardial tissue caused by the preparation technique. M.J.F. Mertens and H.D. Batink

IR PRESSOR RESPONSES IN PITHED RATS ~x-ADRENOCEPTORMEDIATED CA-INFLUX AND CA-RELEASE FROM INTRACELLULAR STORESARE NOT INITIATED BY ~xa- and ~b-ADRENOCEPTORS, RESPECTIVELY B. N i l f f e r t In pithed rats the selective ~-adrenoceptor agonist indanidine e l i c i t s pressor responses predominantly mediated by Ca-influx, whereas the selective ~1-adrenoceptor agonist cirazoline activates both Ca-influx and Ca-release from intracellular stores. Ne investigated whether these two processes are mediated by ~ a - and ~ib-adrenoceptors. The log dose-pressor response curve of cirazoline was only s l i g h t l y shifted to the right by the irreversible selective ~xb-adrenoceptor antagonist chlorethylclonidlne, 12.5 and 25 mg/ kg ( i . v . ) being equieffective. Blockade of the Cainflux component of the pressor response of clrazoline by nlfedipine did not enhance the antagonism of chlorethylclonidine. The effect of cirazollne after pretreatment with chlorethylclonidine displayed the normal s e n s i t i v i t y towards the selective ~1-adrenoceptor antagonist prazosln. The log dose-pressor response curve of Indanidlne was also hardly antagonized by chlorethylclonidlne. The selective ~xa-adrenoceptor antagonist 5-methyl-urapidil (0.05 and 0.15 mg/kg, i.v.) displayed a comparable antagonism towards indanidine- and cirazoline-mediated pressor responses. After pretreatment with nifedipine 5-methyl-urapldil antagonized the pressor effects of cirazoline even s l i g h t l y stronger. In concluslon, in the pithed rat the ~1-adrenoceptormediated Ca-influx and Ca-release from intracellular stores seem not to result from the activation of ~ a and ~b-adrenoceptors, respectively.

In connection with a programme on myocardial ¢~-adrenoceptors we studied the influence of the preparation technique on the outcome of radioligand binding to left ventricular homogenates with SH-prazosin. The experiments were performed either instantly after removal of the heart from the thorax or after 30 minutes of perfusion with Tyrode solution at 37°C according to Langendorff. The perfused hearts were paced at 5 Hz. Left ventricular pressure was measured with a intraventricular balloon-catheter and amounted to 102.3__.2.4mm Hg. The coronary flow was 11.7-+0.6 ml/min. As shown in the table the Bmax-valuesof the perfused hearts were significantly higher, whereas KD values remained uninfluenced by the perfusion. n = 11

B~,~ (fmol/mg protein)

KD (nmol/I)

without perfusion

97.65 _+ 3.27

0.32 _+ 0.03

30 min. of perfusion

116.15 _+ 3.94 *

0.41 _+ 0.03

(* = sign., P<0.05) These results demonstrate that maximal density of SH-prazosin binding sites strongly depends on the preparation technique and on the subsequent protocol. Myocardial al-adrenoceptors appear to be very sensitive to environmental changes which may thus influence pharmacological responses in functional experiments. Dept. Pharmacotherapy, University of Amsterdam, Academical Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Janssen Research Foundation, D-4040 Neuss 21, F.R.G., Dep. of Pharmacology, Academic Medical Centre, ll05 AZ Amsterdam, The Netherlands

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ALPHA RECEPTORS MEDIATING VASOCONSTRICTION IN KIDNEYS AND CONTRACTION IN VAS DEFERENS OF THE RAT CLOSELY RESEMBLE ALPHAIA BINDING SITES IN RAT CORTEX M. Eltze, R. Boer~ and K.-H. Sanders

HOXAVERIN, P~PAVERIN AND VERAP~IL BIND TO ~I" AND ~2-ADRENERGIC RECEPTORS. L.Holmer, B.Effertz, G.Kojda and E.i~oack

The vasodilatatory mechanism(s) of putative 5-HTIA agonists (urapidil, 5-methyl-urapidil, ipsapirone, flesinoxan, 8-OH-DPAT*) and alpha2 a antagonists (WB

4101,*, (+)-niguldipine) was investigated in perfused rat kidneys. The agonists evoked a reversible and spiroxatrine-resistant increase in perfusion of organs preco~stricted by+~xl0~ noradrenaline but not by 27 mMK , 1.5 mMBa or 10 6 M PGFp . ED~ values (-log mol) for renal dilatation, affi~ties~[pK.) at 5-HT 1 (pig cortex), alpha I and alphajR (rat cortex) A _A __ . binding sites and alpha -receptor antagonism (pA2) in rat vas deferens (RVD)o~ the compounds were: Rat kidney 5-HT 1 AlphalA Alpha Drug (-log ED50) (PKi~ (pKi)(pK 2) (PKi ~B 5-Methyl-urapidil Ipsapirone Urapidil

Flesinoxan

10.8 9.3 9.1

9.5 8.8 7.7

9.1 7.7 7.1

9.1 7.9 7.5

7.2 6.0 7.1

8.8

9.0

6.7

7.0

6.7

8-OH-DPAT* 7.5 9.7 5.6 5.3 5.6 W/B 4101"* ii.2 8.5 9.9 9.4 8.3 (+)-Niguldipine 11.3 5.0 10.4 7.5 Spiperone 9.5 6.9 7.6 NO correlation was found between their -log ED for renal vasodilatation and pK. values at 5-HT 50 , IA . . . . . i blndlng sltes. Howevert dllatatlon in rat kldney was significantly correlated with pK. at alpha sites (r=0.986, p<0.001) and pA 2 value~ in ~VD (~0.984, p<0.001), but not with pK? at alphas_ sites (r=0.58, p>0.05). Thus, the vasodi~atation b ~ 5 - H T ~ agon±sts in the noradrenaline-perfused rat kidney ~ mediated by their concomitant alphalA receptor blockade. * 8-Hydroxy-2-[di-n-propylamino]tetralln, ** 2-[2.6dimethoxyphenoxyethyllaminomethyl-l.4-benzodioxane

Papaverin and moxaverin are classified as antispasmodic agents which relax smooth muscle by mostly unkno~ intracellular mechanis~s. Here we present experimental data which strongly suggest interaction of the drugs with ~adrenergic receptor sites. This may partially contribute to their pharmacodynamic activity. Alpha-adrenergic receptors were purified in plasma membranes of guinea pig liver and washed htman platelets in order to discriminate between the el- and ~2-receptor subtypes. Specific binding was defined as total binding minus binding which occurred in the presence of i0 ~mol/l phentolamine and was found to be in tile range of 55-70% for the liver preparation and 80% for the platelets. Cols)etition studies were performed using [3H]-Prazosin [0.5 nmol/l] and [3H]-Yohi~bin [2 nmol/l] as ligands incabeted with guinea pig liver me~ibranes and htman thro~ocytes, respectively. Concentration binding studies of the drugs were tested in both tissues. The table stmmerizes the ICs0-values for the different drugs and alpha-receptor sites. The resalts indicate high affinity, competitive binding of all tested druqs to e-adrenergic receptors without showing any selectivity to one of the ereceptor subtypes. Therefore, it is suggested that the pharuacological effects of the investigated drugs may be partially due to their ~-adrenergic receptor ~ediated activity. This nay be additive if other ~-adrenolytic drugs are used in combination therapy, especially after intravenous application. .

Drugs

H0xaverin Papaverin D,L-Verapamil

,

Byk Gulden Pharmaceuticals~

D-7750 Konstanz,

5.33 12.03 8.62

I0.41 -----

Means of 2-3 different ligand competition binding studies, using 0,01 to 200 ~M concentrations of the antagonists. Institute of Pharmacology, Heinrich-Heine-University, Mourenstr. 5, I)-4000 Dlisseldorf 1

FRG

390 CONTRACTION-MEDIATING a2-ADRENOCEPTORS IN MOUSE BUT NOT IN RAT VAS DEFERENS R. Biiltmann In the vas deferens of the mouse (MVD), the adrenergic response to single pulses is markedly reduced by low concentrations of yohimbine and idazoxan (von Kiigelgen et al., NaunynSchmiedeberg's Arch Pharmaco1340:760-763, 1989). We, therefore, examined the possible existence of postjunctional c~-adrenoceptors in this tissue in comparison to rat vas deferens (RVD). Contractions of the isolated, incubated MVD or RVD were elicited by phenylephrine or U K 14,304 (UK). In either tissue, the response to phenylephrine (EC50 14.3 and 4.8/~M) was not affected by yohimblne 0.1 but was antagonized by yohimbine 1 ~tM (antagonist KR-values 363 and 309 nM) and prazosin 1 nM (Kr~ 0.65 and 0.54 nl~ in MVD and RVD, respectively). In MVD, UI~elicited contraction with a threshold of 10 nM. Prazosin at concentrations of 3 - 100 nM caused a similar degree of antagonism, leaving intact most of the response to low concentrations of UK. Yohimbine 0.1 /~M mainly reduced the response to low concentrations of UK. In the presence of prazosin 10 nM, yohimbine competitively antagonised the effect of UK, yielding a K B of 3.4 nM. In RIND, U K elicited contraction only at concentrations exceeding 0.1/~M. The contractions were unaffected by yohimbine 0.1 ~M but, at up to 10 /zM UK, abolished by prazosin 0.1/~M. When UK was applied before phenylephrine 3/~M in MVD, responses to the latter were enhanced by U K 0.1 and 1/tM (by 29 %) but reduced by U K 10/tM. When low concentrations of phenylephrine were applied before U K 1/.tM, responses to UK were enhanced by up to 600 %. Neuropeptide Y enhanced responses to phenylephrine, but decreased responses to U K in MVD. - The results demonstrate the existence of contraction-mediating a2-adrenoceptors in MVD but not RVD. The receptors seem to belong to the a2Asubtype and may contribute to neurogenlc contractions, especl~i'ly due to potentiation b)' al-adrenoceptors. The a 1- and a2-adrenoceptors differ in their mteraction with neuropeptlide Y. Pharmakologisches Institut, Hermarm-Herder-Strasse 5, D-7800 Freiburg, F.R.G.

Adrenergic receptor alphaI alpha2 [C50 (flmol/l)IICs0(~,mel/1)

392 INCREASED MUSCARINIC PRESTIMULATION OF GUINEA-PIG TRACHEAL STRIPS DECREASES POTENCY AND INTRINSIC ACTIVITY (ISA)OF SYNTHETIC II2-SYMPATHOMIMETICS. COMPARISON OF RELAXATION WITH RECEPTOR BINDING AND ADENYLATE CYCLASE STIMULATION. H. Lemoine 1, C. Overlaek1, H. Worth2 and D. Reinhardt3 lI-Adrenoceptor (BAR) stimulants are widely used as anti-asthmatic drugs in the treatment of reversible airway obstruction. The therapeutic benefit of a liAR-stimulant depends on its ECso and ISA as smooth muscle relaxant and on its l~2-selectivity.We compared the effects of formoterol (F) with those of salbutamol (S) and isoprenaline (I) as standard. Experiments were performed on guinea-pig trachea (relaxation) and lung membranes (binding and adenylate cyclase [AC]). In tracheal rings slightly precontracted with 0.1 #mol/l carbachol (CARB) F and S were full agonists for relaxation as compared to I (ISA=I.00). ECs0'S (-log mol/1) were F:9.6, S:7.8 and I:8.6. However, when tracheal rings were submaximally contracted by 0.6, 1 and 6 #mol/l CARB, F and S exhibited only partial agonistic activity with reduced ISA {F: 0.96, 0.89 and 0.62, resp.; S: 0.88, 0.75 and 0.47, resp.} and reduced ECs0'S {F: 9.0, 8.7 and 8.2, resp.; S: 7.2, 6.9 and 6.5, resp.}. F and S also were partial agonlsts as stimulants of the/~AR-coupled AC. ISA for AC-stimulation (F:0.90, S:0.67) matched ISA for relaxation in the presence of 1 #mol/l CARB. In order to measure B2-sdectivity of drugs heart B1AR and lung I]2AR were specifically radiolabelled by 3H-(-)-CGP 12,177 {(-)~l--(3-t-butylamino-2-hydroxypropoxy)-benzimidazol-2-one} and 3H-ICI 118,551 {erythro-(+)-l-(7-methyliadan-4-yloxy)-3-isopropylaminobutan-2-ol},resp. From inhibition of radioligand binding dissociation constants (KD, -log reel/l) of F and S were determined. KD'S for I~2AR were F:8.12 and S:6.44, KD's for I]IAR were F:5.58 and S:5.11. In conclusion: 1. The great spare receptor capacity of I~AR for relaxation in the presence of 0.1 #mol/l CARB (EC5o<
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NO UNCOUPLING OF BETA 1- AND BETA2-ADRENOCEPTORS IN RAT MYOCARDIUM BY SHORT-TERM EXERCISE E. Werle 1 , W. Fiehn 1 , H. Weicke~

INFLUENCE OF UVA IRRADIATION ON THE EXPRESSION AND FUNCTION OF B~-ADRENOCEPTORS ON PERIPHERAL LEUKOCYTES OF ATOPIC AND NON-ATOPIC PEOPLE. U. Bleise, E. Haen, "B. P r z y b i l l a U V A - i r r a d i a t i o n of h u m a n p e r i p h e r a l m o n o n u c l e a r l e u k o c y t e s (pMNL) h a s been shown to i n h i b i t h i s t a m i n e r e l e a s e i n d u c e d by a n t i - I g E and calcium ionophore ( P r z y b i l l a et al: Pho~odermato]. 4, 7 3 - 7 8 . 1987). Since h i s t a m i n e r e l e a s e is i n f l u e n c e d by t h e g ~ a d r e n e r g i c r e c e p t o r s y s t e m , t h e e x p r e s s i o n and f u n c t i o n of g ~ a d r e n o c e p t o r s on pMNL was s t u d i e d . Venous blood was d r a w n from 7 h e a l t h y v o l u n t e e r s (2 male, 5 f e m a l e s ) and 7 ( u n t r e a t e d ) a~opics s u f f e r i n g from allergic r h i n i t i s (2 male, 5 female). Blood was s e d i m e n t e d for 90 rain in Macrodex. The l e u k o c y t e - r i c h s u p e r n a t a n t was c e n t r i f u g a t e d and f i n a l l y r e s u s p e n d e d in TCM b u f f e r (pH 7.4) in a c o n c e n t r a t i o n o f S x l 0 ~ cells/ml. A l i q u o t s of t h e cell s u s p e n s i o n were e x p o s e d to an UVA r a d i a t i o n of 50 J/cm ~ or 100 J/cm ~, or k e p t under identical conditions, but n o n - i r r a d i a t e d . ~ - A d r e n o c e p t o r d e n s i t y and a f f i n i t y were d e t e r m i n e d in a r a d i o - r e c e p t o r a s s a y with 12 c o n c e n t r a t i o n s of ~z~ I o d o - e y a n o p i n d o l o l (~ ~ ~I-CYP: 1 . 0 150 pmol/l). The i n t r a c e l l u l a r f o r m a t i o n of cyclic a d e n o s i n e m o n o p h o s p h a t e (cAMP) was t e s t e d in r e s p o n s e to 5 c o n c e n t r a t i o n s o f i s o p r o t e r e n o l (10 -~ - 1 0 -" tool/l); cAMP w{~s d e t e r m i n e d by r a d i o immuno a s s a y . No d i f f e r e n c e s were found in t h e n u m b e r and a f f i n i t y o f l ~ - a d r e n o c e p t o r s b e t w e e n t h e c o n t r o l group and t h e atopic s u b j e c t s . H o w e v e r , both b a s a l and s t i m u l a t e d cAMP c o n t e n t were m a r k e d l y lower in pMNL of a t o p i c s u b j e c t s . UVA i r r a d i a t i o n with 50 a n d 100 J/cm ~ h a d no s i g n i f i c a n t e f f e c t on d e n s i t y nor on a f f i n i t y o f l ~ - a d r e n o c e p t o r s . The e x p o s u r e , h o w e v e r , r e s u l t e d in a m a r k e d d e c r e a s e of t h e b a s a l and s t i m u l a t e d cAMP c o n t e n t in both groups. This i n d i c a t e s an UVA e f f e c t on s i g n a l t r a n s d u c t i o n by t h e g a d r e n o c e p t o r - a d e n y l a t e c y c l a s e s y s t e m a t s t a g e s d i f f e r e n t from t h e l i g a n d b i n d i n g s i t e s . The o b s e r v e d d i f f e r e n c e in i n t r a c e l l u l a r cAMP c o n t e n t b e t w e e n h e a l t h y and a t o p i c s u b j e c t s c o r r e l a t e s well with p r e v i o u s s t u d i e s in our l a b o r a t o r y on t h e l ~ R - a d r e n o c e p t o r s y s t e m in a l l e r g i c b r o n c h i a l a s t h m a .

A translocation of cardiac B-adrenoceptors (6-AR) from a presumably intracellular site to the sarcolemmal membrane associated with an increased isoproterenol-stimulated adenylate cyclase (AC) activity has been reported to occur during a 30 min treadmill running of rats (Izawa et al., J. Biochem. 105:110-113, 1989). The 6-AR density is decreased after 2 hrs of endurance swimming exercise (Werle et al., Life Sci, 46:9-17, 1990) indicating a biphasic modulation of cardiac gAR during prolonged exercise. In isolated cell systems high levels of agonists cause a rapid uncoupling of the 6-AR from the guanine nucleotide binding stimulatory protein (G s). The present study was focused on the first t w o minutes of exhaustive swimming exercise with the 6-AR number remaining unchanged: We investigated the receptor - G s - adenylate cyclase coupling in 6 sedentary rats and 7 rats swimming with a weight of 10 % of their body weight, The cardiac membranes were prepared at temperatures _< 4°C. The AC activity (pmol cyclic adenosin monophosphate/mg protein/min, mean+SD) of 2 2 . 8 ± 2 . 2 in control rats ( 2 3 . 9 ± 1 . 7 in exercised rats) was stimulated for 10 rain at 37°C with isoproterenol 10 nM to 2 7 . 5 ± 2 . 9 (30.0±1.6), 100 nM to 4 1 . 6 ± 3 . 2 (44.4±2.3), 1 pM to 4 7 . 2 ± 4 . 2 ( 5 1 . 5 ± 2 . 4 ) and 10 pM to 4 8 . 4 ± 4 . 8 (50.3±2.8). The cAMP levels in controls and exercised rats were elevated by the guanyl nucleotide Gpp(NH)p 100 pM to 1 0 2 . 9 ± 1 3 . 1 and 8 9 , 1 ± 5 . 1 , respectively, and by sodium fluoride 10 mM to 122.9 + 7.2 and 125.5 ± 28,4, respectively. In addition with the help of the ~1- and 62-selective g-AR antagonists CGP20712A and I C I l 1 8 . 5 5 1 no exercise induced effect on 6-AR subtype mediated AC activation could be detected. From these results we conclude that the physiological increase in catecholamines during short-term exercise does not induce a homologous or heterologous desensitization of the AC system in rat heart.

1Central Laboratory and 2Department of Pathophysiology and Sportsmedicine, Medical Clinic and Policlinic, University of Heidelberg, Bergheimerstr. 58, 6900 Heidelberg

Walther-Straub-Institute of P h a r m a c o l o g y and Toxicology. Ludwigs-Maximiliahs-University, Nussbaumstr.26, I)-8000 Miinchen 2, FRG and "1)ept. of Dermatology, L u d w i g - M a x i m i l i a n s U n i v e r s i t y , F r a u e n l o b s t r . 9 - 1 2 , D - 8 0 0 0 Miinchen 2, FRG

394

396

COMPARISON OF I~-BLOCKERS WITH ANCILLARY PROPERTIES BASED ON HEMODYNAMICS IN MAN, EX VIVO RECEPTOR OCCUPANCY AND CYCLIC AMP RESPONSE IN VITRO

IONIC REQtIIRI~MI~NTS FOR THE ABILITY OF IMIPRAMIN~ TO INHIBIT S-HYDROXYTRYPTAMIN~ (S-HT) TRANSPORT IN INTACT PLAT~I.~TS

J. Schloos, K. Breithaupt*, S. Amerschl&ger*, E. Appel, G. Sitzler. and D. Palm The aim of the study was to compare quantitatively the effects of different 13-blockers , i.e. prop ranolol (nonselective, without partial agonistic activity (PAA)), dilevalol (nonselective, B2-PAA) and celiprolol (131selective, 132-PAA), in man and in v i t r o . [n a randomized placebo-controlled, double blind c r o s s o v e r s t u d y 8 healthy male volunteers received 240 mg propranolol, 1000 mg dilevalol, 1000 mg celiprolol and placebo as single oral doses. Heart rate (HR) was measured at rest and a f t e r bicycle ergometry. From plasma samples 131-adrenoceptor (ADR) occupancies using a I~l-receptor assay were calculated as an estimate f o r 131ADR occupancy in vivo. [nfluence of d r u g s on cAMP synthesis was evaluated in v i t r o using rat reticulocytes (132-ADR) and cells from rat submaxillary glands (B1-ADR). A f t e r all g-blockers maximal plasma concentrations were reached 1-3 h a f t e r drug administration yielding 13-ADR occupancies of 90-95 %. At these occupancies the decrease in exercise induced HR amounted to 28 beats/rain (bpm) a f t e r propranolol and dilevalol and 17 bpm a f t e r celiproloh There was a linear relationship between decrease in HR and B-ADR occupancy. Resting HR was slighty depressed a f t e r propranolol (5 bpm) and s l i g h t l y increased a f t e r dilevalot (4 bpm) as well as a f t e r celiprolol (10 bpm). In v i t r o , all t h r e e 13-blockers inhibited concentration dependently the isoprenaline stimulated cAMP synthesis. Only celiprolol caused a concentration dependent stimulation of cAMP synthesis mediated by 131- and 13~-ADR. These results demonstrate t h a t based on comparable 13ADR occupancies qualitative differences of 13-blockers can be evaluated. These can be explained in p a r t by the PAA inherent in celiprolol and to a lesser degree in dilevalol. Zentrum der Ph&rl~kolosie, UrHverslt~tBklinikum, Theodor-Bterr~-Kai 7, 6000 Frankfurt/M. und tZentrum f~ir Kardiovaskul~re Pharm&kologie, Hal:hildenstr. 8~ 6500 Mainz

Christian G e r t e i s It has been s h o w n t h a t the binding of i m i p r a m i n e (1M1) to plat e l e t p l a s m a m e m b r a n e s a b s o l u t e l y requires the p r e s e n c e of Na + (with t w o Na + p a r t i c i p a t i n g in binding) b u t not the p r e s e n c e of C l - o r K + (Rudnick e t al.: P s y c h o p h a r m a c o l Bull 19, 545; 1983). It r e m a i n s to be e s t a b l i s h e d w h e t h e r this holds t r u e also for the f u n c t i o n a l c o u n t e r p a r t of 1MI binding, n a m e l y the inhibition by IMI of 5 - H T u p t a k e into i n t a c t p l a t e l e t s . T h e r e f o r e , r a b b i t p l a t e l e t s w e r e e x p o s e d to various c o n c e n t r a t i o n s of Na + (18-147 m m o l p e r 1; r e p l a c e m e n t by Li*), C1- (16-147 m m o l / l ; r e p l a c e m e n t by m e t h y l s u l p h a t e ) or K + {5-80 m m o l / 1 at 70 m m o l / l Ha+; r e p l a c e m e n t by Li+), and initial r a t e s of 3 H - S - H T (20 nmol/1) u p t a k e w e r e m e a s u r e d in the a b s e n c e and p r e s e n c e of 1MI {3-1000 n m o l / l ) to d e t e r m i n e the ICs0 for u p t a k e inhibition by IMI. 3 H - S - H T u p t a k e in the a b s e n c e of IMI was clearly Na +- and C l - d e p e n d e n t and increased with increasing Na + or C1- c o n c e n t r a t i o n . On the o t h e r hand, increases in the K + c o n c e n t r a t i o n r e d u c e d uptake, with the u p t a k e r a t e being linearly r e l a t e d to the log of the r e c i p r o c a l of the K+ c o n c e n t r a t i o n (r = 0.97; n= 5). Within the r a n g e of Na + and CI- c o n c e n t r a t i o n s t e s t e d here, the ICs0 of IMI (which w a s 17 n m o l / 1 a t 147 m m o l / 1 NaCl) increased 5 . 8 - f o l d when the Na +, and 2.6-fold w h e n t h e C1- c o n c e n t r a t i o n w a s d e c r e a a e d . C h a n g e s of the K+ c o n c e n t r a t i o n had no e f f e c t on the value of 1Cs0 a t all. The d e p e n d e n c e of 1/ICso (i.e., the affinity of the t r a n s p o r t e r f o r IMI) on Na + was c o n s i s t e n t with simple MichaelisM e n t e n kinetics and s h o w e d an a p p a r e n t Hill c o e f f i c i e n t (n H) of unity. H o w e v e r , the 1/ICs0 r e s p o n s e to C1- clearly deviated f r o m M i c h a e l i s - M e n t e n kinetics, giving a n H value of a b o u t 0.5. T h e r e f o r e , our r e s u l t s are c o n s i s t e n t with a single Na + ion being r e q u i r e d for, and a partial C l - - d e p e n d e n c e of, IMl's ability to b l o c k 5 - H T i n w a r d t r a n s p o r t . Hence, the p r e s e n t r e s u l t s obtained in i n t a c t p l a t e l e t s d i f f e r in certain a s p e c t s f r o m t h o s e obtained in p l a t e l e t p l a s m a m e m b r a n e s . P h a r m a k o l o g i s c h e s I n s t i t u t der Llniversit~it Wtirzburg, V e r s b a c h e r Straf~e 9, W - 8 7 0 0 Wiirzburg, Federal Republic of G e r m a n y

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~VIDI~NCE F O R $-HYDROXYTRYPTAMINI~ ($-HT) RBC]~PTOR AGONISTS A C T I N G AS S[IBSTRATHS O F T H H S-HT TRANSPORT B R IN RABBIT PLATBLRTS R. WSlfel and K.-H. Graefe

O-ACYLATED LYSERGOL AND DIHYDROLYSERGOL DERIVATIVES: COMPETITIVE ANTAGONISM OF 5-HT AND ALLOSTERIC MODULATION OF 5-HT 2 RECEPTORS IN RAT TAIL ARTERY H. Pertz and E. Etch

Various 5 - H T r e c e p t o r a g o n i s t s , t r y p t a m i n e s and p h e n e t h y l a m i n e s , which had ICs0 values for inhibition of aH-S-HT u p t a k e ranging from 145 to 24,500 nmol/1, were t e s t e d to d e t e r m i n e t h e i r ability to induce o u t w a r d t r a n s p o r t of aH-5-HT. P l a t e l e t s obtained f r o m r e s e r p i n e - p r e t r e a t e d r a b b i t s were loaded with a H - $ - H T ( m o n o amine oxidase inhibited), w a s h e d and then e x p o s e d for 5 min to various c o n c e n t r a t i o n s ( m u l t i p l e s of ICs0 ranging from 0.2 to 40) of each compound. The c o n c e n t r a t i o n - e f f e c t curves were used to d e t e r m i n e values of b o t h g;max (maximum of induced aH-$-HT e f f l u x e x p r e s s e d in % of the initial ~H-S-HT content) and ECs0 (drug c o n c e n t r a t i o n p r o d u c i n g Emax/2). For the 21 c o m p o u n d s t e s t e d here, a mean ratio of ECs0/IC50 of 2.4 (98% confidence limits: 1.9; 3.1) and a highly s i g n i f i c a n t l i n e a r c o r r e l a t i o n b e t w e e n EC50 and ICs0 (r= 0.903) was found. M o s t of the c o m p o u n d s [including the 5-HT r e c e p t o r a g o n i s t s (±)8hydroxy-2-(N,N-dipropylamino)tetralin, S(+)-ct-methyl-5-HT, 8c a r b o x a m i d o t r y p t a m i n e and $ - m e t h o x y t r y p t a m i n e ] had Emax values (1¢.3- 32.5% per 5 rain) which clearly exceeded t h a t produced By the u p t a k e inhibitor imipramine (6.0% per S rain), i n d i c a t i n g t h a t t h e s e are s u b s t r a t e s of the 5-HT t r a n s p o r t e r ; the o u t w a r d t r a n s p o r t induced by t h e s e c o m p o u n d s was highly s u s c e p t i b l e t o inhibition by imipramine. O t h e r s (e.g., the S-HT r e c e p t o r a g o n i s t s 5 - m e t h o x y - N , N - d i m e t h y l t r y p t a m i n e , 2 - m e t h y l - 5 - H T and S-methylurapidil) s h o w e d Emax values (3.4-11.8% per S min) which did not d i f f e r s i g n i f i c a n t l y from t h a t of imipramine, and hence, can be c l a s s i f i e d as n o n t r a n s p o r t e d inhibitors of the 5-HT t r a n s p o r ter. Em~x for t h o s e s u b s t a n c e s which behaved as s u b s t r a t e s was largely i n d e p e n d e n t of the value of ECs0 b u t n e g a t i v e l y c o r r e l a t e d w i t h the ratio of ECs0/ICs0 (r= -0.740; n= 18), which in case of t r y p t a m i n e and 8 - m e t h y l t r y p t a m i n e approached unity. Hence, some of the c o m p o u n d s used as S-HT r e c e p t o r a g o n i s t s act as s u b s t r a t e s of the S-HT t r a n s p o r t e r and, t h e r e f o r e , may e x e r t i n d i r e c t e f f e c t s by r e l e a s i n g e n d o g e n o u s S-HT. P h a r m a k o l o g i s c h e s I n s t i t u t der LIniversit~it Wiirzburg, Versbacher StraBe 9, W - 8 7 0 0 Wiirzburg, Federal Republic of Germany

Methysergide displays insurmountable antagonism of 5-HT at 5-HT 2 receptors in isolated rat tail artery (Van Nueten et aL, J Pharmacol Exp Ther 218: 217-230, 1981; Left& Marlin, Br J Pharmacol 88: 585-593, 1986; Frenken & Kaumann, this journal 335: 359-366, 1987). We synthesized 12 ergolines (Oacylated derivatives of lysergol and dihydrolysergol; Etch, Arch Pharm (Weinheim) 316, 302-309, 1983; Pertz & Etch, unpublished) in order to evaluate their effectiveness in blocking 5-HT 2 receptors in the same preparation. Contraction experiments were performed isometrically using cylindrical segments of rat tail artery as described by Bradley et aL, Br J Pharmacol 84: 915925, 1985. All compounds produced parallel, dextral shifts of the concentration-effect curves to 5-HT with little or no effect on maximal response. Schild analysis yielded slopes between 0.97 and 1.20 for all compounds. The calculated PA2-values were 7.68 for the weakest and 8.42 for the most potent ergoline derivative in this series. Further experiments with the most potent compound (9.10-didehydro-6-methyl-89-ergoline)methyl R,S-2-methylbutyrate (1__; pA 2 = 8.42 (8.07 - 8.77); slope 0.98 (0.95 - 1.01)) were performed to investigate the mode of action of this ergoline derivative. 1000 nM 1_significantly restored 5-HT induced maximal response depressed by 1 and 10 nM of the insurmountable antagonist methysergide to 90 -+ 1 and 84 + 2 %, respectively. On the other hand the concentration-effect curves in the presence of 1000 nM 1__and 100 nM methysergide were nearly superimposable to the curves in the presence of 100 nM methysergide alone. These results support the model of the allostedc receptor system proposed for 5-HT 2 receptors (Kaumann & Frenken, this journal 328: 295-300, 1985). Ergoline 1_seems to be an activator of 5-HT 2 receptors in rat tail artery. This is remarkable because other members of the ergoline family (e. g. LSD, LY 53857 and methysergide) are deactivators of 5-HT 2 receptors. Insfitut for Pharmazeutische Biologie, Freie Universitt~t Berlin, D-1000 Berlin 33, Kbnigin-Luise-Str. 2-4, F.R.G.

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BOTH SIGUAZODAN AND ROLIPRAM INCREASE POSITIVE INOTROPIC RESPONSES TO 5-HYDROXYTRYPTAMINE IN PIGLET LEFT ATRIA A. J. Kaumann, P. Raval and A. Medhurst

CHIRAL 5-HT 2 RECEPTOR LIGANDS: STEREOSELECTIVITY OPTICALLY ACTIVE ANALOGUES OF KETANSERIN S. El:,

OF

Piglet sinoatrial node possesses 5-HT4-1ike receptors that mediate positive chmnotmpic effects of 5-hydmxytryptamine (5-HT)(This Journal 342:619-621 ). Piglet left atria also contain 5-HT4-1ike receptors through which 5-HT increases both cyclic A M P and contractile force (Kaumann et al. Br.J.Pharmacol. in press). To assess whether the positive inotropic and chronotropic effects of 5-HT are related to cyclic AMP we used the phosphodiesterase (PDE) inhibitors siguazodan (PDEIII) and rolipram (PDEIv). Left atrial strips, paced at 1Hz, and spontaneously beating right atda of piglets were set up in Krebs solution (cocaine 6 p.M, (+)-propranotol 0.41~M, ascorbate 200p.M) at 37¢C. Rolipram 201~M did not modify atrial force or beating rate and siguazodan 201~M caused only occasional and marginal stimulation. However, the combination of rolipram+siguazodan markedly enhanced both force and beating rate to 83+10% of the corresponding responses to (-)-isoprenaline 2001~M, administered to terminate the experiments. Siguazodan and rolipram increased the maximum inotropic responses to 5-HT from 27±3%(n=20) to 49~-4%(n=8) and 69±4%(n=15) respectively o! the (-)-isoprenaline response without increasing the pECs0 of 5-HT. Siguazodan and rolipram marginally increased the maximum chronotropic responses to 5-HT from 63:~8%(n=8) to 79:~7%(n=8) and 79~6%(n=8). In the presence of both siguazodan and roliprem 5-HT increased force and rate from each 83±10% to 101±7%(n=8) and 99±7%(n=8) respectively of the corresponding maximum responses to (-)-isoprenaline; the pEC50 of 5-HT was increased by 1.5 log (tome) and 0.3 log (rate). The results are consistent with a function of both PDE III and PDEIv in piglet left atrium and sinoatdal node. The activity o! both enzymes appears to limit the cyolic AMP increase caused by 5-HT through left atrial 5-HT4-1ike receptors, thereby reducing the positive inotropic responses to 5-HT. The marginal effects of the PDE inhibitors on the positive chronotropic responses of 5-HT suggests that these responses are not necessarily related to cyclic AMP. Alternatively, sinoatrial cells may have a compartment into which 5-HT enhances cyclic AMP and in which PDEs are weakly active or to which the PDE inhibitors have little access.

Enantiomeric potency ratios at 5-HT 2, H 1 and o~1 receptors in favour of the (S) configurated antipodes were 457 (282), 7.6 (4.2) and 1.8 (2.9) for the methyl(ethyl)ated analogues of ketanserin. Interestingly antagonism was nonsurmountable exclusively for (S) configurated __ l b and l__ d , and was quite similar to that observed for the allosteric deactivator ritanserin (Frenken & Kaumann, this journal (1987) 335: 359-366). The maximal response to 5-HT in the presence of 1 nM l~...~b(49 _+5 %) was restored by preincubation with the allosteric activator ketansedn (30 nM; 86 + 3 %; P < 0.001) and also by a high concentration of the mirror image molecule of 1._~b,1._£c(10 p.M; 83 _4-3 %; P < 0.001). Possibly this is the first example that enantiomeric molecules can act as a weak allosteric activator / potent deactivator at the 5-HT 2 receptor in rat tail artery.

SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Hertfordshire AL6 9AR, England.

Fachbereich Pharmazie, Freie Universit~t Berlin, D-1000 Berlin 33, KbniginLuise-Str. 2-4, F.R.G.

Four optically active analogues of ketanserin were synthesized and examined in vitro for antagonistic activity in whole segments o! rat tail artery (5-HT2), rat aorta (~1) and guinea-pig ileum (H1). Affinity estimates are given in the table. Table. pA2-Valuesa and selectivity ratios of ketanserin and enantiomeric 3-[1-alkyl2-[4-(4-fluorobenzoyl)-I -piperidinyl]ethyl]-2,4(1H,3H)-quinazolinediones config, l._.~a l~b l._.q ld l._~e

alkyl

(Ketanserin) (S) methyl (R) methyl (S) ethyl (R) ethyl

5-HT2 9.59 10.2b 7.54 10.0 b 7.56

H1 8.79 7.25 6.37 6.99 6.37

(z1 5-HT2/H1 5-HT2/o~1 7.94 6.73 6.47 6.47 6.01

6 890 15 1050 15

45 2950 12 3470 35

a from Schildregression, slopes not significantly different from unity. b affinity estimated from the rightward shift produced by 0.1 and 0.3 nM of (S) configurated antagonist.

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INTERACTION OF DIHYDROERGOTAMINE (DHE), ERGOTAMINE AND GR 43175 (SUMATRIPTAN) WITH 5-HT1D RECEPTORS. Daniel Hoyer end Philippe Schoefflar.

M0~LE~kTION BY 5-HT~ and 5-HT, RECEPTORS OF 5-HYDROXYTRYP-

DHE end ergotamine are known for meny years to be effective in the acute lreatment of migraine, presumably because of their interaction with a subset of 5HT1 receptors; more recently, it has been proposod that GR 43175 (sumatriptan), a "5-HT~ like" receptor agonist, may also be effective in the treatment of migraine. The affinities of ergotamine, DHE and GR 43175 for subtypes of 5-HT recognition sites hint at the 5-HTl~ receptor subtype to represent the common target (Hoyer et aL, Cephalalgia, 1989, 9, 340). As shown here, the compounds act as marly full agonists at 5-HT~m receptors. Indeed, ergotamine, DHE and GR 43175 inhibit forskolin-stimulated adenylate cyclase activity in calf substentia nigra, a 5-HT1D receptor-methated effect, with the following rank order of potency (see table): ergotamine >_DHE > GR 43175. Effects of DHE, ergotamine and GR 43175 on fomkolin-stimulatod adenylate

cyclase activity in calf substantia nigra: Drags

pEC 5 o (-log molh)

Emax (5-HT = 100%)

n

DHE Ergotamine GR43175

8.30_+0.16 8.49+0.13 7.01+0.28

81 + 3 93 ± 1 96 ± 3

4 4 9

The present data, combined with the similar activity profile of DHE, ergotamine and GR 43175 in a model for neorogenieally-indaced vascular pain (Buzzi and Moskowitz, Br. J. Pharmacol., 1990, 99, 202) end the somewhat preferential interaction of GR 43175 with 5-HT~.D receptors (Schoeffter and Hoyer, N.-S. Arch. Pharmacol., 1989, 340, 135; Waeher et al., Synapse, 1989, 4, 168), suggest activation of 5-HT~D receptors as a common mechanism for DHE, ergotamine end possibly GR 43175, in the acute treatment of migraine. Precllnical Research, 386/525, SANDOZ Pharma Lid, CH-4002 Basel, Switzeflend.

~

(5-n~) ~r.~is~ ~

~h~ w s c u m ~

~m~us~ ~

PIG ILEUM A. Gebauer Various heteroreceptors modulate the release of 5-HT frcm the intestinal mucosa into the portal circulation by a direct action on enterochrcmaffin cells (see e.g. Schw~rer et al., this journal 339: 263, 1989; Rack~ et al., this journal 341: I, 1990). The aim of the present study was to find out whether 5 - H T q a n d 5-HT receptors (cf. Craig et al., this journal 342: 9, 1990f are also involved in the regulation of 5-HTrelease. Segments of the small intestine with their intact vascular supply were perfused in vitro and the outflow of 5-BT into the portal circulation was determined by HPLC with electrochemical detection. (Schw~rer et al., Neuroscience 21: 297, 1987). Drugs were applied via the arterial perfusion medium. All experiments where carried out in the presence of I ~mol/l tetrodotoxin to prevent neuronally mediated effects of drugs on 5-BToutflow. 2-Methyl-5-HT (30 ~mol/l), an agonist at 5-HTq receptors, increased the spontaneous outflow of 5-HT ~ 53Z 14% (p < 0.01; n=5). This effect was prevented in the presence of the 5-HT. receptor antagonis£ ICS 205-930 (I nmol/l), applied 40 ~ n before 2-methyl-5-HT. ICS 205-930 alone d e c r e a s e d t h e spontaneous 5 - H T o u t f l o w b y 41 ~ 15% (I nmol/l) and 59 + 2% (10 nmol/l) (n=3-5). The 5-RT 4 receptor agonist 5---methoxytryptamine (I ~mol/l) decreased the outflow by 42 + 7% (n=3). Micrcmolar concentrations of ICS 205-9~0 enhanced spontaneous outflow of 5-HT by 32 + 8% (I ~mol/l) and 52 + 10% (10 ~mol/l) (each n=3). In--these concentrations I--CS205-930 also blocks 5-HT 4 receptors. The results suggest that the enterochromaffin cells of guinea-pig ileum are equipped with 5-HT d and 5-HT~ autoreceptors which mediate inhibition "(5-HT4) end enhancem~_nt (5-h~3) of spontaneous 5-HT release. Pharmakologisches Institut der Zahlbacher Str. 67, D-6500 Mainz

Universit~t,

Obere

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404

SDZ 205-557, A NEW ANTAGONIST FOR 5-HT 4 RECEPTORS IN THE ISOLATED GUINEA PIG ILEUM

INVOLVEMENT OF THE SEROTONERGIC SYSTEM IN THE ANTINOCICEPTIVE EFFECTS OF TRA~L~DOL B. Driessen, H. Schlitz and W. Reimann.

K.M. Buchheit and R. Gamse A selective antagonist for the newly discovered 5-HT 4 receptor is lacking since the only antagonist available so far, ICS 205-930 (tropisetron), is a far more potent antagonist at 5-MT~- than at 5-MTA receptors. Mere we characterize SDZ ~205-557 (2-methoxy-4-amino-5-chlorobenzoic acid 2-(diethylamino) ethyl ester, SDZ) as the first antagonist which is more potent at 5-HT 4- than at 5-HT 3 receptors in the isolated guinea pig 11eum. SDZ was ~nvestigated in the non-stimulated and in the £ieldstimulated (0.I Mz, 30-40 V, 1 ms) isolated guinea pig ileum longitudinal muscle preparation (GPI) for its affinity for 5-MT&-, 5-HTq-, muscarinic- and histamine receptors. The affinity fo~ 5-HTI- , 5-HT 2- and various other neurotransmitter receptors was Hetermined by binding assays, pA 9 values were calculated by the method of Arunlakshana "and Schild. SDZ was devoid of substantial affinity (pK D values below 5.6) for all receptors investigated except for 5-MTg- and 5-HT 4 receptors. At these receptors SDZ acted asia competitive antagonist without measurable intrinsic activity. In the field-stlmulated GPI, 5-MT,~ receptor mediated responses . to 5-HT and renzapr~de were antagonized by SDZ with pA 2 values of 7.3 and 7.5, respectively. 5-MT4-receptor mediated contractions in the non-stimulated GPI were antagonized with pA 2 values of 7.0, 7.1 and 6.8 (slope i.I) for the agonists 5-MT, renzapride and zacopride respectively. Contractions induced by activation of 5HTR receptors were antagonized with a pA 2 value of 6.2 (sIope 0.66). Conclusion: In the isolated GPI, SDZ 205557 is a much more selective antagonist at 5-HT. receptors than the currently used compound ICS 205-930. 4 Preclinical Research, Switzerland

SANDOZ PHARMA Ltd., CH-4002 Basel,

The centrally acting analgesic tramadol has been shown to pessess noradrenaline uptake inhibiting properties (Naunyn Schmiedeberg's Arch. Pharmacol. 341, RI04, 1990) in addition to its opioid receptor activation. Since the serotonergic system is also involved in the modulation of noxious sensory inputs, we investigated whether tramadol has a serotonergic component in its mode of action. Purified synaptosomes from rat frontal cortex were used for serotonin uptake studies, and frontal cortex slices for serotonin release experiments. For the determination of antinociceptive effects drugs were injected intrathecally in rats and tail-flick latencies following radiant heat were measured. The serotonin uptake was blocked by nitroquipazine and zimeldine with IC50's of 0.4 nM and 0.2 ~M, respectively, thus showing specificity of the uptake. Tramadol inhibited the uptake with an3IC50 of 3.1 ~M. Tramadol enhanced the basal outflow of H-serotonin from frontal cortex slices (by I00 % at 10 ~M) without major effects on the electrically evoked overflow; the effect was strongly reduced in the presence of the serotonin uptake blocker nitroquipazine. Effects by tramadol on serotonergic terminals are compatible with an action as an indirect mimetic. Intrathecal tramadol enhanced tail-flick latencies as did morphine or a combination of by themselves ineffective doses of morphine and desipramine. I.p. injected ritanserin antagonized the antinociception by traraadol but did not affect the other two treatments. The results provide evidence that tramadol enhances the extraneuronal serotonin concentration by displacement of intraneuronal serotonln. This indirect mimetic action seems to be of relevance in vivo since antinociceptive effects of tramadol are specifically antagonized by the serotonin antagonist ritanserin. Gr~nenthal GmbH, Department of Pharmacology, Zieglerstrafe 6, D-5100 Aachen, FRG.

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IDENTIFICATION AND GENE EXPRESSION OF OPIOID PIP,IDES IN IMMUNE CELLS OF INFLAMED TISSUE: FUNCTIONAL ROLE IN ANTINOCICEPTION. C. Stein, R. Przew~ocki, F. Nyberg*, A.H.So Hassan, H. MHller, A. Herz

FUNCTIONAL ALTERATIONS IN THE RAT BRAIN AFTER 13-ENDORPHIN (1-31) AND ITS NATURALLY OCCURING FRAGMENT 13-ENDORPHIN(1-27) A.Ableitner

Our previous studies indicate that endogenous opioids released during cold water swim (CWS) can interact with peripheral opioid receptors to inhibit nociception in the inflamed hindpaw of rats, but that these opioids do not originate from central sources (1,2). This study was designed to identify and localize opioid peptides in inflamed tissue and demonstrate their functional significance in the inhibition of nociception. In rats with Freund's adjuvant-induced unilateral hindpaw inflammation we show that: (i) the levels of immunoreactive B-endorphin (B-EP) and Met-enkephalin (ME), peptides derived from the proopiomelanocortin (POMC) and proenkephalin (PENK) molecules, respectively, are significantly increased in the inflamed paw; (ii) immune cells infiltrating the inflamed tissue are stained intensely with antibodies against B-EP and ME; (iii) POMC and PENK mRNAs are detectable in the same anatomical distribution and (iv) whole-body X-irradiation abolishes CWS-induced antinociception in the inflamed paw. These findings suggest that endogenous opioids are sythesized and released from inflammatory cells upon stressful stimuli to induce localized antinociception. (i) Stein et al., Proc Natl Acad Sci 1990,87: 5935-5939 (2) Parsons et al., Pain 1990,41: 81-93. *Dep. of Pharmacology, University Uppsala, Sweden Dep. of Neuropharmacology, Max-Planck-Institut Psychiatrie, 8033 Martinsried, F R G Supported by DFG and IARS.

The quantitative autoradiozraohic 2-deoxyglucose technique h a s been e[nployed to investizage the functional consequences of mtracerebroventricuq'ar (i.c.v.) ad ,ministration of 13-endorphin (1-31) and its fragment 13enaorohin(1-27). B-endorptiin (1-31) at 8#g (i.c.v.) most notably increased ~:lucose utiIization in regions of the hippocampaI formation, such as the sublculum and the hippocamp.us proper..Within the hippocamp.us proper t.he strongest i.ncreases (up to 200%) were found in tlae s_t.ratu~ radiatum, pyramidale .andoriens of field CA3. The ettects in the ffdrsal part ot the hippocampus proper were less marked as compared to tlae ventral part. Furthermore, significantly increased metaboli.sm after 13.endorphin (1-31-) was measure.d in the latera.l septal n~cl. a.nd regions involved ip .central motor 99ordina.tlon. g#g t~-endorphin (1-27) failed to cause significant alterations in regions sensitive to 13-endorlghin (1-31). At 16t~g, hpwever, th.e fragment, pro~duce,d a, similar pattern as ooserv~ed,witt~ tlae 9omp,iete t~-en..aor~nin,sequen.ce.. laKe.n togemer, these studies tirstlyorovide data in s.,uppor.t, or. an agonistif avtivi~ty o.f 13-en.dprphin (1-27). be.condly, the str.ong.enects otoptg peptiaes on gmcose _utilization, in, tn~e la!ppocampai ~9.rm.atiop sugges.t a~ tunctionm ro~e ot enoogenous opiolOs ~n tlae control or hippocampal excitability.

406 MODULATION OF A 10 DOPAMINE NEURONS BY TONICALLY ACTIVE ENDOGENOUS OPIOID SYSTEMS

Supported by the Deutsche Forschungsgemeinschaft Institut f~ir Pharmakolo~ie, Toxikolo~ie und Pharmazie, Universitat Mtinchen, Ktniginstr. 16, 8000 Miinchen 22

408 G PROTEINS IN THE OPIATE TOLERANT/DEPENDENT GUINEA-PIG MYENTERIC PLEXUS H. Ammer and R. Schulz

R. Spanagel The opposing motivational properties o f # - and k-opioid agonists are mediated by the dopaminerglc mesollmbic system. However, the neuroanatomical sites and also the endogenous 0pioid systems which modulate this reward pathway have not been so well characterized. Therefore we examined this issue by the use of in vivo microdialysis in the rat and the administration of highly selective opioid agoulsts and antagonists. Opioid infusions into the nucleus aectunbeus (NAC) and mlcroinjectious into the ventral tegmental area (VTA) were utilized. Microdialysis probes were inserted into the NAC and perfusates were analysed for dopamine (DA) and its metabolltes: dlhydrox'yphenylacetic acid (DOPAC) and homovanillic add (HVA) using for seperation and quaatitieation a reversed phase HPLC-system with electrochemical detection. Drugs were administered into the NAC via the microdialysis probe by use of a liquid switch. The microinjections into the VTA were made by inserting a 33 gauge injection eannula, the injection volttme being 0.3/~ 1. DAMGO, a highly selective #-agoulst, mieroinjected into the VTA sigttificantly increased DA overflow and metabolltes. However, DAMGO infusion into the NAC did not affect basal levels of DA or its metabolites. VTA microinjections of the #-antagoulst CTOP (D-Pcn-Cys-Tyr-DTrp-Orn-Thr-Pen-Thr-Nl-I2) resulted in dose dependent decreases in DA overflow and metabolism. In contrast, c r o p infusion into the NAC was without effect. The selective k-agoulst U50,488H (trans-3,4-diehloro-N-[2-(pyrrolidinyl)cyclo-hexyl]-benzeneacetamide) microinjected into the V'rA was without any effect on DA overflow and metabolites in the NAC. In contrast, U 50,488H infusion into the NAC resulted in dose dependent significant decreases in DA overflow and metabolism after 40 rain of infusion. Infusions of the k-antagoulst nor-BNl into the NAC produced sigaltlcant dose dependent increases in DA overflow and metabelltes within the NAC. The present results demonstrate that tonic activation of both,/~-receptors in the VTA and k-receptors in the NAC, which are probably presy~aptically located, are required for the maintenance of basal DA release in the NAC. Supported by the Btmdesgesandheitsamt, Berlha Departement of Neuropharmacology, Max-Planek-Iustitut fiir Psychiatrie D-8033 Martinsried, FRG

G proteins are linked to opioid receptors and have been shown to be involved in the development of tolerance and dependence. Using site-directed polyclonal antibodies, G proteins were quantified in particulate membrane fractions of the guinea-pig myenteric plexus by means of Western blotting analysis. In naive preparations Goe and G/~ are most abundant in both nerve somata and terminals, whereas Gi2e and Gse were found at minor levels, mainly associated with somata. Our previous studies have shown that chronic treatment (6 days) of guinea pigs with opioids (#- or z-receptor agonists) or clonidine elevates G protein levels in the guinea-pig myenteric plexus. We found a significant increase of Go~, and G/~ in both pre- and postsynaptic membrane fractions. Here we extend our investigations to additional G protein subunits and to long-term opiate treatment of guinea pigs. In contrast to the data obtained for G,~ and G/~, only chronic stimulation of #opioid receptors caused an elevation of G,,e, and this effect is confined to nerve somata. Chronic exposure'~of~-opioid receptors as well as of e--adrenoceptors failed to affect G |~ -(z Moreover any of the ~nd~cated chronic treatment of the animals faded to alter detectable Gs(~ levels. Long-term exposure (4 weeks) of guinea pigs to #- or ¢-opioids did not result in a further increase in G proteins as compared to 6 day treatment. However, recovery of the elevated G protein concentrations upon withdrawal of the drugs was markedly delayed as compared to preparations exposed for 6 days. These findings contribute to the understanding of opiate tolerance and dependence, as appearance and disappearance of changes in inhibitory acting G protein concentrations do reflect adaptive mechanisms. Stimulatory acting G proteins were not found of critical significance with this respect. •

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Supported by SFB 220 Institut for Pharmakologie, Toxikologie und Pharmazie der Universit&t M0nchen, KSniginstr. 16, W-8000 M0nchen 22

R 103 409

411

THE EFFECTS OF PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE (PACAP) ON PORCINE SMALL INTESTINAL SMOOTH MUSCLE H. Schw~rer, W.E. Schmidt, S. Katsoulis and W. Creutzfeldt

H O E 140 IS A NEW, SPECIFIC, POTENT AND ACTING B2-BR~DYKININ RECEPTOR ANTAGONIST D. w i r t h a n d B.A. S c h S l k e n s

PACAP peptides are new members of the glucagon/secretin/V~ family and stimulate adenylate cyclase activity in rat anterior pituitary cells. PACAP immunoreactivity was observed in nerve fibres and in nerve cell bodies of the intramural ganglia of the porcine and human gut. In the present work we studied the effects of the C-terminally amidated form PACAP-38 synthesized by Fmoc-solid phase strategy on the smooth muscle activity of the porcine small intestine. The muscle was cut in the longitudinal axis and the strips were suspended isometrically (preload 10 nM) in a physiologic salt solution (37~cl in a 3 ml organ bath. The porcine small intestinal specimens showed spontaneous phasic contractions which were not affected by tetrodotoxin {TTx, I ~mol/l), scopolamine (0.3 ~mol/l) or hexamethonium (100 pmol/l). The calcium channel antagonists verapamil and nifedipine (0.03-10 ~mol/l), which block L-type calcium channels, inhibited the phasic contractions, whereas ~-conotoxin (blockade of L- and N-type calcium channels) did not affect the spontaneous contractions. PACAP (0.01-I~mo~ i) concentration- dependently inhibited the phasic contractions; a complete inhibition was observed at 0.3 ~mol/l PACAP. The inhibitory effect of PACAP on the phasic contractions was concentration- dependently antagonized by apamin (0.03-3 ~mol/l), an inhibitor of calcium-dependent potassium channels. The effect of PACAP was preserved in the presence of vasoactive intestinal polypeptide (VIP, 0.01-1 ~mol/l) which by its own on~y inconsistently inhibited the phasic contractions. In conclusion, PACAP affects the spontaneous phasic contractions of the porcine small intestinal muscle via activation of specific PACAP receptors on the smooth muscle cell. In addition, these receptors seem to be linked to ++ + apam~n sens±tlve Ca -dependent K channels.

H o e 140 is a new, p o t e n t , s t a b l e a n d s p e c i f i c B2-bradykinin (BK) antagonist (D-Arg-Arg-ProHyp-Gly-Thi-Ser-D-Tic-Oic-Arg) that exceeded the potencv of the BK antagonist D-Arg[Hyp2,Thi5"8,D-Phe7]BK (DA) synthetised by S t e w a r t b y 3 o r d e r s of m a g n i t u d e . in rive: H o e 140 w a s h i g h l y p o t e n t and long acting against BK-induced depressor responses in a n a e s t h e t i s e d rats. F o u r h o u r s a f t e r s.c. administration of 20 nmol/kg Hoe 140, i n h i b i t i o n w a s s t i l l 60% w h e r e a s t h e i n h i b i t o r y effect of DA (200 nmol/kg s.c.) was not significant at any time. BK-induced bronchoconstriction in anaesthetised guinea pigs assessed via whole body plethysmography was strongly i n h i b i t e d b y H o e 140 (EDs0 13.4 p m o l / k g f o r RL). A f t e r 1 hour, 1 n m o l / k g i.v. still inhibite~ bronchoconstriction b y 60 %. 20 nmol/kg s.c. abolished the BK effect for 2 hours. I n h i b i t i o n w a s s t i l l 50% a f t e r 4 h o u r s . in v i t r o : ~n t h e g u i n e a p i g ileum, Hoe 140 inhibited [ ~ H ] B K b i n d i n g w i t h a n I C 5 0 (l.2nM) c o m p a r a b l e t o u n l a b e l l e d B K (l.6nM) W h e r e a s D A was less potent (85nM). In c u l t u r e d porcine e n d o t h e l i a l c e l l s (EC), H o e 140 a n t a g o n i s e d t h e BK-induced EDRF release (~C50 lOnM) and i n c r e a s e in c y t o s o l i c f r e e c a ± c l u m (IC50 inM). I n E C of b o v i n e aorta, 1 0 0 n M t o t a l l y s u p p r e s sed the BK induced PGI~ release. DA (100nM) showed a weaker antagonlsm. H o e 140 w i l l be a v a l u a b l e t o o l t o i n v e s t i g a t e t h e r o l e of B K a n d may offer therapeutic potential.

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Medizinische Universit~tsklinik G~ttingen, Abt. Gastroenterologie und Endokrinologie, Robert-Koch-Str. 40, D-3400 G6ttingen

H O E C H S T AG; P G U H e a r t - C i r c u l a t i o n ; H 80 03 20 D - 6 2 3 0 F r a n k f u r t / M . 80; F R G

821;

LONG

POB

410

412

NEUROPEPTIDERGIC CONTROL OF INSULIN RELEASE: MODE OF ACTION OF G R P ON ISLET FUNCTION M.A. Wahl, E.A. Landsbeck, R.J. Plehn and E.J. Verspohl

BRADYKININ ANTAGONISTS WITH HIGH POTENCY AND LONG DURATION OF ACTION IN VIVO T. GHesbacher and F. Lembeck

activity underlying the modulating effect of G R P on insulin release.

The novel bradykinin antagonists DArg[Hyp3,ThiS,DTicT,OicS]BK (HOE 140, 1), DArg[Hyp3,DTicT,OicS]BK (11) and [Arg(Tos)l,Hyp3,Thi5,DTicT,Oic8]BK (Ill) were compared with the antagonist DArg[Hyp2,ThiS,8,DPheT]BK(IV) described by Stewart and Vavrek (Adv. Biosci. 65:73, 1987). Compounds I, I! and Ill were 2-3 orders of magnitude more potent than compound IV in vitro and showed a duration of action greatly exceeding the time of presence in the assay preparations (Lembeck et aL, Br. J. Pharmacol., in press). (1) Plasma protein extravasation by BK (500 nmol/kg, i.v.) in duodenum, trachea and urinary bladder of rats was completely abolished by compounds I and 111 (500 nmol./kg) given up to 30 min before BK. Compounds I and 11 showed an agonist activity when given at high doses (5 ~mol/kg). (2) Hypotensive effects of BK (100 pmol, i.a.) in rats were inhibited by short infusions of compounds I and II (300 pmol within 3 min, i.a.) for a period of about /5 rain. 1000 pmol of either compound blocked the effects of BK completely and more than 25 rain were needed to restore the original potency of BK. Compound II1 was less effective by a factor of about 3. Compound IV (1 nmol, i.a.) was completely ineffective. (3) Bronchoconstriction induced by i.v. injection of BK (1 nmol) in anaesthetized guinea-pigs was blocked by i.v. bolus injection of compounds 1 (1 nmol), 11 (1 nmol), and III (2 nmol). The inhibition lasted for at least 20 rain. The inhibitory effect of compound IV (100 nmol) was limited to less than 10 rain. Histamineinduced bronchoconstriction was unaffected by all antagonists, and even extremely high doses of the novel compounds (up to 300 nmol i.v.) did not show any agonist activity. (4) Reflex falls in systemic arterial blood pressure following BK-induced nociceptor stimulation in the rabbit reflex ear preparation were abolished after 5 min infusions of compounds I (15 riM) and 111 (50 riM) (11 and IV not tested). The inhibition lasted for at least 1 hour. A BK antagonist used earlier (LysLys[Hyp3,ThiS,S,DPheT]BIC,,500 nr¢l) showed an inhibition only during its infusion (Griesbacher and Lembeck, Br. J. Pharmacol. 92:333, 1987). The high potency and respectable duration of action of the novel antagonists also under in vivo conditions allows to investigate the role of BK in pathophysiological conditions. Therapeutic applications are likely to be expected.

Department of Pharmacology, Inst. of Pharmaceutical Sciences, EberhardKarls University, A u f der Morgenstelle 8, W-7400 Tiibingen, FR Germany

Depamnent of Experimental and Clinical Pharmacology, Universit~itsplatz 4, A-8010 Graz, Austria.

GRP, a 27 amino acid neuropeptide, is released from nerve endings in the pancreas. It was recently shown that G R P increases insulin release, ~SCa~+ uptake and electrical activity of pancreatic islets. G R P binding sites and the involvement of inositol metabolism have not been investigated yet in pancreatic islets. Binding of

100 pM

[nSI-TyrlS]GRP to colla-

genase-isolated mouse pancreatic islets at 24 *C was time-dependent; maximal binding was 1.65 % of radioactivity per 50 islets; half-maximal binding was achieved at approx. 15 rain; non-specific binding (presence of 1 # M unlabelled GRP) was less than 10 % of totally bound radioactivity. Tracer degradation was less than 13 %. The ICs0 of competition of binding by unlabelled G R P was 2.4 nM; iterative analysis of the data showed one binding site. Binding was specific with respect to the

rank

order

of

GRP

analogues (N-terminus

of

GRP(I-16),

NA-GRP(20-27), and hormones not structurally related to G R P like VIP and galanin. The rank order of G R P analogues on glucose(16.7 mM)-induced insulin secretion (RIA) was similar to their affinity rank order. G R P augmented inositol- 1,4,5-trisphosphate (determined by a mass assay; ECs0 = 0.8 nM). In a virtually Ca2+-free medium 5 nM G R P increased the 4SCaZ+ release of preloaded islets. We conclude that highly specific binding sites for GRP are present in mouse pancreatic islets. They may be involved in the release of Ca ~+ from intracellular stores by inositolphosphates and the known increase in Ca ~+ uptake and electrical

University of Graz,

R 104 413

415

AFFERENT C-FIBRE FUNCTION FOLLOWING ACUTE OR CHRONIC CHEMICAL SYMPATHECTOMY

ACTIVATION OF SUBSTANCE P RECEPTORS MODULATES THE RELEASE OF LOCALLY SYNTHESIZED NERVE GROWTH FACTOR (NGF) FROM RAT SAPHENOUS NERVE NEUROMA. U. Otten, P. Ehrhard, F. Keller, and C. Boeckh °

J. Donnerer, and R. Schuligoi An interaction of the sympathetic efferent system with the sensory afferent system has been demonstrated in neuromas (J/inig, Neurosei. Lett. 111: 309, 1990) and during noxious, tissue damaging stimuli (Coderre et al., J. Neurophysiol. 62: 48, 1989). The aim of the present study was to investigate whether such an interaction also takes place under physiological conditions. To answer this question, a selective chemical sympathectomy was induced in rats either at ueonatal (chronic sympathectomy) or at adult age (acute sympathectomy) using guanethidine or 6-hydroxydopamine and its effects on neuropeptlde content, neuropeptide release and excitability of primary afferent C-fibres Were measured. All 4 treatment protocols lead to a 80 - 90% depletion of noradrenaline from postganglionic sympathetic nerves in heart, spleen and skin. However the neuropeptide transmitter (substance P and calcitonin gene-related peptide) content in afferent C-fibres was found unchanged following acute sympathectomy and increased by 20% in neonatally sympathectomized rats. Afferent C-fibre function was tested with the wiping response to capsaicin instilled into the eye and with the tall withdrawal response from 50°C water. The sensitivity towards capsaicin and towards heat was found to slightly increase following neonatal sympathectomy. The release of sensory neuropeptides from the peripheral terminals was tested by means of plasma extravasation induced either by antidromic saphenous nerve stimulation or by mustard oil application. Only neonatal guanethidine treated rats showed an impairment in plasma extravasation upon antidromic nerve stimulation whereas no changes were observed following the other pretreatment protocols and stimuli used. It is concluded that there is no gross impairment of sensory C-fibre function after selective chemical sympathectomy in the rat.

The most proximal segment of the transected saphenous nerve, forming a stump neuroma provides a preparation suitable for studying mechanisms in NGF synthesis and release. In saphenous nerve neuromata of adult rats a long-term increase in NGF protein was detected by an enzyme-linked immunoassay after nerve transection. There was a rapid 4-fold increase in NGF levels 6h after injury, which reached peak values (10-fold) after 2-4 days and subsequently fell to 2-fold elevated levels within 3 weeks. Quantitative Northern blots showed that NGF mRNA levels increased rapidly, reaching a maximum (up to 30-fold) about 12-18h after transection, indicating that the increase in NGF in response to axotomy is due to local synthesis. In situ superfusion of neuromata with Tyrode solution revealed a continuous basal release of NGF protein which was drastically decreased by substance P (SP) in a dose-dependent manner. Maximum inhibition (85 + 10 %, n = 8) occurred at a concentration of 0.1 I~M. Substance K was less potent than SP. Neurokinin B, as well as other peptides such as calcitonin gene-related peptide, somatostatin, bradykinin and neurotensin at concentrations up to 50 g.M did not significantly affect NGF release. The rank order of potency of tachykinins and SP fragments strongly indicates that SP specifically regulates NGF levels in the microenvironment of regenerating nerve fibers by activation of a SP (neurokinin-1) receptor.

Institut fiir Exp. & Klin. Pharmakologie, Universit/itsplatz 4, A-8010 Graz, Austria

Institut for Physiologie, Universit~.t Basel, Vesalgasse 1, CH4051 Basel ° Pharmakologisches Institut der Universit&t Freiburg, Hermann-Herder-Strasse 5, D-7800 Freiburg

414

416

EFFECT OF PGE~ ON THE PERIPHERAL FUNCTION OF PRIMARY AFFERENTS R. Amann, J.M. Hall*, and D. Mitchell*

IDENTIFICATION OF THE TACHYKININ RECEPTORS MEDIATING SUBSTANCE P ACTIONS LN THE HYPOTHAI.AMUS OF THE RAT C.J. Lebrun *, P. Wende, lL ltoi, U. Stechelings

Prostaglandins are known to increase the excitability of C-fibre afferents in the rabbit (Weinreich D. & Wonderlin W.F., J. Physiol. 394: 415; 1987). Thus, in the isolated perfused rabbit ear with intact neuronal connection, prostaglandin E~ (100 nM) lowered the threshold for bradykinin and eapsalein to elicit a depressor reflex, To investigate the effect of prostaglandin E~ on the peripheral function of primary afferents, the isolated rabbit iris sphincter muscle was used. Electrical field stimulation, bradykinin and capsalcin produced eontraetious which have been shown to be caused by mediator release from primary afferents (Ueda et al., J. Pharmacol. Exp. Ther. 230: 469; ~.984). Tetrodotoxin did not affect the response to bradykimn or capsaiein, but abolished that to electrical field stimulation. Omega-conotoxin reduced field stimulation- and bradykinin-induce.d contractions but not the effect of capsaicin or taehykialus. Prostaglandin Ez (100 nM) enhanced only the omega conotoxin-sensitive responses, but did not influence capsaicin or taehykinin-induced contractions. The results suggest that prostaglandin Ez does not generally facilitate mediator release from afferent terminals. Its effect is limited to stimuh which cause release secondary to depolarization induced Caz+ influx. Dept. Exp. & Clin. Pharmacology, Univ. Graz, Ualv.-Pl. 4, 8010 Graz and * Pharmacology Group, King's College London SW3 6LX, UK supported by FWF P7676M

Substance P (SP) stimulates the hydrolysis of inositul phospholipids in peripheral tissues and in the brain. In mammalian peripheral tissues three tachykinin receptor subclasses (NK1, NK2, NK3) have been identified. The purpose of our study was to pharmacologicallycharacterize the SP receptors in the brain using phospboinositide breakdown as a functional response. SP (NK1 agonist) and Neurokiuln A (NKA) (NK2 agonist) stimulated the phosphoinosifide metabolism in the hypothalamus dose dependently but SP was 100 times more potent than NKA. Th,~ NK2 selective antagonist L-659,877 (eydo(Gln-Trp-Phe-Gly-Leu-Met)) (10-"M) abolished the SF (10-°M) induced phospboinositide metabolism without affecting basal phosphoinosltide breakdown. The NK1 selective antagonist 1.-669,169 (cyclo(Gin-D-Trp(NMe)Phe(R)GIy(ANC-2)Leu-MeO2) stimulated basal p~osphoinusitlde breakdown at concentrations of 10"M and 10"°M but not at 10-°]~1.Tho higher concentration partially inhibited the stimulatory effects of SP (10-°M). SP,~C-terminal fragments such as SP(5-11) (10-6M) stimulated the accumulation of aI"I-inositol phosphates and acted as ~ agoulst because maximal responses wore idantie~l to that fotmd with ~P (10"~VI). SP N-terminal fragments such as SP(1-4) (10"~M) and SP(1-7) (10-"M) wore devoid of activity. Our results suggest that SP C-terminal fragments may be the active component involved in stimulating phospboinositide hydrolysis in the hypothalamus. Furthermore, the tachykinin receptors mediating the actions of substance P in the hypothalamus are different from any subtypes of receptors in the periphery. * Present address: Dept. of Pharmacology, Univ. of Heidelberg, Im Neuenheimer Feld 366, FRG.

CHARAKTERISIERUNG DER SUBSTANZ-P-WIRKUNGSVER MITTELNDEN TACHYKININREZEPTOREN IM HYPOTHALAMUS DER RATTE

R 105 417 EFFECTS OF C- AND N-TERMINAL SUBSTANCE P (SP) FRAGMENTS INJECTED INTRACEREBROVENTRICULARLY ( I C V ) IN CONSCIOUS RATS C.

Tsch~pe,

N. J e s t ,

R. R ~ q h t e r

it has been a l r e a d y e s t a b l i s h e d t h a t sP ( 1 - 1 1 ) in~ected icy in conscious rats induces a cardiov a s c u l a r d e f e n s e r e a c t i o n and a t y p i c a l behavioral r e s p o n s e . I n t h e b r a i n SP ( 1 - 1 1 ) i s e n zymatical]y hydro]yzed to several pept~de fragments, w h i c h may a l s o be b i o l o g i c a l l y active. T h e r e f o r e , we s t u d i e d c e n t r a l e f f e c t s o f SP Cand N - t e r m i n a l f r a g m e n t s on c a r d i o v a s c u l a r and behavioral responses in comparison with those of SP ( 1 - 1 1 ) . We i n ~ e c t e d t h e r e ] l o w i n g C - t e r m i n a l f r a g m e n t s : SP ( 5 - 1 1 ) , SP ( 6 - 1 1 ) , SP ( 7 - 1 1 ) , SP ( 8 - 1 1 ) and one N - t e r m i n a l f r a g m e n t : SP ( 1 - 7 ) icv in conscious chronically i n s t r u m e n t e d r a t s . Data (mean ± SEM) a r e e x p r e s s e d a s t h e a r e a under t h e c u r v e . SP ( 1 - 1 1 ) i n d u c e d a d o s e - d e p e n d e n t ~ncrease of the mean arterial

blood pressure,

heart

r a t e (233±26 bpm-min, 1653±383 mmHg-min a f t e r 50 pmol) and t h e t y p i c a l behavioral response. The C - t e r m i n a l f r a g m e n t s SP ( 5 - 1 1 ) and SP ( 6 - 1 1 ) ~nduced e q u i p o t e n t cardiovascular and b e h a v i o r a l r e a c t i o n s as SP ( 1 - 1 1 ) . The C - t e r m i n a l f r a g m e n t s SP ( 7 - 1 1 ) , SP ( 8 - 1 1 ) and t h e N - t e r m i n a l f r a g m e n t SP ( 1 - 7 ) were d e v o i d o f a c t i v i t y . Our results suggest that the last 6 amino acids of the C-terminal SP-sequence are responsible for e]iciting the typical tachykinin-like cardiovascular and behavioral response to SP. Dept. o~ PharmacoTogy, University Of He~de~berg Im Neuenhe~mer FeTd 266, 6900 HeYdeiberg, FRG

418

SK-N-MC cells co-express 6~-adrenoceptors coupling to stimulation of adenylate cyclase and NPY receptors coupling to mobilization of intracellular Ca ++. Pre-treatment of SK-N-MC cells with the 6-adrenoceptor agonist (±)-isoprenaline enhanced the NPY-induced mobilization of intracellular Ca ++ (assessed by Fura-2 fluorescence). This enhancement was time- (maximal after 24 h) and concentration-dependent (maximal at i0 ~mol/l isoprenaline) and blocked by simultaneous addition of i0 ~mol/l (±)-propranolol. Thus, sensitization of the NPY response required significantly higher concentrations and duration of pre-treatment than homologous desensitization of B~-adrenoceptors (maximal at i00 nmol/l of iseprenaline, assessed as down-regulation of specific [~SI]cyanopindolol binding sites). A similar enhancement of NPYstimulated Ca ++ increases was seen after pretreatment with forskolin. The enhanced Ca ++ mobilization was characterized by a reduced ECho and a simultaneously increased maximal response for NPY. The enhancement was not accompanied by significant alterations in the number or affinity of high affinity [~SI]NPY binding sites. Similarly, we could not detect significant changes of G±-like proteins (as assessed by pertussis toxin-catalyzed ADP-ribosylation). We conclude that isoprenaline enhances NPY-stimulated Ca ++ mobilization in SK-N-MC cells via a cAMP-dependent pathway; this enhancement may occur at a site distal to the G~-protein. Dept. Medicine, University of Essen, Hufelandstr. 55, D-4~00 Essen, FRG

42O

G-PROTEIN COUPLING OF NPY RECEPTORS CELLS. F. Feth and W. Rascher

IN

SK-N-MC

T h e h u m a n neuroblastoma cell line SK-N-MC expresses NPY binding sites of the Yl-type. We have studied the role of G-proteins in the regulation of NPY receptor signalling and binding of the agonist [I=Sl]NPY in these cells. NPY elicited two independent second messenger responses, inhibition of cAMP accumulation (ECho: 1.2 nM) and mobilization of intracellular Ca *+ (ECho: 6.1 nM). Pretreatment of intact SK-N-MC cells with i00 ng/ml pertussis toxin for 24 h completely inactivated all pertussis toxin substrates (as assessed by ADP-ribosylation) and completely abolished the NPY-stimulated second messenger responses. Binding of the agonist ligand [I=~I]NPY in digitoninpermeabilized cells was concentration-dependently inhibited by GTPyS, but even 500 ~M GTPyS did not completely abolish high affinity [I=SI]NPY binding. Saturation binding experiments in the absence and presence of GTPyS demonstrated a reduced number of high affinity binding sites with the remaining sites still having high affinity for the ligand. Pro-treatment with pertussis toxin abolished the inhibition of [I=SI]NPY binding by GTPyS. In nonpermeabilized cells, pro-treatment with pertussis toxin by itself reduced the number of high-affinity [I=~I]NPY binding sites. We conclude that high-affinity [~='I]NPY binding sites represent functional NPY receptors; alterations of the functional state of SK-N-MC G-proteins alter NPYstimulated second messenger responses and agonist binding. Thus, alterations of [~=~I]NPY binding sites do not necessarily mirror alterations of receptor number. Dept. Pediatrics, University of Essen, Hufelandstr. 55, D-4300 Essen i, FRG

Deutscher Titel: Zentrale kardiovaskul~re m i n a l e r S u b s t a n z P (SP)

419 REGULATION OF NPY-STIMULATED Ca ++ MOBILIZATION BY PRE-TREATMENT WITH ISOPRENALINE OR FORSKOLIN M.C. Michel

-

Effekte C- und Fragmente b e i

N-terwachen

NEUROPEPTIDE Y (NPY) POTENTIATES ANGIOTENSIN II (All)-INDUCED VASOCONSTRICTION AND ACCUMULATION OF INOSITOL PHOSPHATES (IP). F.Cressier and K.G.Hofbauer NPY has a direct vasoconstrictor action and potentiates the effects of other vasopresser hormones. To elucidate the type of NPY receptor and the effector mechanisms involved in these effects, we studied the influence of NPY (acting on Yl-and Y2-receptors) and NPY13-36 (acting only on Y2-receptors) on the formation of IP induced by All. We used the rabbit femoral artery to measure vasoconstriction and accumulation of IP (determination of IP1, IP2, IP3 in the presence of 10 mM LiCI, after incubation with [3H]myo-inositol). The vascular effects of All (1 nM) were potentiated by NPY, at a dose (0.05 I~M) which had only a weak direct vasoconstrictor action. NPY13-36 had no effect. All (0.011.0 I~M) induced a dose-dependent formation of IP. NPY (1 ~M) potentiated this effect of All, without having any effect by itself. In contrast, NPY13-36 did not affect the All response. To investigate whether a potentiation of the IP response can be induced by inhibition of adenylate cyclase, we used the ~2-agonist clonidine (0.01-1.0 ~M). Only an additive effect on the All (1 ~M)-induced IP formation was observed with the highest concentration of clonidine which produced a small rise in IP by itself. In conclusion, NPY potentiates the All-induced vasoconstriction and accumulation of IP. This effect is probably mediated by Y1receptors since the Y2-selective fragment NPY13-36 is inactive. Whether the action of NPY on the All-induced IP increase is due to receptor activation or secondary to an increase in intracellular Ca2+ needs to be evaluated. Cardiovascular Research Dept, Ciba-Geigy Ltd., Basel, Switzerland.

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423 I~OLE OF ANGIOTENSIN II IN THE DRINKING RESPONSE AFTER WATER DEPRIVATION

H. STAUSS, TH. UNGER

It ist well known that angiotensin II (ANGII) injected intracerebroventricularly (icy) in rats induces a drinking response. It is not clear, however, whether ANGII plays a role in physiological drinking. Earlier studies employing the ANGII receptor antagonist saralasin failed to provide clearcut evidence. The aim of this study was to investigate whether the drinking response after water deprivation is affected by the more specific ANGII-ATl-receptor antagonist Dup753 (2-nbutyl-4-chloro-5-hydroxymethyl-1-[(2-( lII-tetrazol-5-yl)biphenyl-4-yl)methyl]). Sixteen Wistar rats were instrumented with an icy cannula and an arterial catheter. Water intake was induced by (1) 100ng ANGII icy, (2) 240rig carbachol icy or (3) 24 h water deprivation. Each drinking challenge was preceded by either placebo icy, Dup753 (25/~g icy, n=7), or atropine (10/~g icy, n=9). Water intake was monitored for 30 min after each drinking stimulus. At the end of each experiment, the efficacy of the icy receptor blockade was tested by recording the blood pressure response to icy injections of the respective agonists. The ANGII-induced drinking response (9,0=E1,3ml, means:i:SEM) was completely abolished by Dup753 icy. Similarly atropine icv completely prevented carbachol-induced drinking (5,4=E0,Sml). On the other hand, water intake after ANGII was not affected by atropine icy and water intake after carbachol icv was not affected by Dup753. The drinking response after water deprivation was significantly attenuated by either Dup753 icy (7,2=E0,7ml versus 4,0=E0,4ml, p<0,05) or by atropine icy (10,7:t:l,6ml versus 5,2~-0,9mI, p<0,01). Our results suggest that ANGII as well as the cholinergic system play an important role in the physiological drinking response after water deprivation. *Present address: German Institute for High Blood Pressure Research and Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 6900 Heidelberg, Germany

EFFECTS OF IMMUNOSUPPRESSlVE TREATMENT ON RAT THYMIC ATRIAL NATRIURETIC PEPTIDE 1-126 (ANP 1-126) A.M. Vollmar and F. Colbatzky The presence and synthesis of ANP in various I~/mphoidorgans .such as the spleen, thymus and lymph nodes, hnks ANP to the ~mmunesystem. The following investigation provides further evidence for this notion. ANP-gene expression in the thymus is altered by immunosuppressive treatment. The glucocorticoid dexamethasone (1.5mg/kg rat, s.c.) as well as X-ray radiation (6 Gy) cause profound involution of the thymus (3-4 days after treatment, 78-84% decrease in weight, respectively) which results in a striking increase of mRNA coding for ANP (up to 30 fold). Consecutively, the ANP precursor molecule ANP 1-126 was found to be 4-6 fold elevated as compared to untreated controls. During regeneration of the thymus (10 and 18 days after treatment, respectively) the mRNA coding for ANP and the level of ANP precursor normalize. Immunohistochemical analysis of acute involuted thymus revealed heavy ANP-specific staining in macrophages. As examined by routine- and enzymehistochemistry, this cell population is markedly increased and activated in the thymocyte depleted tissue. In addition, the actively phagocytozing interdigiting cells of the thymic medulla appear to contain the peptide. In comparison, thymus of untreated animals displays only weak staining for ANP in cells dispersed in the cortical area (thymocytes and/or macrophages) and in some medullary epithelial cells. Activation of the phagocytotic system by the cytotoxic action of a glucocorticoid as well as radiation plays an important role for the regeneration of the thymic tissue and, interestingly, seems to be responsible for the strong stimulation of thymic AMP production under immunosuppression. ANP may therefore be involved in mechanisms leading to depletion of thymocytes and more likely to regeneration of the thymus after immunosuppressive actions on the organ. Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, K6niginstr. 16, 8000 Munich 22, FRG.

422 ROLE OF ALPHA- ADRENOCEPTOR STIMULATION IN THE NUCLEUS PARAVENTRICULARIS (PVN) FOR THE VASOPRESSIN (AVP) RELEASE INDUCED BY CENTRAL ANGIOTENSIN II (ANG II). Annette Veltmar. Thilo Stadler and Fatimunnisa Oadri. Evidence from in vitro studies suggests that brain catecholamines are instrumental in the central actions of ANG II such as blood pressure increase, drinking and release of vasopressin from the PVN or nucleus supraopticus through the pituitary gland. Using the microdialysis technique in conscious rats, we have shown that stimulation of specific periventricular ANG II- AT 1- receptors caused an immediate, dose dependent and selective NA increase in the PVN closely associated in time with the increase in blood pressure. We have now investigated the hypothesis, whether the ANG II- induced AVP release into the blood is mediated by alpha- adrenergic receptor stimulation in the PVN by measuring the ANG II- induced AVP release after alpha- adrenergic receptor blockade in the PVN. The alpha2adrenoceptor antagonist, idazoxan (4 nmol), injected bilaterally into the PVN significantly reduced the ANG II (100 ng intracerebroventricularly)- induced AVP release into the blood. Pretreatment via the same route with the alpha1- adrenoceptor antagonist, prazosin (0.7 nmol), also inhibited the ANG IIinduced increase of plasma AVP. These results suggest that stimulation of alpha1- and alpha2adrenoceptors in the PVN contributes to the AVP release induced by periventricular ANG II receptor stimulation. They support the view that some of the ANG II- induced responses are mediated through central catecholaminergic pathways. *Present address: Dept. of Pharmacology, Univ. of Heidelberg, INF 366, 6900 Heidelberg, FRG

424 STRUCTURAL MODIFICATION OF 6-CASOMORPHIN PEPTIDES - CONSEQUENCESON TRANSMITTER UPTAKE PROCESSES E. Kammerer, D.M. Leon*

The opioid peptide 6-casomorphin 1-5 (Tyr-ProPhe-Rro-Gly) underlies a rapid enzymatic degradation in mammalian brain tissue. This metabolic cleavage can be reduced by introduction of Damino acids instead of their naturally occurring L-analogues. In addition to a powerful analgesic action exhibited by the Tyr-containing derivatives, these opioids as well as their corresponding des-Tyr1-analogues possess different effects on rat behaviour induced by depamine or serotonine agonists. To elucidate the possible mechanisms of action in vitro - studies were undertakten to test the synaptosomal uptake of dopamine, norepinephrine and 5~hydroxytryptamine. None of the Tyr-containing 6-casomorphin derivatives did influence the uptake of biogenic amines into synaptosomes derived from hippocampus, striatum or hypothalamus of rat. Some of their corresponding des-Tyr-l-analogues exhibited an inhibitory action on the uptake of norepinephrine and 5-hydroxytryptamine, whereas the uptake of dopamine was uninfluenced by these peptides. The most effective sequences were Pro-D-Phe-ProGly and Pro-Phe-D-Pro-Gly. The inhibitory action seems to be connected with the tetrapeptide structure, because different structurally related tripeptides are ineffective. *Present address: Cuba

Faculty of Medicine,

Matanzas,

Institute of Pharmacology and Toxicology, Medical Academy, Leipziger Str.44, 0-3090 Magdeburg

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DIFFERENT EFFECTS OF COK-8 AND COK-4 ON FOOD INTAKE IN RATS H. rink, H. Bcomgaarden and T. Oft

T.J. Feuerstetn I , 4. Lah~nn 1, W. $eusr~nn 1, V. v. Velthoven2

Cholecystokinin

(CCK) is known to suppress feed]n9 behav i o r in rats. Two CCK receptor subtypes have been d i f f e rentiated: CCK-A receptors, located in the periphery and the CNS, exhibit a high a f f i n i t y f o r CCK-Ss and a bower a f f i n i t y f o r CCK-4, whereas CCK-B sites, widely distributed in t h e b r a i n , d i s p l a y a high a f f i n i t y f o r both CCK f r a g ments. The p r e s e n t s t u d y was designed t o examine the e f f e c t s of s y s t e m i c a l l y g i v e n CCK-8s and CCK-4 in three feeding regimens: - food deprivation (16 hours), - fixed feeding (rats were trained to eat 4 h o u r s / d a y ) , - f r e e l y feeding in the dark phase (7.00-11.00 p.m.). CCK-Ss (t.6, 8, 40 u g / k g i.p,) suppressed dose dependently food intake in deprived rats and In the dark phase. In f i x e d f e e d i n g r a t s CCK-8s (1.6 u g / k g ) induced an increase of food intake. CCK-4 (3.2, 16, 80 ug/kg i.p.) failed to suppress feeding in all regimens, however, the lower dose increased feeding in fixed feeding rats. The data suggest that the hypophagic e f f e c t of systemically given CCK is mediated by the CCK-A receptor. CCK-B receptor may be involved in the hyperphagic response in fixed feeding rats.

Institut fL~r Pharmakologle und Toxikoiogle, Charltd, Humbotdt-Universit~t, PF 140, Berlin O-1040

~CETYLCHOLINERELEASEFROM HUHAN NEOCORTEXTI$$UE

Alzhei mat's dernentia appears to be characterized functionally by a deficit in corticol cholinerglc neurotranami~ton due to the death of choltnergic neuronee. it ts therefore pess~blethat pure antagonists of the presynept~c acotylchol~he (ACh) autoreceptor rney be therepeuUcolly uesful ~n order to d~s~nh~bit ACh re]sees from remaining chel|ner~J|Cnerve terminals. The development of~uch anta(Jon~t~, however, fi rat requ~re~ a clear knowled~jeof these autoreceptors %dtMn humancortex t~ue. Hence,ve attempted to characterize the chol~noceptors modulat~nythe relate of ACh from human neocorte× slices prelahelled v~th 5H-chol~ne, then superfused end stimulated electr~call~J, it has been shovn recent19 that tMs release ~ TT× sensff~ve end dependent on e×trecellular Ca++ vhtch enters the nerve end~s v~o N-type voltage sensitive calcium channels (Feusretetn et el, 1990, J Pherm~col E×p Thor 25Z: 778-78S). In the absence of an ant~chel~neeteraes the concentration-responsecurve of the muscar~nioa~oniat oxotremor~he Vas sh~ffadto the r~ht by atropine t n an exactly paratlol manner. The shapeof rheas curves v ~ compatible ~ith the ~umpUon of a d1rect proportionality between receptor occupenc~jend responsewhich reveslndthe pussibility to estimate the diesociat|on constant of oxotremoHne (pKD=6.76+O.06) at the au.toreceptor by non-linear curve fitting, basedon a funcUon correapond|n~ to the le~ of moss act|on. This estimation ales ~1ie1~1 a biophaes concentration of endogenous~ h of nesrly zero underthe experimental cond|tion~applied which allowed to calculate a res] ~2-ve!us of atropine of 8.56+0.11. Physeattgmtne per es concentratton-dependent]~ decreased, vheroea atropine on]g d the hiOh concentration of 1 ~rno]/] ~]tght]~ d~sinhiMtad the release of ~Ch. The potencies of esveto| mu~cartn|c ante0onlatatn compertsento that of strop1ne a~Inat the endecjermusNoniat/Oh at the autorec~ptor vare a~ased from the1r distnhlbiUon of~Ch release in the pre~enceof phy~ostlgmlne.The rank order of potencies • d not fit ve]] that of a knovn (postsynaptic) muscorinic receptor subt~pe. ~n ~mporta~ finding of the alxPte menUonedpaper, that the release of ~Ch decreasedw~th the o~e of the portents,~ s nov confirmed by analysis of o rnarked]y incree~ number of patients. This ma%lrepresent the siml]erity betvean the normal ayi ny processend a detaujedAlzhe~mat's disesse process. 1Neurophermako]o(jtsches Labor der Neuro]~lschen Universit~tsklinik, c/o Gi~deckeAG, end 2Neuroch~rurtJtscheUniversff~tsk]int k, 7800 Freibury, FRG

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DIETARY CHOBEN~ AND C~OLINE AVAILABILITY INTHEBP~AIN Jr. Kleint A. ~6ppen and K. L~ffelholz

EFFECTS OF LITHIUM AND CARBAMAZEPINE ON MUSCARINIC CHOLINERGIC RECEPTOR PLASTICITY AS SI~JDIED BY AGONIST AND ANTAGONIST INDUCED CHANGES OF RECEPTOR DENSITY IN VITRO AND IN VIVO L. Stoll, D. Riemann, M. Berger~ and W.E. M~ller

In order to support the synthesis of acetylcholine and choline-containing phospholipids, the brain is dependent on the availability of free extracellular choline. Because of the ongoing interest in choline/lecithin precursor therapy for diseases with central cholinergic dysfunctions, we have investigated the effects of dietary choline supplementation and deficiency on choline availability in the rat brain. - Male Wistar rats kept for two weeks on a high-choline diet had elevated oholine levels in the plasma (I4.1 ~M vs. 9.5 ~LM in the control group) as well as in the cerebrospinal fluid (CSF; 15.3 pM vs. 6.8 ~M) and in total brain hemogenate (55.2 nmol/g vs. 28.9 nmol/g). In spite of the high plasma choline level, the arterio-venous difference (AVD) of choline across the brain was strongly negative at -3.3 pM indicating a pronounced net release of choline from the brain. Net uptake of choline by the brain after acute choline aclministration was found to be strongly diminished under these conditions. - In rats kept on a lowcholine diet, the plasma choline levels were decreased to 4.5 pM. However, the CSF choline levels were less affected (5.5 ~M) and were higher than the arterial plasma levels indicating active mobilization of choline by the brain, presumably from choline-containing phospholipids. In agreement with this hypothesis, the brain continued to release choline into the venous blood at a rate of 1.5 nmol/g min. Net choline uptake after acute choline administrationwas unchanged under these conditions. ~ conclude that the brain possesses efficient homeostatic mechanisms which restrict the fluctuations of choline levels in the extracellular fluid to a small range. Pharmakologisches Institut, Universit~t Zahlbacher Str. 67, D-6500 Mainz.

Mainz,

Obere

Although lithium and carbamazepine are widely used for the prophylactic treatment of effective disorders, their precise mechanism of action and their effects on neuronal function remain still unclear. Since it has been suggested that therapeutic concentrations of both drugs might interfere with the phenomenon of receptor plasticity, e.g. the alteration of receptor density as response to periods of chronic over- or understimulation, we have investigated if both drugs affect muscarinic cholinergic receptor (mAChR) plasticity. All experiments were performed using mechanically dissociated neurons of the mouse brain and tritiated N-methylscopolamine (3H-NMS) as radioligand. As observed in experiments with other neuronal cells, incubation of dissociated neurons of mouse brain in vitro with carbachol (i mmol/l) resulted in down regulation of mAChR density by about 30%. Preincubation of the neurons with either lithium (i mmol/l) or carbamazepine (I0 mg/l) nearly completsly abolished the effect of carbachol on receptor density. By contrast, chronic pretreatment of mice with lithium (50 mg/kg, 14 days)or with carbamazepin (I0 mg/kg, 14 days) did not affect mAChR down-regulation by in vivo treatment with diisopropylfluorophosphate (DFP) (I mg/kg, 6 days). Similary, both drugs did not affect muscarinic cholinergic receptor up-regulation by in vivo treatment with scopolamine (I0 mg/kg, I0 days). The data indicate that both drugs can influence specific mechanisms of receptor plasticity but do not generally impair mAChR plasticity. Supported by the DFG (SFB 258) Department of Psychopharmacology and Psychiatric Clinic, Central Institute of Mental Health, D-6800 Mannheim

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IDENTIFICATION OF SPECIFIC BINDING SITES FOR [~H]1,3-DI-O-TOLYL-GUANIDINE ([aH]DTG) IN THE RAT GLIOMA CELL LINE C6-BU-I. A, Georg * and A. Friedl

COMPARATIVE INFLUENCE ON POTASSIUM- OR VERATRIDINE-STIMULATED DOPAMIHE RELEASE FROM RAT STRIATUM SLICES H o - D o F i s c h e r , E. B e r g s t r ~ e r a n d D. D o h r e v

Binding of 1,3-di-o-tolyl-guanidine (DTG), a highly selective ligand for sigma binding sites, and of [aH]N-[1-(2thienyl)cyclohexyl]pipeddine ([aH]TCP), a radioligand specific for phencyclidine (PCP) receptors to C6-BU-1 cells was investigated. Binding of [SH]DTG to C6-BU-1 cell membranes is reversible, saturable (Bmax = 10.5 pmol/mg protein) and of high affinity (Kd = 29 nM). Negligible specific binding of ([aH]TCP) to C6-BU-1 cell membranes demonstrates the absence of PCP receptors in this cell line. Specific binding of [aH]DTG is sensitive to divalent cations like Mg 2* and Ca 2*. The rank order of potency of various PCP and sigma ligands at the [aH]DTG binding site is DTG > (+)-3-(3-hydroxyphenyl)-N-(1 -propyl)piperidine ((+)-3-PPP)) > haloperidol > pentazocine > (-)-3-PPP > PCP > metaphit > (-)butaclamol > (+)butaclamol > (-)N-allylnormetazocine ((-)SKF 10,047) > dizolcipine (MK 801) > (+)SKF 10,047 > ketamine. The drug specifity indicates that these sites are similar to sigma sites labeled in brain tissues by [~H]DTG. A marked difference exists in that C6-BU-1 cells display a reversed stereoselectivity for the benzomorphan opiate SKF 10,047, suggesting that glial [~H]DTG labeled binding sites correspond to a subtype of sigma binding sites. In conclusion, the C6-BU-1 cell line is the first cultured glial cell line demonstrated to have sigma like binding sites.

R e l e a s e of p r e a c c u m u l a t e d radiolaheled dopamine from preparations of b r a i n s t r u c t u r e s is i n c r e a s e d by d e p o l a r i s a t i o n with high external potassium or with ~eratridine. Using a superfusion technique, the p o t a s s i u m i n d u c e d r e l e a s e d e p e n d s n e a r l y t o t a l l y on the c a l c i u m c o n c e n t r a t i o n supp l i e d b y the s u p e r f u s i o n f l u i d . On the c o n t r a r y , a c o n s i d e r a b l e p a r t of d o p a m i n e r e l e a s e i n d u c e d by veratridine d o e s n o t d e p e n d on c a l c i u m p r e sence~ T h e i n f l u e n c e of reserpine (5 m g / k g so., 2~ h b e f o r e k i l l i n g the a n i m a l s ) or m i l d h y p o b a r i c h y p o x i a e x p o s u r e (9 % 0 2 , 18 h) of r a t s a n d the e f f e c t of o u a b a i n I ~ M / I ) p r e s e n t at the s t i m u l a t i o n p e r i o d (30 s) w e r e c o m p a r e d in e a c h c a s e of d e p o l a r i s a t i o n . W h e r e a s p o t a s s i u m s t i m u l a t e d r e l e a s e is h i g h l y influenced by reserpine (vesicular pool)~ verat r i d i n e ~ n d u c e d r e l e a s e is n o t c h a n g e d at a l l . H y p o x i a r e s u l t s in a l o n g - l a s t i n g (days) d e c r e a se of p o t a s s i u m s t i m u l a t e d r e l e a s e , v e r a t r i d i n e i n d u c e d r e l e a s e is o n l y i n f l u e n c e d f o r h o u r s , on the o t h e r h a n d . P o t a s s i u m s t i m u l a t e d r e l e a s e is competitively i n h i b i t e d by o u a b a i n , b u t the d r u g d o e s n o t i n f l u e n c e the v e r a t r i d i n e induced release. T h e r e s u l t s g i v e e v i d e n c e f o r s t r u c t u r e s and mec h a n i s m s i n v o l v e d in transmitter release i n d u c e d b y different k i n d s of s t i m u l i .

*Present address: Institute of Neurobiology, Troponwerke GmbH & Co. KG, Berliner Str. 156, 5000 K61n 80, FRG.

~ n s t i ~ u t e of P h a r m a c o l o g y and Toxicology, Medical University "Carl G u s t a v C a r u s " Dresden~ Lingnerpl. I, 0 - 8 0 1 0 D r e s d e n ~ F R G

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AUTOINHIBITION OF 3H-DOPAMINE RELEASE FROM RABEIT BRAIN SLICES. MARKED DIFFERENCES IN FREQUENCY DEPENDENCE BETWEEN PREFRONTAL CORTEX AND CORPUS STRIATUM. E. Agneter, I. Hoffmann, and L.X. Cubeddu

PHARMACOLOGICAL MODEL FOR CHARACTERIZING ANTIPSYCHOTIC DRUGS USING HALLUCINOGENS ON RATS L. H e t e y

Slices of prefrontal cortex (pf~) and corpus striatttm were prepared, labelled with -H-dopamine (30Ci/mmol; 0.26Nmoi/i, 37°C, 30 min; 0.3~moi/i desipramine present to prevent false labelling of noradrenergic terminals) and superfused with physiological salt solution containing 3~mol/l nomifensine. Electrical field stimulation was applied 8 min, 36 min, 60 min, and 96 min after the start of the collection of 4 min fractions (SlS4; monophasic rectangular pulses, 2 ms, 12V/cm, 18 mA). $I and $2 consisted of 4 pulses, $3 of 16 pulses, and S4 of 64 pulses. Frequencies between 0.05 Hz and 100 Hz were used for electrical stimulation. An additional set of experiments was 3Performed using a single pulse for stimulation of H-overflow (el and $2 only). The D2receptor antagonist sulpiride (3 ~mol/l), when used, was added from 24 min before $2 onwards in all experiments. Stimulation with a single pulse caused a measurable overflow of tritium in pfc and striatum (0.12% and 0.21% tissue tritium, respectively). Sulpiride did not facilitate overflow in the pfc but caused a small increase in the striatum. A similar result was obtained when electrical stimulation was performed with 4, 16 or 64 pulses delivered at 0.05 Hz. Using stimulations with trains of 16 and 64 pulses delivered at different frequencies, maximal facilitation by sulpiride of Hoverflow occurred at 10 Hz in the pfc, whereas in the striatum the best effect was seen at I and 3 Hz. Using stimulations with 4 pulses, the maximal sulpiride-induced facilitation was shifted to 0.3 Hz in the striatum, but remained at 10 Hz in the pfc. The increases caused by sulpiride were consistently lower in the pfc than in the striatum (1.7fold vs 3.9fold at 64 pulses). Dept. of Pharmacology, Universidad de Venezuela~ Caracas, Venezuela, and Institute of Pharmacology, Univ. of Vienna, W~hringer StraBe 13a, A-1090 Vienna.

T h e a c u t e e f f e c t s of d i f f e r e n t h a l l u c i n o g e n s on functional parameters (release, uptake) of t h e dopaminergic (DA) s y s t e m in r a t s w e r e i n v e s t i g a t e d as a p h a r m a c o l o g i c a l model for characterizi n g a n t i p s y c h o t i c drugs. F u r t h e r m o r e , the pathoneurochemical c h a n g e s of t h e s a m e p a r a m e t e r s in p o s t - m o r t e m b r a i n r e g i o n s of s c h i z o p h r e n i c p a t i e n t s w e r e c o m p a r e d w i t h t h e e f f e c t s of h a l l u c i nogens. B a s e d o n d i f f e r e n t ex v i v o a n d in v i t r o e x p e r i m e n t s w i t h h a l l u c i n o g e n s t h e i n f l u e n c e of L a D on both synaptosomal stimulated DA release and high-affinity DA uptake seems to be the most suitable parameter for t h e c h a r a c t e r i z a t i o n of antipsychotic drugs. T h e in v i t r o L S D e f f e c t on D A t e r m i n a l s of t h e N. a c c u m b e n s l i k e i n h i b i t i o n of D A r e l e a s e a n d s t i m u l a t i o n of D A u p t a k e w a s mediated by DA-autoreceptors and 5HT-heteroreceptors, which represents a suitable model for screening and characterizing classical and atypi c a l a n t i p s y c h o t i c drugs. S y n a p t o s o m a l D A r e l e a s e as w e l l as D A u p t a k e in post-mortem N. a c c u m b e n s of s c h i z o p h r e n i c s (Typ I) is s i m i l a r l y changed as a f t e r h a l l u c i n o g e n a p p l i c a t i o n in rats. W h e n e v e r d i f f e r e n t t r i g g e r i n g m e c h a n i s m s occur, t h i s s i m i l a r i t y u n d e r l i n e s the usefulness of the hallucinogen-model for antipsychotics. Bundesgesundheitsamt, i 0 0 0 B e r l i n 33

FIII

7, Pf.

33 00 13,

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ACTION MECHANISM OF OOPAMINE-STIMULATED BRAIN PROTEIN GLYCOSYLATION 8. L66ner, V. Pche&ek*, and R. J o r k *

P~M~EOIEGIC~L ( ~ C I ~ I Z A T I n ~ C~ PRES~I~TIC N - M E ~ Y L ~ ~ (~) ~ ~ ~ ~ ~ ~ (~) ~ ~ ~ ~ ~. ~ ~ H. ~ ~t ~ ~ ~ ~ ~ w ~ 3~_~ ~ ~ ~ ~+-~ ( ~ ~ ~ ) ~ ~ t (~-B) ~ o ~ . ~ ~ ~ o~ ~ g l ~ (~), ~ o n o~ ~ 3~000 ~1/1 ~ ~ 4~ ~ 42~ ~ o~ ~ ~ ~ ~ ~ 3R ~e ~ e~l~. ~ 100 o~ ~000 ~ 1 / ~ ~ 1 ~

D o p a m i n e r g i c a g o n i s t s (dopamine, apomorphine amd ADTN) i n d u c e a d o s e - d e p e n d e n t i n c r e a s e in L - f u c o se i n c o r p o r a t i o n into proteins of various struct u r e s o f r a t and mouse b r a i n both in r i v e and i n vitro. Under i n v i t r o c o n d i t i o n s , ddpamine has no e f f e c t on g l y c o s y l a t i o n of liver proteins. In r a t s , d o p a m i n e - s t i m u I a t e d f u c o s y l a t i o m o f b r a i n p r o t e i n s i s t r i g g e r e d by ( a t l e a s t ) two mechanisms, i . e . (1) v i a an a c t i v a t i o n o f adenyl a t e c y c l a s e c o u p l e d dopamine r e c e p t o r s i t e s (D 1t y p e ) , and (2) v i a a mechanism which appears t o be n o t r e l a t e d t o t r a n s m i t t e r r e c e p t o r but l i n k e d to the 3,4-dihydroxy-phenylethylamine~structure. The D 1 - r e c e p t o r m e d i a t e d dopamine e f f e c t on b r a i n protezn glycosylation appears t o be based on an i n c r e a s e in t h e a v a i l a b i l i t y of "activated" Lfucese since the activity o f f u c o k i n a s e (EC 2.7.1.52) catalysing the initial step i n t h e f o r m a t i o n o f GDP-fucose from which t h e sugar m o i e t y is finally transferred to the accepter proteins was found t o be e l e v a t e d . I t i s assumed t h a t t h e l i n k between D - r e c e p t o r s t z m u l a t z o n and e l e v a t & o n o f f u c o k & n a s e a c t i v i t y i s an a l t e r a t i o n of intracellular "free" CaZ+'ion concentrations. *Present address: Institute o f N e u r o b i o l o g y and B r a i n R e s e a r c h , Academy o f S c i e n c e s , B r e n n e c k e S t r . 6, D-0-3090 Magdeburg Institute o f Pharmacology and T o x i c o l o g y , M e d i c a l Academy Magdeburg, L e i p z i g e r S t r . 44, D-0-3090 Magdeburg

a~l~ ~ ~ ~ ~ t~ ~ ~ ~ ~e~ve, ~t s ~ ~ ~ t ~ o~ ~ (1 ~ l ) ~ G ~ (10 o~ 1 0 0 ~ 1 / 1 ) ~ ~ ~ 1 ~ o~ ~ . ~ ~e p ~ o~ ~ ~ (1 ~ 1 / 1 ) _ ~ 3~ ~ 1 ~ ~ ~0 ~ 1 / 1 ~ d ~ ~ ? ~ - ~ c acld (100 ~1/1), ~ ~ at ~ ~ a ~ G~ s~ o~ ~ e ~ ~ r . ~ ~ o~ ~ + ~.2 ~ 1 / 1 ~ ~e ~ ~ ~d ~ ~e ~ ~ =~ ~ 1 ~ ~ ~ p ~ o~ ~ (10 ~ 1 / 1 ~ ~ ~ ~l ~ ~ ~ st~a~ e ~ o~ ~ ~ ~ ~ ) , ~ 1 ~ ~ ~ o ~ o~ ~ 2 + ~ ~ ~+ ~ ~-H ~l~on. ~e ~ o ~ 3H ~ 1 ~ ~ ~ t ~ o n (~50 2.5 ~ / 1 ~ ~ ~ a t ~ a t ~00 ~ ~1/1:O.49 3 O.08 ~ o~ ~ ~ 3~). ~ e ~ ~ t ~ ~ ~e (~) ~ ~ ~ ~e ~ t ~ ~ ~t~ve ~ ~ ~ g ~ ~ 37849 (D~ (~) - 2 - ~ - ~ y l - 5 - ~ ~ 3 - ~ c ~d; ~2: 6.25). ~ B0~ ( ( + ) - 5 - ~ y 1 - ~ 0 , ~ ~ ~ [a,d]~l~-5,Z0-~), ~ ~ a ~ at ~ Co~ ~ o~ ~ ~ ~ ~ , ~ t ~ t i v ~ y ~ ~ ~ a ~ e ~ o~ ~ ( p ~ ' : ?.17). ~ e ~ _ ~ 3~ ~ , ~ ~ ~e ~ 1 ~ ~ ~ ~ ~C~ ~5 ~ l / 1 , ~ ~ ~ ~ l 32 ~ 100 ~1/1. - It ~ ~ 1 ~ ~t ~ ~ ~ 1 ~ o~ ~ ~ c ~ t ~ o~ ~ ~ ~ . ~ 0£ ~ G~ ~ a p ~ s ~ ~o~ ~ ~d~t ~ t ~ o~ ~ ~ ~ ~ ~ ~ ~ h~. ~ 1 ~ ~ ~ ~ ~ ~ a ~ o~ ~ e p ~ c ~ ~ . ~. ~ 1 . ~v. ~, ~ . 2b, ~5300 ~ ~

434

436

FOR~TION,METABOLISMANDPROPERTIESOF2-SUBSTITUTEDTBIAZOLIDINE-4(R) CARBOXYLICACIDS:A NEWMETABOLICPATBWAYOFBIOGENICANINES-NEUROT~NSMITTERS L., WYodek*,B. Ror~lspacher. R. Susilo, J. Radomski Biogenlc amines like tryptamine, 5-hydroxytryptamine, dopamlneand noradrenaline are oxidized by the monoamlneoxldase [MAO,EC 1.4. 3.4] to the aldehydes which condensate with L-cysteine to the 2-(R)-thiazolidine-4-(R) carboxylic acids (Susilo et al. BBRC14e, 1045, 1987, J. Neurochem.50, 1817, Igse, and 52, 1793, 1989). At least one step of the consecutive pathway is enzymatic. The enzymeshavebeen characterized in brain and )iver mitocbondria and brain and liver cytosol utilizing 2-alkyl and 2-polyhydroxyalkyl substituted 2(R)thiazolldinecarboxylic acids, The for~Idehyde- and indolyl-derivatives were preferentially ~tabolized by mitochondria enzymeswhereasthe carbohydratederivatives were oxidized by the cytosollc enzy~s. Furthert~ore, stereoselectivity was found since the D-xylose-as well as the D-arabinose-derivatives weremetabolized in liver cytoplasmabut not the L-xylose-andL-arabinose-campounds, The findings suggest that manyaldehydes formed in the course of intracellular metabolic pathwaysare trapped by L-cystine. The aim of the reaction is to prevent the chemically active aldehydes from the reaction with macromolecu|esand to oxidize themin a protectedmanner~The result of the reactions are the respective acids.

VEPJ&TRIDINE-INDUCED NA AND CA UPTAKE IN ISOLATED PAT CORTICAL SYNAPTOSONES IS SUPPRESSED BY R 56865 and R 59494 D. Hermelsklrchen, 3. G l e i t z , 3. Urenjak, U. Nebel, A. H i r t h , A. Beile and S. Khan Under ischemic conditions an increase in Na and Ca uptake is thought to play a crucial role in the development of c e l l u l a r dysfunctions. R 56865 ( N - [ l - [ 4 - ( 4 fluorophenoxy)butyl]-piperldinyl]-N-methyl-2-benzothlazolamlne) was reported to be p r o t e c t i v e against Ischemla-induced dysfunctions of various tissues. Since R 56B65 seems to i n t e r f e r e with c e l l u l a r Na and Ca load and the v e r a t r i d l n e - and ischemia-induced increase in Na and Ca uptake show s i m i l a r i t i e s , the e f f e c t of R 56B65 and the structural homologue R 59494 iN-methyl - N - [ l - ( 4 - p h e n o x y b u t y l ) - 3 - p y r r o l l di nyl]-2-benzothiazol amine) on the veratridine-lnduced increase in synaptosomal Na and Ca uptake was investigated. Since mitochond r l a l r e s p i r a t i o n is mainly triggered by the Na/K ATPase a c t i v i t y , the veratridine-induced Na uptake was determined via the corresponding increase in synaptosomal oxygen-consumption. Veratridine-induced Ca uptake was detected by means of 4sCa fluxes. The v e r a t r l d i n e induced Na and Ca uptake was inhibited by t e t r o d o t o x i n (lO -5 M). Nitrendiplne (lO -5 M) and omega-conotoxin GVIA (SxlO-7 M) were i n e f f e c t i v e towards the v e r a t r i dlne-induced Na and Ca uptake. R 56B65 (lO-6-10 -5 M) and R 59494 (lO-6-10 -5 M) c l e a r l y suppressed the veratridine-induced Na and Ca uptake. In summary, the putative anti-ischemic compounds R 56B65 and R 59494 were demonstrated to be potent i n h i b i t o r s of veratridine-induced Na and Ca uptake. The L-type Ca channel blocker nitrendlpine and the N-type Ca channel blocker omega-conotoxln f a i l e d to suppress the veratridlne-induced Na and Ca uptake. I t is suggested, that R 56865 and R 59494 exert t h e i r protective effects against ischemia-induced dysfunctions via reducing both i n t r a c e l l u l a r Na and Ca load.

H\ R~C-" 0 " ! dehyde

HS--FH, -H,0 H \ ~ - - F H , / CH ~ HzN ~COOH L-cy~teine

C CH R/ "~klF(/ ~" COOH 2 T ( R ) - t h i azo I i din(= ~-carboxy/I c ~cld

Inst. f. Nauropsychopharmakologie,FB Berlin, Ulmenallae30, D-IO00Berlin 19, Germany *Inst. of Medical Biochemistry, Medical Academy,Kopernika 7, 31-034Cracow, Poland

OANSSEN RESEARCH FOUNDATION, D-4040 Neuss 21.

R110 437 THE INFLUENCE OF CAPSAICIN ON THE NOCICEPTIVE NEGATIVE MUCOSAL POTENTIAL (NMP) IN RATS N. Thueranf, I. Friedel, C. Hummel and G. Kobal In 1985 Kobal (Pain 22:151-163) described a negative potential, recorded from the human respiratory mucosa after painful stimulation with carbon dioxide (CO2), which correlated with subjective pain sensations. The aim of'the present study was to determine the origin of the negative mucosal potential in an animal model (rat). Additionally, cortical event relatedpotentials were simultaneously recorded. NMPs were recorded after stimulation with different concentrations of CO~. After control stimulation with non-painful hydrogen sulphide no'signals could be recorded. Amplitudes of the NMPs correlated with COT concentrations of the stimulus. Latencies from the onset of tlie stimulus to the onset of the NMP were within 120-500 msec. In order to determine if either functionally unimpaired sympathetic nervous structures or intact C-fibers are requisite for NMPs, the influence of systemic (200 mg/kg capsaicin s.c., cumulative dose, 1% w/v alcoholic capsalcin solution) and local (3 mg in 0.3 ml 1% w/v alcoholic solution) treatment with capsaicin and the influence o f systemic treatment with guanethidine (50 mg/kg s.c., in 0.3 ml 0.9% NaC1) was examined in different groups of rats. The NMP was reduced by systemic treatment with capsaicin and eliminated by local adminstration of capsaicin whereas guanethidine did not eliminate this signal. After administration of the local anesthetic lidocaine (31 mg in 0.3 ml solvent) the NMP was abolished. In all our experiments elimination of the NMPs coincided with elimJnation of event related potentails. In order to exclude a vascular origin of the NMP blood fiow changes were measured. Onsets of blood flow changes appeared significantly later than onsets of potentials. On the basis of these results a sensory neurogenic origin for the NMP is proposed.

439

Department of Pharmacology and Toxicology, University of Erlangen-Nuernberg (Germany), Universit~itsstraBe 22, 8520 Erlangen

As a highly potent uptake inhibitor with negligible affinity to the muscarinic receptor and a favourable pharmacological profile (Teschendorf e t a ] . , this meeting) the (S)-enantiomer of nafenodone is a promising candidate for further developmont. Knoll AG, Department CNS Research, Postfach 21 08 05, D-6700 Ludwigshafen

438

440

LEARNING DEFICITS IN BULBECTOMIZED RATS - AN ANIMAL MODEL FOR ANTIDEPRESSANT-LIKE ACTIVITY G. Grecksch

BASIC PHARMACOLOGY OF THE POTENTIAL ANTIDEPRESSANT LU 43706 H.J. Teschendorf, . H.P. Hofrannn, R,D. Porsolt' and S. Sehult

Following bilateral removal o f olfactory bulbs in rats various behavioural alterations develop, the normalization of which is often used as a test for antidepressant-like activity of drugs. Beside a locomotor hyperactivity bulbectomized rats show deficits in learning tasks. In the literature a retention deficit in passive avoidance learning was mostly described, which we could demonstrate in our experiments, too. This deficit is normalized by antidepressant drugs, only. We tested bulbectomized rats in three different learning models. It was found that the consequence of a bulbectomy on learning and memory is dependent on the learning task. A retention deficit in a brightness discrimination task and a retardation of acquisition of pole jumping behaviour were normalized by the tricyclic antidepressant imipramine and a B-casomorphin peptide with antidepressant-like activity. Since the learning behaviour of naive animals was not influenced or even disturbed by the same drugs it was suggested that the improving effect in bulbectomtzed rats could be a specific antidepressant effect and that the bulbectomy could serve as useful animal model to test antidepressant-like activity. Present address: Institute of Pharmacology Toxicology, Medical Academy M a g d e b u r g . Leipziger Sir. 44, 0-3090 Magdeburg, FRG

and

BIOCHEMICAL STUDIESON THE POTENTIALANTIDEPRESSANTNAFENODONE,ITS ENANTIOMERSAND ITS N-DESMETBYLDERIVATIVES M. Traut, H. Weifenbach and L. Un,~er Using synaptosomes from rat brain'hippocampus, corpus striatum and cortex the influence of nafenodone (LU 39821 = 2-[2-(dimethylamino)ethyl]-2-pheny]-3.4dihydro-1(2H)-naphtslenone-HCl) and its desmethyl derivative LU 39B23 on the neuronsl uptake of noradrenaline (HA), dopamine (DA) and serotonin (SER), respectively have been determined. Rafenodone is shown to be a strong and stereoselective inhibitor of RA uptake with somewhat lower activity at the SER carrier, whereas inhibition of DA uptake is negligible. The N-desmothyl derivative is equally potenf as nafenodone in inhibiting NA uptake, but the effect, in contrast to that of nafenodone, is not stereoseiective. compound

configuration neuronal uptake IC50 [nM]

receptor binding Kl [ ~ ]

HA

DA

nafenodone LU 39821 racemic LU 43706 S LU 43707 R

5-HT

11 7 60

> 1 000 • 1 000 • I 000

N-desmethyl nafenodone LU 39823 racemic LU 49165 S LU 49164 R

6 10 8

000 • 1 IgO > I 000 140 > 1 000 ~ 1 000

M

H1

go 158 370 55 > I 000 > 1 000 240 186 151

846 834 > 1 200 901

By measuring synaptosomal NA uptake ex vlvo in rats nafenodone was shown to be active at least until 6 h after oral dosing. After 24 h the effect had almost vanished. As in vitro the (S)-form is more active than the (R)-form, though the difference is not as pronounced as in vitro. In mice, however, where suppression of a-methy]-m-tyresine-methylester induced NA depletion as a measure of uptake inhibition had been determined, stereose]ectivlty had disappeared or even had been reversed. Binding studies revealed low affinities of nafenodone to moscarinic and HI receptors, the stereoselectivlty being opposite to that of NA uptake; afflnities to all other receptors tested are even lower.

LU 43706, the (S)-enantiomer of nafanodone (2-[2-(Dimethylamino)ethyl]-2phenyl-3.4-dhhydro-l(2H)-naphtalenone-HCl),an inhibitor of the synaptosonmi uptake of noradrenaline and serotonJn (Traut et al., this meeting) was tested in experimental models presumed to be predletive of clinical antidepressant efficacy and in models eharacterising side effects. LU 43706 prevents (]~Dso: 1.7 mg/kg po) and reverses (I~D4.c 15 mg/kg po) reserpine induced hypothermia in mice, being 2 - 3 times more effective than imipramlne. Apomorphine induced hypothermia in mice is inhibited (EDso: 2.5 mg/kg po), too. In rats, higher doses are necessary to inhibit reserpine hypothermia (EDna: 24 mg/kg po), which may be due to lower bioavallability. LU 43706 (10 to 31 mg/kg po) reduces the duration of immobility in a behavioral despair paradigm in mice (tail suspension test). Studies on the sleeping-waking behaviour in eats show a selective suppression of paradoxical sleep (EDna: 0.7 mg/kg po) without affecting the duration of the waking stage. LU 43706 does not decrease blood pressure in pithed and in eouseious rats. In pithed rats there was a mild increase in blood pressure and heart rate. 4 h infusion of LU 43706 to anaesthetized rats results in only minor effects on ECG and blood pressure, whereas an eqnloffeetive dose (regarding uptake inhibition) of desipraminc induces a dear reduction in blood pressure and prolongations of ECG intervals. LU 43706 shows a local anaesthetic effect in the guinea pig wheel test ((ICso: 0.7, imipramlne 0.5, lidoesine 1.3 retool/l). In viva, antieholinergie effects (physosfigmine, carbachol, oxotremorlne antagonism) were not detectable. Due to the high activity in the antidepressant models, the lack of sedative and anticholinergie properties and the minimal cardiovascular effects good ellnJeal efficacy and tolerability are to be expected for LU 43706.

KnollAG, DepartmentCNS Research,Posffach21 08 05,D-6700Ludwlgsh~fen, FRG; "I.T.EJVI,qabo,93,avenuedc Fontainbleau,94270Kremiln-BicStre,France

Rlll

441

443

PHARMACOLOGICAL PROFILE OF FI-4303, A NOVEL ANTIDEPRESSIVE J.M. Planas, C.F. Cartheuser, M. Gil, J. Gimenez, A. Sacrist&n, J.A. Orttz

EFFECTS OF NOOTROPICS AND OTHER PSYCHOTROPIC DRUGS ON SLEEP PARAMETERS IN RATS W. We~zel, T. Wagner, and H. Matthies

As shown previously (Eur.J.Pharm. 183,1468, 1990), FI-4303: (F), e l i c i t s favourable e f f e c t s typical f o r antidepressives in animal models. I t was the aim of t h i s study to f u r t h e r e l u c i date the p r o f i l e of actions of t h i s structure in rats and mice. Results: I . SPOntaneous a c t i v i t y ( a c t l v . meter; holeboard): (F) did not reduce general or exploration a c t i v i t y up to 40 mg/k9 o r a l l y or i . p . whtls Imipramlne (IMI) reduced these a c t i v i t i e s at ~10 mg/kg each. Reduced a c t i v i t y (sedation) occurred at medium doses of Mianserine (MIA) and A m t t r y p t i l i n e (AMI), while Bupropion provoked e x c i t a t i o n . I I . Conduction: In forced swlmmtng ( P o r s o l t ) , (F) had a OE~0 of 24 mg/kg i . p . (Amineptine, AMN: 19; IMI: 21; Maprott]tne, MAP: 27; AMI: 28; MIA: > 50) and was e f f e c t i v e (p (F) > MAP > IMI > MIA > ANN; b) Yohtmbine l e t h a l i t y was potentiated at DE~0s (mg/kg p.os) of: (F) = IMI = AMI (3) > MAP (9) > MIA (28) > AMN (74); c) in Tetrabenazins antagonism, potencies were: (F) > IMI = AMN > AMI = MAP; d) tn Apomorphine hypothermta, the minimum antagonistic dose (p
l_[2-(phenylmethyl)phenyll-4-methylplperaztns-HCl

442 A SIMPLE AND SENSITIVE SCREEN FOR ANXIOLYTIC DRUGS A. Rex and D.N.

Stephens*

The e f f e c t s of a v a r i e t y of drugs, administered acutely, was examined in a c o n f l i c t t e s t , adapted f r o m t h a t o f Bodnoff S.R. et al. (Psychopharmacol. 97; 277, 1989). The test is based upon novelty-induced suppression of feeding in rats. Food deprived rats (24 hours food d e p r i v a t i o n ) were placed in an illuminated open-field containing food and the number of rats beginning to eat in the f i r s t five minutes was recorded. The following drugs increased the number of rats feeding and showed bell-shaped dose response curves: diazepam (peaking at 2.5 mg/kg), abecarnil (0.03 mg/kg), meprobamate (100 mg/kg), 8-OH-DPAT (0.03 mg/kg), ips~pirone (2.0 mg/kg), ritanserin (0.25 mg/kg), ICS 205-930 (3.0 ug/kg), GR 380 32F (1.0 ug/kg), Ilsuride (0.4 mg/kg), morphine (0.3 mg/kg), propranolol (1.0 mg/kg), clozaplne (1.0 mg/kg). Haloperidol, sulpiride, apomorphine, amphetamine and m-CPP were without effect. The results show t h a t t h a t d i f f e r e n t classes of d r u g s which have been shown to reduce anxiety in clinical s t u dies and/or other animal models can be identified in thi~ simple animal test. I n s t i t u t f~ir Pharmakologie und Toxlkologle, Humboldt Universit~t, Berlin, PF 140, 0-1040 Berlin, F.R.G. ~Dept. of Neuropsychopharmacology, Research Laboratories of Schering AG, W-IO00 Berlin 65, P.O. Box 65 03 11, F.R.G

The effects of differen± noofropics (piracetmm, aniracegam, meclofenoxage, pyritinol, methyl~lucamine orotate, nicergoline, dihydroergofoxine) and ogher psychotropic drugs (imipr~mine, ~mphe%aminil) on sleep-waking pattern were invesgigated in rats. Subs±ances were injecged intr~peritoneally immediately before the onset of the 8-hour recording time (8 a.m. - 4 p.m.). In some experiments, after paradoxical sleep (PS) deprivation, sleep-wakefulness cycle was recorded confinuously during 32 hours. We found that some nootropics (meclofenoxate, pirace%~m) increased the duration of PS episodes, whereas ogher (pyri%inol, methylglucamine orotate, dihydroergotoxine) decreased the number of PS episodes. The effecfs of meclofenoxate, piracetam and aniracetam indicated an inverted U-shaped do~e-effect relationship. Injection of noo%ropica in~nediagely following a 2~-hou~ PS deprivation revealed more pronounced and specific effects on PS. Moreover, under the conditions of PS rebound, clear-cu t differences between nootropics and other drugs (imipramine, mmphe9aminil) could be shown. Thus, under special circumstances, the investigation of sleep effects may be a suitable test procedure for the screening of noofropic drugs. Institute of Neurobiology and Brain Research, sir. 6, 0-3090 Magdeburg, Germany.

Brennecke-

444 PENTYLENETETRAZOL (PTZ) - KINDLING IN RATS I N CREASES GLUTAMATE BINDING TO BRAIN MEMBRANES H. SchrSder The s p e c i f i c binding of 3N-l-glutamate to its r e c e p t o r s was i n v e s t i g a t e d on i s o l a t e d crude s y n a p t i c membrane p r e p a r a t i o n s from 15 d i f f e r e n t b r a i n r e g i o n s o f P T Z - k i n d l e d r a t s using an i n vitro filter binding assay. P T Z - k i n d l i n g induced by t e n i n t r a p e r i t o n a a l applications o f 45 mg/kg o v e r a p e r i o d o f 20 days r e s u l t e d i n a s i g n i f i c a n t enhancement o f both c o n v u l s i v e s ~ s c e p t i b i l i t y o f a n i m a l s t o PTZ and t h e s p e c i f i c H - g l u t a m a t e b i n d i n g in h i p p e campus and s e v e r a l n e o c o r t i c a l s t r u c t u r e s . 3B K i n e t i c s t u d i e s i n d i c a t e d t h a t t h e enhanced g l u t a m a t e b i n d i n g t o h i p p o c a m p a l membranes from kindled rats reflects changes in t h e a f f i n i t y of g l u t a m a t e t o i t s b i n d i n g s i t e s r a t h e r t h a n an increase in receptor density. Since g l u t a m a t e b i n d i n g was not a f f e c t e d by acute applications o f c o n v u l s i v e doses o f PTZ (60 mg/kg, i . p . ) , kindling-induced augmentation of glutamate receptor affinity was aaaumed t o be a correlate of plastic changea l i n k e d t o t h e devel o p m e n t o f enhanced e x c i t a b i l i t y of theae brain structures. *Present address: Institute o f Pharmacology and T o x i c o l e g y , M e d i c a l Academy Magdeburg, L e i p z i g e r S t r . 44, D-0-3090 Magdeburg.

Rl12 445

447

ANTICONVULSIVE ]z~'.e;C'rS OF PIRACETAM, MECLOFIR~OXATEAND VINPOCETINEONACUTEANDKINDLEDPENTYLI~'f~r~AZOLE (PTZ) INDUCED SEIZURE G. Keller

DEVELOPMENT OF LONG-LASTING POTENTIATION EFFECTS IN THE DENTATE CYRUS OURING THE TIME COURSE OF KINDLING DEVELOPMENT INDUCED BY PENTYLENETETRAZOL H. Rfithrich

Disturbances of cognitive functions are one of the possible consequences of chronic epileptic disorders as well as long-term therapy with classical antiepileptics. Therefore, subtances with combined anticenvulsant and cognition-enhancing potency are of interest for new ways in the drug-treatment of epilepsies. In the present study we investigated the anticonvulsive effects of three chemically different drugs with well-known psychotropic and cognition-enhancing properties on acute PTZ-induced generalized and low dose PTZ-induced petit mal seizures in comparison to their efficacies on kindling induced seizures. Ccmpared with the anticenvulsive efficiency of classical antiepileptics like carbamazepine and Ca-valprcat piracetam, meclofenoxate and vinpocetine showed very slight anticonvulsive effects in the acute induced seizure models. However, the latter drugs lead to changes in the time curse of onset of seizures and convulsions. In contrast, in the kindling induced seizure model piracetam, meclofenoxate as well ~as vinpocetine depressed generalized convulsions markedly in a dose-dependent manner.

Repeated electrical stimulation can result in the development of electrographic afterdischarges culminating in a generalized electrographic seizure and behavioural convulsion. This phenomenon is called the kindling effect. Kindling can also be induced by repeated administration of subconvulsive doses of convulsants. Previously, we reported that Pentylenetetrazol (PTZ) application caused a long-lasting potentiation of the population spike in the dentate gyrus (DG) in fully kindled rats, whereas control animals showed such a potentiation effect only immediately after PTZ-injection. In the present study, we examined the effects of PTZ (20 mg/kg; every 48 hours) on both the development of PTZ induced kindling and the time course o£ the potentiation effects, respectively. The efficacy of perforant path (PP) transmission to the granule cells was tested in every second kindling session Dy measuring the field potentials recorded in the hilus of the DG following stimulation o f t h e PP a t d i f f e r e n t times a f t e r

Department of Pharmacology and Toxicology, Medical Academy "Carl Gustav Carus" Dresden, Lingnerplatz 1, 0-8010 Dresden, FRG

PTZ-injection in f r e e l y moving r a t s . The c o n v u l s i o n b e h a v i o u r was o b s e r v e d f o r 20 min a f t e r each injection. In summary, we found t h a t t h e d e v e l o p ment of PTZ i n d u c e d k i n d l i n g i s a s s o c i a t e d w i t h t h e d e v e l o p m e n t of a l o n g - l a s t i n g potentiation in t h e p o p u l a t i o n s p i k e evoked in t h e DG o n l y by s i n g l e t e s t s t i m u l i in t h e PP. Institute of Pharmacology and T o x i c o l o g y , M e d i c a l Academy, L e i p z i g e r S t r . 44, 0-3090 Magdeburg

446

448

PENTYLENTETRAZOL-KINDLING DOES NOT INFLUENCE THE PATTERN OF NADPH-DIAPHORASE ACTIVE NEURONS IN EAT FOREBRAIM. I. Seidel*, H.-G. Bernsteln, A. Decker, and M. MUller

THE NMDA RECEPTOR : A LOCUS FOR THE ACTION AN~ICONVULSANT DRUGS IN SPINAL CORD NEURONS

The enzyme reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase exists in distinct subsets of CNS neurons, which are not exclusively associated with a particular neurotransmitter system nor embrace all the neurons using a single transmitter. Though its functional role is as yet very incompletly understood the enzyme is often used as a histochemical marker of certain neuronal subpopulations in the mammalian central nervous system. We intended the present histochemical study in order to prove the behavior of NADPH-diaphorase containing nerve cells in rat brain after pentylentetrazol-kindling. It was revealed that chemical kindling does not influence the number and/or the distribution of enzyme-positive neurocytes in different areas of rat forebrain known to be vulnerable to this kind of experimental epilepsy. Our findings stress once again that NADPH-diaphorase expressing neurons are remarkably resitant to different kind of stimuli capable of damaging other types of nerve cells.

N-methyl-D-aspartate (NMDA) receptors have been discussed to play a role in epileptogenesis, parkinsonism, spastleity and neuronal degeneration. In spinal cord neurons in culture like in other neural cells the NMDA receptor can be activated in a v~ltage dependent manner. These cultured neurons, when treated with picroto~dn, develop paroxysmal depolarizing events that can be attenuated with substances effective against seizures and spasticity. In the present investigation we have examined the role the NMDA receptor plays as a target for drugs that reduce paroxysmal depolarizing events in culture. Carbamazepine and phenytoin, both clinically used in the treatment of seizures, amantadine - in clinical use for the treatment of M. Parkinson - and two of its derivatives, 3-isopropylamantadine and 3,5-diethylamantadine were applied to cultured neurons. The NMDA receptor was activated by pressure-ejection of N-methyl-D-aspartie acid. Recordings were performed in voltage clamp mode in the whole cell configuration at a holding potential of -60 mV. Cultures were continuously superfused with Mg2+ free saline. The antiepileptic agent carbamazepine, in contrast to phenytoin, blocked the peak amplitude membrane current activated by N-methyl-D-aspartic acid in a dosedependent fashion, as did amantadine and its derivatives. The potencies of these drugs to block NMDA receptors were not perfectly matched by their effects on eonvulsant treated spinal cord neurons, because both antiepileptie agents and the amantadine derivatives, though not amantadine itself, had the capacity to curb paroxysmal depolarizing events. It is concluded that the NMDA receptor of cultured neurons may be one target, but by no means the only one for anticonvulsant drugs.

*Department of Neuromorphology, Institute of Neurobiology and Brain Research, Brenneckestr. 6, 0-3090 Magdeburg, FRG

OF

H. Lampe

Department of Pharmacology and Toxicology, Medical School of Hannover, 3000 Hannover 61, Germany. (Supportedby the DFG)

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NMDAAN~AGONISTS SUPPRESS GENERATION AND PROPAGATION OF CORTICAL SPREADING DEPRESSION. D.Scheller. K.Denoler a n d U. H e i s t e r The excitatory amfno acids aspartate (asp) a n d / o r glutamate (glu) can induce cortical spreading depression (CSD), the presumed animal correlate of migraine, as w e l l as c e l l d a m a g e v i a a c t i v a tion of the NMDA receptor. In order to evaluate whether NMDA antagonists were able to suppress CSD and to circumvent the blood brain barrier, two microdialysis (MD) p r o b e s (2 m m l e n g t h , 0.5 mm diameter) were implanted acutely, o n e in a rostral a n d o n e in a c a u d a l r e g i o n of the same hemisphere of a n a e s t h e t i z e d rats, and perfused with artificial cerebrospinal fluid (CSF) to which either ketamine (i m m o l / l , n o n - c o m p e t i t i v e antagonist) or 2-APV (2-amino-5-phosphonovaleric acid, 0.i mmol/l, competitive antagonist) was added. For DC recordings, two microelectrodes (MEs) w e r e a l s o i m p a l e d t o a d e p t h of 1 m m e a c h at a distance of 2 0 0 - 5 0 0 um to one of the MD probes. CSDs were induced in intervals of 6 0 - 9 0 min by switching the perfusate from CSF to a high-K + CSF (128 mmol/l) for 2 min. Subsequent control CSDs showed minor variability. CSDs occurred as a negative DC shift o f 15 t o 20 m V followed by a spontaneous recovery. Propagation velocity was 3.07±0.49 mm/min (mean±SD, n=15). Ketamine blocked generation of CSD in 5 out of 5 experiments and 2-APV blocked C S D in 5 o u t o f 6 experiments w h e n a p p l i e d a t t h e s a m e M D p r o b e as the stimulus. 2-APV blocked the propagating CSDs in 6 o u t o f 12 a p p l i c a t i o n s when applied selectively at the MD probe remote to the KCl probe. From these data we conclude, that the NMDA recept o r p l a y s a n i m p o r t a n t r o l e in g e n e r a t i o n as well as in propagation of C S D , t h u s i m p l i c a t i n g that NMDA antagonism might be a novel intervention with CSD mediated cerebral injury. Janssen Res Found, Raiffeisenstr.8,4040 N e u s s 21

INFLUENCE OF FLUNARIZINE ON THE ANTICONVULSANT ACTION OF COMMON ANTIEPILEPTICS IN M I C E S.J. C z u c z w a r , M. G a s i o r , a n d Z. K l e i n r o k

Department Jaczewskiego

of Pharmacology, Medical 8, P L - 2 0 - 0 9 0 L u b l i n , P o l a n d

School,

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450 ANTAGONISTS

Both, nifedipine and diltiazem (calcium channel inhibitors) strongly potentiated the protective activity of carbamazepine (CBZ) and diphenylhydantoin (DPH) and only nifedipine moderately enhanced that of valproate (VPA) against electroconvulsions in m i c e ( C z u c z w a r et al., Eur. J. Pharmacol. 176:75,1990). We therefore assessed the effects of flunarizine (another calcium channel inhibitor) on the anticonvulsant action of these antiepileptics against maximal electroshock (50 mA; ear-clip electrodes)-induced seizures in mice. The stimulus duration was 0.2 s and the endpoint tonic extension of t h e h i n d l i m b s . Flunarizine u p to 20 m g / k g i.p. (60 m i n before the test) remained without influence upon the convulsive threshold a n d in t h e d o s e of 40 m g / k g raised the threshold significantly f r o m I0 to 1 4 . 8 mA. W h e n c o m b i n e d w i t h the antiepileptics, flunarizine (20 mg/kg) potentiated the protective activity of CBZ, DPH, and VPA, decreasing the respective E D 5 0 v a l u e s f r o m 15.1, 11.3, a n d 233 mg/kg to 7.3, 8.4, and 107.5 mg/kg. Flunarizine (I0 m g / k g ) s t i l l e n h a n c e d t h e anticonvulsant actions of CBZ and VPA. A pharmacokinetic interaction (plasma levels of CBZ, DPH, and VPA were measured by immunofluorescence) does not seem responsible for the observed effects. The results demonstrate that the protective efficacy of common antiepileptics may be considerably improved by combination with flunarizine.

AT

THE

N-METHYL-D-ASPARTATE ISCHEMIC NEURONAL DAMAGE

RECEPTOR COMPLEX

REDUCE

EXCITOTOXIC AND J.H.M. Prehn, M. Self el Nasr and J. Krieglstein. There is considerable evidence suggesting t h a t the release of glutamate and related amino acids during cerebral ischemia might be responsible for the postischemic neuronal death. The N-methyl-Daspartate (NMDA) receptor, a subtype of glutamate receptors, possesses various binding sites apart from the glutamate site, e. g. the PCPbinding site and a strychnine-insensitive glycine binding site. In the present study we evaluated both in vivo and in vitro the ueuroprotective activity of compounds which can block distinct sites of the NMDA receptor. Excitotoxic neuronal damage was induced in primary neuronal cultures of chick embryo cerebral hemispheres by exposing the cultures to 0.5 mM glutamate for 60 min. Eighteen hours later the degree of neuronal damage was determined by means of the trypan-blue dye excluding method and by evaluation of the proteiu couteut of the cultures. It could be clearly demonstrated in vitro that the competitive NMDA-antagonist 2-amino-5-phosphonopentanoic acid and the non-competitive NMDA-antagonists phencyclidine, dizocilpine and memantine, which block the PCP-binding site, as well as the glycineantagonist cycloleucine and also 6,7-dinitro-quinoxaline-2,3-dione (DNQX), which blocks both the glycine site and non-NMDA receptors, can protect neurons against this excitotoxic damage. In order to evaluate the neuroprotective activity of the drugs in vivo, transient forebrain ischemia was performed in male Wistar rats by occluding both common carotid arteries and by lowering the mean arterial blood pressure to 40 mmNg for 10 min. Treatment with noncompetitive N M D A antagonists phencyclidine (2 and 5 mg/kg), dizocilpine (1 mglkg) and memantine (10 and 20 mg/kg) significantly reduced the neuronal damage in the CA1 sector o£ the rat hippocampus after 7 days of recovery. The neuroprotection by cycloleucine (50 mglkg) in the CA1 and CA3 hippocampal sectors was weak but still significant, whereas DNQX (10 mg/kg} just reduced the neuronal damage in the CA3 hippocampal sector. Antagonists at the NMDA receptors are capable of reducing excitotoxic and ischemic neuronal damage , however, it has to be discussed whether the pharmacokinetic properties of some compounds limit their neuroprotective activity in vlvo. Institut fflr Pharmakologie W-3550 Marburg, F.R.G.

und

Toxikoloqie ,

Ketzerbach

63,

CONVULSIONS~ MORTALITYINDUCEDBY INTRAVENOUSLYOR CENTRALLY /~]MINISTEREDNMDAIN MICE: EFFECTSOF SOFECALCI~ ANT~3ONISTS, /WFICONVU~S ~ NMDAANTAGONISTS M. N~Idner and S.S. Chatterjee The glutamate analog N-n~thyl-D-aspartic acid (NMDA) is a selective agodist at the NMDAtype of excitatory amino acid receptors and causes large influx of calci~n ions into CNS-neurons.~ NMDA-receptormediated processes have been i~)licated in a number of physiological and pathological phenon~a such as learning, regulatien of synaptic growth, certain forms of epilepsy, excitotoxicity and hypoxic or ischEmic brain insult etc.. Antagonists of NMDA-receptorsor agents capable of antagonizing NMDA-receptor nL:diated processes are regarded as potential therapeutic agents for the treatment of specific neuronal disorders. Observations in our laboratories indicate that dependingon the route of administration and the dose of NMDAused, specific symptcmesof exaltation, convulsien and eventually mortality can be repreducibly induced in the mice. The specific noncc,petitive NMDA-antagenistdizocilpine (MK-801+) inhibited dose d£1~ently the neurological s)cnptsnes and mortality whereas SOTedihydropyddine type calciun channel blockers _

_

(nifedipi~, nitrendipine, nimodipine, furaldipine) and flunarizine were

more effective against NMDAinduced mortality than NMDAinduced convulsions, lhe anticonvulsants diazepam, valpreic acid, carb~mazepine, diphenylhydantoin and phenobarbital vere only active in preventing tonic seizures. The antitussive agent dextr~methorphanprotects the animals from NMDAinduced tonic seizures and mortality whereas ketamine even in doses up to 50 mg/kg (p.o.) did not i~luence any of tFe NMDAinduced toxicological s)~nptom~s. Except MK-801 which was equiactive in beth of the models, most of the active drugs showedhigher activity against i.v. than against i.c.v. NMDAinduced toxidties. Such experin~mtal observations indicate that the i.v. application of NMDA in mice is not only a sinple and reproducible test for assessing the in vivo activity of NMDAreceptor antagonists but also is suitable for detecting the potential anticonvulsive and/or cerebroprotective action of calciL~ antagonists and other agents. Ibis model also expands the battery of chemically or electrically induced seizure models for characterizing potential anticenvulsants. Deparb~ent of Pharmacology, Dr. Willmar SchwabeGmbH& Co., D-7500 Karlsruhe 41, FRG

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KINDLED RATS ARE MORE SENSITIVE TO P H E N C Y C L I D I N E LIKE BEHAVIOURAL EFFECTS OF NMDA RECEPTOR A N T A GONISTS THAN NON-KINDLED RATS. D. HSnack

THE NO-SYNTNASE INHIBITOR, L-NG-NITRO-ARGININE, INHIBITS LONG TERM POTENTIATION 1N THE RAT HIPPOCAMPUS. C. Bon, G.A. B6hme, J.M. Stutzmann, A. Doble, and J.C. Blanchard.

Selective NMDA (N-methyl-D-aspartate) receptor antagon i s t s h a v e b e e n r e p o r t e d to be p o t e n t i a l c a n d i d a t e s for n e u r o l o g i c a l d i s o r d e r s s u c h as e p i l e p s y , a n x i e t y a n d ischemic b r a i n d a m a g e . T h e r e e x i s t two c l a s s e s of a n t a g o n i s t s for t h e NMDA r e c e p t o r c h a n n e l complex, c o m p e t i t i v e a n d n o n - c o m p e t i t i v e ones. The n o n - c o m p e t i t i v e NMDA a n t a g o n i s t s like p h e n c y c l i d i n e (PCP) a r e l i m i t e d in t h e i r u s e f u l l n e s s b e c a u s e of p s y c h o t o m i m e t i c s i d e - e f f e c t s in humans. We s t u d i e d t h e b e h a v i o u r a l e f f e c t s of CGP 37849 ( D L - ( E ) 2-amino-4-methyl-5-phosphono-3-pentenoate}, the first c o m p e t i t i v e NMDA r e c e p t o r a n t a g o n i s t w i t h p o t e n t o r a l a n t i c o n v u l s a n t a c t i v i t y , in c o m p a r i s o n to MK-801 ( d i z o zilpine), a n o n - c o m p e t i t i v e a n t a g o n i s t , in n o n - k i n d l e d a n d a m y g d a l a - k i n d l e d r a t s (a c h r o n i c model f o r f o c a l seizures with secondary generalisation). PCP-like b e h a v i oural effects were scored using a ranked i n t e n s i t y scale. In k i n d l e d r a t s , 20 m g / k g CGP 37849 i.p. p r o d u c e d t h e s a m e s c o r e s for h y p e r l o c o m o t i o n a n d h e a d w e a v i n g a s 0.1 m g / k g MK-801 i.p. Kindled r a t s e x h i b i t e d h i g h e r b e h a v i o u r a l s c o r e s t h a n n o n - k i n d l e d r a t s , e s p e c i a l l y in c a s e of CGP 37849. T h e b e h a v i o u r a l e f f e c t s could be a t t e n u a t e d or b l o c k e d by s e v e r a l n e u r o t r a n s m i t t e r a n t a g o n i s t s . A h i g h s e n s i t i v i t y of k i n d l e d r a t s to e x c i t a t o r y e f f e c t s o c c u r r e d also a f t e r t r e a t m e n t w i t h m e m a n t i n e , a n a n a l o g of a m a n t a d i n e , w h i c h w a s r e p o r t e d to block NMDA r e c e p tor channels non-competitively. T h e d a t a show t h a t p e r m a n e n t a l t e r a t i o n s of b r a i n f u n c tions as occurring during epileptogenesis, such as induced by k i n d l i n g , c a u s e a h y p e r s e n s i t i v i t y to P e P - l i k e b e h a v i o u r a l s i d e - e f f e c t s of b o t h c o m p e t i t i v e a n d n o n - c o m p e t i t i v e NMDA r e c e p t o r a n t a g o n i s t s , w h i c h c o n t r i b u t e to t h e r e c e n t l y r e p o r t e d i n c r e a s e of NMDA r e c e p t o r f u n c t i o n during kindling. The findings suggest t h a t the t h e r a p e u t i c u s e f u l l n e s s of c o m p e t i t i v e NMDA a n t a g o n i s t s in e p i l e p t i c p a t i e n t s m a y be low.

N i t r i c oxide (NO), produced from the c o n v e r s i o n of a r g i n i n e to c i t r u l l i n e , has been shown to be i n v o l v e d in the s t i m u l a t i o n of c y c l l c GMP f o r m a t i o n by g l u t a m a t e in eerebellar tissue. NO p r o d u c t i o n o c c u r s through an enzyme c a l l e d NO-synthase which has been p u r l f i e d from r a t cerebellum. Immunohtstochemlcal studies have r e c e n t l y d e m o n s t r a t e d t h a t NO-synthase l s a s s o c i a t e d with n e u r a l e l e m e n t s both in the p e r i p h e r a l and c e n t r a l nervous systems. The NO-synthase i n h i b i t o r , k-NG-Nitro a r g i n i n e (L-NOARG), was used to examine the r o l e o£ NO in long term p o t e n t i a t i o n (LTP) in the hippocampus, a s y n a p t i e p l a s t i c i t y phenomenon in which g l u t a m a t e - m e d i a t e d p r o c e s s e s p l a y a c r u c l a l r o l e . Hippocampal s l i c e s were p r e p a r e d and m a i n t a i n e d i n v i t r o a c c o r d i n g to c l a s s i c a l methods. F l e l d EPSPs or p o p u l a t i o n s p i k e s (PS) were evoked in the CA1 s t r a t u m r a d i a t u m or pyramidal c e l l body a r e a f o l l o w i n g low f r e q u e n c y (0.2 Hz) s t i m u l a t i o n of the S c h a f f e r c o l l a t e r a l - c o m m i s s u r a l f l b r e s system a t submaximal i n t e n s i t y . LTP was induced by 2 t r a l n s of high f r e q u e n c y s t i m u l a t i o n (100 Hz, I s d u r a t i o n ) which r e s u l t e d in a p o t e n t i a t i o n of the EPSP s l o p e and the PS a m p l i t u d e l a s t i n g for a t l e a s t 60 min. A p p l i c a t i o n o I 0.1 ~N L-NOARG v i a the s u p e r l u s i o n bath medium, 15 min b e f o r e and 5 min a f t e r tetanic stimulation, r e s u l t e d in a complete blockade of LTP i n d u c t i o n w i t h o u t a l t e r a t i o n of b a s e l i n e synaptic transmission. However, i f L-NOAR6 was a p p l i e d to s l i c e s p r e t r e a t e d I o r 2 hours with a s u p e r l u s i o n medium c o n t a i n i n g 100 ~N L - A r g i n i n e , no i n h i b i t i o n of LTP o c c u r r e d , suggesting that the i n h i b i t o r y e l f e c t of L-NOARG r e s u l t e d from c o m p e t i t i o n with t h e n a t u r a l s u b s t r a t e of NO-synthase. These d a t a s u p p o r t the h y p o t h e s i s t h a t NO f o r m a t i o n i s i n v o l v e d in LTP i n d u c t i o n .

Dept. of P h a r m a c o l . , Toxicol., and. P h a r m a c y , School V e t e r i n a r y Medicine, Bfinteweg 17, D - 3 0 0 0 H a n n o v e r 71

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NON-OPIOID ANTITUSSIVES AND METHADONE INFI//£~CE HIPPOCAMPAL LONG- TEEM M. Kru@ and R. Matthies

E l e c t r o p h y s i o l o g y l a b o r a t o r y , Rh6ne-Poulenc Rorer, C e n t r e de Recherches de V i t r y , 94403 V i t r y - s u r - S e i n e , France.

DIFFERENTIALLY POTENTIATION

K I N D L I N G IN RATS FROM S T I M U L A T I O N OF A HIGHLY SENSITIVE LOCUS IN THE POSTERIOR PART OF THE PIRIFORM CORTEX. U. Wahnschaffe

Recently it has been shown that the glutamatergic N~DA receptor whose activation is essential for the induction of long-term potentiation (LTP) and which is also involved in hypoxia induced neurodegeneration and in epilepsy genesis, can be regulated via additional binding sites, e.g. for phencyclidin and dextromethorphan. Nonopioid antitussives as well as opioids have been reported to possess antiischemic and anticonvulsive properties which can be explained by interaction with the NMDA receptor complex. In order to look whether such sut~ stances, as can be expected, also influence the posttetanic LTP in the freely moving animal, doses of dextromethorphan (40 mg/kg), methadone (2 mg/kg), normethadone (0.1 mg/kg) and codeine (20 rag/ kg) which have been found to be effective in preventing N~DA related neurodegeneration or as anticonvulsives were investigated. It has been demonstrated that dextromethorphan blocks LTP induction and that methadone and normethadone suppress the maintenance of LTP, whereas codeine has no significant influence. There is evidence of an interaction of these substances with the NMJDA receptor complex which perhaps can explain the anticonvulsive action.

Goddard and and coworkers (Exp. Neurol. 25: 295330, 1969) were the first to d e m o n s t r a t e that structures of the olfactory-limbic system, i n c l u d i n g the p i r i f o r m cortex (PC), are highly susceptible to the kindling development. Several lines of evidence have s u g g e s t e d that particularly the PC might be c r i t i c a l l y involved in generation and propagation of seizures. We investigated the susceptibility of different parts of the PC to the kindling d e v e l o p m e n t as c o m p a r e d to that of the b a s o l a t e r a l amygdala (BLA). A locus in layer III of the rostral portion of the posterior PC (PPC) is described, which was more sensitive to electrical stimulations than the caudal part of the PPC, the deep prepiriform cortex, or the BLA. This locus could be r e a d i l y kindled, and seizure t h r e s h o l d s in fully k i n d l e d rats were 60-90 % lower than respective thresholds in rats kindled from other loci. Treatment of fully kindled rats with the antiepileptic drugs diazepam, carbamazepine, phenobarbital, and valproate showed that anticonvulsant effects of these drugs in rats kindled from the rostral PPC were c o m p a r a b l e to effects in BLA-kindled rats, although the rostral PPC tended to be more r e s i s t a n t to a n t i c o n v u l s a n t effects. Unilateral ibotenic acid lesions of the PPC increased the seizure threshold of rats kindled from the ipsilateral BLA. These data suggest that the rostral PPC might represent a generating site within the PC.

Present address: Institute of Pharmacology and Toxicology, Medical Academy Magdeburg, Leipziger Str. 44, ~ 3090 Magdeburg.

Dept.of Pharmacol., Toxicol. and Pharmacy, Sch. of Veterinary Medicine, 3000 Hannover 71, FRG.

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CHRONIC INVESTIGATIONS ON THE ANTICONVULSANT EFFECT OF CLOBAZAM AND ABECARNIL IN AMYGDALA KINDLED RATS: INFLUENCE OF THE EXPERIENCE OF THE ANTICONVULSANT EFFECT ON THE DEVELOPMENT OF TOLERANCE C. R u n d f e l d t and W. LSscher

R E L A T I O N S B E T W E E N I N D I W I D U A L A C T I V I T Y IN A R U N N I N G - W H E E L A N D P H A R M A ~ 0 L O G I C . ~ L E F F E C T S OF DOPAMINERGIC AGONISTS AND ANTAGONISTS IN M I C E OF O N E S T R A I N

B e n z o d i a z e p i n e (BZ) r e c e p t o r l i g a n d s a r e h i g h l y e f f e c t i v e a n t i c o n v u l s a n t s . However, t h e i r long t e r m c o n t i n u o u s use in e p i l e p t i c p a t i e n t s is r e s t r i c t e d b e c a u s e of t h e i r t o l e ranceand d e p e n d e n c e - i n d u c i n g p o t e n t i a l . Durin~ t h e l a s t y e a r s t h e r e h a s been i n c r e a s i n g e v i d e n c e t h a t e n v i r o n m e n t a l cues and l e a r n i n g b e h a v i o u r h a v e an e f f e c t on d e v e l o p m e n t of t o l e r a n c e . T h e r e f o r e we i n v e s t i g a t e d t h e i n f l u e n c e of t h e e x p e r i e n c e of t h e a n t i c o n v u l s a n t e f f e c t on t h e d e v e l o p m e n t of t o l e r a n c e in a m y g d a l a k i n d l e d r a t s . Clobazam, a 1 , 4 - b e n z o d i a z e p i n e , a c t s as a full a g o n i s t and a b e c a r n i l (ZK 112119), a {5-carboline, a c t s as a p a r t i a l a g o n i s t a t t h e BZ r e c o g n i t i o n s i t e . Using a s t a n dardized experimental protocol described elsewhere ( L 6 s c h e r and Schwark, Exp. Neurol. 90, 873, 1985), m a r k e d t o l e r a n c e d e v e l o p e d in c l o b a z a m t r e a t e d r a t s (10 m g / k g 3 t i m e s a d a y for 15 d a y s ) . Using t h e s a m e e x p e r i m e n t a l p r o c e d u r e w i t h a b e c a r n i l (5 m g / k g ~ t i m e s a d a y for 15 d a y s ) we could only d e m o n s t r a t e a p a r t i a l d e v e l o p m e n t of t o l e r a n c e l e a v i n g t h e s e i z u r e s e v e r i t y still r e d u c e d a f t e r two w e e k s of t r e a t m e n t . In t h e s e t r i a l s t h e a n t i c o n v u l s a n t e f f e c t was t e s t e d e v e r y 2 to ~ d a y s 7 t i m e s d u r i n g t h e t r e a t m e n t to follow up t h e d e v e l o p m e n t of t o l e r a n c e . In a s e c o n d s e r i e s of t r i a l s t h e e x p e r i m e n t a l p r o c e d u r e was a l t e r e d to t e s t t h e a n t i c o n v u l s a n t e f f e c t only on d a y 1 and a f t e r 15 d a y s , l e a v i n g t h e t r e a t m e n t s c h e d u l e t h e same. In t h e s e t r i a l s we could d e m o n s t r a t e only a p a r t i a l d e v e l o p m e n t of t o l e r a n c e to t h e a n t i c o n v u l s a n t e f f e c t of clobazam, w h e r e a s with a b e c a r n i l we could n o t d e m o n s t r a te t o l e r a n c e d e v e l o p m e n t a t all. T h e s e e x p e r i m e n t s i n d i c a t e t h a t t h e e x p e r i e n c e of t h e a n t i c o n v u l s a n t e f f e c t s c o n t r i b u t e s to t h e d e v e l o p m e n t of t o l e r a n c e w i t h BZ r e c e p t o r l i g a n d s . It m a y be c o n c l u d e d t h a t t h e d e v e l o p m e n t of t o l e r a n c e to t h e a n t i c o n v u l s a n t e f f e c t of BZ r e c e p t o r l i g a n d s could be s l o w e r in p a t i e n t s in whom t h e t r e a t m e n t s u p r e s s e s t h e s e i z u r e s t o t a l l y t h a n in p a t i e n t s in whom t h e s e i z u r e f r e q e n c y is only r e d u c e d . The d a t a on a b e c a r n i l i n d i c a t e t h a t t h i s n o v e l BZ r e c e p t o r l i g a n d could be a r a t h e r u s e f u l a n t i c o n v u l s a n t drug in h u m a n s .

I n t e r i n d i v i d u a l d i f f e r e n c e s in the r e a c t i v i t y to p s y c h o t r o p i c d r u g s can be s e e n in a n i m a l s and h u m a n s . T h e d e t e c t i o n of the n e u r o b i o l o g i c a l b a c k g r o u n d of t h e s e i n d i v i d u a l - v a r i a b i l i t y is p o t e n t i a l c l i n i c a l r e l e v a n t . In our e x p e r i m e n t s we a n a l y z e d r e l a t i o n s b e t w e e n m o t o r i c p h e n o type - d o p a m i n e r g i c d i s p o s i t i o n a n d r e a c t i o n s to n e u r o l e p t i c d r u g s in m i c e of one s t r a i n . The mice were selected after their locomotor a c t i v i t y in a r u n n i n g - w h e e l . L o w - a c t i v e m i c e (LAM) a n d h i g h - a c t i v e m i c e (HAM) showed c l e a r d i f f e r e n c e s in t h e i r r e a c t i v i t i e s to the d o p aminergic agonists apomorphine, bromocriptine a n d a m p h e t a m i n e r e s p e c t i v e l y to the a n t a g o n i s t s haloperidol and clozapine. Ther@fore a specific f u n c t i o n of the m e s o l i m b i c a n d / o r s t r i a t a l d o p a m i n e r g i c s y s t e m in d i f f e r e n t m o t o r i e p h e n o t y p e s of one s t r a i n can be s u p p o s e d s i n c e d o p a m i n e r g i c t a r g e t s t r u c t u r e s of the u s e d s u b s t a n c e s are d i f f e r e n t .

H.E.

Sehumacher

D e p a r t m e n t of P h a r m a c o l o g y a n d T o x i c o l o g y , M e d i c a l A c a d e m y "Carl G u s t a v C a r u s " , L i n g n e r p l a t z I, 0 - 8 0 1 0 D r e s d e n , F R G

Dept. of P h a r m a c o l . , Toxicol. & P h a r m a c y , School of V e t e r i n a r y Medicine, Bfinteweg 17, 8000 H a n n o v e r 71, F.R.G.

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AGE-DEPENDENT EFFECTS OF PHENOBARBITAL, AMINOOXYACETIC ACID AND HALOPERIDOL ON DYSTONIC DISTURBANCES IN MUTANT SYRIANGOLDEN HAMSTERS A. Richter, G. Fredow

I'NDIVIDUAL DIFFERENT RESPONSES OF APOMORPHINE-TREATED RATS TO HALOPERIDOL AND PIMOZIDE U.Havemann-Reinecke and B.K~cking Apomorphine(AP0;O,5-5mg/kg s.c.)produces oral stereotyped behaviour in rats by stimulation of pos%synaptic DA-receptors. In response to 2m~/kg APO male rats showed individually different motility patterns,each of which could be reproduced in a second test 4 days later.About 4~% of the ra~s tested showed predominantly stereotyped sniffing in combination with an increased locomo%or activity(S(L,G)-rats),the other 60% could be classified as predominantly licking and gnawing rats(L(S,G)-rats)(Havemann et al.,Psychopharm. 90~1986).In the present s%udy the individually dlfferen% types of rats(tested by AP0)were treated with two different DA2-receptor antagonists~Haloperidol (HAL)and Pimozide(PIM).The motility pattern of the S(L,G)-rats was not affected by 0,2mg/kg HAL i.p. in all parameters tested and 0,4mg/kg HAL i.p. showed a weak antagonizing effect only,whereas the motility pattern of the L(S~G)-rats was completely antagonized by injection of 0,2mg/kg HAL.Furthermore in the S(L,G~ rats and the L(S,G)-rats 0,4 and 0,8mg/kg PIM i.p. act in a similar way as HAL(0,2 and 0,4mg/kg)did.However~ in S(L,G)-rats PIM(in both doses)antagonized the motility pattern only at 30-40min post injection of AP0 in contrast to HAL(O,4mg/kg).In L(S,G)-rats the ant~ agonizing effect of £IM seemed to be more pronounced to the stereotyped behavlour and less effective on locomotor activity. To conclude,the L(S~G)-rats seem to be more sensitive to DAergic stimulation and inhibition than the S(L,G)rats. This individually different sensitivity might probably be located on the level of the postsynaptic DA -receptors or might primarily be dependent on un2 ~ . ? known mechanlsms,whlch are controlled by DA-receptor mediated processes.

The m u t a n t h a m s t e r s e e m s to be a s u i t a b l e model for idiopathic dystonia. A g e - d e p e n d e n t dystonic attacks can r e p r o d u c i b l y be i m i t a t e d by stress. Previously, acute p h a r m a c o l o g i c a l s t u d i e s s u g g e s t e d t h a t an i n b o r n d e f i c i e n c y of t h e GABAergic s y s t e m is i n v o l v e d in m u t a n t h a m s t e r s (LOSCHER et al., 1989, M o v e m e n t Disorders 4: 2 1 9 - 2 3 2 L Drugs w h i c h i n c r e a s e GABAergic t r a n s m i s s i o n , s u c h as phenobarbital (PhB) and aminooxyacetic acid (AOAA), e x e r t e d p o t e n t a n t i d y s t o n i c e f f e c t s . H o w e v e r , also h a l o p e r i dol (HP) d e c r e a s e d t h e s e v e r i t y of d y s t o n i c d i s t u r b a n c e s (FREDOW and SCHMIDT, 1989, N a u n y n - S c h m i e d e b e r g ' s Arch. P h a r m a c o l . 339, Suppl.). Recently, c h r o n i c p h a r m a c o l o g i c a l examinations h a v e shown t h a t PhB p a r a d o x i c a l l y h a s prodystonic effects during chronic treatment, whereas AOAA s e e m s to be i n e f f e c t i v e , In c o n t r a s t to t h e a c u t e i n v e s t i g a t i o n s , which were c a r r i e d out b e t w e e n 81. a n d 35. d a y of life, c h r o n i c t r e a t m e n t w i t h PhB and AOAA s t a r t s d on d a y 21.. This s t u d y i n v e s t i g a t e d t h e a g e - d e p e n d e n c e of a c u t e e f f e c t s on d y s t o n i c d i s t u r b a n c e s . PhB (20 m g / k g i.p.), AOAA (20 m g / k g i.p.) and HP (0.5 m g / k g i.p.) were a d m i n i s t e r e d on 21. ( d a y of w e a n i n g ) a n d 31. d a y of life. FhB was s i g n i f i c a n t l y more e f f e c t i v e on d a y 31. t h a n on 21. d a y of life. AOAA r e t a r d e d t h e s e v e r i t y of d y s t o n i c a t t a c k s only d u r i n g t h e f i r s t h o u r a f t e r a d m i n i s t r a t i o n b u t f a i l e d to i n f l u e n c e t h e maximum s e v e r i t y of d y s t o n i c a t t a c k s on d a y 21., w h e r e a s t h e s e v e r i t y was s i g n i f i c a n t l y d e c r e a s e d on 31. d a y of life. HP showed a n t i d y s t o n i c e f f i c a c y b o t h on 21. and on 31. d a y of life. T h u s , a c u t e e f f e c t s on d y s t o n i c d i s t u r b a n c e s by m a n i p u l a t i o n s of t h e GABAergic s y s t e m seem to be a g e - d e p e n d e n t , w h e r e a s t h e a c u t e a n t i d y s t o n i c e f f e c t of HP was i n d e p e n d e n t of t h e a s e of m u t a n t h a m s t e r s . This m a y i n d i c a t e t h a t t h e d e v e l o p m e n t of t h e i n h i b i t o r y GABAergic s y s t e m is r e t a r d e d in m u t a n t d y s t o n i c h a m s t e r s . Dep. of P h a r m a c o l . , Toxicol. and P h a r m a c y , School of V e t e r i n a r y Medicine, Biinteweg 17, 3000 H a n n o v e r , F.R.G.

Psychiatric Hospital of the University of GSttingen, yon Siebold-StraSe 5, D-3400 GSttingen

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LONG-TERM EFFECTS OF BCH 3 2 5 ( P R O - D - P H E - P R O - G L Y ) ON APOMORPHINE-INDUCED BEHAVIOURAL RESPONSES Ch. RAUCA

THE ANTI-PARKINSONIAN EFFECTS OF T R A N S D E R M A L (+)-4-PROPYL-9-HYDROXYNAPHTHOXAZINE (+PHNO) IN MPTP-TREATED MARMOSETS K.W. Lange, P.-A. L6schmann, P. Jenner, and C.D. M a r s d e n

Following 0.04

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of b e h a v i o u r in rats c o u l d be o b s e r v e d . a single intrastriatal application still inhibited significantly the induced rotations.

rotational 5 days after this p e p t i d e apomo~ p h i n e -

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p o t e n t i a t e d the a p o m o r p h i n e - m e d i a t e d hypoactivity on c l i m b i n g behavl.our in m i c e 20 and 40 m i n a f t e r adminisuFation. Nhereas at a later date no chanQes of t h i s b e h a v i o u r a l reaction could be found, ~e observed that the apomorphine h y p e r a c t i v i t y on c l i m b i n g b e h a v i o u r was d e c r e a s e d 2 te 5 d a y s a f t e r a s i n g l e p e p t i d e a p p l i c a t i o n . Botl~ types of e x p e r i m e n t suggest a long-lasting e f f e c t of BCH 3 2 5 ~ h i c h was p o s s i b l y u n r e l a t e d to the p r e s e n c e of the p e p t i d e in the brain. Institute Academy,

of

Pharmacology

Leipziger

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Toxicology,

0-3090

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Magdeburg,

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O r a l a d m i n i s t r a t i o n of t h e d o p a m i n e a g o n i s t (+)4-propyl-9-hydroxynaphthoxazine (+PHNO) r e v e r s e s the parkinsonian m o t o r s y m p t o m s p r o d u c e d b y lmethyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) in m a r m o s e t s a n d is e f f e c t i v e in p a t i e n t s w i t h P a r k i n s o n ' s d i s e a s e . We h a v e e x a m i n e d the t r a n s d e r m a l e f f e c t s of + P H N O in the m a r m o s e t . Common marmosets were rendered parkinsonian by treatment with MPTP. In comparison with a control solution, t o p i c a l a p p l i c a t i o n of + P H N O (50, i00, 150 ~g) s o l u t i o n to a 1 cm 2 a r e a of a b d o m i n a l s k i n i n c r e a s e d the l o c o m o t o r a c t i v i t y in a dose-dependent manner. .In a second experiment, I00 ~g +PHNO were applied to d i f f e r e n t - s i z e d areas of skin (i cm 2, 4 cm2) . The larger area produced a large and brief effect whereas the smaller area produced a smaller and m o r e p r o l o n g e d effect. These results confirm the anti-parkinsonian effects of +PHNO and demonstrate that dermal application of + P H N O p r o d u c e s effects in t h e c e n t r a l n e r v o u s system. The q u a n t i t y of the d r u g a p p l i e d t o a c e r t a i n a r e a of s k i n a p p e a r s to determine t h e d u r a t i o n of d r u g r e s p o n s e w h i l e t h e size of the a r e a of skin c o n t r o l s the size of the response. U n i v e r s i t y D e p a r t m e n t of Neurology, I n s t i t u t e P s y c h i a t r y , D e n m a r k Hill, L o n d o n SE5 8AF, UK

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EFFECTS OF STIMULATION OF PUTATIVE DOPAMINE AUTORECEPTORS IN COMPARISONWITH BLOCKADE OF EITHER 0--1 OR D-2 POSTSYNAPTIC RECEPTORS ON EEG IN RATS W. Kropf and K. K u s c h i n s k ¥

QUALITATIVEALTERATIOI~SOF THE RESPONSETO SELF-ADMINISTEREDETHANOLIN BEHAVIORALLYDEPENDENTRATS J. WOLFFGRAMM The development of an addiction can be studted in a rat model of alcoholism {1). In a free choice situation the animals estab]fsh an individually stable pattern of ethanol consumption which is modified by external and internal factors. This period of "controlled" drag tntake lasts for 6 - g months, i t is succeeded by a "behavioral dependence" which is characterized by a loss of control. In the rat, this state is irreversible, it persists 9 months of drug abstinence (1). Using this model i t was investigated whether ethano] (ETOH) has the same effects on the behavior of mole Nistar rats being either in the state of controlled drinking or of behavioral dependence. Two groups of rats were tested (N=12, each, age-matched). One group had received ETOUfor 8 weeks (RE), the other group for 36 weeks (LE). The last 12 weeks before the experiment the rats received no alcohol Then ETOHwas offered in the homecages as well as during a 15 min test in a novel environment in which the behavior of the rat was registered by means of an infrared beam monitoring syst~ ("ACTIFRAME"). The animals had access to tap water, 5 Voi % and 20 Vol % ETOH. The tests started 5 days before ETOH was reoffered. At this time, all the bottles in home and test cages contained water. A control group of 12 animals (KO) received water over the whole course of the experiment. In contrast to ME, LE rats revealed signs of behavioral dependence. They consumed high doses of ETORand maintained a preference even when ETOHwas adulterated with quinine. The individual ETOH intake during a test was not correlated to the consumption in the home cages, i t varied over a wide range (0 - Z g/kg). Thus it was possible to compare the individual ETOHdose taken voluntarily during the test with subsequent changes in the behavior. ME revealed the typical pattern of ETOHactions (slight excitation with low doses depressant effect with high doses). A tolerance to the sedative effects developed in ME from the f i r s t to the fourth day of testing. In the behaviorally dependent LE rats, the pattern or response was qualitatively changed, Extrem]y ]om doses of 0,1 - 0,2 g/kg exhibited strong sedative effects. Rapid and slow locomotion, rearing and smell stationary movements were depressed. With higher doses the effect switched towards an excitation, This inverse pattern of ETOHactions showed no signs of tolerance. I t is suggested that the differences between LE and ME reflect general changes in the rewarding properties of ETONwhich are associated to alcohol addiction. (I) Wolffgranr~ J.& Heyee A. Social behavior, dominance, and social deprivation of rats determine drug choice. Pharmecol. Biochem. Behav., in press

Low doses of apomorphine or quinpirole and both low and high doses of ta]ipexole produce hypokinesia, ptosis and yawning (suggesting sedation), probably due to an activation of dopamine autoreceptors with resulting impairment in dopaminergic t r a n s m i s s i o n . In contrast~ haloperidol~ which ~ i n I y b l o c k s D--2 receptors, o r Sch 23390 ( [ R - ( + ) - 8 - c h l o r o - 2 , 3 , 4 , 5 - t e t r a hydro-3-methyl-5-phenyl-lH-3-benzazepin-7-ol]hemimaleate) an antagonist o f D-1 receptors, produce hypokinesia, b u t e x c e p t i n l a r g e doses, less signs o f sedation. I t was s t u d i e d , w h e t h e r c o r t i c a l EEG alterations correlate w i t h t h e s e b e h a v i o u r a l effects. Apomorphine ( 0 . 0 2 o r 0 . 0 5 mg/kg s . c . ) , q u i n p i r o l e ( 0 . 0 5

mg/kg) or talipexole (0.02 or 0.5 mg/kg) produced i n creases i n t h e power i n a l l o f the frequency bands, except b e t a - 2 , w i t h t h e most pronounced increase in the d e l t a band. In contrast, haloperidol (0.1 mg/kg i . p . ) or SCH 23390 (0.2 mg/kg i . p . ) l e d t o l i t t l e increases in the delta band~ but more pronounced increases in the alpha-2 band. These results suggest that activation of putative dopamine autoreceptors p r o d u c e s b e h a v i o u r a ] and EEG patterns different from those produced by blockade o f either D-1 o r D-2 postsynaptic dopamine receptors. in this Intrastriatal EEG recordings demonstrated t h a t region, the delta activation after a p o m o r p h i n e ( 0 . 0 5 mg/kg) was even more pronounced than over the cerebra] cortex.

Supported by a grant of the Deutsche Forschungsgemeinschaft (Ku 395/4-1). I n s t i t u t e of Pharmacology and Toxicology, Faculty o f Pharmacy, U n i v e r s i t y o f Marburg, K e t z e r b a c h 63, D-3550 Marburg, F.R.G.

Inst. f. Neuropsychopharmako]ogie,FU Berlin, g]~enallee 30. D-IO00 Berlin 19

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ADDITION OF CAFFEINE ENHANCESTHE PREFERENCEFOR PARACETAMOLBUT NOT FOR ACETYLSALICYLICACID IN THE RAT A. HEYNEAND C, ROHM Self-administration of some psychoactive drugs m a y lead to abuse and dependence. I t has been suspected that continuous consumption of non-optold analgesics has similar consequences, in particular when caffeine i s added. By means of an animal model of voluntary intake, the influence of forced administration of caffeine (C) on free choice of paracetamo] (P) or acetylsalicylic acid (ASA) was studied. The substances were offered in the drinking fluids. Two groups of Wistar rats (n = 8, each) had the choice between water and different concentrations of P or ASA (I,2 and 4 g/l). Rats of two other groups had the same choice, but C (0.5 g/I} was added to all solutions (including the water). To detect a hypothetlc preference for P and/or ASA deriving from their prlncipa] (analgesic) actions, the rats were submitted once a week to a distressing enviroamental situation. The normal L/D-cycle of 12 h/12 h was disrupted by a 36 h lasting light-phase. During this period a11 the animels increased significantly the intake of analgesics by 20 - 50 % in relation to the other days. Only minor differences among the groups were observed. During the "unstressed" time of the g weeks lasting choice period significant differences among the groups were found. In general, P-solutions ~ere more preferred than ASA. The addition of C had no influence on the choice of ASA. The rats consumed only small doses with or without C (11 mcJ/kg/d and 12 mg/kg/d, resp,). In contrast, intake of P was significantly modified by addition of C. During the f i r s t week, the rats took high deses of P (140 mg/kg/d without C and 136 mg/kg/d with C). Afterwards P-rats reduced their intake whereas the C+P-rats maintained a high preference for P. They consumed three times more of P than the animals without access to C (P: 48 mg/kg/d, C+P= 143 mcJ/kg/d). I t is suggested that the combination of C and P exhibits specific actions different from those of the single compounds. The preference for E+P cannot be due to a choice of C, since the intake of C is forced. I t is also unlikely that i t derives fro~ analgesic effects since these effects concern P as well as ASA. Thus, the mixture of P and C may provide a certain kind of psychotropic reward which reinforces further drug intake. During withdrawal C-pretreated animals revealed a transient increase of algesic threshold (+ 15 %) and a decrease of body weight. Animals which had received on]y the analgesic showed no signs of withdrawal. After a drug-free period of 4 weeks, the same solutions as before were reoffered. There were no indications for a behavioral dependence of the rats on the analgesics with or without C.

~'FiCACY OF CEREE~OPROTECTIVE SUBSTANCES IN MANAG~4ENT OF THE CYTOTOXIC BRAIN OED~4A K. Andreas

The hexachlorophene-induced cytotoxic brain oedema can be used as experimental model of brain damage. Because of the clinical importance of the brain oede~na the hexachlorophene-produced brain injuries are suited for testing of cerebroprotective potential substances. The primary target of this neurotoxic substance is the cell membrane. Hexachlorophene causes disruption of myelin lamellae and vacuolation. Rats received orally hexachlorophene 240 mg/kg a day in liquid diet during three weeks. Thereafter the motor behaviour was disturbed, the water content of the brain and the sodium content were inereased, and the potassium content was decreased. Light microscopically vacuolation of the white matter in the cerebellum was observed. The irritation of the coordinative motor response obsezved in an especially developed test, the water content and the histological alterations were used to characterize the hexachlorophene-induced injuries in stDdies to evaluate potential cerebroprotective substances. Besides substances with known cerebroprotective effects some ncotropics were examined. Present address: Institute of Pharmacology and Toxioology, Medical Academy "Carl Gustav Carus" Dresden, Lingnerplatz I, O-8010 Dresden, FRG

Supported by a grant of the Bundesgesundheltsamt, I o s t i t u t for Arzneimittel. Inst. f . Neuropsychopharmekologie, FU Berlin, H1menallee 30, D-IO00 Berlin 19

466

468

I N F L U E N C E OF C E R E B R A L I S C H E M I A ON S H A K I N G B E H A V I O U R I N D U C E D BY Q U I P A Z I N E AND E L E C T R I C A L S T I M U L A T I O N OF THE P E R F O R A N T P A T H IN THE RAT J. Schmidt and E. L i p p e r t

AMPHIBIANS AS ALTERNATIVE EXPERIMENTAL MODELS? ABNORMAL DEVELOPMENT OF THE CENTRAL NERVOUS SYSTEM (CNS) INDUCED BY VALPROIC ACID Axel Oberemm, Frank Kirschbaum

S h a k i n g b e h a v i o u r in rats occurs in r e s p o n s e to a v a r i e t y of p h a r m a c o l o g i c a l stimuli and by e l e c t r ~ cal s t i m u l a t i o n of s e l e c t e d parts of the brain, p a r t i c u l a r l y the h i p p o c a m p u s . G r a n u l e cell discharges are n e c e s s a r y for the i n d u c t i o n of shaking b e h a v i o u r . N e u r o n s in the h i p p o c a m p u s are h i g h l y v u l n e r a b l e to i s c h e m i c insult. Brief bilateral c a r o t i d a r t e r y o c c l u s i o n has been shown to p r o d u c e m a r k e d d e l a y e d n e u r o n a l damage in the h i p p o c a m p u s , e s p e c i a l l y in the CAt sector. Ischemia-induced hippocampal neurodegeneration is a w e l l - e s t a b l i s h e d m o d e l r e l e v a n t to a n a l y z e cerebral i s c h e m i a and the e f f i c i e n c y of c e r e b r o p r o t e c t i v e drugs. This i n v e s t i g a t i o n aimed to test the f u n c t i o n a l c o n s e q u e n c e s of i s c h e m i a - i n d u c e d h i p p o c a m p a l n e u r o d e g e n e r a t i o n on s h a k i n g behav i o u r in rats. I s c h e m i a was i n d u c e d by c l a m p i n g both c a r o t i d a r t e r i e s for 60 min, or for 10 min in c o m b i n a t i o n w i t h a d e c r e a s e of the m e a n arterial b l o o d p r e s s u r e to 40 mm Hg, r e s p e c t i v e l y . T h e pharmacological (5 m g / k g q u i p a z i n e ip.) and elect r i c a l by s t i m u l a t i o n of the p e r f o r a n t path at 10 Hz for ~0-20 sec (0,1 msec imp. dur., 0,1 - 0,5 mA) i n d u c e d s h a k i n g b e h a v i o u r was e s t i m a t e d I, 3, 5, 7, 14 and 21 days after ischemia. C e r e b r a l i s c h e m i a leads to a m a r k e d d e c r e a s e of evoked s h a k i n g b e h a v i o u r i n d i c a t i n g f u n c t i o n a l d i s t u r b a n c e s of h i p p o c a m p a l t r i g g e r e d p r o c e s s e s . P r e l i m i n a r y e x p e r i m e n t s d e m o n s t r a t e the p o s s i b l e value of this m e t h o d in c h a r a c t e r i z a t i o n of cereb r o p r o t e c t i v e drugs.

Testing for embryofeto-toxicity and especially teratogenicity as part e r a safety evaluation has, up till now, exclusively been done m studies using whole mammalian animals. We have initiated studies to assess the possibility of using non-mammalian systems as models for revealing the potency of chemicals to induce abnormal development. Here we present data on studies performed with early stages of amphibian development. Previous attempts made by other laboratories have not been very successful in this direction. After establishing several models for monitoring embryonic development in amphibia (using: Ambystoma mexicanum, Xenopus la~is, Hyperoliusviridiflavus taeniatus), we standardized the procedures and defined the biological endpoints to be evaluated. As the first set of test substances we analysed the potential of valproic add (VPA) and some derivatives to induce abnormalities with these systems. This antiepileptic drug causes exencephalies (exe) in mice and is highly suspected to induce spina bifida (spb) in man. Results of our studies show for the first time the induction of typical abnormalities in amphibia which very much resemble the defects seen in mammals. The most obvious and persisting type of abnormal development of the CNS (exe and spb) occurred in Ambystoma following a pulse exposure to 5 and 10 mM VPA during gastralation (stages 9-12 in controls). Similar abnormalities were observed in Xenopus and Hyperolius, but in these species the effects are mainly observable during the early developmental stages and less uniform than in Ambystoma. The data presented appear promising and justify a closer evaluation of this system. Many more studies and especially systematic studies are necessary to validate the usefulness of these

D e p a r t m e n t of P h a r m a c o l o g y and T o x i c o l o g y , Medical A c a d e m y "Carl G u s t a v Carus", L i n g n e r p l a t z I, 0-8010 Dresden, Germany.

developmentalmodels. Institut f~r Toxikologie und Embryopharmakologie, Freie Universit~t Berlin, Gatystr. 5, D-1000 Berlin 33

R 118 471

469 IRREGULAR SCATCHARD PLOTS BINDING BY SERUM PROTEINS R. W a l t e r a n d A. S c h e r b e r

IN

SULFAMERAZINE

Conspicuous Scatchard plots of sulfamerazine b i n d i n g by H S A w e r e o b s e r v e d . T h e r e g u l a r curve of d e c r e a s i n g quotient r/cf (moles b o u n d d r u g p e r mol p r o t e i n a b o v e the free f r a c t i o n ) with increasing r w a s a l t e r e d to a t w o - p h ~ e curve. At low c o n c e n t r a t i o n s (to a b o u t i0. M) the bound fraction i n c r e a s e d m o r e r a p i d l y t h a n the free f r a c t i o n from w h i c h an increasing r e g i o n of the curve r e s u l t e d . T h e m a x i m u m r / c f r a n g e d at a b o u t r = 0 . 1 to 0.~ m o l / m o l . A r e g u l a r d e c r e a s i n g curve was registered at h i g h e r c o n c e n t r a t i o n s . The described p l o t s r e m a i n e d w h e n u s i n g H S A or s e r u m of c o n t r o l persons. Pretreatment of the p r o t e ~ n w i t h the d e f o l d i n g a g e n t u r e a (0.02 a n d 0.8 M) or w i t h the s t r o n g l y b o u n d p h e n y l b u t a z o n e (0.1 a n d i m_M) c a u s e d o n l y q u a n t i t a t i v e but not q u a l i t a t i v e c h a n g e s . T h e use of s e r u m of u r a e m i c p a t i e n t s r e s u l t e d in s i m i l a r d e c r e a s e d plots. P r o b a b l y the f l e x i b i l i t y of the a l b u m ~ n m o l e c u l e as well as intramolecular interactions and conformational changes during ligand binding must be t a k e n into a c c o u n t . T h e r e s u l t s s h o w e d t h a t b i n d i n g of s u l f a m e r a z i n e by s e r u m p r o t e i n s at low t h e r a p e u t i c concentration w a s less t h a n expected from extrapolated regular binding curves. T h e free fraction w a s r e l a t i v e l y h i g h e r t h a n at t h e r a p e u t i c concentrations. U r e a in the pathophysiological range decreased the b i n d i n g curves slightly only but phenylbutazone did this at a g r e a t e r e x t e n t e v e n at t h e r a p e u t i c levels.

DIALYSABILIT¥ OF DIMETINDFI~K MALEATE (FENISTIL R) FROM BLOOD AFTER INTRAVF~OUS INJECTION U. T11ei~, I. Kuhn, N. Yetzelsberger, P.W. Lficker Fenistil R is a drug often used ill dialysis patients to treat skin itching. To settle the question of dialysability of dimetindene maleate 4 ml Fenistil R injection solution was administered intravenously to n=3 healthy, male volunteers. Ten minutes later 100 ml venous blood was taken and 25000 I.E. Liquemin R was added. The blood was pumped through a dialyzing module of cuprophan hollow fibres with an effective surface of 35 cm 2. In distances of ten minutes samples of blood and dialyzer solution were taken and analyzed for the dimetindene concentration. A factor of 285,7 was used to calculate the clearance per

m2 .

A mean clearance of 38 ml/min*m 2, sd i0,2 for a CV% o£ 26,8 was found. This is comparable to the well known clearance of theophylline, so that it can be considered that dimetindene maleate is eliminated during haemodialysis of patients. Institut ffir klinische Prof. Dr. Lficker GmbH, D-6718 Gr~nstadt

Pharmakologie Richard Wagner

Bobenheim Str. 20,

D e p a r t m e n t of C l i n z c a l P h a r m a c o l o g y Ernst Moritz Arndt University Klinikum Fleischmannstr. Gr'eifswald, 0-2200, FRG

470 DETERMINATION OF 5-FLUOROURACIL IN HUMAN LIVER TISSUE C.Jochheim*, P.Janning*, U.Marggraf*, M.Linscheid*, F.Hasse #, D.L6hlein ~ 5-Fluorouracil (5-FU) is widely used in the chemotherapy of human carcinomas of the gastrointestinal tract. To avoid the undesired secondary effects, oral application has recently been displaced by regional therapy. Since the sample material available for the studies is very limited, extremely sensitive procedures using micro methods for the determination of small amounts of 5-FU in human blood plasma and liver tissue are required. Therefore, sensitive and selective detection methods based on C-18 reversed-phase (RP-18) HPLC and microHPLC with UV-absorbance and fluorescence detection were developed. From the liver tissue (typically 200 mg), the drug was extracted with ice-cold Acetonitrile after homogenization. A further clean-up has been obtained by TLC on silicagel plates with a mixture of n-Hexane, Ethylacetate, Formic acid and water as the mobile phase. After elution of the 5-FU spot with methanol and evaporation to dryness the residue was used for derivatization reactions. The fluorescence method uses BrMmc (4-Brommethyl-7-methoxycoumarin) as the derivatization reagent. The samples of liver tissues from patients with tumors were carefully separated into the different types (tumor, directly adjacent tissue, healthy tissue) and we have found the level of the drug inside of the tumor tissue higher by a factor of c. 1000 in comparison to adjacent tissue outside of the tumor. Very recently, the time consuming TLC step has been replaced by column switching and the number of merits of the new method and first results with tissue using this simplified procedure will be given.

"ISAS [nstitut fiir Spektrochemie, Bunsen-IgErchhoff-Str. 11 4600 Dortmund 1 #Sti~dtische Kliniken Dortmund, Beurhausstr. 40, 4600 Dortmund 1

472 A NOVEL NON-INVASIVE LACTOSE-[I~C]-UREIDE BREATH TEST DEMONSTRATES DRUG EFFECTS ON GASTROINTESTINAL TRANSIT TIME. H. K. Berthold, W. Heine and P. D. Klein Glycosyl ureides have recently been suggested as a new class of noninvasive stable isotopically labelled markers to determine gastrointestinal transit time (Heine et al., in press). Five h after bolus ingestion of 1 g of lactose-[13C]-ureide (equivalent to 160 mg of 13C-labelled urea), 13COz begins to appear in breath. Peak exhalation of label occurs at about 8-10 h and the signal returns to baseline after about 15-16 h, as determined by gas-isotope-ratio mass spectrometry. The effect results from enzymatic degradation of the substrate by colonic bacteria and is entirely abolished after sterilization of the intestine by a standard presurgical procedure using neomycine and e~thromycine. The time of peak ~3CO~appearance reflects substrate arrival in the transverse colon and microbial degradation. To determine whether drugs with known effects on gastrointestinal motility can modify the course of label appearance in breath, we administered an oral dose of metoclopramide (10 rag) or loperamide (16 mg) 1 h before the labelled substmte to increase or decrease upper gastrointestinal motility, respectively. Metoclopramide advanced both the onset and the time of peak response by approximately 1 h while loperamide broadened the peak and delayed the time to reach peak excretion by about 5 h. This novel stable isotope breath test should be useful in the diagnosis of gastrointestinal motility disorders and intestinal bacterial overgrowth. Because of its ability to reflect changes in intestinal motility rapidly, it may be a valuable tool in the development of new drugs. The lactose-[l~C]-ureide is non-invasive and involves no radiation exposure. Its acceptance by patients makes it eminently suitable for studies in normal volunteers or patients with higher risks, such as infants and children. USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, U.S.A.

Rl19 473

475

INTERACTION OF T H E SELECTIVE BETA B L O C K I N G D R U G TALINOLOL (TA) AND SULFASALAZINE (SU) IN T H E GASTROINTESTINAL T R A C T OF H U M A N B.Terhaag, U.Lange, H.Sahre, R. O e r t e l

T H E ABSORPTION OF P A R A C E T A M O L F R O M DIFFERENT REGIONS OF T H E H U M A N SMALL INTESTINE Th. Gramatt~, K. Richter aud K. Feller

SU is u s e d n o t o n l y for d r u g t h e r a p y , b u t a l s o for characterizing oro-caecal transit time (octt). In o r d e r to c h a r a c t e r i z e the o c t t of TA, b o t h d r u g s w e r e a d m i n i s t e r e d t o g e t h e r , bec a u s e one r e l e v a n t i n t e r a c t i o n of SU w i t h digo x i n is r e p o r t e d (Juhl RP, Clin. Pharm. Ther. 20:387, 1976). M e t h o d s : T h e a b s o r p t i o n of T A was i n v e s t i g a t e d without and together w i t h SU in ii h e a l t h y y o u n g v o l u n t e e r s in a d o s e of 50 m g (TA) a n d 4 g (SU). TA was a n a l y z e d b y an H P L C - m e t h o d . The pharmacokinetic parameters were analyzed by conventional methods. R e s u l t s : W i t h o u t 8U, the tma x of T A is 2.9 h (range: 1 - 4.5 h), a n d the h a l f life a n d M R T were calculated to be 15 h (range: 7.4 22.6 h) and 22.3 h (range: 11.6 - 58.3 h), respectively. In the p r e s e n c e of SU, T A c o u l d be f o u n d o n l y in s e r u m of 3 i n d i v i d u a l s , but was n o t d e t e c t a b l e or in the r a n g e of the limit of d e t e c t i o n in s e r u m of the o t h e r i n d i v i d u a l s . The pharmacokinetic analysis was not p o s s i b l e in all of t h e s e i n d i v i d u a l s . Conclusion: The r e a s o n of i n t e r a c t i o n is the adsorption ( v e r i f i e d b y in v i t r o e x p e r i m e n t s ) of T A b y SU. T h e r e f o r e , SU s h o u l d not be g i v e n t o g e t h e r w i t h T A b u t a l s o not w i t h o t h e r b e t a b l o c k i n g drugs. I n s t i t u t e of C l i n i c a l P h a r m a c o l g y , Medical Academy "Carl-Gustav-Carus", F i e d l e r Str. 27, 8019 D r e s d e n , F R G

High initial peak concentrations of p a r a c e t a m o l (PAR) do not c o r r e s p o n d to b e t t e r a n a l g e sic e f f i c a c y . Thus, s u s t a i n e d r e l e a s e PAR m a y be an i m p o r t a n t f o r m u l a t i o n in c l i n i c a l p r a c tice (STROM et al., 1990, J. Clin. Pharmacol., 654). In this study the absorption of PAR from d i f f e r e n t r e g i o n s of the s m a l l i n t e s t i n e was evaluated as a p r e r e q u i s i t e for r a t i o n a l drug d e s i g n - b y u s i n g the i n t e s t i n a l s t e a d y state perfusion technique with three lumen tubes. Preliminary results (three healthy volunteers) reported here revealed similar rates of a b s o r p t i o n (mg / cm x hr) from the duodeno-jejunal junction (1.4; i.I; 1.3), from d i s t a l j e j u n u m (1.2; 1 . 5 ) ' a n d from ileum (i.i). In the same way, b l o o d levels m e a s u r e d simultaneously during perfusion p e r i o d s disp l a y no local d i f f e r e n c e s r e g a r d i n g the shape of c o n c e n t r a t i o n - t i m e p r o f i l e s as w e l l as the AUC-values calculated. PAR absorption rates c o r r e l a t e to w a t e r t r a n s p o r t w i t h i n the int e s t i n a l s e g m e n t s u n d e r s t u d y (p = 0.01). R e s u l t s r e c o m m e n d P A R to be a s u i t a b l e c a n d i d a t e for s u s t a i n e d r e l e a s e f o r m u l a t i o n . I n s t i t u t e of C l i n i c a l P h a r m a c o l o g y M e d i c a l School, F i e d l e r s t r 27, 8019 D r e s d e n , G e r m a n y

474

476

THE E F F E C T OF B I L L R O T H I I - G A S T R E C T O M Y ON THE PHARMACOKINETIC OF A S O L I D OR LIQUID ORAL D I P Y R I D A M O L E P R E P A R A T I O N IN MAN B. T r a u s c h , M. T r a u s c h

SERUM PARAMETER~ OF BISMUTHSUBNITRATE M. Mateblowski, D. Kuhn and P. Topfmeier

Billroth II-gastrectomy causes changes of physiology of the u p p e r g a s t r o i n t s t i n a l t r a c t (pH, t r a n s i t time). D i p y r i d a m o l e was u s e d as a model drug, b e c a u s e of it's physicochemical properties: base ( p K a - v a l u e : 6,1) and p H - d e pendent octanol:water partition coefficient (pH 2: 0,85; pH 7: 9000) and w a t e r s o l u b i l i t y (pH 7: 8 pg/ml). Dipyridamole concentrations were d e t e r m i n e d by s p e c t r o f l u o r i m e t r y in s e r u m of 5 m a l e p a t i e n t s (group I, 37-63 years, 67-84 kg, > 2 y e a r s post g a s t r e c t o m y ) a n d in a group of 5 h e a l t h y m a l e v o l u n t e e r s (group II, 24-27 years, 68-84 kg) a f t e r oral a d m i n i s t r a tion as a s o l i d f o r m and a solution (pH: 8-8,5) in a d o s e of 225 mg. group I g r o u p II preparation solid liquid solid liquid cm~ (~g/ml) 0 , 2 6 " x 3,1 2,5 3,4 t ..... (min) 132"x 17~ 74 61 A U C = ~ ( ~ g * m i n * m l -:L) 5 8 " x 360 460 487 t - T e s t (p<0,05) * g r o u p I s o l i d - g r o u p I l i q u i d x g r o u p I s o l i d - g r o u p II s o l i d o g r o u p I l i q u i d - g r o u p II s o l i d Conclusion:_Billroth I I - g a s t r e c t o m y has an imp o r t a n t e f f e c t on b i o a v a i l a b i l i t y of d i p y r i d a mole preparations. For d r u g s w i t h comparable physicochemical properties such changes in pharmacokinetics are p o s s i b l e . It s h o u l d be taken into consideration when drugs with a s m a l l t h e r a p e u t i c d o s a g e r a n g e are used. P r e s e n t adress: I n s t i t u t e of C l i n i c a l kology, M e d i c a l A c a d e m y "Carl G u s t a v F i e d l e r s t r . 27, 0 - 8019 D r e s d e n , F R G

PharmaCarus",

Within a series of controlled trials several pharmacological parameters of ULKOWIS R, [Bismuthsubnitrate; BSN] a therapeutic active preparation for the treatment of gastritis and peptic ulcer, was evaluated. Serum levels: Bismuth [Atomic absorbance] The extent to which bismuth is absorbed following single and multiple administration of [BSN] was investigated in healthy male subjects. After ingestion of 700 resp. 2100 mg BSN blood samples were taken after 30 min, then hourly for 8 h, thereafter in 2-h-intervals until 24 h p.a.. For both dosages the bismuth concentration in all blood samples were less than the detectable limit. After uptake of 3x700 mg/day for 9 days and a final uptake of 700 mg blood samples were analyzed in 2-h-intervals for 24 h. Here the absorption of phase, app. 6 h, is follwed by an elimination phase during which a 12-16 h plateau is formed. After 24 h the bismuth concentration in all samples was below the limit again. These results indicate that only t!aces of orally administrated BSN in the form of ULKOWIS K tablets were absorbed resp. enter the systemic circulatory system. In patients [n>3000] after treatment for 3-4 weeks, 10502100 mg BSN/day, average bismuth levels of 3-4 ~g/l were found. Ground level: 0-2.6 ~g/l before application. The overall maximum level in any trial was 21.3 ~g/l. Thus all values range below the alert level of 50 ~g/l. Serum levels: Nitrate. Methaemoqlobin NO significant increase was found neither in nitrate nor in methaemoglobin concentrations in any trial. Surprising methaemoglobin levels were found to be often decreased after therapy with BSN. Average nitrate concentrations were 3.8±1.3 mg/l before and 4.0±1.9 mg/l after 4 weeks treatment [2100 mg/day]. Cimetidine patients showed comparable values: 3.4±1.6 mg/l before and 4.6±1.9 mg/l after therapy. *Medical Department, Marburg, FRG

Temmler Werke, Temmlerstr.

2, D-3550

R 120 477

479

COMPARATIVE BIOAVAILABILITY OF THREE ORAL FORMULATIONS

ENHANCED BIOAVAILABILITY OF CICLOSPORINE (CYA) AFTER ORAL APPLICATION OF SANDIMMUN R i.v. TO PATIENTS ON CHRONIC CYA TREATMENT. H.iven 1L, R.Preuss2L, R.Wacke 1R, R.Bast2R

OF 8-METHOXYPSORALEN *

8. Quednow and T. Walther In the phototherapy o f p s o r i a s i s , t h r e e i n t e r n a t i o n a l l y introduced 8-methoxypsoralen p r e p a r a t i o n s were t e s t e d f o r their bioavailability. A f t e r s i n g l e doses o f 30 mg 9-methoxypsoralen each in s o f t - g e l a t i n capsules (1), h a r d - g e l a t i n capsules (2) and t a b l e t s (3), the serum c o n c e n t r a t i o n p a t t e r n s were determined in nine p a t i e n t s over 8 hours by means o f an HPLC method. The b i o a v a i l a b i l i t y parameters determined did not suggest t h a t bioequivalence e x i s t e d . Hence, dosage as a f u n c t i o n o f the s p e c i f i c p r e p a r a t i o n i s d e s i r a b l e t o obtain s i m i l a r serum l e v e l s and, thus, a comparable p h o t o t h e r a p e u t i c e f f e c t , 8ioavailability

parameters (Median) o f G e r o x a l e n ® ( 1 ) ,

Oxsoralen@)(2) and

Mopsora~en(~(3) 1

2

3

AUC [rig. m1-1. h - i ]

293,0

i61,8

135,8

C max

~47,0

~04,0

98,0

1,5

1,5

1

bg.ml- 1

t

max

1

[hi *

Department o f Dermatology, U n i v e r s i t y o f Leipzig L i e b i g s t r . 21, 0-7010 Leipzig

The intestinal absorption of CyA is incomplete, ranging between 10 and 60% in man. Experiments in rats showed that the bioavailability of CyA from Sandimmun i.v. when given orally was nearly twice that from the oral application form. The ratio of emulsifier to CyA (g/g) which is 3.2 for the oral and 13 for the i.v. preparation was considered responsible for the increased bioavailability (Iven,Harries, Naunyn-Schmiedeberg's Arch. Pharmacol. 341; R7, 1990). We now extended these studies to patients with kidney grafts. So far, 6 patients with a stable kidney graft function receiving CyA orally on a twice daily schedule took part in the study. During three consecutive days after the morning dose, 9 blood samples were collected at appropriate time intervals from an indwelling catheter. On day 1 and 2 the patients took their usual Sandimmun solution, on day 3 Sandimmun i.v. was given orally once, taking into account the difference in concentration between Sandimmun oral and i.v. solution. Whole blood CyA concentrations were determined with the specific Sandoz 3H-RIA and AUC was calculated using the trapezoidal rule. AUC 0-12h (ng*h*m1-1 ) Pat. Sandimmun oral Sandimmun i.v. A B C increase* I(L) 915 1163 +27.1% II(L) 2774 2080 3571 +28.7% Ill(L) 2298 2183 2508 + 9.1% IV(L) 2235 2671 3064 + 14.7% V(R) 1805 2018 3123 +54.8% VI(R) 3953 3506 4588 + 16.1% * as compared to the higher of the two control AUCs Under control conditions AUCs may differ considerably (A;B), however, in all 6 patients AUC was highest after oral dosing of Sandimmun i.v.(C) with a mean increase of 25.1 ± 6.7%. These results so far confirm our observations in rats.

I n s t i t u t e o f C l i n i c a l Pharmacology, Medical Academy o f Magdeburg, L e i p z i g e r Str. 44, 0-3090 Magdeburg, FRG

Institute of Pharmacology (1) and Clinics for Internal Medicine (2) of LObeck (L) and Rostock (R) University, D-2400 LObeck

478

_48O

PHARMACOKINETIC OF NICOTINE AFTER TOPICAL APPLICATION S.Caspary, BoKeller-Stanislawski, T.Huber, P.-G.Merz

INTERACTIONS WITH CYCLOSPORIN A: INDUCERS AND INHIBITORS OF ITS METABOLISM EVALUATED WITH LIVER MICROSOMAL INCUBATIONS. S. Bauer1'2, S. Olaizola-Horn2, M. Reisinger~, H. HOller1

Transdermal nicotine systems (Nicotinell R 30) were applied daily to the skin of healthy nicotine-dependent smokers for 4 days. Blood samples were taken for the measurement of nicotine and cotinine in plasma using capillary gas chromatography with nitrogen detection. The plasma concentration-time profiles of nicotine and cotinine and the pharmacokinetic parameters Cmax, tmax, AUC and the elimination-half life were determined under steady-state conditions (results see table below). Nicotine levels after application of the 30 cm 2 patch were in accord with published values for I0, 20 and 2x20 cm 2 patches. The elimination half-life of nicotine after removal of the patch was longer than reported values obtained after i.v. application.

Table I: Pharmacokinetic parameters of nicotine after NicotinellR 30 in the 4th dosage period Parameter Nicotine Cmax tma x AUC tl/2 Cotinine AUC tl/2

Mean

±

SD

16.0 8.5 254 4.2

± ± ± ±

2.8 2.4 74 2.2

ng/ml h ng*h/ml h

4028 22.7

± ±

1852 6.4

ng*h/ml h

Dep. Clin. Pharmacoloqy, University Hospital Frankfurt, Theodor-Stern-Kai 7, D-6000 Frankfurt/Main 79

Clinically relevant drug-interactions with the immunosuppressant cyclosporin (CyA) are mainly due to interactions with its metabolism. Cytochrome P-450111A isoenzymes have been described as CyA metabolizing enzymes (Kronbach et al., Clin Pharm Ther 43: 630). As shown here, also compounds like 8-naphthoflavone (Nf) result in the induction of specific reactions of CyA metabolism. Antihypertensives are often nessessary comedication for CyA treated patients, therefore, their influence on CyA elimination is of interest. Methods: Induction was evaluated in liver microsomes from male SpragueDawley rats after pretreatment (50 mg/kg i.p.; 4 days). Inhibition of CyA metabolism was tested with human liver microsomes. The 1 ml microsomal incubations were adjustet to microsomal protein concentration of 2.2 mg/ml and substrate concentrations between 4 and 160 nmol/ml were used. Inhibitor concentrations were between 0.01 and 1.5 ,umol/ml. Results: While the predominantly CyA metabolizing enzymes belong to the cytochrome P-450111Afamily, also isoenzymes belonging to family I contribute to CyA metabolism, as shown in the table by the respective Vmax values (means of 5 different preparations, pmol/mg protein/min). Pretreatment Metab. 1 Metab.17 Metab,18 Untreated 4 2 2 Dexamethasone (Dx) 9 5.5 2 B-Naphthoflavone (Nf) 3.5 2 5.5 Dx and Nf 17.5 9.5 7 The inhibitory effects of the following calcium channel blockers were evaluated: Diltiazem, Verapamil, Nicardipin, Nitrendipin, Nifedipin, Nimodipin, Nis01dipinand Diperdipin. Significant inhibition was determined for all these substances, with Ki values between 5 and 80 ~uM. Significant clinical interactions have been observed for Diltiazem, Verapamil and Nicardipin. Institute l~r Klinische Pharmakologie: 1 Klinikum Steglitz, Freie Universit&t Berlin, ~ Charitd, Humboldt-Universit~t, Berlin.

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CICLOSPORIN(Cs) AND ITS METABOLITES(CsM) IN A COMBINATION THERAPY WITH AZATHIOPRINE(Aza) AND PREDNISOLONE(P) IN KIDNEY ALLOGRAFT RECIPIENTS J.$. Bleck ~, V.Kliemz, U.Christians 3, H.Repp4, U.Frei z

GENOI~fPIC DETERMINATION OF CYTOCHROME P-450IID6, GLUTATHIONE S-TRANSFERASE ~, AND N-ACETYLTRANSFERASE IN LUNG CANCER PATIENTS I. Roots, J. BrockrnSller, N. Drakoulis, M. Beland, K. Grof~, D. Grot~

Triple drug therapy (Aza,Cs,P) may be a successful regime in patients with clinical signs of Cs-toxicity. It was the aim of this investigation to study metabolic pattern of Cs and its CsM in patients treated with Aza/Cs/P (strived trough level I00 ng) in comparison to Cs/P treated kidney allograft recipients(strived trough level 130 ng). In 40 patients, 19 treated with Aza/Cs/P (Aza:l.74±0.37 mg/kg/d, Cs:4.0±1.2 mg/kg/d, P:7.5mg/d) and and 21 treated with Cs/P (Cs:4.3±1.5 mg/kg/d, P:7.5 mg/d) Cs and 17 CsM in blood(trough level) and 24h-urine were measured by HPLC. The groups were comparable in age, sex and time after transplantation. Results: There was no difference in liver function, but serum creatinine concentrations were significantly elevated in the Aza/Cs/P-group (2~8±99 vs. 168±77 ~mol/l, p(0.013). Although both groups reached the different Cs trough level as desired(Aza/Cs/P:99±10 vs. 128±6 ng/ml,p<0.007) the required Cs-dose was comparable. Both groups differed in metabolic pattern in the trough level for CsM AMI(Aza/Cs/P: 121± 14.8 vs. Cs/P:I91±I2.5 ng/ml, p~0.04), AM9 (Aza/CS/P:48.3±7.4 vs. Cs/P:68.7±7.4 ng/ml7, p<0.01) and AMIc (Aza/Cs/P:29.6±8.2 vs. Cs/P: 9.0±5.0 ng/ml, p<0.01) with a tendencially increase of more hydrophilic CsM (AM19, AMIA, AMI4N). As a result of a worse kidney function the urinary excretion was decreased in the Aza/Cs/P-group for Cs (333±60 vs. 735±98 ~g/d, p< 0.0003) and ZCsM(1616±317 vs. 4781±639 ~g/d, p<0.0006). ~ummary: The study shows an influence of Aza on Cs metabolic pattern. A different Cs-pharmacokinetic may explain the difference of Cs-trough-levels in comparable Cs-doses. * Abt. Gastroenterologie und Hepatologie~, Abt. Nephrologiez, Abt. Allgemeine Pharmakologies, Abt. Abdominal u. Transplantationschirurgie4 , Medizinische Hochschule Hannover, Konstanty-Gutschow- Str.8, 3000 Hannover 61,FRG

Previous studies showed that the poor metabolizer (PM) phenotype of debrisoquine hydroxylase (cytochrome P-450IID6) and the high activity trait of glutathione S-transferase (GST) m u occur less frequently among lung cancer patients than expected from respective controls. These phenotypes might represent protecting host factors. N-Acetyltrans±erase polymorphism obviously had no influence on lung cancer susceptibility. Possible pitfalls of phenotyping procedures can now be overcome by genotyping using leukocyte genomic DNA. Restriction fragxnent length polymorphism (RFLP) with a cDNA gene probe of cyt. P-450IID6 was analyzed in 81 tung cancer patients. Mutant alleles were specified by use of allele-specific polymerase chain reaction 0PCR; Helm and Meyer, Lancet 336: 529). Genotype of GST isoenzyme g was analysed by RFLP patterns and correlated with invitro activity of trans-stilbene oxide glutathione corrugation. RFLP patterns discriminate slow and fast genotype of N-gcetyltransferase as well as the heterozygotes. Results: Five phenotypical PM's of debrisoqu~ne were found (6.2 %). One PM could be unambiguously recognized by his 11.5/11.5 kb alleles. The other PM's could only be characterized by PCR, in two cases as 29B/29B, in one case as 29A/29B and one 44/29A mutant. Among extensive metabolizers, 42 patients had homozygous 29 kb wild type alleles, and 32 were heterozygous with defective alleles 29B, 29A, 44, or 11.5 kb. A gene dose effect resulted in higher debrisoquine 4hydroxylase activities in the homozygotes. The phenotypically bimodal distribution of GST m u activity only partly correlated with genotypicai patterns, presumably due to presence of polymorphic GST~. Lowactivity GST m u phenotypes are prevailing among lung cancer patients. A good correlation of pheno- and genotyping of N-acetyltransferase was obtained. These new genetic techniques proved powerful tools for molecnlar-epidemiological studies. Institut fiir Klinische Phannakologie, Klihikum Steglitz, Freie Universit~it Berlin, Hindenburgdamm 30, D-1000 Berlin 45

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PHARMACOKINETIC INTERACTIONS BETWEEN NOCLOPROST AND ANTIPYRINE, SULFAMETHAZINE, AND ACETYLSALICYLIC ACID G.Franke, W.Sieqmund~ M.Zschiesche The p r o s t a g l a n d i n d e r i v a t i v e n o c l o p r o s t (NP, as c l a t h r a t e ) w a s f o u n d to be a p o t e n t i a l cytoprotective drug in preclinical and hitherto c l i n i c a l e v a l u a t i o n s . W i t h the f o l l o w i n g s t u d y was t e s t e d w h e t h e r a s i n g l e 400 ~g oral d o s e of N P i n f l u e n c e s the a b s o r p t i o n and fate of the test c o m p o u n d s a c e t y l s a l i c y l i c a c i d (ASA, i000 mg), antipyrine (AP, 15 mg/kg), and s u l f a m e t h a z i n e (SMZ, 500 mg). P h a r m a c o k i n e t i c t e s t s w ~ t h concomitant AP and S M Z w e r e p e r f o r m e d in 16 h e a l t h y m a l e v o l u n t e e r s (7 r a p i d [RA] a n d Q s l o w a c e t y l a t o r s [SA], 2 1 - 2 4 years, 65-85 kg) 30 m i n a f t e r t h e y h a d t a k e n o r a l l y a p l a c e b o or NP, resp. P h a r m a c o k i n e t i c s w i t h A S A w a s i n v e s t / g a t e d in 8 h e a l t h y m a l e v o l u n t e e r s (22-25 years, 6 2 - 7 9 kg) u n d e r the s a m e c o n d i t i o n s . ASA, s a l i c y l i c acid, and s a l i c y l u r i c a c i d in p l a s m a and urine, AP in s e r u m and nor-AP, h y d r o x y m e t h y l - A P , and 4 - h y d r o x y - A P in urine, and SMZ and N - a c e t y l - S M Z in s e r u m a n d u r i n e w e r e m e a s u r e d w i t h h p l c and p h o t o m e t r i c m e t h o d s , resp. NP did neither influence signi'ficantly the o x i d a t i v e d i s p o s i t i o n of AP, the h y d r o l y s i s and g l y c i n c o n j u g a t i o n of ASA, nor the N - a c e t ¥ 1 a t i o n of SMZ, H o w e v e r , there is e v i d e n c e that the cytoprotective drug diminished the a b s o r p t i o n r a t e s of b o t h the b a s i c AP and the a c i d i c ASA and SMZ. T h e s e r e s u l t s w e r e m o r e p r o n o u n c e d in SA then in RA. The sum of pharmacological e f f e c t s of N P on the g a s t r o i n t e s t i n a l t r a c t as well as the properties of the vehicle ~-cyclodextrin might be reasons for the observations. Department of Clinical Pharmacology, Ernst Moritz Arndt University, Fleischmannstra~e, 0 - 2 2 0 0 G r e i f s w a l d , FRG

~ REDUCES HEPATIC BLOOD FLOW IN HUMANS M. ziehmer, U. Grundmann, H. Raahimi, P. Altmayer, and H. P. B~ch* Volatile anesthetics markedly influence tissue perfusion. It is assumed that hepatic blood flow (HBF) may also b e affected. The usual noninvasive dye methods for the determination of HBF in humans are suffering eminently from intra- and interindividual variability. In the present study an attempt has been made to reduce this variability by two consecutive administrations of indocyaninegreen (ICG) and b y simultaneous determination of cardiac output (CO) using the noninvasive bioimpedance method. Preliminary studies in volunteers revealed intraindividually no difference between the two consecutive HBF measurements: the ICGclearance (estimated by the area under the curve) was related to the CO. In order to investigate the effect of volatile anesthetics on HBF healthy patients undergoing minor orthopaedic surgery obtained a bolus of 0.3 mg/kg ICG i.v. and blood samples were taken after 2,4,6,8,10,12 and 14 min. A general anesthesia was induced w i t h etomidat, fentanyl and suxamethonium chloride. After tracheal intubation anesthesia was maintained with either halothane or isoflurane (i MAC) in O2/air (FiO2=0.4) . At steady state, verified by the endexpiratory concentration of the respective volatile anesthetic, usually 45 min after the first ICG application, a second bolus of 0.3 mg/kg ICG was injected and blood samples withdrawn. ICG concentration in serum was determined photometrically at 800 nm. In isoflurane-anesthesia CO decreased, but HBF remained almost unchanged, whereas under halothane both, CO and HBF, were significantly reduced to 60 % of the preanesthetic value. Klinik f~r Anaesthesiolgie und Intensivmedizin, Institut f~r Pharmakologie und Toxikologie* der Universit~t des Saarlandes, D-6650 Horaburg

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INTRA- AND INTERDAY VARIABILITY OF THE CLEARENCE OF INDOCYANINE GREEN (lOG). J. L6tsch, A. Fricke

INFLUENCE OF AZATHIOPRINE-TH~'RAPY ON DIffERENT CYTOCHROME P-450 DEPENDENT BIOTRANSFORMATION REACTIONS M. Hippius, F. Pakodi. M. Gassel. K. A b e n d r o ~ h ,

The clearance of ICG is commonly used for the determination of hepatic blood flow. The objective of the present study was to investigate the variability of ICG clearence in respect to the possible use for estimation of influence of a given drug on hepatic blood flow. Pharmacokinetics of lOG was investigated in eleven healthy volunteers (mean age 26 years) in a dose of 0.5 mg/kg body weight by i.v.-bolus-injection three times a day (with an interval of 2 hours) on two different days. Between the two days, a time period of three weeks elapsed. The ICG-serum-levels were determined by spectrophotometric analysis at 780 nm wavelength. The mean values (±GD) of ICG-clearence [ml/min] for each subject were 181.5~46.11, 204.8±34.1, 242.0±49..9, 199.8±57.6, 184.6±43.6, 173.4±27.2, 164.6±78.5, 135.7±6.4, 114.9±29.1, 146.6±24.8 and 155.0±10.9 at the first day and at the second day 194.9±12.8, 184.6±55.7, 260.6±25.6, 189.1±29.7, 223.7±9.0, 160.1±28.1, 241.8±26.6, 279.5±100.6, 254.8±54.8, 295.9±134.0 and 283.0±31.7. Large intra- and interindividual variations were observed, both between different ICG-administrations on the same day (coefficient of variation for the hole group 28.5% (individual range 4.7-47.7) and 30.1% (4.0-45.3) for the first and the second day, respectively) and between the two days (coefficient of variation 0.4-80.3%) . Therefore, we conclude that due to these large variations, the ICG-clearence cannot be used for evaluation of the influence of a drug on hepatic blood flow.

Azathioprine is a potentially useful antileukaemic and immunosuppressive agent in the therapy of ~eumatoid arthritis. Despite s t u d i e s o f a z a t h i e p r i n e l i v e r d a m a g e in ~nlmal m o d e l s a n d s p o r a d i c c a s e r e p o r t s in humans, no clear evidence for azathioprine hepa~etoxioity in h u m a n s c o u l d be p r o v i d e d . T h e r e f o r e stI~dies were p e r f o r m e d to d e t e r m i n e the i n f l u e n c e o f an a z a t h i o p r i n e t h e r a p y on biotra.nsI~z~ation reactions characterized by m o d e l substances. Test s u b s t a n c e s f o r c y t o c h r o m e P - 4 5 0 d e p e n d e n t reactions are caffeine .for the $methylcholanthrene inducible subtype, and metamizole for the phenobarbital inducible s u b t y p e o f c y t o c h r o m e P-450, S u l ~ h a d i m i d i n e was used for the determination of acetylation reactions. D-glucaric acid excretion was m e a s u r e d as an e n d o g e n o u s c o m p o u n d which s e r v e d as an indicator of induced state of drug m e t a b o l i z i n g c a p a c i t y o f the liver. These i n v e s t i g a t i o n s were c a r r i e d out b e f o r e d r u ~ a d m i n i s t r a t i o n a n d a f t e r 10 days t r e a t m e n t with a d a i l y d o s e o f 150 m g a z a t h i o p r i n e . The effect of azathioprine on the model s u b s t a n c e s is d i f f e r e n t : Total c l e a r a n c e a n d elimination half-life of caffeine before and after treatment are not significantly different. The serum level of 4-methylaminoantipyrine is reduced, the elimination half-life is retarded si~nlficantly. The e x c r e t i o n o f D - g l u c a r i c a c i d is induced.

Institute Clincal Pharmacology, Medical ~chool of Dresden, Fiedlerstra~e 27, 8019 Dresden, Germany

Present address: Department of Clinical Pharmacology, Friedrich Schiller ~h~iversity. B a c h s t r 1 8 , 0-6900 Jena

488

486 ~OCKTAIL OF MOIM~ SUBSTANCF~ BIOTRAA~FO[~I4TION ~ A C I ~ A. H o f f ~ m , L. Henschel , A. Huster, ~aul, ~ d J. T ~ c k e n b r ~ t

"

TO R.

Eb~flMATE T~pfer,

H,

A cocktail of model substances is presented to characterize various liver enzymes. Metamizol and caffeine were used for phenobarbital inducible and for 3-methylcholanthrene inducible cytochrome P-450, respectively. The acetylation and hydroxylation phenotype were characterized using sulfadimidine and debrisoquine. The investigations were done on 11 healthy male subjects and 16 patients wit~ various liver diseases. First, each subject received the four substances alone (metamizol: 1000 rag, caffeine: 200 rag, sulfadimidine: 500 rag, and debrisoquine: 10 rag). Then the cocktail was given. Venous blood was sampled before and up to 24 hrs after drug administration. Sulfadimidine and 4-acetylsulfadimidine serum and urine concentrations were measured s~trophometrially, caffeine and 4-monomethylaminoantipyrine serum concentrations were determined by H~LC, as well as debrisoquine, f~e calculation of pharmacokinetic parameters was applied to a method described by Partier & Mayersohn, For statistical calculation Wilcoxon"s ranlc test was used. The limit of significance ist p
C E F T R I A X O N E C O N C E N T R A T I O N IN S E R U M A N D C S F W I T H NORMAL AND IMPAIRED BLOOD-BRAIN BARRIER IN BORRELIOSIS-THERAPY.

M . S t o l l , A . H a a s s , F . S6rgel°,W. T6njes,G. Holzer, K. S c h i m r i g k Borreliosis is i n c r e a s i n g and a simple,noni n v a s i v e s u f f i c i e n t t h e r a p y w i t h a n t i b i o t i c s is necessary to avoid late neurologic manifestations. 62 p a t i e n t s w i t h b o r r e l i o s i s w e r e t r e a t e d w i t h c e f t r i a x o n e (ix2g) d u r i n g i0 d a y s . T h e c e f t r i a x o n e s e r u m and CSF c o n c e n t r a t i o n s w e r e m e a s u r e d 2 h and 4 h a f t e r the first i n t r a v e n o u s i n f u s i o n and 24 h a f t e r the last a p p l i c a t i o n . In p a t i e n t s w i t h a n o r m a l b l o o d - b r a i n - b a r r i e r (BBB) (n=14-25) the CSF c o n c e n t r a t i o n i n c r e a s e d from 0.14 +- 0.03 (2h) to 0.28 +- 0.05 (4h) a n d 0.43 + - 0 . 0 5 (24 h) ~ g / m l . T h u s the m i n i m a l CSF ceftriaxone concentration of the longterm t r e a t m e n t was still 7 times h i g h e r t h a n the m i n i m a l i n h i b i t i o n c o n c e n t r a t i o n of 0.06 ~g/ml. The i m p a i r m e n t of the BBB was d e t e r m i n e d b y the serum albumin to CSF albumin ratio. In c o r r e l a t i o n to the d e g r e e of the BBB i m p a i r m e n t the c e f t r i a x o n e CSF c o n c e n t r a t i o n i n c r e a s e d to m e a n v a l u e s of 0.12 (2h),0.90(4h) and 1.47 (24 h) ~g/ml (n=5-13), w h i c h is 4 h and 24 h a f t e r the a p p l i c a t i o n 3 times h i g h e r than in p a t i e n t s w i t h a n o r m a l BBB (p<0.03; p<0.01). Thus the c e f t r i a x o n e d o s e of 2 g / d a y r e a c h e d sufficient high CSF concentrations for the t r e a t m e n t of b o r r e l i o s i s w i t h and w i t h o u t an i m p a i r e d BBB. D e p a r t m e n t of N e u r o l o g y , U n i v e r s i t y of Saarland, 6650 H o m b u r g / S a a r , F R G I B M P - I n s t i t u t e ° for b i o m e d i c a l and p h a r m a c e u t i c research,Harrichstr. 5,8500 N ~ r n b e r g , F R G

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HEPATIC FREE CLEARANCE OF CEFTR][AXONE IN CRITICAL CARE PATIENTS WITH ACUTE RENAL FAILURE G. Heinemeyer, J. Link * and V.Meschede ~

DOSE D E P E N D E N C Y OF T H I A M I N PHA/LMACOKINETICS L. Harnisch, M. Looby T h i a m i n dose d e p e n d e n c y was i n v e s t i g a t e d in 6 h e a l t h y male volunteers following a c o m b i n a t i o n of m u l t i p l e p a r e n t e r a l l y and orally administered doses. Initially a i0 mg dose was i n f u s e d over 1.5 hrs. S u b s e q u e n t l y a 500 mg solution was i n g e s t e d daily for 6 days, followed by a 40 mg/4 hrs infusion. Finally a 5 g solution was i n g e s t e d for a further 6 days followed by a 120 mg/4 hrs infusion. Both t h i a m i n and total thiamin (Th, TMP, TDP, TTP) were a s s a y e d in p l a s m a and b l o o d cells by HPLC for each i.v. kinetic, and the final oral dose of each phase respectively. Previous studies d e m o n s t r a t e d that over a p l a s m a c o n c e n t r a t i o n of 300 nmol/l the kinetics were nonlinear. Therefore each i.v. kinetic time course was fitted individually u s i n g a sum of exponentials. Total clearance was found to range from 1100 m l / m i n for the lowest to 500 m l / m i n for the h i g h e s t i.v dose. W~nen however, all p l a s m a c o n c e n t r a t i o n s under 300 nn~ol/l were fitted together over the w h o l e time course, a clearance of 900 m l / m i n was calculated. This effect demonstrates the nonlinear nature of the cumulative kinetics. N o n l i n e a r oral r e a b s o r p t i o n is also evident since the steady-state concentrations do not increase p r o p o r t i o n a l l y to the given dose. The rate at w h i c h t h i a m i n c o n c e n t r a t i o n s in the b l o o d cells decline from the steady-state plateau reflects the body's c a p a b i l i t y to retain or store thiamin. The terminal half live for total thiamin in the b l o o d cells was 55 hrs c o m p a r e d to only ii hrs for thiamin itself. This c l e a r l y illustrates the role of thiamin phosphates in the storage process. Institut for Klinische Pharmakologie, K l i n i k u m Steglitz, H i n d e n b u r g d a m m 30, D-1000 B e r l i n 45

Clerarance of c e f t r i a x o n e (CTX) s t r o n g l y depends on renal and non-renal ( b i l i a r y ) clearance of t h e f r e e f r a c t i o n . In c h r o n i c renal f a i l u r e , dosage must not reduced as f r e e clearance remains unchanged due to increased hepatic excretion. In eleven p a t i e n t s with acute renal f a i l u r e , plasma levels cumulated o v e r a period of seven days of CTX administration (Arch. Pharmacol. 332, Suppl., R96). Pharmacokinetic reevaluation by f i t t i n g all c o n c e n t r a t i o n s and u r i n e data revealed s i g n i f i c a n t correlation of c r e a t i n i n e clearance with renal (CIren=0,14 ClCrea+2,2; r=0,951, p
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G e n t a m i c i n drug m o n i t o r i n g in premature newborns u s i n g N O N M E M p o p u l a t i o n parameters w. Weber, K.L. Rost, M. Nitz, G. Kewitz*

DRUG BINDING KINETICS IN TYPE I DIABETIC PATIENTS W. N6rner, A. Preissner and N. Rietbrock

Drug m o n i t o r i n g is essential for drugs w i t h a n a r r o w therapeutic range. During a drug therapy reliable estimates of p l a s m a c o n c e n t r a t i o n s can be a c h i e v e d u s i n g b a y e s i a n forecasting, if p o p u l a t i o n parameters are available. In this study p o p u l a t i o n parameters of g e n t a m i c i n in premature or n e w b o r n infants w i t h sepsis were c a l c u l a t e d by the s y s t e m a p p r o a c h e m p l o y i n g t h e c o m p u t e r p r o g r a m m NONMEM. The advantage of the system a p p r o a c h is that only system b e h a v i o u r has to be modelled. This m e t h o d requires linear kinetics, i.e. v a l i d i t y of the s u p e r p o s i t i o n principle. A m o n o e x p o n e n t i a l unit i m p u l s e - r e s p o n s e was assumed. C o n c e n t r a t i o n - t i m e - f u n c t i o n s were d e s c r i b e d as the s u p e r i m p o s e d responses to one or more step responses. A l t h o u g h the a d m i n i s t r a t i o n times are arbitary, samples have to be taken at least one hour after the last dose. T r o u g h and peak levels of g e n t a m i c i n in 88 newborns were m e a s u r e d by TDX and were u s e d to calculate p o p u l a t i o n parameters. The w e i g h t of the newborns r a n g e d from 0.82 to 4.93 kg w i t h a m e d i a n of 2.65 kg. The parameters means and their c o e f f i c i e n t of v a r i a t i o n were: d i s t r i b u t i o n v o l u m e (Vd): 0.72 (12%) i/kg clearance (Cl) : 0.78 (40%) ml/min/kg A t~ of 9.6 h w i t h 95% confidence interval b e t w e e n 8.9 and 11.3 h was c a l c u l a t e d from V d and CI. *

Institute of C l i n i c a l Pharmacology, Department of neonatology, K l i n i k u m Steglitz, Free U n i v e r s i t y of Berlin, H i n d e n b u r g d a m m 30, D-1000 Berlin 45

The non-enzymatic glycation of serum albumin lowers the in vitro albumin binding of numerous drugs e.g. warfarin, salicylic acid and tolbutamide (i-3). The importance of this phenomenon in patients with elevated blood glucose levels is uncertain. The binding kinetics of dans~isarcosine, a probe for binding site II on serum albumin, was determined with the stopped flow method in 18 insulin dependent diabetic patients. In addition, the concentrations of glycated serum albumin, total serum albumin, blood glucose and free fatty acids were measured, The association velocity and affinity constants of dans¥1sarcosine in more than 50 % of patients were markedly decreased (k2:157 ± 76 s-l, KA: 2.21 ± 0.32 I/mol) (versus 200 ± 32 s-I and 4.84 ± 1.72 i/mol for healthy subjects) whereas the dissociation velocity constant was unchanged (k_2:20.1 ~ 0.32 s-I vs. 18.4 ± 3.9 s-l). k2 and KA were negatively correlated with the levels of glycated albumin and free fatty acids. These data are strong evidence that the effects of albumin glycation and the presence of free fatty acids on the drug binding of diabetic patients in vivo will be additive. (i) W. W6rner eta]. (1990) Unpublished results (2) K.A. Meirish et al. (I982) J. Pharm. Soc. 71, 235 (3) S. Tsuchiya et ai.{1984) Biochem. Pharmacol. 33, 2967 Abt. f. KIinische Pharmakologie, Universit~tsklinikum Frankfurt, Theodor-Stern-Kai 7, 6000 Frankfurt~Main 70

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IDENTIFICATION OF CYTOCHROME P-450 ISOZYMES INVOLVED IN THE METABOLISM OF VERAPAMIL H.K. Kroemer, Ph. Beaune ~, CJ. Henderson~, C.R. Wolff and H. Heidemanna

SIMULTANEOUS DEIERMINATION OF THE I.V. AND P.O. DISPOSmON OF THE ENANTIOMERS OF RACEMIC 1,4-DIHYDROPYRIDINE CALCIUMANTAGONISTS BY MEANS OF STABLE ISOTOPE METHODOLOGY AND CHIRAL PHASE CHROMATOGRAPHY. Fischer, C., MOck, W.*and Heuck, K.*

Verapamil is a calcium channel blocker widely used in the treatment of hypertension, supraventricular arrhythmias and angina pectoris. The drug undergoes extensive and stereoseleetive first pass metabolism in man. The major metabolic pathwaysare N-dealkylation [formation of 2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile; D-617], N-demethylafion [formation of norverapamil] and O-demethylation [formation of 2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dlmethoxyphenyl)-6-methyl-2-1sopropyl-6-azaoctanitrile and 2- (3,4-dimethoxyphanyl)8-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile]. The individual cytochrome P-450 isozymes involved in the metabolism of verapamil have not been identified yet. We therefore investigated the maximum rate of formation of metabolites in the presence of 640 gMof S- or R-verapami/ in microsomes obtained from ten different human livers. The rate of formation was then correlated to the expression of the individual P-450 isozymes as determined by Western blotting. A highly significant correlation was observed for expression of F-450II/A3/4 and formation rate of norverapamil (r = 0.91, p < 0.001 for S-verapamil; r =0.81, p<0.001 for R-verapamil) and D-617 (r =0.77, p<0.05 for Sverapamil; r = 0.94, p < 0.001 for R-verapamil). Subsequent studies carried out in microsomes of 3 different human livers using an inhibitory polyclonal antibody directed against the P-450IIIA family showed inhibition of norverapamil formation (66.3+-10.4% and 71.3+-3.3% after incubation with S- and R- verapamil, respeetlvely) and D-617 formation (65.6 -+11.2% and 70 + 14.3% after incubation with S- and R- verapamil, respectively). Formation of O-demethylation products was not affected. Antibodies directed against P-4501/D6, the enzyme involved in polymorphic sparteine/debrisoquiae oxidation did not affect the rate of Ndealkylation, N-demethylation or O-demethylation. It can be concluded that P-450II/JL3/4 contributes to the metabolism of verapamil. Verapamll therefore has the potential to interact with other drugs which are substrates or inducers of the same enzyme. Supported bythe Robert-Bo~c~-Foundation, Stuttgart. Dr. Margarctc Fischcr.Bosch-lnstitut L KJJn. Pharmakol., Auerbacizstr. 112. 71700Stuttgart 50, FRG ~INSBRM LIT~, CHU Neekcr-Enfants-Maladeg 155 rac de Vaugirard, 75730 P a ~ F~ance Impeffal Cancer R~carch Fund, Dcpt Biochcmist~, George Squam, Edinburgh EH89XD, UK Dcpt Internal Mcd, 1. Me.dLednische Klini~ Sehittcnhclmstr.12. 2300 IOcl, FRG.

The application of stable isotope technique has been proven to be an effective methodology to investigate absolute and relative bioavailability. In particular, the disposition of drugs which exhibit extensive presyetemic elimination like 1,4--dihydropyridine compounds (DHPs) can be studied by simultaneous i.v. (stable isotope labelled) and p.o. (unlabelled) administration and subsequent measurement by mass selective detection (GC/MS). Intraindividual variation are avoided using this study design. Most of the DHPs are chiral and clinically used as the racemate. The enantiomera can substantially differ with regard to their pharmacological activity. Therefore, an enantiosalective assay is essential for meaningful interpretation of plasma concentration effect curves. In order to determine the absolute bioavailability of nimodipine enantiomers in humans, the following proceeding was performed: simultaneous i.v. (stable isotope labelled) and p,o. (unlabelled) administration of rac. nimodipine and measurement of serum concentrations by the combination of two methods, chiral phase HPLC to saparate the enantiomers and subsequent quantification per GC/MS to differentiate between i.v. and p.o. doses. Highly sensitive and specific detection was achieved by measurement in negative ion chemical ionization mode monitoring the molecular ions. The accuracy of the method was assessed by adding Sand R-nimodipine concentrations to blank serum samples with an enantiomeric excess of the R-enantiomer ranging from 0 to 0.89% e.e.. Calibration curves in the range from 0.1 to 40 ng/ml/enantiomer were established. Limit of quantification was 0.1 ng/ml serum/enantiomer. In conclusion the combination of stable isotope technique with chiral phase HPLC provides an useful tool to study the stereoselective disposition of racemates following their simultaneous administration either by two routes or two preparations. Dr. Margin Rzcher-Bo~h-ln~tutfor 1OlnlB:hePhamud(ologle,Auefbsche~.112, D.TQ00 8tutlgaa ~0, *B~yzrA@,D-~00 Wupperud I1~1~~ l y w~ ~uppomdbylh~ ~ ~ Founde~on,8tuUoaaa~l BayerAGI,Wuppert~.

494

496

STEREOSELECTIVE PHARMACOKINETIC5 OF ORAL NITRENDIPINE IN ELDERLY HYPERTENSIVE PATIENTS WITH VARYING DEGREES OF RENAL FUNCTION

ENANTIOSELECTIVE DISPOSITION OF R- AND S-NIMODIPINE AF'rER SIMULTANEOUS ORAL AND INTRAVENOUS ADMINISTRATION K. Sporckmann, M. Eiehelbaum

A. H a l a b i * ,

P.A. Soons, D.D. Breimer, and W. K i r c h *

Nitrendipine, a c h i r a ] dlhydropyridine calcium entry blocker, i s used as the racemate f o r the treatment of hypertension. Plasma concentrations and systemic a v a i l a b i l i t y of the (S)-enantiomer are almost twice of t h a t of the (R)-enantiomer. Apparently c o n f l i c t i n g data have been published on the influence of chronic renal f a i l u r e (CRF) on the pharmacokinctics and haemodynamic e f f e c t s of racemic n i t r e n d i p l n e in man. Therefore, the stereochemical aspects of n i t r e n d i p i n e pharmacokinetics in CRF were studied by us. Plasma samples of 7 e l d e r l y p a t i e n t s with chronic renal f a i l u r e (CRF) and 6 c o n t r o l subjects (mean c r e a t l n i n e clearance ~0 and 97 ml/min r e s p e c t i v e l y ) , obtained in a previously published study (Eur J Clin Pharmacol 1989; 36:#33-37) were reanalysed for the enantiomers of n i t r e n dipine. Racemic nitrendipine (20 mg) was administered once-daily f o r 7 days. Over the l a s t dosage i n t e r v a l (2# h) the mean AUC of (S)- and (R)-nitrendlpine were increased in CRF with 132% and 85% r e s p e c t i v e l y . The observed doubling of h a l f - l i v e s and increases in Cmax dld not reach s i g n i f i c a n c e due to high v a r i a b i l i t y within each group. In both groups n i t r e n d i p i n e exhibited s t e r e o s e l e c t i r e pharmacokinetlcs (AUC, Cmax) but h a l f - l i v e s of enantiomers were s i m i l a r . As index f o r s t e r e o s e l e c t i v i t y , the mean S / R - r a t i o of AUC in CRF (2.68) was not s i g n i f i c a n t l y (p=O.15) d i f f e r e n t from the r a t i o in c o n t r o l subjects (2.07). Altered pharmacokinetics of oral n i t r e n d i p i n e in CRF was confirmed, but no change of s t e r e o s e l e c t i v i t y in nitrendipine

kinetics

Nimodipine (N) is a calcium channel blocker with 1,4-dihydropyridlne structure. Its distribution in bral~ is higher than that of nlfedlplne. N dilutes cerebral blood vessels at couslderably lower concentrations than those required for dilatation of peripheral blood vessels. N is currently approved for the prevention of cerebral vasospasm in patients with subarachnoid hemorrhage and for its beneficial effects in patients with organic brain syndrome. In addition, N has shown potential to prevent neurological deficits after acute ischemie stroke and to improve chronic eerebrovascular diseases. N is clinically used as a raeemate. The S-enantiomer is at least an order of magnitude more potent than the R-anantiomer considering their pharmacological vascular effects in animal models (Towart et al. 1982). The pharmacoklnetic data so far reported are based on total drug conCentrations not taking into aCcount the composite nature of the drug. N undergoes extensive ftrst-pass metabolism and as a consequenCe has a low bioavailability (F) after oral adm;n;~txatinn. So far it has not been addressed whether first-pass metabolism and disposition of the Nenantiomers is stereuselective. We have studied the disposition of R- and S-N in 6 healthy male volunteers (age 52.7 ---7.6) after simultaneous adminlstradon of intravenous (2rag stable labelled IsCaisN1) and oral (30rag) N. By using the method of Fischer et al., a combination of ckiral HPLC and GC-MS, it is possible to assess the disposition of the enantiomers after i.v. and p.o. adm;ni~trafion. After i.v. infusion the pharmaeekineties of R- and S-N exhibited multicompartment characteristics. The systemic clearance of R-N was 2.23 ±0.41 l/rain and that of S-N was 2.28 -+0.23 l/rain. Following oral adm;Mstrafion F was low. PronounCed differenCe in F of the enantiomers were observed. F for R-N was on average 22.3 ---6.3 % and for S-N 3.5 ±1.7 %. Conclusion: lq undergoes extensive stereoseleetive first-pass metabolism after oral administration favoring the clearanCe of the more active S-anantlomer. These data explain why oral doses exceeding the i.v. dose 10-20 times have to be administered to achieve comparable plasma concentrations of the more potent S-enantiomer. \ ~ H~HC~OOC COOCH~-CH~.OCH~ ~

was observed.

* P r e s e n t address: I . M e d i z i n i s c h e K l i n i k , C h r i s t i a n A l b r e c h t s - U n i v e r s i t ~ t , K i e l , Germany D i v i s i o n o f Pharmacology, Center f o r BiG-Pharmaceutical Sciences, Leiden, The Netherlands

niraodipin¢

H~C HC

z

CH

~ * ehiraleadoon al., Arznoim.-Forseh.32(I),4, 338-346(198~} The studywas supportedby the RobertBoschFoundationand by BayerAG, Wuppertal. Lit.: R.Towart et

1~. Margarete I~saher.Bosch-/nstitut f~r K/in. Pharmakologie, Auerbachstr. 112, 7000 Stuttgart 50

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499

SUSTAINED-RELEASE NIFEDIPINE IN HYPERTENSIVES: 24-H-MONITORING OF BLOOD PRESSURE, HEART RATE, PHARMACOKINETICS AND PLASMA cAMP. B.Lemmer, G.Nold a n d R . K a i s e r

DISPOSITION OF DIPYRONE AND ITS M E T A B O L I T E S IN MAN: INFI;JEN(~ O F P H E N O B A R B I T A L T R E A T M E N T A N D ~:~'~'~L~ OF CIGA-

ks~'i'K SMOKING

A. Balogh, A. Bode, K. Hoffmann

In 12 h y p e r t e n s i v e p a t i e n t s the p h a r m a c o k i n e t i c s a n d t h e e f f e c t s on h e a r t r a t e (HR), blood p r e s s u r e (BP) a n d plasma cAMP were s t u d i e d a f t e r a 7 - d a y s t r e a t m e n t (20 mg at 8 °o a n d at 19 ° ° ) with a s u s t a i n e d - r e l e a s e n i f e d i p i n e (NIF, Cordicant r e t a r d S ) . Hemodymeunic e f f e c t s were monitored with a p o r t a b l e automatic BP d e v i c e ( P h y s i o p o r t R ) , d r u g e f f e c t s were compared to c i r c a d i a n control v a l u e s . Nifedipine a n d t h e main metabolite conc e n t r a t i o n s were determined by gaschromatography, plasma cAMP b y r a d i o a s s a y . The n o n - l i n e a r f i t t i n g p r o gram PHARMFIT (Mattes et a l . , in p r e p . ) was u s e d for r h y t h m a n a l y s e s . In c o n t r a s t to a i m m e d i a t e - r e l e a s e NIF (Lemmer et a l . , Eur J Pharmacol 183:521,1990) no s i g n i f i c a n t t i m e - d e p e n d e n t d i f f e r e n c e s were found in t h e p h a r m a c o k i n e t i c s (Cmax, tmax, AUC) of NIF a f t e r 8°0and 19°°-dosing. Hemody-namic p a r a m e t e r s w i t h a n d without d r u g treatment displayed significant rhythms, improvement of fit was o b t a i n e d when f i t t i n g to a 24ha n d 1 2 h - p e r i o d s i m u l t a n e o u s l y . The 24-h-means in s y s t o lic a n d d i a s t o l i c BP were s i g n i f i c a n t l y r e d u c e d (p<0.0001) and in HR s i g n i f i c a n t l y i n c r e a s e d (p<0.01) b y NIF, d a i l y p e a k v a l u e s a n d a m p l i t u d e s were not i n f l u e n c e d . Whereas BP was also r e d u c e d d u r i n g s l e e p , no i n c r e a s e in HR was f o u n d . A h i g h l y s i g n i f i c a n t 24h- a n d 1 2 h - r h y t h m was f o u n d in plasma cAMP with a n d w i t h o u t NIF, mean v a l u e s w e r e in t h e r a n g e of 7.7-21.5 pmol]ml; p e a k cAMP c o n c e n t r a t i o n s were o b s e r v e d a r o u n d 10 ° ° , t r o u g h v a l u e s at 2-3 °0 with a d a i l y a m p l i t u d e of +20% of t h e mean, NIF did not c h a n g e t h i s r h y t h m . Aside from d e m o n s t r a t i n g t h a t t h i s NIF p r e p a r a t i o n r e d u c e d h i g h BP t h r o u g h o u t 24h of a d a y , a s u i t a b l e f i t t i n g p r o c e d u r e for e v a l u a t i n g 24-h-BP r e c o r d i n g s is p r e s e n t e d . Zentrum d e r Pharmakologie, J . W . G o e t h e - U n i v e r s i t ~ i t , T h e o d o r - S t e r n - K a i 7, F r a n k f u r t ] M 70.

Dipyrone is an effective analgesic, antipyretic and antiinfla~natory drug which is rapidly hydrolysed to 4-methylaminophenazone (MAP) and further metabolized to 4-aminophenazone (AP), acetylaminophenazone (AAP) and formylaminophenazone (FAP). We determined the plasma concentration of MAP and tl~ elimination of main metabolites mentioned above in urine: I. in healthy nonsmokers (n = 22) 2. in healthy smokers (n = 10) 3. in volunteers before and after phenobarbital treatment (300 mg for 3 days) (n = 9) In smokers the elimination of MAP (tl/~ = 2.07 _+ 0.07) is significantly more rapid than in''~on-smoking subjects (tl/p = 3.15 + 0.25). However, the, elimination rate of maih-metabolit~s (total amount of AP + FAP + AAP) is unchanged. Phenobarbital can induce the elimination of MAP. After phenobarbital treatment over 3 days the clearance of MAP in serum is increased by 54 % and the amount of the I~in metabolites in urine is higher in 3- and 6-hour collecting-perieds (+59 % and +28 %, respectively). In 24-hoururine the total amount of metabolites is the same as that oberserved in the absence of phenobarbital treatment. Institute of Clinical Pharmacology Bachstr. 18 O - 6900 Jena

498

5OO

INFLUENCE O F INDOMETAOIN AND A O E T Y L S A L I C Y L I C A C I D ON THE A N T I H Y P E R T E N S I V E E F F E C T I V E N E S S OF NIFEDIPINE W. S z i e g o l e i t , G. K r a u s e , and V. E c k e r t

DIFF~NT PH~0KIDETICS (F ORALIS(~I~E-5-MONONIIIRAIE IN P A T ~ S ~I~ I ~ C ~T F ~ A. ~ i ~ r , S. P e ~ r ~ , ~ . ~ i ~ , R. S i ~ H.M. ~ I ~

Nonsteroidal anti-inflammatory drugs, which inprostaglandin biosynthesis, h a v e b e e n rehibit ported to modify the antihypertensive effectiveness o f b e t a - a d r e a o c e p t o r blocking a g e n t s and other antihypertensives. Hitherto it is not whether a similar interaction exists with clear, calcium antagonists. Therefore, we investigated t h e i n f l u e n c e of i n d o m e t a c i n a n d a c e t y l s a l i c y l i c a c i d (ASA) on t h e e f f e c t o f n i f e d i p i n e m o n o t h e r a p y i n 43 a m b u l a t o r y p a t i e n t s suffered from essential hypertension (WHO s t a g e I or II). After 3 days without treatment the patients rec e i v e d an i n d i v i d u a l oral dosage of nifedipine ( i n most c a s e s 30 m g / d ) f o r 42 d a y s . A t d a y s 15 t o 28 f o l l o w i n g treatment was a d d e d : i n d o m e t a c i n ( 7 5 mg/d o r a l l y ) in group I (13 females, 9 mal e s ; mean a g e 6 3 . 9 y e a r s ) and ASA ( 1 . 5 g / d o r a l ly) in group II (16 females, 5 m a l e s ; mean a g e 61.6 years). Nifedipine alone lowered systolic and d i a s t o l i c blood pressure in standing, sitt i n g and l y i n g position. Its effect on s y s t o l i c (sigb l o o d p r e s s u r e was r e d u c e d by i n d o m e t a c i n nificantly only in standing position) and i n tensified by ASA ( s i g n i f i c a n t l y i n s t a n d i n g and lying position). But t h e e f f e c t of nifedipine on diastolic b l o o d p r e s s u r e was n o t c h a n g e d s i g n i ficantly by b o t h i n d o m e t a c i n and ASA. I n t e r a c EGG p a r a m e t e r s and s y s t o l i c tions on h e a r t r a t e , time intervals w e r e n o t f o u n d . The o b s e r v e d i n teractions on s y s t o l i c blood pressure in hypertensive patients may h a v e p r a c t i c a l relevance. Division of Olinical Pharmacology, Department of P h a r m a c o l o g y and T o x i c o l o g y , Martin Luther University, Leninallee 4 , D-O 4020 H a l l e

l~i~-5~itra~ ( I ~ ) is a willy u ~ ~ g in t ~ t~rapy of ~ ~ t di~a~ ~ i ~ offers a hi~ bi~vail~illty a~ ~ l l in~ri~ivi~al variati~s in p l a ~ levels. ~ s , a ~ ~sa~ ~ i ~ ~ ~ v i ~ to a~ieve effective p l a ~ ~ n t r a t i ~ s ~d ~ r ~ t l y avoid~ p ~ l ~ of nitra~ ~ e r ~ ~ i ~ is as~c i a ~ wi~ hi~ ~ ~ - f l ~ t l ~ ~ n t r a t i ~ s . T~ a ~ of ~ i s s ~ y was to invcsti~te ~ ~ i ~ t i c s of I ~ in ~tients with d i f f e ~ t ~ g ~ s of ~ t failu~ a f a r a ~ ~ i ~ infa~ti~. ~ ~ntral v e ~ s p ~ s ~ ( ~ ) was ~ d ~ f o ~ initiati~ of t~rapy as a ~ r of g l ~ l ~ i a l f~ti~. ~p I i~l~d 9 ~tien~ wi~ ~ l (~ 6 ~ H20; ~ l l i p cla~ I) ~ (2.8 ~ 0.6 ~ ~0)( ~ + ~M). ~ II @ s ~ s ~ of 8 ~tien~ wi~ eleva~d (> 6 ~ ~9; Kil~ip cla~ II-III) ~ (I0.# C 1.1 ~ ~0). After oral ad~nistrati~ of 20 ~ I ~ , b l ~ was ~ at 0,~0,~, ~ , # 5 , ~ , ~ ~n a~ 2,~,#,6,8 h. I ~ was ~ i ~ in p l a ~ usi~ a g a ~ t o gra~ic ~ . (~onp I

( ~ -~ 6 ~P

m

Group I I

to)

66~ + ~ ~ 33 ~_ 6

O.lg + O.OI

(~

> 6 m

to)

#75 ;~2 n-" 55 ~ _ 9

~Iml min

O,lg + O.OI

~ = peak plas~ concentration; t = ti.~ at peak concentration; as.~ conoentrationat 8h~ ke =Pelim[natlonrate ~stant. cSh = We co~ludo that in patientswith iscbemic heart failure the phannaeckinetics of oral IS~N are significantlyaffected by the h~nodynemie dysfunction. We observed a ~ b c e d peak plasma concentrationand a prolonged eliminationof iSH4. Pemodyrk~miestudies have to show whether in view of these findings different dosage regimenswith higher single doses and longer time intervals should be reommended. I. MedizinischeKlinik, Chcistian-Albrechts-Universit~t, Schittenhelmstr. 12, D-2300 Kiel I.

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503

O N S E T OF HEMODYI~AMIC E F F E C T S TO G L Y C E R Y L T R I N I T R A TE F O L L O W I N G TRANSDERF~A3 A N D S U B L I N G U A L A D M I N I S TRATION

CONTINUOUS AND INTERMITTENT DIURETIC TREATMENT IN CHRONIC MOOERATE CONGESTIVE HEART FAILURE K. Cao, 8.D. Gonska, K . J . Fehske, H. K r e u z e r

A. Wiegand,

R. Bonn, K.H. Bauer,

E. J~hnchen

Early treatment of patients suffering from myocardial infarction with low doses of glyceryl trinitrate (GTN) seems to improve clincal outcome (Jugdutt & Warnica; Circulation 78:906, 1988). A sublingual dose followed by a transdermal administration could therefore be an appropiate dose regimen under ambulatory conditions. Thus, we investigated the onset and the time course of hemodynamic effects following either sublingual or transdermal administration of GTN as well as the combined administration. 12 healthy male volunteers received in a double blind, placebo controlled, 4-fold crossover design GTN sublingually (0.4 mg), and/or a transdermal delivery system (TDS; 15 mg GTN/32 cm2/24h), and placebo. The hemodynamic effects were measured by the a/b-ratio of the digital pulse (ratio of systolic peak/dicrotic wave), heart rate and blood pressure under orthostatic conditions. Plasma concentrations of GTN and its metabolites were determined frequently. Following GTN-TDS the onset of effect on a/b-ratio was obtained 15 ± 13 minutes after application. The mean corresponding plasma concentration of GTN was 133 ± 175 pg/ml. Blood pressure and heart rate were affected somewhat later (20 to 30 minutes). Following sublingual GTN, hemodynamic effects peaked already 3.5 ± 0.9 minutes after administration with corresponding plasma concentrations of 2514 ± 839 pg/ml GTN, respectively. Thus, the combination of a single sublingual stroke Of GTN and a transdermal delivery system of GTN provides both, a rapid onset as well as the maintenance of hemodynamic effects.

Diuretics are cornerstones in the treatment of congestive heart failure (CHF). However, long-term continuous administration leads to an activation of the renin-angiotensin-aldosterone system which is already stimulated by reduced tissue perfusion resulting from CHF. Furthermore, a number of metabolic complications can be associated with diuretic therapy. Therefore, a reduction of the dosage might be desirable. In order to compare the effects of continuous and intermittent diuretic monotherapy, an open, controlled study was performed. In randomized order, 20 pts with moderate CHF (NYHA II-III) r e c e i v e d f o r 3 weeks e i t h e r 2x3 mg o f t h e l o o p d i u r e t i c p i r e t a n i d e per day (Group A) or 3x6 mg p i r e t a n i d e per week (Group B). At t h e b e g i n n i n g o f t h e s t u d y , t h e 2 groups did not differ with respect to clinical symptoms and hemodynamic or l a b o r a t o r y f i n d i n g s . A f t e r 3 weeks o f t r e a t m e n t , 24h u r i n e volume had s i g n i f i c a n t l y inc r e a s e d i n both groups and s u b s e q u e n t l y body w e i g h t was reduced ( - 1 . 6 kg and - i . 7 kg; both p < O . 0 5 ) . The clinical symptoms had m a r k e d l y improved in 6 pts in each g r o u p . E c h o c a r d i o g r a p h i c a l l y d e t e r m i n e d hemodynamic p a r a m e t e r s r e v e a l e d a 9% i n c r e a s e in f r a c t i o n a l s h o r t e n i n g i n Group A (p
Abteilung fur Klinische Pharmakologie, Rehabilitationszentrum, S~dring 15, D-7812 Bad Krozingen

Department of C a r d i o l o g y , Germany

502

5O4

University

of Goettingen,

EVALUATING ORAL NITROGLYCERIN SPRAYS WITH DIGITAL PULSE PLETHYSMOGRAPHY (DPG). K.A. Erb ~, C. de Mey.~,

~-'r~CT OF ~

M.I. Leschinger ~, M. Kroll~, G.K. Wolf~, G.G. Belzk

J,Pr~er,

Comparative analyses of nitroglycerin (NTG) formulations via pharmacokinetics are complicate d by NTG's rapid and complex clearance and bio-analytical constraants. We used DPG to assess the pharmacodynamic equivalence of 2 NTG-sprays for acute oral use: a new chlorofluorcarbon- (CFC)-free (NT) vs a oresently available CFC-eontalning spray (NR, Coro-Nitro ~, 3M Medica GmbH, Borken, FRG). Nine male and 6 female healthy volunteers were studied on 3 different days receivingeither 0.8 mg NT, 0.8 mg NR or NT-matched placebo (PL) in a double-blind period-balanced cross-over design. DPG was measured before dosing (time -5,-3,-1 rain) and then at 1, 2,4,6,8,10,12,15,20,25,30,35,45,60,75,90,105,120 min after dosing. NTG caused an increase of the systolic wave (a) and a deepening of the dicrotic notch (c). The time course of the median changes of the c/a-ratio from predosing baseline for NT matched that for NR and both were clearly distinguishable from PL. The effects were summarized by the minimum in the time course (Rmin), its timing (Tmin) and the area under the time course during the 1st hr after dosing (A(60)); means [range] and the Least Signifikant Difference LSD) as derived from analysis of variance, are presented below :

~ i n g ~ e ~ s t i s ~ e ~ c p ~ s e afte~ heart o ~ a t i o n w i ~ increased s ~ e t i c outfit(i) a ~tual interde~ndence ~ t ~ e n ch~ging adEenergic state ~ d ~ i n t a i n ~ inhibition of phosph~iesteras~III is exacted. The study group co~rised 20 ~tients ( A ~ ) w i ~ no previous d o ~ n t ~ i~i~nt of ventri~lar ~ c t i o n (Cl 2.3 ~0.4 i / ~ n ~ ; ~ 7.4 ~2.4~g).By direct c o ~ r i s o n of ~ e corres~nding h a e ~ c values -~ita e n o x ~ n e (2x ~ l u s 0.5 ~ g b.w. ,continuous in~sion 5 mcg~g/~n) ~ n u s control g ~ p (n =i0)-, ~ e d e v e l o ~ n t of ~ e phar~ c ~ c effect ~ s evaluated ~ t i ~ t i v e l y . ~ter ~ing off c a r d i o ~ o ~ r y ~ss (~B) ~ e increase in CI i ~ c e d ~ ~ e d ~ g reached a ~ i ~ l value afte~ 30 ~ n ~ d faded ~e~eaft~r al~st to control values f~om +0.9 ~0.3 to +0.i i / ~ n ~ afte~ 60 ~ n . In a second s l ~ phase(5 h) ~ e ulti~te steady state ~ s achieved (~I 3.8 ~0.1 ,contri~t~on of enoxi~ne +0.71 ~0.08 1 / ~ n ~ ; +20.6 +1.7%,>6hr)wi~ no ch~ge in heaEt rate. ~ e a u ~ tation-of stroke v o l ~ index (delta ~ : +8.8 ~1.3 ~ ; +25.3 ~2%) oc~red c o n c ~ t ~ t ~ a decrease in syste~c vas~la~ resist~ce (delta ~ : -253 ~14 d ~ e s xsec x ~ ; -24.4 ~i.1%). ~ e t r ~ s i t o ~ dlsap~ar~ce of ~ e ~sitlve inotropic ~ d vas~ilato~ effect of ~ e d ~ g ~ ~nde~ndent f r ~ vari~les like heart rate(86~4 b ~ ) ~ d ~ a~terial pressure(67~7~g) or from ~ e plash-concentration of enoximone (442 ~31 n ~ ) , ~ i c h re~ined const~t. High concentrations of e n d ~ e n ~ s noIadrenaline in ~e ~st~B ~ri~(901~83 ~ / ~ ) are ch~ging ~ e ~ 1 ~ c e ~en adrenergic receptors ~ d enoxi~ne ~nduc~ i~ibition of PDE -III. ~ d i t i o ~ l l y ~ e res~nsiveness ~ y ~ m~ified ~ a t r ~ s i t o ~ sensitization of adenylyl ~clase (2) f o l l ~ n g gloW1 ~ocardial isc~e~a. 1. ~ g e l ~ ~ et al J.~orac.Cardiovasc.Surg.86,608(1983) 2. StraBse~ ~ et al C i ~ l a t i o n 82(suppl II),II-23 (1990)

PL

NR

NT

LSD

Rmin [%]

- II.0 - 33.4 - 34.8 10.98 [-35.9,-4.0] [-44.7,-19.6] [-56.9,-15.3]

Tmin

[minI

37 [ lO,12o]

A(60), [%.mini

+ 5 -1147 -1163 [-701 ,+613] [-1738,-635] [-1823,-143]

10

[ 4, 30]

10

[ 5, 35]

253.5

The 90% cI of the (NT/NR)-ratio of Rmin was 88 to 120%, and of A(60), 79 to 123%. It is concluded that NT was equivalent with NR

for therapeuticallyrelevant pharmacodynamicresponses. 1: Center of Cardiovascular Pharmacology,ZckaPha, Maiaz, FRG. 2: lnstitut fiir Klinischa Forschung, Biometrie uad Informatik, Heidelberg, FRG. 3: 3M Medica GmbH, Borken (Westfalcn), FRG.

PO~TISCR]~IIC TRANSITORY DIMIN~JTI~ OF ~ W.~r,

~

~III

~l~

~pt. of Cardiov~s~a~ Surge~, ~dring 15, ~7812 ~ ~OZING~

IIk~]IBITORY

~ I ~ I C

R.Gie~ke, W.B~ett, ~

~ .

D . B ~

Re~ilitationszent~, (~G)

R 127 505

507

INTRAVENOUS ENOXIMONE (SELECTIVE PDE-III-INHIBITOR) I N N E W B O R N S W I T H T H E R A P Y - R E F R A C T O R Y POSTOPERATIVE ~ARDIAC LOW-OUTPUT-STATES (LOS} G.Hausdorf-, N.Friedel, H.A.Dieterich, J.J.Fell, H.T.DreBler, A.Werneyer, R.Hetzer, P.E.Lange

HYPOKALAEMIC EFFECT OF FENOTEROL IN PREGNANT WOMEN R. Hildebrandt, H. Weitzel

Enoximone was administered intravenously with a l o a d i n g d o s e o f 1 . 0 m g / k g o v e r I0 m i n f o l l o w e d b y a n i n f u s i o n o f IO ~ g / k g / m i n a f t e r 30 m i n t o 16 n e w b o r n e s with postoperative, catecholamine-refractory cardiac low-output-states as add-on to high-dose catecholamine treatment. In all children mortality-risk was clinically judg e d t o b e h i g h e r t h a n 80%. H e m o d y n a m i c paramet e r s w e r e i m p r o v e d in 12 r e s p o n d e r s , 9 (75%) of these survived. Enoximone-therapy failed in 4 non-responders which all died subsequently. Hemodyn.data

(x ± SD) o f E n o x i m o ~ e - r e s p o n d e r s : baseline infusion signif. CARDIAC INDEX 0 . 9 1 ± 0.63 3 . 2 9 ± 0.67 p< 0 . 0 0 1 ( m l * m i n - ~ m -2) S V I ( m l * m -~) 5.9 ± 4.6 2 1 . 9 ± 6.9 p< 0.001 46 ± I0 49 ± 6 n.s. M A P (mmHg) R A P (mmHg) 15 ± 3 ii ± 3 p< 0 . 0 0 1 LAP (mmHg~ 18 ± 6 i0 ± 3 p< 0.01 HR ( m i n -~) 1 6 5 ± 26 1 6 0 ± 16 n.s. AVDO 2 1383 ± 328 660 ± 165 p< 0 . 0 0 1 (ml-O2/ml blood)

I t is c o n c l u d e d , that Enoximone can be a highly effective agent in management of catechol~minerefractory LOS in neonates, but further experie n c e is n e c e s s a r y to evaluate the definite role in pediatric intensive care medicine. *Present address: German Heart Augustenburger P l a t z I, D - 1 0 0 0

Center Berlin

Berlin, 65, F R G

Fenoterol is a B-2-adrenergic drug used in the treatment of premature labour. It has been shown to cause hypokalaemia. However, two questions remained unanswered: I. Is the hypokalaemic effect of Fenoterol concentration-dependent? 2. Does hypokalaemia persist throughout therapy? To answer these questions, we measured plasma potassium (K) and Fenoterol plasma levels (F) in pregnant women undergoing tocolytic therapy for threatening premature delivery. Group i. Fenoterol was administered intravenously (constant rate infusion: 1.3/2.0 ~g/min, n=lb) and F and K were determined before and 20,40,60,90 and 120 min after the start of the infusion. Group 2. F and K were measured once in 44, 9, 42 and 37 pregnant women without Fenoterol, on day 2, on days 3 - I0 and on day ii or more of Fenoterol therapy respectively. K was determined by KlinaFlame (Beckmann) and F was measured by RIA. Group i. K dropped in all subjects (before: 4.24 _+ 0.53 mmol/l, 120 min: 3.00 _+ 0.38 mmol/l). A weak correlation could be established between the hypokalaemic effect (K,before - K,120 min) and F in steady-state (r=0.53, PK0.05). F had reached steady-state by 120 min. Group 2. No correlation between K and F could be detected within each of the groups on Fenoterol therapy. Mean values of K on day 2 were lower than without Fenoterol (3.72 + 0.52 mmol/l (day 2) vs 4.17 _+ 0.5] mmol/l (without), p<~.05, U-test). However, on days 3 - i0 as well as after more than ii days of treatment K was not different from mean values of K without Fenoterol. Our results show that the hypokalaemic effect of Fenoterol is initially concentration-dependent. However, tachyphylaxia occurs within a few days of continous treatment. Frauenklinik, UKS, FU, Hindenburgdamm 30, I000 Berlin 45

506

5O8

DO ACTIVE METABOLITESOF EENOTEROLEXIST ? L ~reiss-N0wz0h0ur,B. Kruedewagen, A. Schulz,U. Gundert-Remy

I N V E S T I G A T I O N OF T H E I N T E R A C T I O N B E T W E E N THE A N T I ARRHYTHMIC DRUG LNC-834 AND THE ORAL ANTICOAGULANT D R U G W A R F A R I N IN H E A L T H Y V O L ~ N T E E R S D. T r e n k , W. M ~ h r k e , M. S e i b e r l i n ~ , L. W a r t h

Opinions on the clinical effectiveness of 0ral treatment with betasympathumimetlcfencter01 are divergent. The aim of this studywas to evaluate the different effectof 0ral vs. iv application of fen0terol on heart rate, systolic and diastolic blood pressure (bp), potassium and glucose plasma concentrationon the basis of same fenoter01plasmalevels. In a cr0ss-0vertrial20 n0n-pregnant,healthywomen wererandomly give~3xl0mg fen0tersiin tabletform at time 0, 30' and 60', fen0tar01infusionwith s rate of 1 ~g/min.for 4 hours and placebo with a washoutperiod of i week between each. Bloodsampleswere takenevery 15 min. for the first 2 hours, every30 min. fur the following2 hours, and pharmac0dynamicmeasurementswere made at the same time. Plasma levels 0f fen0ternl and its metaboliteswere determined by radiuimmun0assay. A significant increase in heart rate, systolic bp and glucose plasma concentrationand a significant decreasein diastolic bp and pntassium plasma concentrationwas observedwith both 0ral and iv administration of fen0terul. However,at the same blood level the pharmac0dynamiceffectswere more pronounced after oral administrationthan after iv application. At the same time, the concentration of the metabolites, fen0terul-glucur0nideand -sulfatide, was s~gnificantlyhigher when fenntar01 was given orally,whereas fen0terolplasma !e~el was in the samerange withboth routes.Biuavailabilityof fenuterolwas 0,5%~ Taking intoaccount the higher effectivenessafter0ral administrationof fenuterol,we concludethat, indeed,the metsb01itesof fenoter01are act!re.

The i n t e r a c t i o n of the n e w q u i n i d i n e - l i k e a n t ± a t rhythmic drug LNC-834 ((9S)-10,11-dihydro-6-methoxy-cinchonan-3,9-diol hydrogen sulfate pentahyd r a t e ) a n d the o r a l a n t i c o a g u l a n t drug warfarin w a s i n v e s t i g a t e d in a r a n d o m i z e d c r o s s - o v e r f a s h ion in i0 h e a l t h y m a l e v o l u n t e e r s . Prothrombin time and plasma concentration of w a r f a r i n w e r e d e t e r m i n e d u p to 144 h o u r s f o l l o w i n g a d m i n i s t r a t i o n of a s i n g l e o r a l d o s e of 0.36 ± 0.03 m g / k g warfarin without and w i t h c o n c u r r e n t treatment w i t h L N C - 8 3 4 (700 m g b . i . d . ) for a p e r i o d of n i n e d a y s in total. C o n c u r r e n t a d m i n i s t r a t i o n of L N C 834 d e c r e a s e d significantly the a r e a u n d e r the plasma concentration-time c u r v e of w a r f a r i n ( 1 1 7 , 8 8 9 ± 2 5 , 0 1 0 vs. 1 2 5 , 2 9 4 + 2 2 , 3 1 4 n g * m l / h r ; p = 0 . 0 4 8 8 ) . Thus, a s i g n i f i c a n t ~ n c r e a s e in a p p a r ent oral c l e a r a n c e (CL/f) of w a r f a r i n in the p r e s e n c e of L N C - 8 3 4 w a s d e t e r m i n e d (3.98 ± 0.63 vs. 3.71 ± 0.50 m l / m i n . ; p = 0 . 0 4 8 8 ) . L N C - 8 3 4 h a d no e f f e c t e i t h e r on the m a x i m a l a n t i c o a g u l a n t e f f e c t nor on the t o t a l a n t i c o a g u l a n t e f f e c t per d o s e as d e t e r m i n e d b y the i n t e g r a t e d e f f e c t . H o w e v e r , adm i n i s t r a t i o n of L N C - 8 3 4 at a d o s e of 700 m g b . i . d . o v e r a p e r i o d of t h r e e d a y s i n c r e a s e d s i g n i f i c a n t ly the p r o t h r o m b i n c o m p l e x a c t i v i t y b e f o r e a d m i n istration of w a r f a r i n (PCA: 9 5 . 8 1 ± 1 0 . 8 3 vs. 8 5 . 0 2 ± 1 2 . 4 7 % of n o r m a l ; p = 0 . 0 1 9 5 ) . The m e c h a n i s m of t h i s p r o c o a g u l a n t e f f e c t is u n k n o w n yet, b u t an i n d u c i n g e f f e c t on s y n t h e s i s a n d / o r a c t i v a t i o n of c l o t t i n g f a c t o r s b y L N C - 8 3 4 s e e m s l i k e l y a n d c o r r e s p o n d s w i t h the e n h a n c e m e n t of the m e t a b o l i s m of w a r f a r i n .

Department of Clinical Pharmuc01ogy,Bundesgesundheitsamt,SeestrasseIO, I000 Berlin 65, Germany

A b t e i l u n g fur K l i n i s c h e P h a r m a k o l o g i e , R e h a b i l i t a t i o n s z e n t r u m , S ~ d r i n g 15, D - 7 8 1 2 B a d K r o z i n g e n

R 128 509

511

PHARMACOKINETICS OF INTRAVENOUS QUINAPRILAT FOLLOWING RISING SINGLE AND MULTIPLE DOSES

THE D~£~/~4INATION OF RENIN, A / ~ O N E AND C ~ I U M IN THE P E R I P H ~ ] ~ IN VARIOUS FORbiS OF A ~ I A L HYPERTENSION K. Farker~ G. Stein~ M. H~ller~ A. B6rner

M. Neub*, U.F. Legler* Quinaprilat (QT) is the active metabolite of quinapril, an orally active nonsulfhydryl ACE inhibitor. The pharmacokinetics of QT were investigated in two doubleblind, placebo-controlled rising dose tolerance studies in healthy volunteers. In the course of the single-dose (SD) study, f o u r groups of 6 subjects received 1, 2.5, 5, and 10 mg single IV bolus doses f o r pharmacokinetic assessment. In the multiple-dose (MD) sfudy, five groups of 6 subjects received 0.5, 1, 2.5, 5, and 10 mg IV bolus doses QID ( e v e r y 6 hours) f o r 48 hours. Serial plasma samples were taken over 24 hours (SD) and 72 hours (MD), urine samples were collected over 24 hours (SD) and over 12 hours following last dose (MD).~ Plasma and urine samples were assayed f o r QT using a validated radioimmunoassay method. Plasma and urine QT concentrations were examined by established noncompartmental methods. QT was well tolerated in both studies, Following SD and MD, mean AUC values exhibited dose proportionality over the whole range, Mean apparent elimination half-life values were in the range of 1.7 to 2.~ hours (SD) and 1.8 to 2.2 hours (MD). QT total plasma clearance was between 62 and 80 ml/min (SD) and between 58 and 80 ml/min (MD). The apparent volume of distribution of QT was approximately 11 liters. Cumulative u r i n a r y excretion of QT exhibited dose p r o p o r t i o n a l i t y over the whole range in both studies as well. * GSdecke Research Institute,

The renin-angiotensin-aldosterone system affects b l c ~ presstu~e, retention o f w a t e r and scx~ium by t/~e kidneys, ~ the secretion of aldosterone. Peripheral venous renin and aldosterone levels were measured in 57 patients with renoparenchymatous, 23 patients with renovascular and 111 patients with essential hypertension. In addition, calc i ~ concentrations (total, ionized) were measured in these three groups. Basal (supine) and stimulated (2 h upright posture and 40 mg furosemide i.v.) plasma renin activity and aldosterone concentration were determined by radioir~nuno assay. The classical n~)del of renin independent hypertension occurs as one form of renovascular hypertension. %"he determination of basal (sensitivity 48%, specificity 81%) and stimulated activity of renin (sensitivity 82%, specificity 31%) is not suitable as a discriminatorymethcd for the classification of renovascular hypertension. ~'nerewere no abnormalities in the total calcium concentration but the ionized calcium concentration was significantly decr~_ased in all three groups. The conclusion of this study is: Because of the differences in sensitivity ~ specificity, the [:~.ripheral venous renin determination should not be used as a routine method in the diagnostics of hypertension in amb u l a t o ~ patients. Institute of Clinical Pharmacology, BachstraBe 18, 0-6900 Jena, FRG

University

Jena,

Mooswaldaltee 1-9,

D-7800 Freiburg

510

512

PHARMACOKINETIC INTERACTION BETWEEN ORAL BUNAZOSINE AND CIHETIDINE IN HEALTHY HALES. S.Harder, F.Th~rmann T~e pharmacokinetic disposition of the ~l-blocking agen~ bunazosine (B) was investigated in 15 healthy males (age 21-34y) before and under concomitant cimetidine (C) treatment. On day l, a 2mg-tablet of B was given p.o. and blood samples were drawn 0-24h. C treatment (2*400mg/d) was started on day 2 and was maintained to day 8. On day 7, a second 2mg-tablet of B was administered and blood samples were drawn 0-24h. Supine blood pressure was recorded on day I and day 7. B was analyzed in serum by a HPLC-method. All subjects were phenotyped for debrisoquine hydroxylation using dextrometorphan, one subject was identified as a poor metabolizer. Pharmacokinetic parameters of B obtained on day I and day 7 were compared intraindividually by WILCOXONS matched pairs si~ned rank test. table: pharmacokinetic parameters of B {mean±sd, N=15) parameter day i day 7 Cmax (ng/ml) 16.9±7.5 19.3±5.8 n.s. tmax (h) 0.83±0.45 1.35±1.02 p<0.05 AUCo_oo(ng*h/ml)# 47.3~16.9 68.4±17.9 p<0.001 t/2abs (h) # 0.22±0.21 0.49±0.32 p<0.001 t/2B(h) # 2.56±0.83 2.90±1.09 n.s. # c a l c u l a t e d by p h a r m a c o k i n e t i c p r o g r a m ~OEREP Increase in AUCo-oo may reflect the slight, but insignificant increase in Cmax and t/2~. The increase in t/2abs and tmax suggests delayed absorption of B under C treatment, which might be caused by a decreased dissolution of the weak base B under increased gastric pH. A slight, but significant reduction in diastolic BP was measurable daring 0-4 h after B application on day i and during 0-6 h on day 7. However, intraindividual comparison of BP-profi]e yielded no differences between the two investigations. Abt.Kliniscbe Pbarmakologie, Universitfitsklinik Frankfurt, Theodor-Stern-gai 7, D-6000 Frankfurt/Main-70

PHARMACOKINETICS AND PHARMACODYNAMICS DIFFERENT DOSES OF URAPIDIL-E AFTER INFUSION ORAL APPLICATION. G. Q u a b e c k , K. Nelson, J. S t o r c k , R. K i r s t e n

OF AND

U r a p i d i l decreases blood p r e s s u r e b y b l o c k i n g p e r i p h e r a l a, receptors and activating serotonimA (SHTIA) receptors. In t h i s s t u d y t h e e f f i c a c y a n d s a f e t y of u r a p i d i l in 1 2 hypertensive patients and the correlation between pharmacokinetlcs and pharmacodynamics was examined. In an open 4-periodic study with placebo-run -in, u r a p i d i l (100 m g / 2 4 h ) w a s i n f u s e d d u r i n g t h e f i r s t period. In the following 3 periods the patients received retard capsules of I00 mg in the morning, then 75 mg twice a day and finally 2 x i00 mg in the morning, each for 14 days. Blood pressure, heart rate und plasma urapidil c o n c e n t r a t i o n was m e a s u r e d a t t h e l a s t d a y of e a c h period. There was a good correlation between dose and the maximum plasma concentration. The diastolic blood pressure decrease was in good correlation ~o the urapidil plasma concentration, as well. The minimal diastolic blood pressure decrease over 24 h was always more than -13 m m H g during capsule application (Table I). T a b . i: Maximal p l a s m a u r a p i d i l c o n c e n t r a t i o n (c®ax) a n d m a x i m u m a n d minimum d i a s t o l i c blood p r e s s u r e d e c r e a s e ( s i t t i n g ) (DBPD®ax, DBPDm,~) d u r i n g u r a p i d i l t r e a t m e n t . infusion lOOmg Cmmx (ng/ml) DBPDamx (mmHg) DBPDmia (mmHg)

394 -18

oral administration lOOmg 2 x 7 5 m g 200mg 322 -19 -13

402 -22 -13

747 -23 -17

Dep~menCof ClinicalPharm~'o~o~,[Jniversi~yof P~nkfu~Theo@r-S~em-I~7, 6900Fmkfm~/w

R 129 513

515

RACEMIC VERSUS HALF DOSED OPTICALLY PURE (S)PROPRANOLOL UNDER STEADY STATE CONDITIONS: BETA-BLOCKADE, PLASMA CONCENTRATIONS AND EFFECTS ON THYROID HORMONES METABOLISM K Stoschitzky, W Lindner*, G Egginger*, B Obermayr-Pietsch and W Klein

TONIC VS. PHASIC PAIN: DOSE-RELATED ANALGESIC EFFECTS OF KETOPROFEN Hummel T., Huber H., and Pauli E.

In a randomized, double-blind, cross-over exercise study, 10 male healthy volunteers (aged 24:t:3 years) were administered orally 40mg (R,S)- and 20rag (S)-propranolol.HC1, respectively, t.i.d, for 7 days devided by a drug free interval of 1 week. During 8 hours after the last intake of either drug exercise testing was performed at intervals of 2 hours and 10 blood samples were taken. The mean rate pressure product was decreased (p<0.0001) by both drugs to the same extent - (R,S) -18.8% and (S) -19% - indicating equal beta blocking effects. The mean plasma concentrations of (S)propranolol were higher than the ones of the corresponding (R)enantiomer - (R) ll.6ng/ml and (S) 14.9ng/ml (p<0.05). When 20rag (S)-propranolol.HC1 was administered the mean (S)propranolol plasma concentration (13.4ng/ml) was slightly lower than after administration of 40rag of the racemate but the difference did not reach a significant level. After 7 days of drug intake the triiodothyronine/thyroxine ratio was decreased (-25%, p<0.0001) and the reverse-triiodothyronine plasma concentration was increased (+18%, p<0.005) by (R,S)propranolol whereas the optically pure (S)-enantiomer did not influence these parameters indicating that only (R)-propranolol inhibits the conversion of T4 to T3. In conclusion, (R)- and (S)-propranolol are two drugs with different .pharmacodynamic and pharmacokinetic properties: 0t)-propranolol is a pure T4-T3-conversion inhibitor, (S)-propranolol is a pure beta adrenoceptor antagonist (further effects are due to both enantiomers) and the plasma concentrations of the (R)- and (S)-enantiomers are different after oral application. Since there is an effective method available to separate the optically pure enantiomers at a large w.ale and at reasonable costs they should be preferred to the currently used racemic mixture. Medizinische Universit~tsklinik, A-8036 Graz, *Institut t'fir Pharmazeutische Chemic, Karl Franzens Universitht, A-8010 Graz

The aim of the study was to demonstrate a dose-related analgesic effect of ketoprofen (placebo, 50, 100 and 150 mg i.v.) in a human experimental pain model. 18 young, healthy volunteers were investigated. The study was conducted in a double-blind, randomized, controlled design. After an initial training session the subjects participated in four experimental sessions, in which either placebo or one of the three doses of ketoprofen were administered. The session was divided into three measuring sequences (before, 30 and 120 min after administration of drug). In each of these, 45 phasic painful carbon dioxide stimuli (duration: 200 msec, interval: appr. 40 sec, concentrations: 45, 52, and 58% v/v CO2) were randomly presented to the nasal mucosa. Additionally, 20 acoustic stimuli (300 msec duration, bu~'sts, 2 kHz, 105 dB HL) were presented to the left ear, in order to control non-specific; central nervous effects. Cold, dry air was used for tonic painful stimulation. During measuring sequences it was continuously applied to the nasal mucosa. EEG was recorded from 8 positions referenced to linked earlobes. A linear relationship between the serum levels and the administered doses became apparent. Ketoprofen dosedependently reduced the subjective estimates of the tonic pain. At the same time an increase in the subjective estimates of the phasic pain was observed in connection with increased amplitudes and a decrease in latencies of the pain-related evoked potentials. It was established that ketoprofen reduced the intensity of the tonic pain only to a certain degree, which indicates a ceiling-effect of the analgesic action of ketoprofen in this pain model. Department of Pharmacology and Toxicology. University of Erlangen-NDrnberg. Universit&tsstr. 22, 8520 Edangen. FRG

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516

ACETYLSALICYLIC ACID: KINETIC AND CNS-EFFECTS IN HUMANS B. Bromm and E. Scharein

SPECIFIC EFFEC]SOF BENZODIAZEPINESON HUMAN VISUAL RECEP'rlVE FIELDSIN LIGHT AND DAI~K ADAPTA'IION. M. C-roner, F. Walder, 1~.Groner, H.U. Fisch

Acetytsalicytic acid (1000 mg orally) was investigated with a noninflammatory experimental pain model, in a placebo-controlled, double-blind repeated measures study (N = 32). Phasic pain was induced by intracutaneously applied electrical pulses of constant current. The nociceptive responses measured were, pain ratings, cerebral potentials and EEG delta power in response to ~he stimuli. In addition, spontaneous EEG, auditory evoked potentials and reaction times were evaluated to determine unspecific effects. Blood samples were taken to monitor the plasma concentrations of the active agents. Acetylsalicylic acid produced clear analgesic effects in all pain relevant target variables. The effects increased with postmedication time, becoming significantly different from placebo 90 min after medication (p < 0.01). At this time the pain ratings were reduced by 4%, the pain related cerebral potentials by 15%, and the stimulus induced delta power of the EEG by 17%. These findings suggest a central action of acetylsalicylic acid by attenuation of experimentally induced nociceptive activity. No influences could be observed upon auditory evoked potentials, spontaneous EEG and reaction times, i.e., acetylsalicylic acid did not change the vigilance level. The plasma concentration of acetylsalicylic acid reached mean values of 2.5 =t=2.4 l~g/ml within 25 min after oral medication, which remained constant during the entire postmedication period of 105 min. In contrast, the concentration of the metabolite salicylic acid increased steadily reaching mean values of 32.0 =~16.8/*g/ml at the end of the investigated period. Though both, the analgesic efficacy and the concentration of salicylate increased with postmedication time, no correlations were found between individual plasma levels and effects. Institute of Physiology, University Hospital Eppendorf 2000 Hamburg 20, Martinistr. 52

In the cat, bicuculline decreases the activity of the inNbitory surround of motion-sensitive retinal ganglion cells. In humans, center and inhibitory surround of visual receptive fields were determined with subthreshold summation (Wilson, H.R., Vision g4es.]8, ]978). Stimuli were three vedical parallel lines displayed for 120 ms on a CI~T.The contrust of the flanking lines was 3/8 of the contrast of the central line. Dependent variable was the contrast threshold of the central line, independent variable the distance b e l ~ e e n the central and the flanking lines. In control experiments, no learning effect or circadian variations were observed. In light-adapted subjects (SS), the strongest increase of contrast threshold was found in the inhibffory pad of the visual receptive fields (0.24° of visual angle, p<0.0]) after 7.5 and ]5, but not 4rag Midazolam (M) p.o., indicating a dose-effect relationship. After six hours, predrug values were obtained. After dark~3daptation (8 SS) no effect of M on visual receptive fields was found. The control drugs Atropine ( l i n g i.v.), Sulpiride (]00mg i.m.), and Levodopa (]00mg) + Benserazide (25mg) p.o, had no effect. Conclusions: Specific actions of GABA observed on single retinal ganglion cells in the cat may be assessed with the GABA-potentiating M with non-invasive methods in humans. In dark-adapted SS no free GABA may be available in the synapses and M may therefore have no effect. The specific effects of M (and possibly all BDZ) on the visual system may explain the visual distortions sometimes reported in BDZ withdrawal. Depts. of Psychiatry and *Psychology, University of Berne, Murtenstr. 2], 30]0 Berne, Switzerland.

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INHIBITION OF CHOLINESTERASES IN VITRO IN HUMAN BRAIN AND ERYTHROCYTES BY GALANTHAMINE T.Thomsen, J.P.Fischer, U.Bickel, B. Kaden and H.Kewitz

AGONIST PATIENTS

Introduction: Galanthamine (NIVALIN, Waldheim, Vienna, Austria) is currently inveslJgated in the symptomatic treatment of Alzheimer's disease. It is a reversible and rather selective inhibitor of acetylcholinesterase and might act via restoration of the cholinergic deficit at the synaptic site in the brain. Methods: A series of concentration-response experiments was performed to evaluate the inhibition of acetylcholinesterase (EC 3.1.1.7) in human brain tissue from 4 patients who underwent neurosurgical removai of a brain tumor and erythrocyte samples prior to induction of anaesthesia by galanthamine. In addition, post-mortem human brain tissue was gained within 28 h from 5 individuals without history or histological evidence of neurologicai diseases. The enzyme activity was measured radiometricaily as published previously [LifeSci 46, 1553-1558, 1990]. Results: Acetylcholinesterase in the human brain tissue of neurosurgery patients and in the post-mortem tissue was almost equally inhibited by gaianthamiae (GAL) with IC50 values of 2.9 and 3.6 /zmol/L. No relevant differences were observed between the frontal cortex and hippocampt~s region of the brain post-mortem tissue. GAL concentrations between 0.8 and 20.4 #mol/L referred to an inhibition range of 20-80 % catalytic activity. The respective acetylcholinesterase inhibition in erythrocytes disq~layed a similar concentration response slope which was, however, 10-fold less potent. Conclusion: The inhibition of acetylcholinesterase in the brain might not easily be estimated by measuring ex vivo inhibition of acetylcholinesterase in erythrocytes.

EMD

~9980 IN

-

A THE

NOVEL DOPAMINE TREATMENT OF

WITH NEGATIVE SYMPTOMS K . W i e d e m a n n , A . L o y c k e and J.-C.

AUTORECEPTO~ SCHIZOPHRENIC

Krieg

B a s e d u p o n r e c e n t o b s e r v a t i o n s that d o p a m i n e autoreceptor agonists might improve negative s y m p t o m s the c o m p o u n d EMD 49980 ( 5 - h y d r o x y - 3 (4-phenyl-l,2,3,6-tetrahydropyridil-(1)butyl)-indol) was administered in an open clinical trial to p a t i e n t s suffering from schizophrenia (according to international classification criteria). Ten patients participated in the s t u d y and r e c e i v e d the test d r u g o r a l l y for u p to 28 days in a d o s e r a n g e from 0.5 to 9.0 mg/day. In four p a t i e n t s a s i g n i f i c a n t a m e l i o r a t i o n ( r e d u c t i o n of the i n i t i a l s c o r e of n e g a t i v e s y m p t o m s b y 40 % o r more) was observed; one patient improved slightly, four patients remained without i m p r o v e m e n t of psychopathology.' W o r s e n i n g of p s y c h o t i c s y m p t o m a t o l o g y was o b s e r v e d in one patient. T r e a t m e n t w i t h EMD 49980 did not lead to any extrapyramidal symptoms which are characteristic for t r a d i t i o n a l n e u r o l e p t i c s . As was to be e x p e c t e d from the p h a r m a c o l o g y , plasma prolactin concentrations were s i g n i f i c a n t l y r e d u c e d four h o u r s a f t e r oral a p p l i c a t i o n of a s i n g l e dose of the drug. Max-Planck-Institute for Psychiatry, K r a e p e l i n s t r a B e i0, D - 8 0 0 0 M ~ n c h e n , F R G

Insfitut fiir Klinische P "harmakologie, Klinikuin Steglitz, FU Berlin, Hindenburgdamm 30, I000 Berlin 45, FRG

518

520

PERIPHERAL CIRCULATING LYMPHOCYTES AS MODEL SYSTEM TO STUDY PHOSPHOINOSITIDE SIGNALLING IN HUMANS T. Schubert

EFFECTS OF BATHING ~ITH NELISSA ADDTTTVE ON PHYSIOLOGTCAL PARAMETERS AND PSYCHOLOGICALSCALES IN HEALTHY SUBJECTS AND PATIENTS WITH NERVOUSNESS/TNSOMNIA A CONTROLLED STUDY. B.Uehleke and H. P±schel

Activation of the phosphoinositide cycle mediated by cell surface receptors like the alpha-I adrenoceptor, serotonin 5-HT2- and muscarinic receptors leading to the synthesis of several inositol phosphates represents a well established mechanism of neuronal signal transduction. Since the observation that lithium affects this transducing mechanism by inhibiting myo-inositol-l-phosphatase and other inositol phosphatases phosphoinositide metabolism may represent an interesting target for the action of antidepressants and other psychoactive drugs. Because of the inaccessibility of the central phosphoinositide system to experimental research in man, we investigated the possible use of circulating peripheral lymphocytes as model system to study phosphoinositide metabolism in humans. Using (3H)-inositol-labeled human lymphocytes, formation of inositol phosphates was found as a specific response to the chemotactic formylpeptide N-formyl-methionyl-leucylphenylalanine (fMLP) and could not be induced by several neurotransmitter agonists like carbachol, epinephrine, and serotonin, fMLP stimulated formation of inositolmono- and inositolbisphosphates as well as higher inositol phosphates could be significantly augmented in the presence of lithium ions in therapeutic plasma concentrations (ED50 0.5 mmol/l). The atypical antidepressant (-)-oxaprotiline (levoprotiline) seems also to specifically interact with inositol phosphate generation by reducing intracellular inositol and augmenting accumulation of inositol bisphosphates. Supported by a Grant of BMFT Department of Psychopharmacology, Central Institut of Mental Health, D-6800 Mannheim (FRG)

Sedative effeete of etheric o i l s of meliesa or cymbobogon ( c i t r o n e l l o i l ) have been demonetrated in animal medele. We performed a randomized controlled craeaaver study versus "placebo bath" with same ealour and foam in 22 healthy aabjecte (high and low-dose) and in 26 patients with nervousness/insomnia. Rhyaiological parameters (E.C.G., heart rate, breath rate, blood preeuraa, temparatures, E.M.G.) have been registered during $0 min rest (R1), 2g min. bathing (D) with 37 %, and 10 min. rest (R2) again with a modular bioayetem. Psycholegical feeling was registered with analogue scaled questionaire (medified EWL-N) before and a f t e r bathing. Quality of sleep was evaluatad with SF-A f o r the nights before and a f t e r bath. Tanua of muacul~e daltoideus showed s i g n i f i c a n t higher degree of r e l a x a t i o n with verum in both patient groupe. The f a l l of blood preaure during the bath eeeme to be decreased with e i t r o n e l l o i l , which might be interpreted as an analeptic e f f e c t . Evaluation of psychalegical scales showed s i g n i f i c a n t differences batwean gro~pa in change of relaxation and tiredness. There are s i g n i f i c a n t dese~dependent d i f ferences in the change of the dimensions activity, irritation, and d e a a c t i v i t y . In eonclusien, herbal bath additives with c i t r o n e l l - o i l seem to exert sedative affects in healthy prabanda aa well ae in patiente with nervousness / insomnia. Sabaatian Kneipp Farschung L. Oberh~uSeretr. i D- 8939 Bad W~riahofen

R 131 521 ENHANCEMENT INFECTIONS B.

Braeunig,

OF R E S I S T A N C E IN U P P E R R E S P I R A T O R Y BY E C H I N A C E A PURPUREA RADIX M.

Dorn,

E.

Knick

In a m o n o c e n t r i c placebo-controlled, doubleb l i n d s t u d y , a 50 % e t h a n o l i c e x t r a c t of Echinacea p. w a s s t u d i e d in p a t i e n t s with U.R.I. 60 p a t i e n t s received 900 mg drug/die ( d o s e II), 60 p a t . 45.0 m g / d i e ( d o s e I) a n d 60 p a t . p l a c e b o . Selected symptoms and clinical findings were observed a f t e r 4 a n d 10 d a y s . In a l l c a s e s visible effects of d o s e I a l r e a d y c o u l d be s h o w n after 4 days, however no d i f f e r e n c e a f t e r 10 d a y s in c o m p a r i s o n to p l a c e b o . Statistical evaluation reveiled a significant e f f e c t of d o s e II in comparison to p l a c e b o a n d to d o s e I in a l l studied parameters. The reactions of l y m p h o c y t e s and granulocytes s e c m to m a k e d i f f e r e n t i a t i o n between viral and bacterial infections possible. Dose-dependent e f f e c t s of E c h i n a c e a on c l i n i c a l parameters c o u l d be s h o w n in U . R . I . In c o n c l u s i o n an e t h a n o l i c e x t r a c t of E c h i n a c e a p. is a b l e to i m p r o v e s y m p t o m s and findings in U.R.I.

Scientific Consulting P h a r m a Dr. m e d . M. D o r n , Im S c h l e n k e r t la, D - 6 2 5 0 L i m b u r g / L a h n in c o o p e r a t i o n w i t h Fa. F i n k a n d S a l u s - H a u s .