R1

1 EVALUATION OF COMBINED EFFECTS IN DOSERESPONSE STUDIES WITH THE AID OF "ALLFIT" AND THE X.2 GOODNESS-OF-FIT STATISTIC G. PSch, P. Dittrich, S. Holzmann An improved method for the evaluation of combined drug effects by means of dose-response curves (DRCs) is described, for which the programm ALLFIT and the chi-square goodness-of-fit statistic was used. In essence, observed DRCs were compared with the computed theoretical DRCs for additivity and independence. A drug A was tested in the absence and presence of a fixed concentration of a drug B in organ-bath experiments with smooth muscle strips from bovine coronary arteries and tracheal muscle. Data of insecticidal effects were taken from Tammes (Neth J Plant Path 70" 73-80, 1964). The results of such experiments are expressed in terms of percent of maximum response. Median values of effects at each concentration of A (in the absence and presence of B) were determined rather than mean values in order to allow a statistical comparison of observed with expected frequencies above or below median theoretical DRCs. Interactions not different from additive point to actions of drugs at the same site inasmuch as they significantly differ from independent interactions. Examples: SIN-1 (3-morpholino-sydnonimine) in the presence of nitroprusside-Na and nitroglycerin, respectively (relaxation of coronary arteries); parathion in the presence of malathion (insect mortality). Overadditive combinations, i.e. interactions significantly exceeding additivity (as evident from numerous examples) reflect differences between the sites of action. This is obvious regardless of whether or not they significantly differ from independence. For example, salbutamol in the presence of verapamil (relaxation of tracheal muscle) does not significantly differ from DRCs of independent interaction, whereas DRCs of isoprenaline in the presence of papaverine (relaxation of coronary arteries) significantly exceed independent DRCs which indicates sequential synergism. Inst. f. Pharmakodynamik u. Toxikol., A-8010 Graz, Univ. PI. 2

2 AN ~ C SYST~Z4 FOR FORCE ~ ON CS~MICALLY-SKINNED ~ S C L E FIBERS. H. schiehlen, A. Wagenhals, W. Gerstenberg and G. Trieb Measurements on chemically-skinned fibers are used to study the effects of c ~ on Ca++-induced contractior~ of cardiac smooth and/or skeletal muscle fibers. Generally, skinned muscle preparations are very stable which make them suitable for long-running experiments. Obviously, the efficacy of such a test system can be i~proved when measLzrements additionally can be performed overnight. Tnerefore, we developed a fully-autc~atic system which measures and analyzes force develo[mnent and co~tantly controls bath changes. The system consists of three main c(mponents: 1-8 rotors with contraction sensors and control devices, an amplifier unit with multichannel XY-writer and an HP 1000-A 400 c ~ with AD-converter. The HP i000 computer controls up to 8 rotors with 20 bath positions each. This is achieved by a RS 232 interface. The analog signals from the amplifier unit are digitized by the HP i000, evaluated and saved in a compressed manner on disk files. The positioning sequence and the control of the duration of ixmnersion of the fibers in the various bathes are variable and can independently be chosen for each rotor. Ccmputer listings document the kind of graphical and statistical data analysis. The system can be used for routine screening of cc~npounds as well as detailed pharmacological studies on preparations of different muscle tissues. M e a ~ t s on skinned cardiac fibers will be demonstrated. Biologische Informatik, A Forschungsadministration, Dr. ICarlTncmaeGmbH, D-7950 Biberach/Riss, FRG.

3 ON THE TIME-COURSE OF THE EFFECT AND OF THE TISSUE BINDING OF ASTEMIZOLE IN GUINEA-PIG TAENIA COLI H.Giinzler and A,ZieEler In the course of an investigation of newer histamine antagonists, astemizole and terfenadine were found unique with respect to the rate of onset of the histamine antagonism tested in isolated guinea-pig taehia coil The antihistaminic effect became evident only with some latency (15- 20 rain, astemizole 0.2 - 0.5 lflVl) and proceeded even than when the drug has been removed from the bath medium before the antagonistic action became obvious. Once estabfished the effect was maintained for a rather long period of time ( > 3 hours) and a restitution of the control histamine sensitivity could not be achieved any more. The ongoing increase of the histamine antagonism after removing the drug from the bath medium may be explained by an initial binding of the drug to sites different from the histamine receptors. In order to test this hypothesis the time course of the onset of astemizole's action was studied in isolated guinea-pig taenia coli in the presence of drugs (bupivacaine, loperamide, lidocaine) supposed to compete with astemi~le for sites of initial binding. Bupivacaine and loperamide each in a concentration which by itself did not affect the histamine sensitivity were able to accelerate the onset of antagonism but they did not influence its magnitude. In the presence of bupivacaine (1 pM) or luperamide (l Bid) the half maximal effect of astemizole (0.5BM) was reached 15 minutes after applying the antihistamine whereas it to(~ 45 minutes under control conditions. Lidocaine up to 10 pM had no influence. Even very high concentrations of diphenhydramine did not affect the final response to astemizole as obvious from the size of the antihistaminic effect 30 minutes after the removal of dipbenhydramine which is similar to that obtained under control conditions, i.e. studying astemizote alone. Under identical conditions, binding experiments in guinea-pig taenia coil using ~H-astemizole were performed. Astemin)le is highly accumulated by the tissue yielding a tissue medium ratio of ~ 100 after 2 hours of incubation. The aceumnlation could be depressed by 50% by treatment with bupivaeaine (10 vM), it was, however, not influenced by diphenhydramine (10 pM) under otherwise identical conditions. The results concerning the kinetic of the histamine antagonism and of the tissue binding studies are in favour of the hypothesis of an iidtial binding of astemizole by the tissue before receptor occupation. Abt. Pharmakologie, Universit/tt Kiel, HospitalstraBe 4-6, 2300 Kiel

4

BIOEQUIVALENCE OF VERAPAllIL PRODUCTS: VARIABILITY AFTER REPETITIVE APPLICATION N.Siewertl,S.Harder2,P.Th~rmann2,I.Wmisshard2.T.Huberl, G.Stenzhornl,B.Scheide]l, H.Blumel After repetitive single dose administration of a commercially available immediate release verapamil tablet formulation (80 mg) of one identical production batch at two different days b i o a v a i l a b i l i t y was investigated in 16 healthy male volunteers. Blood samples were drawn and PR-intervals were measured simultaneously up to 24 h after application. Verapamil and norverapamil were determined in plasma by HPLC. Bioequivalence was tested by calculating 90 %-confidence intervals for AUC and Cmax considering a l l possible randomised crossover combinations. For a simulated "2-way-crossover-design" relative b i o a v a i l a b i l i t y of verapamil was evaluated as 103 % for AUC and 96 for Cmax with confidence intervals of 78 - 122 % for AUC and 77 - 119 % for Cmax. tmax-values were not sign i f i c a n t l y different for both applications (~ = 1.0 vs. 1.1 h). Concentration-effect-analyses of the PR-intervais by linear model show consistent results for slope m (~0.45 vs. 0.41) in both applications. Calculated confidence intervals already reach or even exceed the generally accepted bioequivalence limits of 80 - 120 % relative b i o a v a i l a b i l i t y . This may be due to intraindividual variation of f i r s t pass and/or intralot variation of drug quality. I t can be concluded that these findings concerning the a p r i o r i - v a r i a b i l i t y of verapamil reference product's b i o a v a i l a b i l i t y have to be regarded for design and conduction of further as well as for evaluation of previous b i o a v a i l a b i l i t y studies. 1) 2)

Zentrallaboratorium Deutscher Apotheker, Ginnheimer Str. 20, D-6236 Eschborn. Abteilung Kiln. Pharmakologie, Universit~tsklinik Frankfurt, Theodor-Stern-Kai 7, D-6000 Frankfurt

R2 5 APPARENT DOSE AND TIME-DEPENDENT DRUG DELIVERY OF TIMOLOL FROM A NEW TDS It Bonn, W. Cawelio, A. So-Jansen, and H.-M. Wolff

The goal of this study was to evaluate the apparent dose of the g. blocking drug timolol (TIM) from a transdermal delivery system (TDS) and the drug delivery as a function of time. In an open randomized diuical study 12 healthy volunteers received a new TIMTDS for different time-periode (3, 6, 9,12,18 and 24 hours application interval). After application each TDS was removed and the residual drug content of TIM was measured using HPLC. The apparent dose was determined as the difference of the initial potency (31.7 +- 0.7 rag/16 cm2, n = 10) and the residual potency in the used patch. After the 24-bout-period the apparent dose was 131 ± 3.2 rag. The drug delivery was dependent on the time. In the first 6 hours of the application about 80 % of the dose was delivered to the sldn. The difference in drug delivery in the first 12 h and the following 12 h was statistically significant (2 p < , 0.001). There was more drug delivered to the sldn in the first 12 h but with no difference if these 12 h where during the day (9.$ ± 1.6 m~12 h) or daring the night (9.9 ± Z.lm g ~ h).

SCHWARZ PHARMA AG, Department of Clinical Pharmacology and Pharmaeokinetics, Department of T r s , Mlttelstr., D-4019 Monheim, FRG

7 CYfX~SPORINE: ~NHANCED INTESTINAL ABSORPI~ON IN RATS FROM THE INTRAV~q>3S SOLUTION H. Iven and Anna Harries Intestinal absorption of cyclosporine (CyA) is ~ncomplete with large inter- and intraindividual variations of bioavailability. In pilot experiments to study the influence of food on CyA absorption in a rat model, we realized that the bioa~ailability of CyA f r ~ the solution for i.v. application exceeded that frown the oral solution. Four groups of six rats were given 14 mg/kg CyA dissolved in 10 ml/kg instant drinking chocolate by gastric gavage either frcxn the ccamereial solution for i.v. or from the solution for oral application and either fed or starved (12 h). Blood (160 ~i) was repeatedly collected until 12 after dosing frcm the cut end of the tail and whole blood CyA concentrations were determined by a specific HPLCprocedure. After 12 h the animals were killed and liver samples obtained for CyA determination. Statistics: ANOVA, Scheffe-Test for significance of differences between groups. Geometric means (n = 6). of pharmacokinetic parameters.

CyA i.v.sol. starved CyA i.v. sol. fed CyA oral sol. starved CyA oral sol. fed

C-max (~g/ml)

t-max (h)

AUC 0-12 (~g x h/ml)

Liver (12 h) (~g/g)

1.960

2.3

13.0

5.5

1.390

I .7

9.0

8.3

0.890

1.4

5.4

3.0

0.694

2.4

4.8

3.6

After absorption, in all four groups CyA concentrations in blood declined with a half-life of 6 to 7 h. Feeding did not significantly influence bioavailability, however, from the i.v. solution absorption was by a factor of 2.1 higher than frcm the oral solution. This is also reflected by the residual CyA concentrations in the liver. The more ~ favourable ratio emulsifier to CyA (g/g) which is 3.2 for the oral and 13 for the intraver~us preparation may be responsible for the increased bioavailability. Institut f'dr Pharmakologie, Medizinische Universit~t zu L'dbeck, Ratzeburger Allee 160, D-2400 L'dbecM

8 NO PHARMACOK1NETIC INTERACTION BETWEEN ISOSORBIDE-$-NITRATE AND ACETYLSALICYLIC ACID

INTERSPECIES DIFFERENCES IN BINDING OF DRUGS TO SKIN N.M.Khan Ghilzai, E.Haen,H.Kurz

.%.Heydt-Liman, U. F. Legler, H. B6kens and D. Lutz

Little is known about the reversible and non-specific binding of drugs to tissues. This however, could influence considerably the pharmacokinetics and pharmacodynamics of a drug. Binding to human skin was recently reported (Walter & Kurz, 1989). The purpose of the present investigation is to study the species differences in binding of drugs to skin. Binding of chlorpromazine, desipramine, diazepam, imipramine, propranolol, indometacin, phenytoin, salicylic acid, warfarin to guinea-pig, pig and rat skin were determined by equilibrium dialysis with 10-5 mol/l, pH 7.4 in 0.125 mol/l phosphate buffer. The dialysis was performed at 4°C for 20 h. Drug concentration was determined by liquid scintillation counting. Acidic drugs were bound from 18.36 % to 78.52 %, but only salicylic acid showed significant difference in its binding to different species (guinea-pig and p i g p < 0 . 0 5 , guinea-pig and r a t p<0.05). Basic drugs were bound from 73.20 % to 94.87 t. Chlorpromazine, desipramine, diazepam, imipramine show significant difference in their binding to different species (guinea-pig and pig p<0.001 for diazepam, p<0.05 for imipramine, guinea-pig and rat: p
The aim of this study was to determine the relative bioavaliabflity of isosorbide-$-nitrate (IS-8-N) and acetylsalicylic acid (ASA) when coadminlslrated in comparison with the individual drugs. For this purpose 50 mg IS-S-N (A) and 700 mg ASA (B) were administered separately or in combination (C) to 12 healthy volunteers in a randomized triple cross-over design. The pharmacokinetie parameters tmax, emax, and AUC(0-t) were calculated from the plasma concentrations of the IS-S-N, ASA and salicylic acid. There were no statistical signitirant differenees between both treatments (A/C or B/C). The 98% confidence intervals for the quotients of Cmax and AUC were within accepted limits of equivalence. It was concluded that the coadministration of IS-$-N and ASA does not influence the bionvailability of these drugs.

SCHWARZ PHARMA AG, Department of Clinical Pharmacology and Pharmaeokineties, Mltteistr., D-4019 Monheim, FRG

Walther-Straub-Institut f~r Pharmakologie und Toxikologie, Nu~baumstr.26, D-8000 M~nchen 2, FKG

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ELIMINATION OF MEPIVACAINE AND BUPIVACAINE BY LUNG P. KOrbe, W.P. Geng,~D. K i e t z m a n n , F. F r a n z m a n n a n d H. Foth

PHARMACOKINETICS OF CARBAMAZEPINE P R O I C A C I D IN T H E C A T E. D r e i m a n n

AND

VAL-

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

Local anaesthetic d r u g s (LA) such as mepivacalne and bupivacaine are widely used for peridural anaesthesia (PDA). Already I0 - 15 rain after peridural instillation plasma concentrations of 2.3 + 0.5 pg/ml (mepivacaine) and 1.2 +__ 0.2 pg/ml (bupivacaine) are reached in the central venous blood of p a t i e n t s . T h e e x t e n t of p u l m o n a r y f i r s t - p a s s e x t r a c t i o n is e s s e n t i a l for t h e risk of L A - i n d u e e d c a r d i a c or c e n t r a l n e r v o u s t o x i c i t y . I s o l a t e d r a t l u n g s p e r f u s e d with 2 pg LA/ml a t 12 ml/min e x h i b i t c l e a r a n c e v a l u e s of 0.18 + 0.03 ml/min ( m e p i v a c a i n e ) a n d 0.49 +__ 0.06 ml/min ( b u p i v a c a i n e ) and e l i m i n a t i o n half-lives of 8.2 _+ 2.3 h (mepivacaine) and 3.5 _+ 0.3 h (bupivacaine). Pulmonary metabolism is minor when compared with the corresponding values of isolated rat liver with clearance values of 10.4 _+ 0.6 (mepivacaine) and 8.2 _+ 1.2 m l / m i n ( b u p i v a c a i n e ) . H o w e v e r , v o l u m e s of d i s t r i b u t i o n in lung (VD) with 8 ( m e p i v a c a i n e ) and 22 ml ( b u p l v a c a i n e ) p e r g of wet w e i g h t i n d i c a t e a major a b i l i t y of lung to r e t a i n t h e d r u g s . This e f f e c t is also v i s i b l e in s i n g l e - p a s s p e r f u sion of t h e r a t lung with b u p i v a c a i n e showing a Vo of 9 ml/g wet w e i g h t and a m e a n r e s i d e n c e time (MRT) of 82 + 18 sec c o m p a r e d with 30 +_ 6' sec for a p p a r a t u s control. At a l o w e r e d blood flow of 6 ml/min t h e d i s t r i b u t i o n of b u p i v a c a i n e into t h e lung is e n h a n c e d d e p i c t i n g a VD OC 14 ml/g of wet w e i g h t a n d a MRT of 127 sac c o m p a r e d to 58 sac in controls. The MRT of t h e m a r k e r s u b s t r a t e s for i n t r a v a s c u l a r d i s t r i b u t i o n , *4C-inulin, a n d for t o t a l w a t e r s p a c e , 3H20, were 59 sec and 58 ± 2 sec, r e s p e c t i v e l y . Major p u l m o n a r y e x t r a c t i o n r a t e s of m e p i v a c a i n e and b u p i v a c a i n e a r e o b s e r v e d for p a t i e n t s d u r i n g PDA i n d i c a t i n g t h a t t h e lung r e t a i n s c o n s i d e r a b l e a m o u n t s of t h e s e local anaesthetics. Institut fiir Pharmakologie und Anaesthesiologie, Universit~it 40, 3400 Gbttingen

Toxikologie Gbttingen,

und *Zentrum ffir Robert-Koch-Str.

Carbamazepine and valproic acid are often u s e d in the t r e a t m e n t of e p i l e p t i c d i s o r d e r s , but no p h a r m a c o k i n e t i c d a t a are a v a i l a b l e for the cat. S u c h d a t a are d e s i r a b l e b o t h for the t r e a t m e n t of f e l i n e e p i l e p s y a n d for the u s e of c a t s in e p i l e p s y r e s e a r c h . So s i n g l e doses of c a r b a m a z e p i n e (20 mg/kg p.o.) a n d v a l p r o i c a c i d (40 m g / k g i.v.) were administered to c a t s a n d the c o n c e n t r a t i o n s in plasma determined by HPLC and gaschromatographic methods, respectively. The plasma concentrations time-course for valproic acid could be described by a biexponential equation with elimination half live of 5 . 1 ± 0 . 4 h, for c a r b a m a z e p i n e we f o u n d a monoexponential equation with elimination h a l f l i v e s of 1 1 . 1 ± 1 . 8 h for the u n c h a n g e d drug and 22.8 ± 5.2 h for the active m e t a b o l i t e c a r b a m a z e p i n e epoxid. The individual pharmaeokinetic data were calculated, a n d on t h i s b a s e a d o s e r e g i m e n for c o n t i n u o u s treatment with both drugs was studied in order to maintain therapeutic p l a s m a c o n c e n t r a t i o n s in cats. 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 S c h o o l of V e t e r i n a r y M e d i c i n e F r e e U n i v e r s i t y of B e r l i n K o s e r s t r . 20 D - J 0 0 0 B e r l i n (West) 33

I0

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CELLULAR KINETICS AND EFFECTS OF PREDNIMUSTINE COMPARED TO ITS COMPONENTS CHLORAMBUCIL AND PRENISOLONE M. Malek, E. Musch, A. Werner, E. H6gl and H. J. Dengler.

PHARMACOKINETICS OF MORPHINE AND ITS SURROGATES: EFFECT OF SIMULTANEOUSLY ADMINISTERED NALTREXONE AND MORPHINE ON THE PHARMACOKINETICSAND PHARMACODYNAMICSOF EACH IN THE DOG PETER LANGGUTH*, PATRICIA J. KHAN+ AND EDWARD R. GARREI-I-+

Prednimustine (PM), the 2 1 - p r e d n i s o l o n e {P) e s t e r of c h l o r a m h u c i l (CLB), has d e m o n s t r a t e d different antitumour activity and toxicity compared to its c o m p o n e n t s . We i n v e s t i g a t e d the c e l l u l a r p h a r m a c o k i n e t i c s of PM v e r s u s C L B + P. E x p e r i m e n t a l t u m o u r cell l i n e s as w e l l as lymphocytes separated from patients with CLL were incubated w i t h e i t h e r PM or C L B + P. T h e cells exposed to PM exhibited higher and longer-lasting concentrations of PM and C L B c o m p a r e d to c e l l s i n c u b a t e d w i t h C L B + P. The alkylating potency ( = concentration-time i n t e g r a l s of all alkylating a g e n t s ) was not significantly different in both incubation series ( Z PM + C L B = 1087 nmol/ml.min v e r s u s CLB + phenylacetic acid mustard = 878 nmol/ml.min). Lymphocytes from healthy volunteers and lymphoma patients exhibited a m o r e p r o n o u n c e d u p t a k e of P M than of CLB. Thus, the r a t i o n a l e of a f a c i l i t a t e d cellular uptake of the h o r m o n e c o n j u g a t e PM might he confirmed. As an experimental approach in rive, PM and C L B w e r e i n j e c t e d at equivalent doses to rats bearing transplanted HH-9 m a m m a r y c a r c i n o m a s . PM was f o u n d in b l o o d and within the t u m o u r c e l l s and showed a longerlasting concentration profile than CLB combined with a higher inhibition of the malignant c e l l s in the t u m o u r c o l o n y - f o r m i n g

There were no dramatic modifications of the pharmacokinetics in the dog of iv bolus doses of 0.5, 2.7 and 5 mg/kg morphine by coadministedng iv 5 mg/kg naltrexone as bolus injections over 15-20 sec. and 12.3 mg/kg by continuous infusion. Morphine's terminal half-life, clearances, apparent volumes of distribution (except for that of the central compartment), percentages of drug and conjugated metabelite excreted in urine and bile did not differ significantly by paired "t" test (probability (P)>0.05 for rejection of the null hypothesis of no difference) when naltrexone was coadministared. There were no statistically significant (by "t" test) modifications of the plasma pharmacokinetics in the dog of iv bolus doses of 5 rng/kg naifrexone with and without morphine c.,oadministrationexcept for the coefficient of the second (or terminal) exponential of the sum that fitted the plasma concentration-time data of naltrexone. Although morphine coadministration did not significantly affect the terminal half-life of naitrexone, its clearances or apparent volumes of distribution by "t" test of the differences between averages (with each dog equally weighted), drug ¢oadministrationdid significantly affect the traction of naltrexone dose secreted into bile as conjugate (fB), the fraction of the dose excreted as conjugate in the urine, and the fraction excreted elsewhere (f'B). Although naltrexone reversed the central action of morphine in affecting monitored pupil diameterS, it did not antagonize the peripheral effects of morphine in pertubating renal and bilianj flow rates. This led to a larger fraction of the naltrexone dose being metabolized to conjugate on morphine coedministration. Since less naifrexone conjugate was renally and biliary excreted initially, due to morphine inhibition of the initial renal and biliary processes, naltrexone conjugate plasma concentrations were higher when morphine was coadministered.

assay.

* Supported by Land Nordrhein-Westfalen. Medizinische Universit~tsklinik, Sigmund-Freud-StraSe 25, D - 5 3 0 0 B o n n 1

*Dept. of Pharmaceutical Technology, Swiss Fed. Inst. Tech. (ETH), CH-8092 Z0rich, Switzerland, +The Beehive, College Of Pharmacy, University of Florida, Gainesvgle, FL 32610-0494, USA

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13

15

PHARMACOKINETICS OF ISOXSUPRINE IN THE HORSE AND ITS PHARMACOLOGICAL EFFECTS ON BLOOD VISCOSITY. B. Lubczyk (i) and A. Hashem (2) .............................................

CHANGES OF QUINOLONES (PEFLOXACINE) LEVELS IN SERUM AND TISSUES OF RATS UNDER THE INFLUENCE OF LIGHT. C. Tesseromatis, M.Loukissar G.Symeenoglou, and A.Pantazidis.

Isoxsuprine, an adrenaline related agent, is used in the treatment of a variety of ischaemic disorders because of its action on the peripheral blood vessels, relaxing their smooth muscles and lowering the blood viscosity. In equine medicine, isoxsuprine is widely used in the treatment of navicular disease and other movement disorders. The aim of the present study was to determine the pharmacokinetic parameters after oral and intravenous administration of isoxsuprine and to elucidate its pharmacological effects on the blood viscosity. The plasma half life was found to be 0.73 ± 0.25 hours after oral and 1.31 ± 0.15 hours after intravenous administration. Isoxsuprine decreased the blood viscosity in a plasma concentration related manner both after oral and intravenous administration. After intravenous administration some cardiovascular effects occured, but no side effects were observed after oral administration.

(I) Clinic of EqUine Diseases and General Surg~y, School of Veterinary Medicine,Fr~University0fB~lin.0e~zenw~ 19b.D-1000~rlin 37 (2) Depart~ntof Pharmac01~yand T0xicol0gLSchoolof VeterinaryMedicine, FreeUniversity0fBerlin.Keserstr.20. ~1000Mrlin 33

Quinolones (gyrase inhibitors, enzlnne needed for the replication of DNA), particularly the new generation, are chemotherapeutics with wide therapeutic spectrum, large distribution volume and advantageous properties. The proteinbinding of pefloxacine is 30%. The aim of the study is to investigate wether the light influences the Fharmacokinetic of quinolone and if there is a relationship between the s k i n p h o t 0 s e n s i t i v i t y and the overlocalisation of the substance to it. 30 wistar rats were used (weight 120±5 g.) divid: ed into three groups A,B,C (n= 10 animals in each group).The animals of group A lived in cages under normal conditions of lighting. The animals of group B lived in cages under ultraviolet lighting 24 h a day. The animals of group C lived in complete darkness 24 h a day. Pefloxacine was given i.m. 11mg/kg every 8 h for 48 h to obtain a study state concentration in plasma. The animals were sacrificed 2 h after the last administration. The level of pefloxacine was estimated in serum, skin and femur of the animals. The concentration of pefloxacine was estimated from the measurement of the inhibition zone of E. coli (Bennet 1966). The results demonstrate that pefloxacine levels in serum, skin and femur were decreased in the group of light exposure whereas group C (darkness) had increased levels. Evidently the metabolism of pefloxacine was faster in the light exposure groups. Department of Exp.Pharmacology, Athens 115 27 , Greece.

Medical School,

14

16

PHARMACOKINETICS OF (R)- AND (S)-CARVEDILOL AND THEIR GLUCURONIDES IN RATS WITH PORTOCAVAL SHUNT AFTER RACEMATE DOSAGE E. Stahl, U. Baumgartner*, D. KrauS, H. Spahn, J. Sch61merich*

A NEWANTICANCERAGENT- S 12363: PRE-CLINICAL SCREENING M. Berlion*, P. Deloffre, G. Lavielle, A. Pierre, G. Seurre

Carvedilol is a non-selective 8-adrenoceptor blocking agent with vasodilating activity. The B-blocking activity mainly resides in the S-enantiomer, while an u1adrenoceptor blockade was found for both enantiomers. The lipophllic compound carvedilol is highly metabolised to its glucuronides and Odesmethylcarvedilol. In order to determine pharmacokinetic parameters in dependence on reduced liver function, rats with a portocaval shunt were used as a model for cirrhotic liver disease. The concentration-time curves in the rats were compared with those of control animals. Both groups received a 10 mg/kg p.o. or i.v. dose racemic carvedilol. Plasma, urine and partly bile samples were collected over six hours. The carvedilol enantiomers were determined after alkaline extraction and derivatisation with R-(+)-phenylethyl isocyanate by HPLC with ituorimetdc detection. The carvedilol glucuronides were determined after cleavage with 8-glucuronidase. After p.o. as well as after Lv. administration the plasma concentrations of the R-enantiomer exceeded those of the S-enantiomer in both groups significantly. In comparison to the control group the plasma concentrations of both enantiomers increased (up to 100%) in rats with portocaval shunt while the differences between the stereoisomers decreased. The increase in the concentrations was more pronounced for the S-enantiomer, the actual 8adrenoceptor antagonist. Furthermore the pharmacokinetic parameters in rats with portocaval shunt showed less interindividual variability. Parent R- and Scarvedilol and their glucuronides were extensively excreted into the bile with preference for the S-enantiomers of both compounds (up to 5 fold). After p.o. administration the total amounts excreted into the bile were up to t0 fold higher than after i.v. injection. The results support the assumption that the low bioavailabUity (20-25%) of carvedilol is a result of an extensive first-pass effect by the liver with preference for the S-enantiomer of the parent drug as well as of the glucuronide. Pharmakologisches Inatitut fQr Naturwissenschaftler, Johann Wolfgang Goethe-Universit&t, Theodor-Stem-Kai 7, Geb&ude 75A, D-6000 Frankfurt/M. *Medizinische Universit&tsklinik, Hugstetterstr. 55, D.7800 Fraiburg

The new anticancer agent S 12363 (chemical name: 1 - [3 (04- deacetyl - 3 - demethoxycarbonylvincaleukoblastyl) carbonylamino] - 2 - methyl propylphosphonic acid, diethylester) is a vinca-alkaloid derivative characterized by the presence of an aminophosphonate bioisoster of valine which enhances its intracellular uptake by the tumorous cell. This compound shows a high stereospecificity which leads to three main advantages. 1. It exhibits a very potent cytotoxic activity: - an in vitro activity 25 to 80 times increased compared to reference compounds (microculture tetrazolium assay), - a considerable antitumoral activity in vivo in particular on the P388 leukemia, L1210 leukemia, B16 melanoma, 38 colic adenocardnoma models and human colic adenocarcinoma CX-1 with optimum doses 10 to 20 times lower than those of the reference compounds, - a spectrum of activity on human cell lines in vitro close to vinblastine, - a pronounced antimetastatic activity on 3 disseminated models: the P388 leukemia inoculated subcutaneously, the M5076 reticulum sarcoma and the B 16 melanoma inoculated intravenously, a large therapeutic index, a lower cross resistance towards resistant cell lines in vitro and in vivo than reference compounds. 2. Its mechanism of action, which is more specific than that of the reference compounds, involves the inhibition of microtubules assembly and is characterized by: a more rapid cytotoxic activity (x 3 to 10), a more efficient blockage of the cells in M phase, a magnitude (double at concentration 10 to 20 times less) and a kinetic (x 8) of spiraiization highly specific without expressing detectable neurotoxicity (as it has been shown in a preliminary toxicological study in animals receiving repeated administrations). 3. These remarkable advantages in terms of posology and cellular targeting are tightly bound to its highty stereospedficity leading to: - the same level of pharmacological activity at a dose 1 000 times less, as compared to its epimere, - an in vitro cytotoxic activity 20 to 70 times more potent, - a cross resistance 1.5 to 3.5 less, - a spirilization markedly different -

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*Present address: I.R.l.Servier, 27 rue du Pont, F - 92200 Neuilly/Seine

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19 INTERACTION OF ETRETINATE AND CORTICOSTEROIDS EPIDERMAL M E T A B O L I S M M. Kietzmann and D. Lubach*

17 PHARMACOLOGICAL PROPERTIESOF THE NEWANTI-CANCER DRUG FOTEMUSTINE: CLINICAL IMPLICATIONS J. P. Bizzari*, V. Cour and M. 8r0ch Fotemustine is a new 2-chloroethyl nitrosourea with an amino ethyl phosphonic acid grafted onto a nitrosourea radical in order to facilitate its cell penetration (especially in c a n c e r ceils) aiming at improvement of its antitu mor activity, and - to increase the rate of its passage across the blood-brain barrier (optimal octanol/water partition = log P of 1.25) aiming at treatment of cerebral metastases.

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@ In pharmacology, this drug has been tested on the tumor panel screen of the National Cancer Institute and shows considerable antitumor activity both in vitro and in vivo in experimental models of melanoma, cerebral tumors and visceral metastases. Its mechanism of action, although qualitatively identica) to that described for other nitrosoureas, basically differs from BCNU (the reference compound) by an inhibition of DNA synthesis at a later step, by more significant changes in cell cycle kinetics at lower dose and by fewer single strand breaks in the DNA and lower carbamoylating activity. Toxicity for the liver and the immunological system is reduced compared to BCNU. @ In toxicology, studies performed on mouse, rat, dog and monkey confirm the hematotoxicity observed with this class of agents without any new specific fotemusti ne related toxicity. Compared to BCNU, fotemustine has a lower genotoxic activity, a tower mutagenicity in the Ames test, reverse mutation test and micronudeus test and a lower transforming effect in the cell transformation test. @ In pharmacokinetics, studies conducted in mouse, monkey and dog confirm the excellent distribution of fotemustine (refleCting the lipophilia of the molecule), the very intense chemical reactivity (short half-life) and the rapid and intense uptake of fotemustine in the tumor tissue. Based on these results, this compound entered into clinical research and is being investigated in patients with malignant melanoma and brain tumors. * Present address: Institut de Recherches Internationeles SERVIER,27 rue du Pont, F-92200 Neuilly/Seine, France

Interactions between etretinate (ETR) and glucocorticoids were examined in female NFLRI-mioe. Using various dosages, mice (n - 6 per group) were treated orally with ETR and subcutaneously with prednisolone (PRE). The substances were also administered together (ETR/PRE). Additionally, ETR was combined with topically administered fluocinolone acetonide ointment (FLU, 0,025 %). After 3 to 21 days of treatment, tail skin was sampled. The workup of skin specimens was followed by the measurement of the incorporation rate of 3H-thymidine, 3H-thymidine triphosphate~ ~H-leucine, OH-histidine and a mixture of leC-amino acids. Additionally, keratin protein fractions were examined electrophoretically. Within some days, PRE (5 to 25 mg/kg) diminished the incorporation rate of 3H-thymidine and 14Camino acids. ETR (2 to 5 mg/kg) induced an initial increase of the ~H-thymidine incorporation rate. Compared to control values, the ETR/PRE combination induced no significant changes. Confirming these results, antagonism was found also between ETR and topically administered FLU. The anti-keratinizing potential of ETR was documented by electrophoresis of the keratin proteins, while FLU remained without any effect. ETR induced changes of keratin pattern were not influenced by FLU either. The r e s u l t s demonstrate an antagonism of glucocortiooids and ETR. The clinical importance of these findings remains to be examined. Institut fur Pharmakologie, Toxikologie und Pharmazie, Tier~rztliche Hochschule a~d *Hautklinik Linden, D-3000 Hannover

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18 POLIDOCANOL AS INHIBITOR OF PBP2' SYNTHESIS STAPHYLOCOCCUS AUREUS S°Spaltenburg, H.Keppeler, and W.Bruns

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Methicillin resistance in S.aureus can be suppressed with polidocanol (PDO), a dodecyl polyethyleneoxid ether (W.Bruns et al., Antimicrob. Agents Chemother.27(1985)632-639). Resistant strains produce an additional penicillin-binding protein (PBP2') with low affinity for penicillins. PBP2' can take over functions of the other PBPs, thus becoming the only one essential for bacterial growth in presence of penicillins. It may therefore be a target for the action of PDO. The influence of PDO on the expression of PBP2' was studied with the methicillin-resistant S.aureus 5814R. Its MIC of 2000 mg/l of methicillin was reduced to 2 mg/l of methicillin in the presence of 100 mg/l of PDO. PDO alone was not inhibitory for this strain up to 10 g/l. PBPs were analyzed by SDS-PAGE and fluorography after labelling of cells with 3H-benzylpenicillin. Besides the usual PBPs strain 5814R produced the additional PBP2' which was only detectable with high concentrations of ~H-benzylpenicillin (100 ~mol/l). The synthesis of PBP2' was inducible by penicillins. By growth in presence of PDO the production of PBP2', uninduced or induced, was strongly inhibited. With 25 mg/l of PDO inhibition was more than 50%, This could be increased to about 80% by raising the PDO concentration to 100 mg/l. In contrast, influence of PDO on the synthesis of the other PBPs was negligible. In this way it was possible for the first time to suppress methicillin resistance in staphylococci by rather specific inhibition of PBP2' synthesis. Institut fHr Pharmakologie der Universit~t zu K~in, Gleuelerstrasse 24, D-5000 K61n 41

STRAIN-RELATED DIFFERENCES IN THE EFFECTS OF BEZAFIBRATE ON SERUMLIPIDS AND HEPATIC PEROXISOMESIN RATS U. Werner+, K. Beier, A. V61kl, F. Hartiq* and H,D. Fahimi Since most hypolipidemic drugs including bezafibrate (B) induce proliferation of peroxisomes (Pc) in rodents and i t has been suggested that the l i p i d lowering effect and the peroxisomal (Pc) proliferation are causally related, we investigated the effects of B (10 and 50 mg/kg/d, 7 d) on serum lipids and hepatic Pc in male rats of Sprague-Dawley (SD) and Lewis (L) strains. In SD rats B lowered the serum cholesterol (CH) markedly at a dosage of 10 mg/kg/d, whereas in L rats a comparable effect was observed at 50 mg/kg/d. Serum triglyceride (TG), however, were lowered more significantly in L than in SD rats. The l i v e r weights were increased in a dose-dependent manner in both strains, with the L rats showing a stronger response. The reduction of CH and TG in l i v e r was maximal in the low dosage group in L rats but showed dose-dependency in SD rats. The increase in hepatic carnitine-acetyl-transferase a c t i v i t y was dose-dependent in both strains. A similar effect was observed also for relative specific a c t i v i t y (RSA) of hepatic 8-oxidation in L rats, whereas in SD rats an elevation was found only in the high dosage group. In untreated control rats RSA of catalase is about 50% higher in L than in SD rats. In SD rats treated with 10 mg/kg/d i t increased but returned to control values in the group treated with 50 mg/kg/d. In L rats RSA of catalase was unaffected in the low dosage and was reduced in the high dosage group. Morphometric analysis revealed higher volume density of Pc in untreated L than SD rats with corresponding increases after B treatment. These observations demonstrate that the hypolipidemic effects induced by B vary significantly in different rat strains and do not correlate with the proliferation of hepatic Pc indicating that there is no direct relationship between these two effects. +ASTA Pharma AG, Dep. of Pharmacology, Frankfurt; Inst. of Anatomy and Cell Biologie I I , Univ. Heidelberg; *Medical Res. Dept. Boehringer Mannheim ,6800 Mannheim 31 (all FRG)

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UPTAKE AND METABOLISM OF NUCLEOSIDES IN RAT LIVER N.Frijus-Plessen, H.C.Michaelis, M.R.Steinwachs and G.F.Kahl At present, the thymidine (AZT)

pyrimidine derivatives 3'-azido-3'-deoxyand 2',3'-dideoxycytidine (ddCyt) are the

most active pyrimidines used for the treatment of the HIV infection Other modified nucleosides as 3'-flu0r0-3'-de0xythymidine (FT) ale0 e x h i b i t potent c y t o s t a t i c a c t i v i t y in

vitro. We studied the elimination k i n e t i c s of FT, ddCyt and AZT in rat liver and compared these d a t a with those of the c y t o s t a t i c pyrimidine 5 - f l u o r o - 2 ' - d e o x y u r i d i n e (FUDR) which is e x t e n s i v e l y metabolized by the liver. At an i n i t i a l concentration of 4 l~M, FT is eliminated from the perfusion circuit of isolated rat l i v e r with low clearance v a l u e s of 0.13 + 0.03 ml/min and long elimination h a l f - l i v e s of 13 +_ 4 h. Very similar elimination v e l o c i t i e s are observed for ddCyt (5 - 7 laM) with clearance v a l u e s of 0.18 + 0.03 ml/min and elimination h a l f - l i v e s of 8 + I h. The corresponding parameters for AZT indicate a f a s t e r hepatic e l i mination: clearance amounting to 0.71 ml/min and elimination h a l f - l i v e s amounting to 2 h. Removal of FUDR markedly exceeds these r a t e s with 10fold higher clearances (7.4 + 1.3 ml/min) and shorter elimination h a l f - l i v e s (0.05 + 0.02 h). Nucleoside uptake across the cellular membrane was studied in i s o l a t e d hepatocytes. An i n t r a - to e x t r a c e l l u l a r concent r a t i o n ratio of unmetabolized ddCyt of 0.6 + 0.1 is observed at concentrations of 80 - 200 tiM. The corresponding v a l u e s for 3H-AZT (70 - 200 llM) are s l i g h t l y lower (0.4 + 0.1). The t o t a l AZT-derived r a d i o a c t i v i t y reached equilibrium r a t i o s of 1.0 + 0,1 i n d i c a t i n g t h a t AZT metabolites r e p r e s e n t about 60 % of i n t r a c e l l u l a r a c t i v i t y . However, for FUDR the fraction of c a t a b o l i t e s exceeds 90 % of the t o t a l i n t r a c e l l u lar r a d i o a c t i v i t y with almost no d e t e c t a b l e i n t r a c e l l u l a r FUDR. Our r e s u l t s demonstrate t h a t hepatic elimination of FT, ddCyt and also AZT is limited by uptake and metabolism. This is in c o n t r a s t to the r e s u l t s obtained with FUDR, a pyrimidine d e r i v a t i v e containing an unmodified ribose.

METABOLISM OF CIPROFLOXACIN IN MAN: STRUCTURAL CHARACTERIZATION OF AN UNKNOWN METABOLITE. W. Raasch and F. Kees

Ciprofioxacin is a new 6-fluoro-7-piperazino-4-quinolone carboxylic acid with a broad spectrum of antibacterial activity. In vivo, ciprofloxacin is partially metabolized, and four metabolites (M1-M4) have been identified in man (Gau et al., Arzneim.Forsch./Drug Res. 36 (II), 1545 (1986)). In addition, traces of further unknown metabolites were found (c.f. Borner und Lode, Infection, 14 (Suppl. 1). 54 (1986); Myers and Blumer, J. Chromatogr., 422, 153 (1987)). The plasma concentration-time course of one such metabolite has been qualitatively described in patients (Kees et al., Arzneim.-Forsch./Drug Res. 39 (I). 523 (1989)). This unknown metabolite has been now isolated from urine of patients by high-performance liquid chromatography, and characterized by its chromatographic, spectrophotometric and electrochemical behaviour. As chemical structure an intermediate form between the two known metabolites M3 and M1 with cleaved piperazin-ring is proposed. The amount found in urine was estimated to be less than 1 % of ciprofloxacin doses administered. Department of Pharmacology, University of Regensburg, Universit/itsstraJ3e 31, D-8400 Regensburg, FRG.

I n s t i t u t fiir Pharmakologie und Toxikologie, Universit~it GSttingen, R o b e r t - K o c h - S t r a s s e 40, D-3400 GSttingen

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M E T A B O L I C D I S P O S I T I O N OF R A C E M I C B E X O B A R B I T A L AND OF ITS E N A N T I O M E R S IN THE ISOLATED P E R F U S E D RAT L I V E R U N D E R B T H ~ O L LOAN B. Stahlhacke, R. Sch~ppel Acute e t h a n o l load has g e n e r a l l y b e e n shown to inhibit b a r b i t u r a t e h y d r o x y l a t i o n and inactivation - e x c e p t that of h e x o b a r b i t a l (i). E l i m i n a t i o n kinetics of h e x o b a r b i t a l in rats was not a l t e r e d by a m o d e r a t e dose of ethanol, and sleeping time in these experiments was only slightly p r o longed (2). - This interaction has been studied in detail using the i s o l a t e d p e r f u s e d rat liver preparation. E l i m i n a t i o n of r a c e m i c h e x o b a r b i t a l and of its R - / S + - e n a n t i o mere (3) and the formation of three m a j o r h y d r o x y l a t e d metabolitee have been followed in the p r e s e n c e of ethanol

ETHYL ETHERS OF 2-NAPHTHOL AND ?-QUINOLINOL IN COMPARISON TO THAT OF UMBELLIFERONE AS SUBSTRATES OF THE MURINE HEPATIC MONOOXYGENASE SYSTEM Feil F, Tegtmeier M, Netter KJ The O-deethylatlon of 7-ethoxycoumarin (7EC) and 7-hydroxylaton of coumarin, both leading to umbelliferone as product, are model reactions for the measurement of cytochrome P-450 dependent monooxygenase activities. This study presents physicochemical and metabolic properties of the ethyl ethers of 2-naphthol and 7-quinollnol (i.e. 2EN, 7EQ) as structural analoges of 7EC. A b o v e a pH of 9 the free phenols show a strong increase in their fluorescences compared to their ethers. This allows a selective determination of the products using a fluorlmetric technique according to Aitio (1978). The wavelengths of the m a x i m a of excitation and emission measured at a pH of 10.5 in glycine-NaOH buffer (pH 12 for naphthols in phospate) are: umbelliferone (390/440; 16.6), 7-quinolinol (370/510; 45), 2-naphthol (355/420; 67). The third parameter refers to the intensity of emission of the phenolates in comparison to those of the ethyl ethers measured under the same conditions. The E X / E M wavelenghth pairs of the ethyl ethers are: 7EC (340/390), 7 E Q (330/380) and 2 E N (325/350). The ethers were tested as substrates of the hepatic microsomal monooxygenase of three different strains of untreated male mice: B6, D2 and NMRI. Using a substrate concentration of 10-4M (5.10-4M for 2EN) the phenolic products were quantified fluerlmetrlcally after a reaction time of 10 rain (2 rain for 2EN). The highest molecular activity expressed as "nmol product/nmol P-450.min" is found for 7 E Q (24 NMRI, 45 B6 and 78 D2) followed by 7EC (11, 17, 57) and by 2 E N (7, 13, 10). D2 mice of a genetically high coumarin 7-hydroxylase activity show high deethylase activities towards 7 E Q and 7EC, however, they poorly metabolize 2EN. This clearly indicates an independence of these enzymatic activities. O n the other hand. 2 E N does not seem to be a suitable substrate, since the 2-naphthol formed acts as a strong inhibitor of the metabolic reaction (app. KI about IO-~M). E n z y m e kinetic parameters with 2 E N were therefore determined indirectly. Dept. PharmacoL,Philipps-University, Lahnberge, 355 Marburg

(initial concentration range: 60 raM). Parent drugs and metabolites in the perfusate were determined using a h.p.l.c.-assay. - Kinetics of racemic hexobarbital as well as both of its enantiomers have been found unaltered by ethanol, confirming the above results (found in vivo). Further, peak concentrations of 3'-keto-hexobarbital and of 1,5-dimethyl-barbiturie acid were significantly reduced by ethanol. However, peak concentrations of 3'-a-HO-hexobarbital and 3'-g-HO-hexobarbital, the primary hydroxylation products, were found to be raised by up to 85 and 45% of controls, respectively. - Testing both enantiomers in the system, kinetics of primary hydroxylated (3'-HO-hexobarbital) and of secondary (3'-keto-hexobarbital) metabolites were differently affected in the R- and S+-series reflecting a complex pattern of metabolic interaction with ethanol. - In conclusion, ethanol fails to inhibit the primary hydroxylating step in the disposition of hexobarbital, but does inhibit subsequent reactions to occur within the hepatic cytochrome-P-450 isoenzyme system. i. Sch~ppel,R.; Petruch,F. Arch. Pharmacol.270, R-129 (1971) 2. Kuthe,C.; Sch~ppel,R. Arch. Pharmacol. 311, R-16 (1980) 3. R-/S+-Hexobarbital was a gift of Prof. Dr. Knabe, Dept. Pharmaceut. Chem., Universit~t des Saarlandes. Inst. f~r Pharmakol. & Toxikol., Techn. Universit~t D-33OO Braunschweig, MendelssohnstraBe i

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EFFECTS OF Coh-LOCUS INDUCERS ON THE DEETHYLATION OF THE ETHYL ETHERS OF 2-NAPHTHOL, 7-QUINOLINOL AND UMBELLIFERONE IN MICE H a h n e m a n n B, L e g r u m W Earlier reports described the murine coumarin 7-hydroxylase a s b e i n g i n d u c i b l e more s e l e c t i v e l y b y t h e h e t e r o a r o m a t i c c o m p o u n d s a m i n o t r i a z o l e (AT), p y r a z o l e (PL), p y r a z i n e (PN) and the heavy metals cobalt and indium than by standard i n d u c e r s PB a n d MC ( H a h n e m a n n e t al., A r c h T o x i c o l 13 Suppl, 297, 1989). In a d d i t i o n t w o s t r u c t u r a l a n a l o g e s of 7-ethoxycoumarin (7EC), 2-ethoxynaphthalene (2EN) and 7 - e t h o x y q u i n o l i n e (7EQ) w e r e i n t r o d u c e d a s s u b s t r a t e s of t h e cytochrome P-450 dependent O-deethylase measuring f l u e r i m e t r i c a l l y t h e f r e e p h e n o l i c p r o d u c t s (cf. Fell e t al.; M a n g o u r a e t al., t h i s i s s u e ) . The p r e s e n t s t u d y i n v e s t i g a t e s t h e i n d u c t i v e e f f e c t s of t h e C o h - l o c u s i n d u c e r s in c o m p a r i s o n w i t h t h o s e of s t a n d a r d i n d u c e r s on t h e O - d e e t h y l a t i o n r a t e s of t h e t h r e e e t h y l e t h e r s a n d o n t h e 7 - h y d r o x y l a t i o n o f c o u m a r i n . Male B6 mice (6 p e r g r o u p ) w e r e p r e t r e a t e d a s follows: AT, PL, PN (200 m g / k g b . w t . , i,p.), CoC12 (40 m g / k g , s.c.), PB (80 m g / k g , i.p.), MC (30 m g / k g , i.p.) d a i l y f o r two d a y s a n d i n d i u m s u l f a t e (100 m g / k g , s.c.) o n c e f o r t w o d a y s . As e x p e c t e d t h e m e t a b o l i s m s o f 7EC a n d c o u m a r i n a r e i n creased after the Coh-inducers, while the standard inducers h a r d l y a f f e c t t h e i r m e t a b o l i s m . For, 7EC t h e fold o f i n d u c t i o n w a s : PL(3.5)> Co,PN(3.0)> AT(2.5)> In(2.4)> MC(1.6)> PB(1.4). T h e d e e t h y l a t i o n of 7EQ is s i m i l a r l y b u t l e s s e n h a n c e d b y t h e i n d u c e r s : In(2.4)> PL(2.3)> PN(2.1)> Co(2.0)> AT(1.9)> MC(1.7)> PB(1.5). On t h e o t h e r h a n d t h e d e e t h y l a t i o n of 2EN is p r a c t i c a l l y n o t i n f l u e n c e d b y t h e i n d u c e r s t e s t e d . T h e r e f o r e 7EQ is t h e f i r s t e x a m p l e o f a s u b s t r a t e n o t b e i n g a c o u m a r i n d e r i v a t i v e of w h i c h t h e m e t a b o l i s m is e n h a n c e d after Coh-inducers. H o w e v e r , in c o m p a r i s o n to 7EC a n d c o u m a r i n t h e s p e c i f i c i t y of 7EQ t o w a r d s C o h - l o c u s d e p e n d e n t m o n o o x y g e n a s e s is s m a l l e r . 2EN although structurally a n a l o g o u s to 7EC - is n o t s u i t a b l e to d e t e c t a C o h - t y p e of induction. Dept. P h a r m a e o l . , P h i l i p p s - U n l v e r s i t y , L a h n b e r g e , 3 5 5 M a r b u r g

IMPAIRMENT BY DIETHYLENEGLYCOLMONOETHYLETHER OF RAT LIVER MICROSOMAL DRUG-METABOLIZING ENZYMES F.E.Beyhl*, A.Aydogmu~**, E.Arinq** .......................................................... The effects of diethyleneglycolmonoethylether (DEG) on microsomal, cytochrome P-450-dependent mixed-function oxidases catalyzing ethoxycoumarin O-deethylation, methylayapanine O-demethylation, p-nitrophenol hydroxylation (a substrate specific for a particular, ethanol-inducible cytochrome P-450 isozyme), ethylresorufin O-deethylation (a substrate specific for cytochrome P-448), and aniline p-hydroxylation (a type II substrate) as well as of microsomal, NADPH-dependent cytochrome c and neotetrazolium reductases and a series of glucuronyltransferases (GT I, with methylumbelliferone as the substrate; GT II, with 4-hydroxybiphenyl as the substrate; GT III, with phenolphthalein as the substrate) were studied in rat liver microsomes in vitro. DEG decreases mixed-function oxidase activities concentration-dependent but only weakly (ICo0s between 0.02 mM and 1.5 M), and also the activities of cytochrome c reductase (ICL0 about 1.2 M) and of GTs I and II (IC~0 about 45 and I00 mM, reap.). Both neotetrazolium reductase and GT III as well as glueose-6-phosphatase (an enzyme not involved in drug metabolism) are not affected by DEG, even in concentrations higher than 0.3 M. This kind of impairment of enzymic activity which can also be observed with high concentrations of glycerol and dimethylsulphoxide, is a phenomenon quite different from "ordinary" ("true") enzyme inhibition where ICs0 will lie far below i0 mY.

* Hoechst AG, D-6230.Frankfurt/Main (FRG) ** Dept.of.Biol.Sciences, Middle East Technical University Ankara (Turkey)

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VARIATION OF THE SIDE CHAIN OF 7-ALKOXYQUINOLINES AND THE INDUCTION OF THEIR DEALKYLATION BY Coh-LOCUS INDUCERS IN MICE M a n g o u r a SA, M a y e r RT*, H e u b e l F Recently Hahnemann and Legrum (this issue) reported that inducers of the coumarin 7-hydroxylase (Coh) a r e a b l e to e n h a n c e t h e m e t a b o l i s m of 7 - e t h o x y q u i n o l i n e (7C2Q = 7EQ) besides that of 7-ethoxycoumarin. The deethylation of 2-ethoxynaphthalene, however, remained unaffected. Cohlocus inducers include N-containing heteroaromates as a m i n o t r i a z o l e (AT), p y r a z o l e (PL) a n d p y r a z i n e (PN) a n d t h e h e a v y m e t a l s c o b a l t a n d i n d i u m . T h e s e c o m p o u n d s selectively induce the monooxygenases depending upon the Coh-locus ( H a h n e m a n n e t al., A r c h Toxieol 13 Suppl. 297, 1989). In o r d e r t o e s t a b l i s h t h e o p t i m a l s u b s t r a t e t h e p r e s e n t s t u d y investigates the dependence of the 7-n-alkoxyquinoline O - d e a l k y l a t l o n on 1) t h e c a r b o n n u m b e r of t h e e t h e r s i d e c h a i n ( n = l to 6) a n d on 2) t h e p r e t r e a t m e n t o f m a l e D2 mice w i t h C o h - i n d u c e r s . T h e p r e t r e a t m e n t s c o n s i s t e d of two i.p. a d m i n i s t r a t i o n s o f AT, PL, PN, (200 m g / k g b.wt.) o f CoClz (40 m g / k g s.c.) d a i l y f o r t w o d a y s a n d of 100 m g / k g of i n d i u m s u l f a t e (s.c.) o n c e f o r t w o d a y s . A f t e r a r e a c t i o n t i m e o f 10 rain ( s u b s t r a t e conc. lO-4M) the product 7-quinolinol was q u a n t i f i e d f l u o r l m e t r i c a l l y a t a pH of 10.8. In c o n t r o l mice maximum O-dealkylation r a t e s o c c u r w i t h 7Czq a n d 7Csq ( b o t h 47 nmol 7 - q u i n o l i n o l / n m o l P - 4 5 0 . m i n ) , w h i l e e l o n g a t i o n of the chains diminishes the metabolic rate gradually. Pret r e a t m e n t b y PB or MC o n l y s l i g h t l y i n c r e a s e s t h e 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 . H o w e v e r , t h e i n d u c e r s o f t h e C o h - l o c u s I n c r e a s e u p to 4 - f o l d t h e d e a l k y l a t i o n of 7C,Q a n d 7czQ (PN>Co>PL>AT>control>In), w h i l e t h a t of l o n g e r s i d e c h a i n s is d i m i n i s h e d (PL. AT) or u n a l t e r e d (PN, Co). K i n e t i c s t u d i e s r e v e a l t h a t t h e a f f i n i t y a n d vmax of t h e d e a l k y l a s e s are i n c r e a s e d t o w a r d s 7 C , q a n d 7C2Q a f t e r C o h - l n d u c e r s a n d PB, b u t n o t a f t e r MC. A f f i n i t i e s t o w a r d s 7CaQ a n d 7C4Q a r e lowered generally. Therefore it is possible that the m e t a b o l i s m o f 7C, q a n d 7C~Q is c o n t r o l l e d b y t h e g e n e s of the Coh-loeus. Dept. P h a r m a e o l . , P h i l i p p s - U n i v e r s i t y , L a h n b e r g e , 3 5 5 M a r b u r g *US H o r t i c u l t . R e s e a r c h Lab., 2 1 2 0 C a m d e n Road, O r l a n d o FL

Tyramine Metabolism in G a s t r o i n t e s t i n a l (Gl) E p i t h e l i a l C e l l s of the Guinea Pig. M. S c h w e n k I, E. N i l s s o n 2, E.K. S c h m i d t ~ Introduction: In p a t i e n t s on M A C i n h i b i t o r (MAOi) therapy, the intake of tyramine-rich m e a l s can lead to d a n g e r o u s r i s e s of b l o o d pressure. T h i s is due to e x c e s s i v e a c c u m u l a t i o n of n o r e p i n e p h r i n e w i t h i n a d r e n e r g i c n e u r o n s . MAOI a l s o p r e v e n t i n a c t i v a t i o n of t y r a m i n e in the G I - t r a c t . T h i s adds to the i n c r e a s e d p r e s sot s e n s i t i v i t y . H o w e v e r , it is not k n o w n h o w m u c h e a c h cell type of t h e s e o r g a n s c o n t r i b u t e s to d e a m i n a t i o n . Methods: C e l l s w e r e i s o l a t e d f r o m the stomach, small intestine, large i n t e s t i n e and liver of m a l e g u i n e a pigs (200 g). V i a b i l i t y w a s c h e c k e d by o x y g e n c o n s u m p t i o n m e a s u r e m e n t and e l e c t r o n microscopy. Cells were incubated with tyramine (euM) and the c o n v e r s i o n to h y d r o x y p h e n y l acetic a c i d w a s s t u d i e d b y HPLC. Results: All cell t y p e s d e a m i n a t e d t y r a m i n e to HPAA. But the r a t e s d i f f e r e d c o n s i d e r a b l y . The h i g h e s t r a t e s (nmol x m g prot -I x m i n -I) w e r e o b s e r v e d in h e p a t o c y t e s (43). In the s t o m a c h , the h i g h e s t r a t e s w e r e s e e n in p a r i e t a l c e l l s (40) f o l l o w e d b y m u c o u s cells (17) and chief cells (12). The a c t i v i t y in small i n t e s t i n a l cells w a s c o n s i d e r a b l y lower (4). C o l o n o c y t e s s h o w e d h i g h e r a c t i v i t y (21), A d d i t i o n of b r o f a romine, a s e l e c t i v e i n h i b i t o r of M A O - A at a d o s e of 500 u M i n h i b i t e d m o r e t h a n 90% in all cell t y p e s (93% in h e p a t o c y t e s ) . C o n c l u s i o n : C e l l s f r o m all p a r t s of the G I m u c o s a c o n t r i b u t e to d i f f e r e n t d e g r e e s to the first p a s s d e g r a d a t i o n of n u t r i t i o n a l t y r a m i n e . ~Abt. Allg. P h a r m a k o l o g i e , MHH, Hannover = H u m a n p h a r m a k o l . Inst., C i b a - G e i g y , T ~ b i n g e n .

R8 29

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C O F A C T O R R E Q U I R E M E N T A N D INHIBITOR PROFILES O F C A R B O N Y L R E D U C I N G E N Z Y M E S IN KIDNEY O F RODENTS. U. Oppermann and E. Maser

S T E R E O S E L E C T I V E G L U C U R O N I D A T I O N O F S- A N D R - N A P R O X E N BY 8 G L U C U R O N O S Y L T R A N S F E R A S E S

K i d n e y c y t o s o l a n d m i c r o s o m e s of f e m a l e W i s t a r r a t s a n d NMRI mice w e r e i n v e s t i g a t e d in o r d e r to e v a l u a t e and characterize carbonyl reduction, using the ketone metyrapone (MP). In p r e v i o u s i n v e s t i g a t i o n s we f o u n d t h a t MP is r e d u c e d in l i v e r c y t o s o l a n d m i c r o s o m e s b y t w o d i f f e r e n t e n z y m e s , w h i c h d i f f e r in c o f a c t o r r e q u i r e m e n t , i n h i b i t o r s e n s i t i v i t y a n d i m m u n o l o g i c a l h o m o l o g y . While t h e c y t o s o l i c e n z y m e w a s characterized as a ketone reductase, the microsomal enzyme t u r n e d o u t to be a n a l d e h y d e r e d u c t a s e w i t h r e s p e c t to i n h i b i t o r c l a s s i f i c a t i o n of c a r b o n y l r e d u c t a s e s . Moreover, t h e latter was competitively inhibited by 3-oxosteroids. Investigating the kidney, known as one major carbonyl r e d u c t i o n s i t e in m a m m a l s b e s i d e s t h e l i v e r , we f o u n d : (1) C y t o s o l i c M P - r e d u c i n g e n z y m e s s t r i c t l y r e q u i r e NADPH, w h e r e a s m i c r o s o m a l M P - r e d u c t a s e s c a n u t i l i z e e i t h e r NADH or NADPH a s e l e c t r o n d o n o r , r e s e m b l i n g t h e s i t u a t i o n in l i v e r . (2) C y t o s o l i c M P - r e d u c t a s e s are inhibited by quercitrin s u g g e s t i n g t h e s e e n z y m e s to be k e t o n e r e d u e t a s e s . In contrast microsomal MP-reductases are affected by phenobarbitone, indicating that these proteins are aldehyde r e d u c t a s e s . T h i s is a l s o f o u n d in l i v e r . (3) C y t o s o l i c s p e c i f i c a c t i v i t y in mice is 2 - f o l d h i g h e r in k i d n e y t h a n in l i v e r , w h e r e a s ra~ k i d n e y h a s o n l y h a l f of t h a t in r a t l i v e r . H o w e v e r , m i c r o s o m a l M P - r e d u c t i o n is low in k i d n e y c o m p a r e d to t h a t in l i v e r of b o t h s p e c i e s . (4) 8 - o x o s t e r o i d s inhibit only weakly, suggesting that a n d r o g e n i n a c t i v a t i o n is n o t t h e p h y s i o l o g i c a l f u n c t i o n a s i t s e e m s to be t r u e f o r l i v e r m i c r o s o m a l M P - r e d u c t a s e . (5) A n t i b o d i e s r a i s e d a g a i n s t m i c r o s o m a l m o u s e l i v e r MPr e d u c t a s e s h o w no i m m u n o r e a c t i v i t y w i t h a n y f r a c t i o n of r a t or m o u s e k i d n e y , a l t h o u g h t h e r e is a n t i g e n i c c r o s s r e a c t i v i t y b e t w e e n t h e l i v e r m l c r o s o m a l p r o t e i n s of r a t a n d mouse. In c o n c l u s i o n we f o u n d a s i m i l a r s i t u a t i o n in k i d n e y a s in liver concerning cofactor requirement and diagnostic inhibitor p r o f i l e , b u t k i d n e y a n d l i v e r M P - r e d u c t a s e s s e e m to p l a y d i f f e r e n t r o l e s in p h y s i o l o g i c a l m e t a b o l i s m . 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 , L a h n b e r g e , 855 M a r b u r g

PURI-

In a previous study we reported of the carbonyl reductase activity in several species using the enantiomers of warfarin (W) and some of its analogues as substrate (Eermans JJR, Thijssen HHW, Biochem Pharmacol 38: 3365-3370, 1989). The reduction of the acetonyl side-chain of W is performed by both microsomal and cytosolic enzymes, displaying substrate- as well as product stereoselectivity. Based on the similar pattern observed in the response on the substitution of the 4'H of W the cytosolic reductases in the various species (including man) are probably closely related. A rabbit liver cytosolic W reductase has been purified 130 fold, using ammoniumsulfate fractionation, DEAE chromatography and gel-filtration. The enzyme displayed very low affinity towards (weak) anion- and cation exchangers and is sensitive for biodegradation during processing at O°C. The formation of W alcohols was assayed by HPLC. The reductase exhibited strong substrate- as well as product stereoselectivity: i.e. the R-enantiomers were reduced to the RS alcohols. The reaction had a pB optimum of 7.0. The S-enantiomers, being reduced to the SR alcohol, proved to be a very poor substrate for the reductase: alcohol formation rates were 400 to 5,000 fold higher for the R- than for the S-enantiomers, depending on the W analogue used. S-enantiomers were very weak inhibitors of the reduction of the R-enantiomers (less than 10Z at an inhibitor concentration of 2.0 mM and a substrate concentration of 0.2 mM). Dept. of Pharmacology, University of Limburg, P.O. 616, 6200 MD Maastricht, the Netherlands

Multiple i s o z y m e s of t h e g l u c u r o n o s y l t r a n s f e r a s e (GT) family have been characterized, with little information a b o u t t h e i r s t e r e o s e l e c t i v i t y . Racemic n a p r o x e n , o n e o f t h e n o n s t e r o i d a l a n t i - i n f l a m m a t o r y d r u g s w h i c h is m a i n l y e x c r e t e d a s a c y l g l u c u r o n i d e , w a s s e l e c t e d a s GT s u h s t r a t e to s t u d y i t s s t e r e o s e l e c t i v e g l u c u r o n i d a t i o n b y p h e n o barbital(PB)- and 3-methylcholanthrene(MC)-inducible rat l i v e r i s o z y l a e s . On c h r o m a t o f o c u s s i n g o f P B - i n d u c e d s o l u b i l i z e d m i c r o s o m e s , t w o n a p r o x e n GT f r a c t i o n s w i t h d i f f e r e n t s t e r e o s e l e c t i v i t i e s w e r e e l u t e d a t pH 8.7 a n d pH 7.8. The f i r s t f r a c t i o n p r e f e r e n t i a l l y g l u c u r o n i d a t e d S n a p r o x e n (S/R r a t i o = 1.6) a n d t h e pI 7.8 f r a c t i o n p r e f e r e d t h e R - i s o m e r (S/R r a t i o = 0.7). C h r o m a t o f o c u s s i n g of MC-induced solubilized microsomes resulted in the s e p a r a t i o n o f 2 n a p r o x e n GT f r a c t i o n s a t pI 9.4 a n d pI 8.7 w i t h S/R r a t i o s o f 0.2 a n d 0.8, r e s p e c t i v e l y . The c h a n g e of t h e S/R r a t i o in t h e pI 8.7 f r a c t i o n from 1.6 w i t h P B m i c r o s o m e s t o 0.8 w i t h M C - m i c r o s o m e s c o u l d b e e x p l a i n e d b y t h e p r e s e n c e of R - n a p r o x e n GTMc in t h i s f r a c t i o n in a d d i t i o n t o S - n a p r o x e n GTeB, k n o w i n g t h a t GTs a r e p r e s e n t as oligomers. Interestingly, kidney microsomes preferentially glucuronidated R-naproxen with a high degree of s t e r e o s e l e c t i v i t y (S/R r a t i o = 0.2). T h e r e s u l t s s u g g e s t the existence of at least 3 stereeselective naproxen-GTs in r a t l i v e r , (1) R - n a p r o x e n GTMc (S/R r a t i o = 0.2) w h i c h is p r o b a b l y i d e n t i c a l t o t h e p r e v i o u s l y c h a r a c t e r i z e d MCi n d u c i b l e p h e n o l GT; (2) S - n a p r o x e n GTes (S/R r a t i o = 1.6), a n d (3) R - n a p r o x e n GTes (S/R r a t i o = 0.7}. S u p p o r t e d b y t h e A l e x a n d e r yon H u m b o l d t F o u n d a t i o n a n d the Deutsche Forschungsgemeinschaft. I n s t i t u t e o f T o x i c o l o g y , U n i v e r s i t y of T f i b i n g e n , W i l h e l m s t r a B e 56, 7 4 0 0 T f i b i n g e n , F.R.G.

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3O KETONE REDUCTION OF WARFARIN AND ANALOGUES: PARTIAL FICATION OF A LIVER CYTOSOLIC CARBONYL REDUCTASE J.J.R. Hermans, B.H.W. Thijssen

Mohamed E1 Mouelhi and Karl Walter Bock

Box

IO-HYDROXY METABOLITES OF AMITRIPTYLINE IN HUMAN URINE: SEPARATION OF ENANTIOMERS AND IDENTIFICATION OF QUATERNARY N-GLUCURONIDES U. Breyer-Pfaff, E. Nusser, B. Benher, K. Nill, and A. Prox E- and Z-lO-hydroxyamitriptyline (E- and Z-10-OH-AT) are racemic alcoholic metabolites of the antidepressant amitriptyline (AT). Their enantiomers were separated by HPLC as diastereomeric derivatives using R-(+)-a-methoxy-~-trifluoromethylphenylacetyl chloride (Mosher's reagent). Conjugated metabolites were isolated from the urine of patients receiving AT treatment by a combination of solidphase extraction, HPLC and TLC. By NMR and mass spectrometry, N-glucuronidee of E- and Z-10-OH-AT and of translO,11-dihydroxy-ATwere identified in addition to the previously described O-glucuronides of E- and Z-IO-OH-AT and -nortriptyline and AT-N-glucuronide (AT-N-Glue). The quaternary ammonium glueuronides proved to be resistant to acid hydrolysis, while the 10-OH compounds formed IO,lldehydro-AT-N-Gluc by the elimination of water. In urine samples from three patients, 35-60 % of conjugated 10-OHAT was found in the form of N-glucuronides. These conjugates can be produced by E- and Z-lO-hydroxylation of AT-N-Glue as well as by N-glunuronidation of IO-OH-AT as was shown by analysis of urinary metabolites in a volunteer given AT-N-Glue by i.v. infusion and in a separate experiment E-10-OH-AT orally. While E-IO-OH-AT excreted in patient urine in free form or as the O-glucuronide consisted primarily of the (-)-enantiomer, the N-glucuronide contained comparable quantities of the two enantiomers. Z-10-OH-ATwas analysed in one patient and an excess of the (+)-isomer was found in the unconJugated, total conjugated and N-glucuronidated metabolite. Racemic E-IO-OH-AT ingested by a volunteer underwent oxidative metabolism to a large extent; only ii % ef the (-)- and 2.4 % of the (+)-enantiomer was excreted in free form and as the glucuroaide. Institut fur Toxikologie der Universit~t, D-7400 Tfibingen; R. Chem. Forsnhung, Dr. Karl Thomae GmbH, D-7950 Biberach

R9 33 GLUTATHIONE ENHANCEMENT IN V A R I O U S MOUSE ORGANS AND PROTECTION BY GLUTATHIONE ISOPROPYL ESTER AGAINST LIVER INJURY

35 S T U D I E S ON T H E N E W A N T I O X I D A N T SELENOENZYME, PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE IN T H E M O U S E

Stefan Uhlig

FRANK WEITZEL

The important role of gtutathione for many cellular functions is evident. Today more than 20 glutathione dependent enzymes are known. Therefore means of manipulating the glutathione status in vivo as well as in vitro are of practical pharmacological interest. Since the tripeptide is strongly hydrophilic it does not readily diffuse into cells. A new and promising way is the use of glutathione esters, which are hydrophobic enough to diffuse into cells, where they supposedly are cleaved by unspecific esterases. In order to verify this concept, we studied the pharmacokinetics and pharmacodynamics of glutathione isopropyl ester. Intraperitoneal administration of glutathione isopropyl ester to fasted, mate NMRI mice led to a dose dependent increase of glutathione in various organs. Four hours after administration of 1 g/kg glutathione isopropyl ester the following increases were found'." liver 166%, lung 164%, heart 121% and brain 111%. Spleen, kidney, muscle, serum and blood cell glutathione was not affected by the treatment. Pretreatment with glutathione isopropyl ester was found to protect against paracetamol- or allylalcohol-induced liver damage. Following treatment with the ester a significant correlation between protection against liver damage and enhancement of liver glutathione content was obtained. The dose dependence of this protection was studied.

Peroxidized lipid has been implicated in central mechanisms of cytotoxicity, malignant growth and ageing of cells. Recently, phosp.hatidyl choline hydroperoxide was shown to initiate tumors m mouse liver. A new selenoenzyme was discovered which preferentially reduces phosphatidyl choline hydroperoxide and acts only poorly on H 2 0 2. We therefore studied the distribution of this enzyme (PH-~Px) in various mouse organs and its dependence on the dietary selenium supplementation. An assay suitable for the determination of PH-GPx in biological samples was developed. The following specific activities were found: liver: 8,0 +- 0,5; lung: 20,3 +- 1,7; heart: 8,2 --+ 0,6; kidney: 15,7 ± 1,2 (mU/mg, --- SEM, n=5). The "classical" GPx had a markedly different organ distribution with specific activities which were 65-, 9-, 6- and 12-fold higher, respectively, in the various organs. During t30 days of dietary selenium deficiency the activity of the "classical" GPx dropped to undetectable levels. In contrast, the main organs of these animals retained considerable activities of PH-GPx. These findings together with a complementary subcellular distribution of these two selenium-dependent GPx suggest that the newly discovered PH-GPx as a membrane- associated protein acting preferencially on interfacial lipid hydroperoxide substrates plays a major role in antioxidant deiens.e.

Biochemical Pharmacology, Faculty of Biology, University of Konstanz, D-7750 Konstanz, FRG

Biochemical Pharmacology, Faculty of Biology, University of Konstanz, D-7750 Konstanz, FRG.

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REINVESTIGATION OF S- CARBOXYMETHYL- L - CYSTEINE METABOLISM IN HUMANS BY NOVEL STABLE ISOTOPE TRACER STUDIES C.O. Meese a, D. Specht b, D. Batge b, H. Wisserb, P. Fischer c, J. Oesselmann d, and A. Gerding d

METABOLISM OF NITRENDIPINE IN VITRO ON RAT LIVER MICROSOMES IN PRESENCE OF VARIOUS DIHYDROPYRIDINE COMPOUNDS WITH A HOMOHISTAMINE SUBSTITUTED ESTER GROUP

A large variability has been reported for the human metabolism of the mucolytic agent S-carboxymethyI-L-cysteine (1), with respect to interindividual sulphoxidation capacity (Mitchell SC et al,, Br. J. Clin. Pharmacol.18: 507, 1984). Some of the metabolites on which this argument is based, however, are normal food constituents, or may easily have been formed by autoxidation during work-up or chromatography. A novel stable isotope technique has been developed, therefore, to monitor the metabolites, formed specifically from 1, directly from untreated human urine. The synthetic isotopomers of 1 (Scarboxy[ is C]methyl-L-eysteine, 13C - I ) and of S-methyl-L-cysteine (2) (S-[ISC]methyl-L-cysteine, XaC-2), a presumed intermediate metabolite of 1, were administered orally to human subjects. The labelled species, excreted in the urine are monitored by lSC NMR spectroscopy. Since in the known metabolites of ISC-1 and 18C-2 the label is retained, all renally excreted metabolites can thus be identified and quantified directly. The novel analytical principle is supplemented by new HPLC and GC/MS procedures which allow assessment of amino acids and carboxylic acids, respectively (Specht D et al., J. Clin. Chem. Clin. Biochem. 26: 769, 1988; Meese CO et al., Eur. J. Clin. Pharmacol. 36(Suppl): AI51, 1989). Significant amounts of eysteinyl sulphoxides (> 2.5% of the dose [375 or 750 rag] during a 0-8h collection period) or traces of enzymatically formed l a c - 2 could not be discovered this way. Instead, labelled thiodiglycolic acid (4-20%), thiodiglycolic acid sulphoxide' (1-6%), and 3-(carboxymethylthio)lactic acid (~ 2%) were characterized as the major degradation products, besides unchanged excreted drug (14-25%). For the reported metabolic pathway of 2 (Mitchell SC et al., Xenobiotica 14: 767, 1984), only a few of the postulated metabolites could be confirmed by the 13C labelling technique and additional breath analysis (XZCO~) by mass spectrometric isotope ratio measurements. The hitherto accepted metabolic pathway of 1, and the pharmacological consequences of a presumed polymorphic sulphoxidation, have to be revised. Supported by the Robert-Bosch-Foundation Stuttgart nDr. Margarete Fischer-Bosch-Institut for Klinische Pharmakologie, Auerbachstr. 112, D-7000 Stuttgart 50.- bAbt. Kiln. Chem., RobertBosch-Krankenhaus Stuttgart.- CInst. f. Org. Chemie, Univ. Stuttgart.aFinnigan MAT GmbH, D-2800 Bremen

E. PreuS and R. B~cker Dihydropyridine compounds undergo various oxidative reactions on microsomal enzymes including formation of the corresponding pyridine, the side chain hydroxylation (at position 2 or 6 of the DHP), and the ester cleavage (F.P.Guengerich, R.B~cker, J.Biol.Chem. 263:8168 1988; F.P.Guengerich, L.A.Peterson, R.B~cker, J. Biol.Chem. 263:8176, 1988). Test compounds with dihydropyridine structure and homohistamine substitution in one of the two ester moieties did not undergo one of the known metabolic pathways of dihydropyridine compounds. The rate of the pyridine formation on rat liver microsomes was less than I% compared to that of nitrendipine-pyridine formation from nitrendipine using the same microsomes. When nitrendipine was coincubated in vitro with dihydropyridine test compounds with the above mentioned substitution the formation of nitrendipine-pyridine was inhibited concentration dependent. The pyridines of the test compounds did not inhibit the pyridine formation of nitrendipine but they inhibited the oxidative ester cleavage of nitrendipine-pyridine. Lehrstuhl fur Toxikologie und Pharmakologie Universit~t Erlangen-N~rnberg Universit~tsstraSe 22 D8520 Erlangen

der

RIO 37 INTERACTION OF H2-ANTAGONISTS ON PHASE ( I I ) - M E T A BOLISM IN ISOLATED RAT AND GUINEA PIG HEPATOCYTES K.O. Weber, F. M ~ l l e r , and F,R. Ungemach

39 SUBCUTANEOUS CONTINUOUS INFUSION AS A METHOD TO STUDY PRENATAL TOXICITY OF VIRUSTATICS IN RATS

Renate Thiel, Ute Rahm, Klaus Duwe, Brigitte Biirkle

The e f f e c t o f H 2 - a n t a g o n i s t s on phase ( I I ) m e t a b o lism i s s t i l l c o n t r o v e r s i a l . Therefore cimetidine (C), r a n i t i d i n e (R), and f a m o t i d i n e (F) were i n v e s t i g a t e d f o r t h e i r a b i l i t y t o i n h i b i t conjugat i o n r e a c t i o n s in f r e s h l y i s o l a t e d hepatocytes from r a t and Guinea pig (Gp). A f t e r p r e i n c u b a t i o n f o r 3Omin with v a r i o u s c o n c e n t r a t i o n s o f C (0.2 3.2mM), R and F (0.1, 0.5, 1.0mM), oxazepam (Ox, O.ImM) o r mebendazole (M, O.01mM), both drugs c u r r e n t l y b e l i e v e d t o be e l i m i n a t e d e x c l u s i v e l y by g l u c u r o n i d e f o r m a t i o n , were added t o the c e l l suspension. V i a b i l i t y did net d i f f e r from c o n t r o l groups w i t h i n a 3h incubation p e r i o d under these c o n d i t i o n s . A n a l y s i s o f Ox or M and t h e i r metabol i t e s b e f o r e and a f t e r enzymatic h y d r o l y s i s o f the conjugates was performed by HPLC. Whereas no s i g n i f i c a n t e f f e c t f o r C, R and F on g l u c u r o n i d a t i o n o f Ox could be demonstrated in hepatocytes from Gp, Ox was l a r g e l y metabolized o n l y by a phase ( 1 ) - r e a c t i o n in the r a t . Mebendazole conjug a t i o n was s i g n i f i c a n t l y a l t e r e d by C but not by R and F, as could be demonstrated p r e v i o u s l y . In the Gp, M was found t o be o n l y a minor candidate f o r both g l u c u r o n i d a t i o n and s u l p h a t i o n which were a p p a r e n t l y not i n f l u e n c e d by any o f the H2a n t a g o n i s t s t e s t e d . Thus, i t can be suggested t h a t the i n h i b i t o r y e f f e c t o f C on c o n j u g a t i o n i s ( 1 ) n o t a g e n e r a l phenomenon f o r a l l d r u g s and (2) restricted to glucuronidation but not to sulphation.

Several nucleoside analogues which are currently used as virustatie agents reveal prenatal toxic effects. One important question is whether the teratogenic action is related to the peak concentrations or to steady-state concentrations of the applied drug. We used two methods of parenteral constant drug application with two virustatic agents in pregnant rats. (1) In our studies with aciclovir osmotic minipumps (Alzet, model 2ML1; filling volume: approx. 2 ml) were used. Due to the limited solubility of the drug even with 2 minipumps per animal only daily doses of up to approx. 40 mg/kg body wt could be applied (appl~cation period: during organogenesis, from day 7 on). Blood was taken (from a tall veto) on day 13 of pregnancy. Plasma concentrations of aciclovir measured by HPLC ranged from 8.0 - 99.5 mg/1. This wide range of plasma concentrations shows one limitation of this method. Furthermore, the end of the application period cannot be clearly defined. (2) In order to avoid these disadvantages a new method for s.c. application was developed in our laboratory. As an example, we present here results obtained after application of 1000 Zidovudine mg/kg body wt (10 ml of the commercially available product, RetrovirK) during 24 h on day 10 of gestation. Blood samples were taken at the end of the application period. The concentrations of zidovudine were 12.4 +_2.Tmg/h mean -+ S.D. (range: 6.9 - 14.7). This method shows the opportunity to deliver relatively high and precise amounts of drug solutions over defined time periods. It is possible to pump continuously or intermittently for several days. From our experience we suggest that the constant s.c. injection method is superior to the minipump application for studying problems in toxicology.

Present

lnstitut fi2r Toxikologie und Embtyopharmakologie, Freie Universit~t Berlin, Garystr. 5, D-IO00 Berlin 33

address:

Institute

of

Pharmacology

and

T o x i c o l o g y , FB Vet. Med., FU B e r l i n , K o s e r s t r . 20 D-IO00 B e r l i n 33 (Supported by DFG)

38 A MICRO-METHODOF LIQUID/SOLID/LIQUID-EXTRACTION FOR GAS-CHROMATOGRAPHIC ANALYSES W. O~nges, H. Muno, T. Coban and St. Esser Silica gel, the surface of which modified with hydrophobic organic entities, 40-#m-particles: "reversed phase material" or rpm, is presently a widely used medium for the transfer of or- ganics from aqueous or biological samples. A standard technique in pesticide trace analysis consists of extracting 2 l of water with 4 g of C18-modified silica gel, treatment of the rpm-column with methanol, reduction of the desorbate to 1 ml and injection of 200 #l into an HPLC with UV-diode array detection, G. Werner, DVGWSchriftenreihe Wasser, 65 67 (1989). In capillary gas-chromatography (GC) the maximum injection volume is I #l: Only a small part of such a rpm-extract can thus be investigated. Therefor the analysis can only be carried out using very sensitive and specific systems as GC with nitrogen sensitive detection or with MS in the single-monitoring mode, while GC-MS (full-scan spectrum) and GC-FID are not sensitive enough for the sub-ppb range. For the above sorption operations we have now developed a micro methode, including: desorption, drying the desorbate and reduction of the organic solution to 5 #l. With GC/FID we can new determine organics in water from toluene to butylphthalate in 0,1 ppb concentrations.This is of special interest for environmental control. Good blanks and recoveries even after 200,O00fold enrichment result from auxiliary techniques as the cleaning of solvents and glassware. The method can also be applied to the solution of further problems, for instance for GC-investigations of biological fluids for organic compounds in the ppm-range. I n s t i t u t fur Kernchemie, Universit~t Mainz, SaarstraSe 21, D-6500 Mainz, Deutschland

40 PREDICTION OF METABOLIC TRANSFORMATION RATES OF SUBSTITUTED ALKENES. G. Csan&dy Substituted alkenes are metabolized by Cytochrome P-450 enzymes to epoxides, which may be mutagenic and carcinogenic. A model was developed which describes the metabolic transformation rates of ethene, fluoroethene, l,l-difluoroethene, chloroethene, l,l-dichloroethene, cis- and trans-dichloroethene, trichloroethene, perchloroethene, propene, isoprene and butadiene on the basis of molecular parameters. To evaluate the model, metabolic rate constants, experimentally determined by inhalation pharmacokinetics with rats were taken from the literature and were transformed to an apparent metabolic rate constant (k-r~). The model is based on the assumption that metabolic epoxidation can be described as an electrophilic reaction. To obtain a measure for the different reactivities of the compounds ~ -electron densities (d( ~ )), normalized by the corresponding HOMO-energy were used. The HOMO-energies and z electron densities were calculated with a quantum chemical method based on the Minor Neglect of the Diatomic Overlap. The HOMO-energies were corrected by including the ionization potentials (Ip) into the calculation. To describe the different binding properties of the alkenes to the enzyme, the dipole moment (~) was introduced as a weighting factor. With a=0.011, b=9.73, c=0.00S as model parameters our model is described by: a d(z) i k~ Pp = ~i + C IPi - b m

The model was applied to predict the metabolic rate of isobutene in rats. Our results show that molecular parameters can be used to predict metabolic transformation rates. G. Csanady, Institut far Arbeitsphysiologie, Universit~t Dortmund, Ardeystr. 67, D-4600 Dortmund.

Rll

41

43

URINARY EXCRETION OF ISOPHORONE METABOLITES BY MALE RATS

CYTOTOXICITY OLEFINS

R. Thier~ D.G, Xu Isophorone (3,5,5-trimethyl-2-cyclohexen-l-one) is an important intermediate in chemical industry and is used as a solvent in agriculture and industry. It has been found in finished drinking water and in fish tissues. In a two-year bioassay, isophorone has been shown to cause a low incidence of renal tumours in male but not in female rats, A DNA-hinding-study with rats of both sexes indicated that this carcinogenic effect was not due to a direct genotoxicity of isophorone. An epigenetic mechanism, such as interference with the metabolism of sex hormones or an influence of alpha-2uglobulin, a specific protein of male rats, was therefore sugested as the cause for the observed tumors. The object of the investigations presented here was to evaluate the urinary excretion of metabolites of isophorone. Male rats were dosed orally by garage with 500 mg isophorone per kg body weight dissolved in neutral oil over a peri~ of nine days. The final application was done with C-labeled isophorone. The control group received neutral oil only fo~ the first eight days and radiolabeled isophorone in oil (500 mg/kg) on the last day. Urine and feces were collected for 14 days. The excretion of isophorone was determined by liquid scintillation counting The urine was separated by molecular weight gel filtration chromatography. Four peaks were detected by UV monitoring (280 run) The main peak eo-eluted with protein (BCA assay). The components with low molecular weight (below i0000 Da) were prepared by centrifugal filtration, Chromatographic analysis by fluorescence TLC showed differences in elution profile between treated rats and the controls. The results confirm the suspected interference of isophorone with the urinary excretion of proteins such as alpha-2uglobulin and sex hormones.

G. BIRNER,

Institut ff~r Arbeitsphysiologie.Ardeystr. 67. Dortmund. FRG

OF

MERCAPTURIC

ACIDS

OF

HALOGENATED

F. SCHWERTFEGER AND A. KOCHLING

Renal proximal tubular cells are a target for toxicity of many xenobiotics. Haloalkene induced toxicity may be caused by reactive intermediates formed by several metabolic pathways. Bioactivation by glutathione (GSH) leads to metabolites considered to be responsible for selective nephrotoxicity. GSH-adducts are cleaved to cysteine conjugates, the precursors of B-lyase formed acylating agents and the corresponding mercapturic acids (MA) which are excreted with urine. We developed a method to analyse MA in urine by solid-phase extraction and determined the contribution of MA to nephrotoxic effects on freshly isolated rat renal tubular cells. N-acetyl-S-dichlorovinyl-cysteine, N-acetyl-Strichlorovinyl-cysteine, and N-acetyl-S-pentachlorobutadienyl-cysteine (0.2-0.5 mM) reduced cell viability (defined by the trypan blue exclusion technique) from more than 80% to 10-30% after 3h. Preincubating the cells with the B-lyase inhibitor aminooxyacetic acid (AOAA), with bis-(p-nitrophenyl)-phosphate, an inhibitor of N-deacetylation, or probenecid, an inhibitor of organic acid transport, clearly decreased the cytotoxic effects. The results suggest that MA are transported into proximal tubule cells and are deacetylated yielding cysteine conjugates which are cleaved to reactive intermediates by B-lyase. The lack of toxicity of N-acetyl-S-(2chloropropenyl)-cysteine supports the hypothesis that chlorine i n x -position to sulphur is necessary to form toxic intermediates. Institut fur Toxikologie, Universit~t W~rzburg, Versbacher Str. 9, D-8700 W~rzburg, FRG

42

44

REDUCTIVE M E T A B O L I S M OF I,I,I-TRICHLOROETHANE H. D~rk, Oh. Klessen und H. Frank

DICHLOROACETYLENE - EVIDENCE FOR RENAL BIOACTIVATION BY GLUTATHIONE CONJUGATION REACTION

M. Koob, W. Kanhai and D. Henschler

M e t a b o l i s m of l,l,l-trichloroethane is slow compared to other chlorocarbons used as industrial solvents; its hepatotoxicity is moderate. In order to investigate whether the low toxicity is a direct consequence of slow metabolism, inhalation experiments with male Sprague-Dawley rats have b e e n performed. Uptake reflecting m e t a b o l i s m after e q u i l i b r a t i o n to a concentration of about 300 ppm (MAK = 200 ppm) was shown to be only 17 u m o l / k g - h for both untreated and phenobarbitalp r e t r e a t e d animals. In both cases acetylene was exhaled upon exposure to the solvent. To our knowledge, this is the first time that acetylene has b e e n identified as metabolite; the compound has been ascertained by gas chromatography, mass spectrometry and silver acetylide formation. It is thought to be formed from l,l,l-trichloroethane by reductive dehalogenation and subsequent elimination of hydrochloride. Furthermore, uptake of l , l , l - t r i c h l o r o e t h a n e is accelerated upon reduction of the oxygen partial pressure in the respiratory atmosphere, again an indication for reductive metabolism; at the same time, exhalation of acetylene is increased. Reductive metabolism is often associated with "lipid peroxidation", but exhalation of alkanes was only slightly increased. In vitro m e t a b o l i s m of l,l,l-trichloroethane with rat liver microsomes under reduced oxygen partial pressure (i mm Hg) also entails formation of acetylene. H i s t o p a t h o l o g y and histochemistry of the livers of rats do not exhibit any sign of cytotoxicity after exposure at concentrations of about 2000 ppm for a week, 8 hours per day.

Dichloroacetylene (DCA), a potent and selective nephrotoxin in rodents, undergoes bioactivation by conjugation reaction with glutathione (GSH) and further processing of the formed diohlorovinylglutathione (DCVG). The current hypothesis involves GSH conjugation of DCA in the liver, translocation of the formed DCVG to the kidney with bile and enteric reabsorption or with blood. M e t a b o l i s m of DCVG in the kidney by ~ - g l u t a m y l transpeptidase and dipeptidases results in S-(l,2-dichlorovinyl)-L-cysteine (DCVC). D C V C is metabolized to reactive intermediates assumed to be responsible for nephrotoxicity. In vivo metabolism studies (inhalation of lO0~Mol DCA within lh) showed an equivalent distribution of metabolites in urine (N-acetyl-DCVC) and faeces (DCVG). Two explanations are possible: i) equivalent amounts of DCVG formed in liver are either transported to the kidney or eliminated via faeces; ii) DCA undergoes hepatic as well as renal GSH conjugation. DCVG formation proceeds at the same rate with hepatic and renal microsomes. To support our hypothesis that DCA is bioactivated in the kidney the bile duct of male/female Wistar rats was cannulated prior to exposure of 100#Mol DCA within lh. Under these conditions the bile and urine S-conjugates were eliminated w i t h i n the first 3h starting from time of exposure. DCVG excretion with bile amounted to 7.7#Moi, N-AcDCVC in urine to 7.4~Moi. These experiments suggest that both the liver and the kidney form DCVG and that reabsorption of DCVG from the intestine is not accounting for the nephrotoxicity.

Institute f~r Toxikologie und Anatomic, Medizinische Klinik, Universit~t Tiibingen, 7400 T~bingen

Institut fur Toxikologie, Universit~t W~rzburg, Versbacher Str. 9, D-8700 W~rzburg, F.R.G.

R12 45 SULFUR CONTAINING pRORE&CTIVE INTERMEDIATES: HYDROLYSIS AND M U T A G E N I C X T Y OF HALOVINYL 2-NITROPHENYL DISULFIDES Dirk-Achim M~ller, Gudrun Urban and Wolfgang Dekant

47 SPECIES DIFFERENCES IN ACRYLATE H.-J. Wiegand

THE METABOLISM OF n-BUTYL

Chemical cleavage of the sulfur-sulfur bond in halovinyl and fluorealkyl 2-nitrophenyl disulfides is expected to yield halovinyl and fluorcalkyl thiols identical to those formed by cysteine conjugate B-lyase catalyzed cleavage of the corresponding cysteine S-conjugates. To study the potential use of disulfides as precursors for these thiols, whose transformation to acylating agents is most likely responsible for cysteine S-conjugate mutagenicity, we determined the mutagenicity of several halovinyl and fluoroalkyl 2-nitrophenyl disulfides and identified products formed by hydrolysis of these disulfides. 1,2,3,4,4-Pentachlorobutadienyl 2-nitrophenyl disulfide, 1,2,2-trichlorovinyl 2-nitrophenyl disulfide, l-fluoro-2,2-dichlorovinyl 2-nitrophenyl disulfide and 1,2-dichloro-3,3,3-trifluoropropenyl 2-nitrophenyl disulfide were mutagenic in nitroreductase deficient strains of Salmonella typhimurium TAI00; as haloalkyl cysteine S-conjugates, l,l-difluoro2,2-dichloro 2-nitrophenyl disulfide and l-chloro-l,2,2trifluoroethyl 2-nitrophen[1 disulfide were not mutagenic. Hydrolysis of 1,2,3,4,4-pentachlorobutadienyl 2-nitrophenyl disulfide and l-chloro-l,2,2-trifluoroethyl 2-nitrophenyl disulfide in presence of diethylamine resulted in tetrachlorothiobutenoic acid diethylamide and chlorofluorothionoacetic acid diethylamide. The differences in mutagenicity between halovinyl and fluoroalkyl disulfides are most likely responsible to their different abilities to react with DNA-constituents. Products formed from the mutagenic 1,2,3,4,4-pentachlorobutadienyl 2~nitrophenyl disulfide modified 2'-deoxyguanosine-3'-monophosphate as detected by 32phosphor-postlabeling, whereas products formed from the non-mutagenic l-ehloro-l,2,2-trifluoroethyl 2-nitrophenyl disulfide did not result in detectable 2'-deoxyguanosine-3'-monophosphate modification. Institut f~r Toxikologie, Universit~t W~rsburg, Versbacher StraBe 9, D-8700 W~rzburg, F.R.G.

The metabolismof n-butyl acrylate (BA) was investigated in laboratory animals (rat, mouse, guineapig, rabbit) and in man. The kinetics of BA elimination in blood and liver was studied by head space analysis and enz3nnatic tests in vitro using diluted samples of blood, plasma, erythrocytes, liver cytosol, and microsomes. In the blood of the rodents, BA was metabolized with half live times (tl/2) of 3.7 (rat), 4.3 (mouse), 1.6 (rabbit), and 2.3 min (guinea pig). However, in humanblood t l / 2 of BA was 37.6 min. Further analysis revealed that in rodent plasma BA was rapidly hydrolyzed by alkyl ester specific carboxylesterases (ti/2 between 2.0 and 13.4 min), whereas in human plasma these enzymes were missing. Only a minor hydrolyzing activity was observed (tl/2 = 89.3 min), probably due to the butyryl cholinesterase. In suspensions of erythrocytes t i / 2 was 4.5 (rat), 10.0 (mouse), 5.0 (rabbit), 8.9 (guinea pig), and 29.9 min (man). In the erythrocytes of all species studied, BA reacted with non protein sulfhydryl groups (NPSH). The NPSNbinding was characterized by pseudof i r s t order reaction constants of 0.106 (rat), 0.063 (mouse), 0.103 (rabbit), 0.047 (guinea pig), and 0.012 (man). In addition to the NPSH binding other metabolic processes like protein binding and hydrolysis were observed in erythrocytes. In the liver, all species exhibited a high carboxylesterase activity corroborating published data on the rapi4 hydrolysis of acrylate esters in the liver. The results indicate that after tnhalative or dermal exposureBA possibly persists a longer time in humanblood comparedto rodent blood, resulting in a higher steady state concentration of intact BA.

46

48 PERMEATION OF GASEOUS ETHYLENE OXIDE THROUGH SKIN OF RATS AND GUINEA PIGS IN VITRO P.E. Kreuzer and K. Dorst

THE GLUCURONIDATION OF THIAARENES C.Augustin ~, A.Schmoldt ~, J.Jacob 2, G.Grimmer 2

ON

As previously shown the sulfur-containing polycyclic aromatic hydrocarbons (S-PAC, thiaarenes) are metabolized slightly different than the corresponding homocyclic PAH. Thiaarenes with a central thiophene ring are predominantly sulfoxidized to sulfoxides and sulfones but show additional ring oxidation, whereas those compounds with a peripheral thiophene ring only show ring oxidation. In the present investigation the intention was to find out whether the thiaarene-hydroxymetabolites were substrates for rat liver UDP-glucuronosyltransferases. Therefore a two-step assay was developed. In the first step the thiaarenes were incubated for cytochrom P450 dependent oxidation. The metabolites formed were extracted and reincubated in a second assay for enzymatic glucuronidation using ~'C-UDPGA as cosubstrate. Thiaarenes with a three ring system were not at all glucuronidated, irrespective of the S-position. Among S-PAC with more than three arene rings those with a high sulfoxidation rate were glucuronidated two- to fourfold higher than the low sulfoxidized compounds. From these results it can be supposed that sulfoxidation may play an activating more than an inhibiting role in the glucuronidation of hydroxy-thiaarenes. i Institut fur Rechtsmedizin der Universit~t Hamburg, Butenfeld 34, 2000 Hamburg 54 2 Biochemisches Institut fur Umweltcarcinogene, 2070 Gro~hansdorf

HQIs AG, P.O. Box 1320, Ps/Biologie-Toxikologie, 4370 Marl, FRG

Ethylene oxide (EO) is a major industrial intermediate and is widely used for sterilisation procedures. Since a number of employees may be exposed to EO under various conditions the substance may be absorbed not only by inhalation but by skin uptake too. Therefore, the aim of this work was to determine the kinetics of transdermal permeation of gaseous EO using skin of rats and guinea pigs. We developed an all glass closed skin permeation system based on the commercial available "LGA system". The system was divided by the exposed skin in an upper gas chamber and a lower acceptor chamber containing Dulbecco's minimal essential medium, pH 7.4. Temperature was kept constant at 25°C in the gas chamber and at 37°C in the acceptor chamber. T h e concentrations in the gas phase (100, 300, 1000, 3000 ppm) were maintained constant (_+ 15%) by repeatedly substituting the loss of EO gas due to skin uptake. The acceptor medium in the lower chamber was replaced by fresh medium in time intervals of one or two hours. The exposed acceptor media were transferred into 25 ml vials and the amount of EO taken up was determined gas chromatographically using a head space method. After a lag-time of 1-2 h, the rates of EO permeation across skin were constant and were linear related to the exposure concentrations for both species. These rates were 1.5 times higher in freshly prepared rat skin compared to that of guinea pigs. The permeability constants according to Fick (cm/h) were 0.34 for rats and 0.22 for guinea pigs. The permeation rate through skin of rats increased 1.4 times after storage at -20°C for several weeks, the permeability constant being 0.47 crn/h. The data obtained, showing that gaseous EO permeates skin of two species may serve as a basis for extrapolation to man. GSF, Institut fiJr Toxikologie, D-8042 Neuherberg

R13 49 ESTIMATION OF HUMAN CANCER RISK DUE TO EXPOSURE TO ETHYLENE AND ETHYLENE OXIDE B. Denk and J.G. Filser

51 METABOLISM AND TOXICOLOGY OF 2-METHYLPROP~NE (ISOBHT~YE) : COMPARISON TO OTHER ~ . R.J.Laib, U.Hindermeier,*M.Cornet and*V.Rogiers

Ethylene (ET) and ethylene oxide (EO) are widely used industrial chemicals. Furthermore, ET is ubiquitous in the environment. It has been shown to be metabolized to EO which was mutagenic and carcinogenic in animals. Therefore, a carcinogenic risk has to be deduced for ET too. In this work human cancer risk of EO and its metabolic precursor ET is estimated based on the internal dose of EO. The latter was calculated by means of pharmacokinetic data as the product of concentration and exposure time. For risk estimation we chose two different approaches. The first was based on an allometric species scaling, the second on the "rad equivalence factor" according to Ehrenberg and coworkers (Ehrenberg, International Atomic Energy Agency, Vienna, 1980). In the first case the risk observed in a long term inhalation study with rats was linear extrapolated to low concentrations and allometric scaled to man considering the species specific lifetimes. In the second approach a "rod equivalence factor" for EO was determined by means of the "rat liver foci bioassay'. This factor, defined as the relative carcinogenic effectiveness of gamma-rays as reference standard, was transferred to man. Cancer mortality of man due to radiation exposure was given by reports of the United Nations Scientific Committe on the Effects of Atomic Radiation (1977), and of the Strahlenschutz-Kommission(1988), respectively. The cancer risk connected with exposure to 1 ppm EO 40h/wk for 45 years (equivalent to the american threshold limit value) was estimated allometrically to be 104.10-4. Using the rad equivalence approach the estimated risk for this exposure is somewhat higher: between 170.10 -4 and 850.10 -4. Using pharmacokinetic parameters of ET and EO for man the corresponding ET exposure concentration was calculated to be 20-70 ppm (40h/wk for 45 years). According to our results, for ET too a threshold limit value should be recommended wich can be based on an accepted risk for EO. Since ET is produced endogenously in humans, an endogenous cancer risk is inevitable. Based on our results it is estimated to be 1.25.10-'4, allometrically, and 2.1.10 -4 to 10.4.10 -4 using the rad equivalence factor.

Data on metabolism and toxicology of 2-methylpropene (MP) , used in the production of rubber chemicals, fuel additives~ plastic polymers and adhesives, are lacking. In analogy to other alkenes, MP should be metabolized by cytochrome P450 enzymes to its epoxide 2-methyl-1,2-epoxypropane fMEP). Further metabolism of MEP by epoxide hydrolase or spontaneous hydrolysis should lead to 2-methyl-1,2-propanediol.A cc~parative investigation of inhalation pharmacokinetics in rats and mice indicates,that metabolism of MP is saturable in both species. The maximal metabolic elimination rate of MP in rats is about 320~ol/h/kg b.w. and thus faster than for the other alkenes investigated. After pretreatment of the animals with pyrazole or diethyldithiocarbamate,metabolism of MP is nearly ccmpletely inhibited. When MP is incubated in head space vials with rat or mouse liver microsomes and an NADPH-regenerating system,formation of MEP can be d~monstrated. During exposure of rats in a closed exposure system to high concentrations of MP, MEP is exhaled by the animals and can be identified by GC/ MS in the air of the system. Although MP is metabolized faster than butadiene, ethylene or propene, exhalation of the corresponding epoxide is minor for MP when ccspared to the other alkenes. Investigation of kinetics of acid catalyzed hydrolysis for various ethylene oxide derivatives resulted in the highest hydrolysis rates for MEP. Our data support the metabolic scheme for MP suggested above. The high metabolic rate of MP determined experimentally by inhalation pharmacokinetics with rats is in agree~-nt with the metabolic rate predicted by our QSAR studies for this alkene molecule. The authors thank the BG-Chemie for financial support.

GSF, Institut fiir Toxikologie, D-8042 Neuherberg 50 PHARMACOKINETICS AND ENDOGENOUS PRODUCTION OF ISOPRENE IN HUMANS M. Hartmann and W. Kessler Isoprene (2-methyl-l,3-butadiene) is mainly used in the industrial synthesis of elastomers. Furthermore, it is the major endogenous hydrocarbon exhaled by animals and man. It was genotoxic in mice in vivo which may be due to its metabolism to reactive epoxides as shown in mouse liver microsomes. Therefore, a certain carcinogenic risk due to metabolites of isoprene has to be taken into account. In man, the pharmacokinetics of isoprene and the rate of its endogenous production and metabolism have not yet been evaluated. We investigated these parameters in six volunteers (5 males, 1 female) using a closed spirometer system (15.6 1 including lung volume). The initial gas concentrations of isoprene were 0 ppm, 8 ppm and 50 ppm. Concentration-time courses of inhaled and exhaled isoprene were measured gas chromatographically up to 3 h. Using pharmacokinetic data previously obtained by means of a 2compartment model in rats and mice (Peter et al., Tox. Letters 36, 9, 1987), we predicted concentration-time curves for man. An allometric procedure based on the body surface (= b.w. 2/3) was carried out. Curves extrapolated from rats fitted the measured data better than those extrapolated from mice. Within the measured concentration range, first order kinetics were observed. Clearance of metabolism related to the atmosphere was 81 Vh. Since in these studies the pulmonary ventilation rate was about 320 I/h, the pulmonary retention was 25%. As in rats and mice, only a limited accumulation in the body of about 3 times the actual atmospheric concentration was observed; metabolism of inhaled isoprene was limited by the transport to the metabolizing sites rather than by metabolic capacity. The endogenous production rate was calculated to be 0.15 lamol/(h'kg) body weight. These data will serve as a base for risk estimation of isoprene if required. GSF, Institut Flir Toxikologie, D-8042 Neuherberg

Institut f~r Arbeitsphysiologie, Abt.f'dr Toxikologie und Arbeitsmedizin, ArdeystraBe 67, 4600 Dortn~nd, FRG *Free University Brussels, Dept.of Toxicology, Laarbeeklaan 103, 1060 Brussels, Belgium •

52 STUDIES ON THE ACTIVITY OF SOME HEPATIC CYTOCHROME P450-DEPENDENT MONOOXYGENASES IN CHICKENS (Ga//u~ d o ~ ) Barbara Heim'ich-Hirsch and Dorothea H o f m a n n Sex-dependent differences in the activities of hepatic monooxygenases are well known for the rat, but not significant in several other species. We therefore studied the activities of 3 monooxygenuses: Aldrinepoxidase (AE), 7-E,tho~-O-deethylase (ECOD) and 7-Ethoxyresorafm-OMeethylase (EROD) in liver microsomes, as well as the 1'450 content, from 2-year-old male and female chicken. males (n=3)

females (n=9)

female (*)

P450 320_+ 33 175_+ 25 310 AE 1221_+ 55 405_+ 67 1708 ECOD 5979 + 557 3317 _+ 394 5544 EROD 116 _+ 28 17 + 2 15 values given as Mean _+ S.D. P450: p m o l e s eytochrome P450/mg protein AE, ECOD, E R O D : pmoles product/rain x mg protein Although both sexes could not be studied to the same extent in this investigation, up till now the data dearly indicate differences between the two sexes. In males enzyme activities and P450 contents were distinctly higher than in females. The difference was most obvious for the E R O D activity. Furthermore, within the group of females one particular individual (*) appeared to differ in P450 content, A E and E C O D activity from the others, exhibiting values rather comparable to those of males. The reason for this is not yet known. Studies supported by grants from the Bundesmlnlsterium fiir Forschung und Technologic (0318785A). Institut fiir Toxikologie und Embryopharmakologie, Freie Universitiit Berlin, Garystr. 5, D-1000 Berlin 33

R14

53 CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST CYTOCHROME 1'450 FROM RAT AND MARMOSET MONKEYS ( Callithrix jacchus ) Rothin Pramanik-Strehlow, Ilse Lass, Norbert Hinz Monoclonal antibodies were produced against microsomal cytochrome P450 fractions derived from rats and marmosets treated with either g-naphthoflavone (BNF) or 3-methylcholamhrene (MC). When tested with an enzyme-linked immtmosorbent assay (ELISA) against purified P450 fractions from 2,3,7,8-Tetrachlorodibenzo-pdioxin (TCDD) induced animals, the MABs clearly differed in species cross-reactivity and in the reactivity to the two P450-subforms (1) catalysing the highest EROD activity and (2) exhibiting the highest binding capacity for t4C-TCDD (Table): RAT P450: I (EROD) 2 (14C-TCDD)

M J h R M O S E T P450: 1 (EROD) 2 (14C-TCDD)

MAB-No. 1-BNF-R 2-BNF-R 4-BNF-R 1-BNF-M 2-MC-M

+++ + ++++ +++

+-

+++ +++ +++ +++ +

+++ +++

+++ +++ ++++ +++

55 A CHARACTERIZED P450IIB GENE IS EXPRESSEDAT HIGH LEVELS IN SEBACEOUSGLANDS BUT NOT IN THE LIVER. T. Friedber 9, M. Grassow, P. Siegert Oligomer probes for characterized genes of the P450IIB family were used to study the expression of these genes in various tissues. I n t e r e s t i n g l y one of these genes (P450gene 4) for which no corresponding protein product had as yet been i d e n t i f i e d coded for a mRNA which was found in the preputial gland of both sexes but not in the l i v e r or any of the f i v e other tissues tested. The P450-gene 4 RNA encoded a protein which was recognized by antibodies d i rected either against p u r i f i e d cytochrome P450IIB1 or against defined cytochrome P450IIBI or B2 peptides. The P450 gene-4 protein product appeared to have no cytochrome P450 dependent enzyme a c t i v i t y . P450-gene 4 was expressed at high levels in rats of various age grops and was not inducible by Aroclor 1254. This is the f i r s t report showing that a member of the P450IIB family is expressed exclusively and at high levels in a extrahepatic organ. This finding opens up the p o s s i b i l i t y of studying the factors regulating the tissue specific expression of the members of the P450IIB family. I n s t i t u t fur Toxikologie, Johannes Gutenberg-Universit~t Mainz, Obere Zahlbacher StraBe 67, D-6500 Mainz

Antibodies 1-BNF-R and 2-MC-M inhibited the ethoxyresorufin-Odeethylase (EROD) activity of TCDD induced rat liver microsomes more than 80%; the other forms were somewhat less effective in this respect. Using histo-immunfluorescence techniques it was easily possible to localize the antigens within the liepatocytes in sections of TCDDinduced rat livers with the MABs 1-BNF-R and 2-MC-M. Supported by a grant (0318785A) from the Bundesministerium fiir Forschung und Technologie, and from Deutsche Forschungsgemeinschaft (Sfb 174). Institut fiir Toxikologie und Embryopharmakologie, Freie Universitiit Berlin, Garystr. 5, D-1000 Berlin 33

54

56

CHARACTERIZATION OF CYTOCHROMES P-450 DERIVED

PROSTAGLANDIN H SYNTHASE CATALYZED FORMATION OF C A T E C H O L E S T R O G E N S AND REACTIVE INTERMEDIATES G.H. Degen! J. Foth, and A. Frevb@r~er

FROM UNTREATED AND TCDD-TREATED MONKEYS (Callithrix ]acchus) Michael Kastner

MARMOSET

Three forms of cytochrome P-450 (P450) were purified from liver mierosomes of marmoset monkeys (Callithrix jacchus) induced with a single dose of 300 ng 14C-TCDD/kg body wt. Comparison of ethoxyresorufin-O-deeth~lase (EROD), benz~.loxyresorufin-Odeethylase and aldrin epomdase activities, as well as spectral properties suggest that one of these forms is constitutive. Two forms are apparently TCDD-inducible and deserve special attention: one has a high EROD activity while the other has a lower EROD activity but appreciable capacity to bind TCDD. EROD activities (pmol product per nmol P450 per min) in the presence of cumene hydroperoxide, TCDD binding (molar ratios of 14C-labeUed TCDD per P450), wavelength maxima of ferrous-carbonyi complexes, and molecular weights from SDS-PAGE are summarized as follows: P450 form

P450-1 P450-2 1'450-3

E R O D rate

1.3 54.6 5.7

TCDD/P450

0.03 0.06 6.2

'~ max

mol. wt.

450.4 447.5 448.5

47 000 52 500 53 500

Binding of TCDD by Form 3 seems to be tight over all stages of chromatographic separation. On the other hand, removal of excess non-tomc detergent concentrations by means of hydroxyapatite apparently results in dissociation of this snbstrate. Supported by grants from the Bundesministerinm fiir Forscliung und Teclmologie (0318785A and 07VDX 019). Institut for Toxikologie and Embryopharmakologie, Freie Universitgt Berlin, Galystr. 5, D-1000 Berlin 33.

P r o s t a g l a n d i n H synthase (PHS) has been shown to m e t a b o l l i c a l l y activate stilbene and steroid estrogens by means of its peroxidase activity. This study is aimed to elucidate the nature of the reactive intermediates and the m e c h a n i s m of P H S - c a t a l y z e d e s t r o g e n oxidation. Incubation of r e g i o s p e c i f i c a l l y t r i t i a t e d estradiol with m i c r o s o m a l enzyme in the p r e s e n c e of arachidonic acid or h y d r o g e n p e r o x i d e resulted in c o n s i d e r a b l e release of 3H from p o s i t i o n s 2 and 4 (40 and 26 ~) of the A ring indicative of aryl hydroxylation. The P H S - m e d i a t e d catechol estrogen formation has been c o n f i r m e d by product a n a l y s i s using TLC, HPLC and GC/MS. The following m e c h a n i s m is p r o p o s e d for the PHScatalyzed aryl h y d r o x y l a t i o n on the basis of these and further data: p h e n o x y radical initially formed by one e l e c t r o n abstraction, respectively a mesomeric C - c e n t e r e d radical triggers formation of an orthohydroperoxide; this is reduced by PHS or n o n e n z y m a t i c a l l y to the corresponding alcohol which tautomerizes to the catechol product, a process d r i v e n by reestablishment of a r o m a t i c i t y w h i c h involves release of 3H from ortho-tritiated analogs. PHS oxidized catechols e.g. 2 - h y d r o x y e s t r o n e further to the ortho-quinone as d e m o n s t r a t e d by spectroscopy. By comparison, P H S - m e d i a t e d aromatic hydroxylation of d i e t h y l s t i l b e s t r o l was minor; an initially formed p h e n o x y radical is more r e a d i l y o x i d i z e d to a para-quinoid intermediate because of the conjugated s t i l b e n e system. Supported by the Deutsche F o r s c h u n g s g e m e i n s c h a f t Institute of T o x i c o l o g y and Pharmacology, SFB 172, V e r s b a c h e r Str. 9, D-8700 W ~ r z b u r g

R15 59 Formation of reactive oxygen species during the thiol-mediated activation of 1,4-hydroquinone, aminophenols and paracetamoL

57 NADPB-DEPENDENTMICROSOMALOXIDATIONOF ALDEHYDES G. Cans and d. Werringloer A highly sensitive HPLC-procedure was developed f o r the determination of aldehydes in biological samples which is based upon t h e i r derivatisation with 3-methyl-2-benzothiazolone hydrazone (Gans et al., Naunyn-Schmiedeberg's Arch. Pharmacol. 339:R15,1989). Application of this new procedure to the investigation of biotransformation reactions catalyzed by rat l i v e r microsomes revealed a marked inhibitory effect of semicarbazide on both the dealkylation and the denitrosation of N-nitrosodialkylamines (NDAA) at low substrate concentrations. Comparative studies on the recovery of aldehydes, however, disclosed a marked enhancement by NADPHof the time-dependent loss of aliphatic aldehydes, except formaldehyde, from the incubation media when both semicarbazide and NDAAwere absent. The reactions responsible f o r this enhancement were found to be of enzymatic nature and dependent on the presence of molecular oxygen; they proved to be highly sensitive to inhibition by carbon monoxide and indazole as well as NDAAand aliphatic alcohols. Further, they were found to be accelerated after pretreatment of rats with isopropanol, an inducer of P-450 f i e f , rather than phenobarbital. Underthese conditions half maximal veloc i t i e s of the NADPH-dependentdecomposition of aliphatic aldehydes were reached at concentrations as low as ]O to 15 ~M. The formation of carboxylic acids as products of this novel metabolic pathway was established with butyraldehyde as a substrate and analysis of butyric acid by HPLC a f t e r i t s derivatisation with the fluorescence label 4-bromomethyl-7-methoxycoumarin (DUnges, Anal. Chem. 49: 442,1977). Based on these results i t is suggested that the cytochrome P-45O-dependent microsomal alcohol o x i d i zing system (Teschke et a l . , J. Biol.Chem. 250:7397,]975) is functional also as an aldehyde oxidizing system which may be termed MALOSin analogy to MAOS.

K.-G. Eekert and P. Eyer Evidence

is accumulating

that thioether formation of phenolic

compounds represents some risk for sensitive target organs. Recently, we found that synthetically prepared thiocthers of 4-aminophenol (4-AP) produced more ferrihemoglobin (HbFe3.) than the parent compound: Eckert et M., Arch. Toxtcol., Suppl. 13, 287-290 (1989). Hence, some phenolic thioethers apparently bear a higher pro-oxidant character than their congeners. Therefore we Investigated reactions of various glutathione S-conjugates from 1,4-hydroquinone (HQ), 4-AP, 4-dimethylaminophenol, and paracetamol (NAPAP) in the presence of oxyhemoglobinIn detail. The most active thioethers producing HbFe3+ were the all-substituted derivatives which formed significantly more HbFe 3+ than the unsubstituted compounds. Unexpectedly, the 3-glutathionyl derivative of NAPAP produced HbFc3+ which was not observed with NAPAP. Reaetive oxygen species were involved in HbFe3+ formation by the aminophenol thioethers, since superoxide dismutase (SOD) and eatalase (CAT) diminished HbFe3+ formation In an additive manner. Such an effect was not observed with the parent aminophenols. In contrast, SOD enhanced I-IbFe3+ formation by HQ and its thioethers, whereas CAT diminished that effect. These data point to different redox potentials of the semiquinone radicals Involved: In the HQ series, superoxide radicals mainly reduce the serrdquinones thereby withdrawing them from HbFe 3+ formation. With the aminophenol thioethers, the presumed lower redox potential allows superoxide radicals to oxidize the parent thioethers which increases the rate of HbFe3+ production.

Institut f~r Toxikologie, gberhard-Karls-Universit~t, Wilhelmstr. 56, D-7400 T~bingen 1

Walther-Straub-Institut fiir Pharmakologie und Toxikologie, Ludwig-Maximliians-Univarsit~it,D-8000 Mlinehen 2, F.R.G.

58

60 MACROPHAGES AND NEUTROPHILS AS POTENTIAL SOURCES OF REACTIVE OXYGEN IN HEPATIC ISCHEMIA/ REPERFUSION INJURY. H. Jaeschke and A. Farhood

Toxicity and Metabolism of Hydrogen Peroxide in Cultures of Rat Hepatocytes H. Desel, A. Kastien, M. M~ller, and R. Kahl ........................................................

~-

Hydrogen peroxide is produced by activated neutrophils zn an oxidative burst and may be responsible f o r l i v e r damage during inflammation. Rat hepatocytes were cultured f o r 48 h and treated with millimolar amounts of H~02. The concentration of H~02 in t h e medium (> 500 times the t o t a l cell volume) was monitored by a luminol chemiluminescence assay. H20z concentration decreased exponentially with a h a l f - l i f e of 25 min. The decomposition is slowed down by a factor of 3 in the presenceof azide. The data indicate that hepatocytes are able to clear Hz02 very e f f e c t i v e l y from a culture medium, predominantly by the action of catalase. With a time course similar to that of the decay of H202, a release of thiobarbituric acid reactive substances into the medium is observed indicating l i p i d peroxidation. At H202 concentrations < I mmol/1 no release of intracell u l a r enzymes is observed. I f more than I mmol/1 H202 is applied, a dose-dependent release of lactate dehydrogenase is detected 30 min after the start of the incubation. Glutamate dehydrogenaseis released another 30 min later only when the hepatocytes are treated with more than 10 mmol/l H202. Our experiments show that cultured hepatocytes can tolerate high H202 concentrations. Although l i p i d peroxidation is detected at submillimolar H202 concentrations, functionally important membrane damage occurs only at concentrations exceeding I mmol/l. The damage is only expressed when Hz02 in the medium has already been decreased to concentrations in the micromolar range. Abt. Klinische Pharmakologie, Universit~t Gbttingen, Robert-Koch-StraBe 40, 3400 G6ttingen

Reactive oxygen species (ROS) are thought to be involved in the pathogenesis of ischemia/reperfusion injury. Previous data showed a significant extracellular generation of ROS during the reperfusion period after hepatic ischemia in pivo (H.Jaeschke, Free Rad.Res. Commun,, in press). To test if neutrophils (PMN) or tissue macrophages (Kupffer cells, KC) are the source of ROS formation, the liver was subject to various time periods of partial no-flow ischemia followed by reperfusion for 1 h. Plasma GSSG concentrations were usedas index of ROS formation and the number of neutrophils were counted in tissue sections (50 high power fields). Basal plasma GSSG eonc. of 1.9-+0.3 #M GSH-eq. increased significantly only during reperfusion oath values of 10.5-+1.6 (45'I) and 24.2-+2.4 (60'I) after 60 rain. The number of PMN in hepatic tissue (50 HPF) increased from 14-+3 in controls to 61-+ 13 (45'I) and 101 -+19 (60'I) in the posfischemic tissue after 60 rain of reperfusion. Plasma GSSG levels after 2 h ischerrda reached values up to 70.1 --.7.8/zM but PMN counts increased only to 56-+12. Tissue GSSG content increased by 100% only in the postischemic tissue while PMNs also accumulated in the non-ischemic tissue during reperfusion: 35 ± 12 (45'I), 67-+ 18 (60'I), 152_+25 (120'I), indicating a general activation of PMN during the reperfusion period. Activation of KC with galactosamin or retinol pretreatment consistently enhanced plasma GSSG conc. during the reperfusion period by 100-300%, while inactivation of KC with methyl palmitate significantly suppressed plasma GSSG levels by more than 40%. Conclusion: The data indicate that the extracellular oxidant stress during reperfusion after hepatic ischemia in r,7'vo is caused predominantly by Kupffer cells rather than neutrophils. Center for Experimental Therapeutics, Department of Medicine (HJ) and Department of Pathology (AF), One Baylor Plaza, Baylor College of Medicine, Houston, Texas 77030, USA.

R16 61 PARAMAGNETIC METABOLITES FORMEDDURING HYDROXYLAMINEINDUCED METHEMOGLOBINFORMATION K. Stolze and H. Nohl

63 TUMOR FREQUENCY

Compounds i n i t i a t i n g Met-Hb formation are supposed to produce paramagnetic side products. This assumption is based on the fact that redox-cycling of the heme-iron is a one electron transfer step. Several paramagnetic reaction intermediates have been found to be involved in the Met-Hb formation by hydroxylamines. The s t a b i l i t y of the primary reaction product, the n i t r o x i d e radical NRIReO', increased from the transient unsubstituted specie~ (RI=R~=H) and the monomethyln i t r o x i d e (R~=H,R~=CH3) to the d i ~ e t h y l n i t r o x i d e (RI=%= CH~) which w~s stable for more than one hour. A low-sp~n f e r r i c Met-Hb complex was formed from a l l observed species, whereas the hemoglobin-nitric oxide adduct was only formed from unsubstituted hydroxylamine. An additional species was detected with N-methylhydroxylamine which formed the B-aminonitroxide CH~NO'CH~NH~ as a dimerization product. The investigation r~veale~ p~rallels as well as d i f f e r ences in the reaction path of the three hydroxylamine compounds.

W. H U B E R

Supported by the Oesterr. Fonds zur F~rderung der wiss. Forschung Inst. Pharmacol. and T o x i c o l . , Vet.Med.Univ. of Vienna, Linke Bahngasse I I , A-I030 Vienna

AND OXIDATIVE

DAMAGE IN RATS OF

D I F F E R E N T A G E A F T E R T R E A T M E N T W I T H NAFENOPIN,

PEROXISOME

A

PROLIFERATOR

Institut f.Tumorbiologie Vienna, Austria

& Krebsforschung

Nafenopin and other peroxisome proliferators are carcinogenic in the livers of rats without showing genotoxicity. According to a hypothesis the mechanism of tumorigenesis is based on the enhancement of peroxisomal 6-oxidation. Subsequent increases in hydrogen peroxide and lipid peroxidation are supposed to lead to cancer development. Our data are put in relation to this hypothesis. We compared: I) treated rats with controls and 2) two groups of equally treated rats with an age difference of 42 weeks. Our basic observation was tumor enhancement by nafenopin, much more pronounced in the older animals. We investigated inhowfar this difference between the age groups was reflected by changes in the following hypothetic carcinogenic parameters: i) PEROXISOMAL B-OXDATION: treated vs. control: increase, approx. 10-fold; no difference related to age 2) MALONDIALDEHYDE: treated vs. control: reduction, approx, down to two thirds; no difference related to age 3) FATTY ACIDS: treated vs. control: reduction in linoleic and docosahexenoic but no change in arachidonic acid, not typical for lipid peroxidation; no difference related to age The observed higher tumor incidence in the old animals thus cannot be explained by changes in any of our investigated parameters.

62 THE HEPATOCARCINOGENIC ACTION OF THE PEROXISOME PROLIFERATOR NAFENOPIM MAY BE EXPLAINED BY PROMOTION OF SPONTANEOUSLY INITIATED LIVER CELLS B. K r a u p p - G r a s l

Peroxisome inducing agents, such as nafenopin (NAF), produce hepatocellular carcinoma in longterm animal bioassays. Carcinogens of this type have displayed neither genotoxicity in various assays nor tumor initiating capacity. We studied the effect of NAF on tumor formation through promotion of "spontaneous" foci, which a r e known to appear in livers of rats in the course of aging. NAF was fed for 55 to 59 weeks to two groups of rats. One of them represented young animals that were 13 weeks of age at the start of treatment. The other group received the first dose at the age of 56 weeks. NAF produced numerous adenoma and carcinoma in old but very few in young animals. A similar result though less pronounced was seen with phenobarbital (PB). The difference in incidences and multiplicity of liver tumors between young and old animals is consistent with N A F as a strong promoter of spontaneously initiated liver cells. Most of the foci and tumors seen in NAF-treated livers were of weak cytoplasmatic basophilia and in general phenotypically quite different from those detected after PB. A similar subtype of foci was observed recently when initiation with aflatoxin Bl was followed by NAF-treatment. This suggests that tumor promotion by NAF involves a specific, hitherto neglected subtype of foci. Present address: Institute for TumorbiologyCancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna

64 EFFECT OF DEHP AND NAVENOPINON LIVER WEIGHT AND SOME RELEVANT HEPATIC CARBOHYDRATEMETABOLISING ENZYMES C. Einig, E. Eigenbrodt, and U. Gerbracht The effect of di(2-ethylhexyl)phthalate (DEHP) and Nafenopin on l i v e r growth and changes of carbohydrate metabolising hepatic enzymes have been investigated. Both agents are peroxisome p r o l i f e r a t i n g drugs which exert carcinogenic effects in r a t l i v e r . Female Wistar rats were given a single dose of Aflatoxin BI followed by nafenopin(lOO mg/kg b.wt) for 6,16 or 64 weeks, respectively. In a second study female Sprague-Dawley rats were treated with several doses of di(2-ethylhexyl)phthalate for 9 consecutive weeks subsequent to a single dose of diethylnitrosamine (DEN). The biological endpoint evaluated were the l i v e r weight and the a c t i v i t i e s of pyruvate-kinase (PK), fructose-l.6-bisphosphatase (FDPase), malic enzyme (ME), glucose-6-phosphatedehydrogenase (G6PDH) and NAD(H)-dependent cytosolic glycerol-3-phosphat-dehydrogenase (G3PDH). Biochemical i n v e s t i gations indicate that DEHP as well as Nafenopin increased l i v e r weight and altered the metabolic enzyme pattern in the l i v e r . A decrease of pyruvate-kinase and FDPase j u s t as an enhancement of malic enzyme, G6PDH and G3PDH was observed following treatments with both substances. In the dosedependent experiment with DEHP an increase in r e l a t i v e l i ver weight ( r a t i o of l i v e r weight to body weight) and an a l t e r a t i o n of the investigated enzyme a c t i v i t i e s were found. In the time-dependent study with a d a i l y dose of lOOmg nafenopin/kg b.wt. no changes of r e l a t i v e l i v e r weight and enzyme a c t i v i t i e s were registered between 6, 16 and 64 weeks. These results indicate that the a l t e r a t i o n s of carbohydrate metabolism correlates well with the l i v e r weight. Inst. fur Veterin~r Biochem. Universit~t GieBen, Frankf u r t e r s t r . 100, 6300 GieBen, FRG

R17

65 EFFECT OF MOUSEHEPATITIS VIRUS INFECTION ON DIETHYLNITROSAMINE-INDUCED MOUSEHEPATOCARCINOGENESIS K. Zuber, W. Nicklas, R. Schmitt and M. Schwarz Mouse h e p a t i t i s virus, serotype OHM (MHV4), induces massive l i v e r cell damage during the acute phase of inf e c t i o n a f t e r i . p . i n j e c t i o n which is followed by l a t e n t i n f e c t i o n without c l i n i c a l signs. In the present study the e f f e c t of virus infection on chemically induced mouse hepatocarcinogenesis was investigated. In the f i r s t experiment which was designed to study i n t e r ference of virus with tumor i n i t i a t i o n groups of male B6C3F1 mice were infected with MHV4 on day 43 a f t e r b i r t h . Seven days l a t e r , at the time point of maximum DNA synthesis in l i v e r , 5 mg/kg body wt. d i e t h y l n i t r o samine (DEN) were administered by single i . p . i n j e c t i o n . In the second experiment, where possible promoting e f f e c t s of virus infection were studied, DEN was given on day 15 and MHV4 on day 43. Control groups were given e i t h e r DEN or MHV4 alone. Mice were k i l l e d at 24, 28 and 32 weeks a f t e r DEN treatment and the number of macroscopically v i s i b l e tumors per l i v e r was counted. No l i v e r tumors were observed in animals treated with MHV4 alone. In contrast, DEN led to a time-dependent increase in the number of animals carrying l i v e r tumors and in the number of tumors per animal. Quite unexpectedly, a s i g n i f i c a n t decrease in l i v e r tumor response was observed at all time points investigated in mice treated with the combination of DEN and MHV4, independent of whether the virus was given p r i o r to or a f t e r carcinogen administration. The mechanisms of t h i s virus interference with chemical hepatocarcinogenesis are unknown. Changes in cell ploidy status and a persistent decrease in the number of binucleated l i v e r cells observed in MHV4-infected mice may play a role. Our results demonstrate that virus infections can i n t e r f e r e with the outcome of chemical carcinogenicity studies. German Cancer Research Centre, I n s t i t u t e of Biochemistry, Im Neuenheimer Feld 280, 6900 Heidelberg, F.R.G.

66 SELECTIVE SYNTHETIC DNA PROBES FOR RAT AND HUMAN PHENOL GLUCURONOSYL TRANSFERASE P. Mfinzel and E. RShrdanz

Previously it was found that the level of phenol glueuronosyl transferase (GT), one isozyme of the GT enzyme family, is increased in rat liver foci and liver nodules produced by several hepatocarcinogenesis models. To elucidate the mechanisms responsible for altered GT levels at cancer prestages, D N A probes for phenol GT were synthesized. The D N A probes were also used to study the gene homology of phenol GTs in different tissues and species. Since GT isozymes differ mostly in the coating sequence for the NH2-terminal region a 280 bp D N A probe (rat: nucleotide 71-350, human: nucleotide 74-353) was synthesized and amplified by the polymerase chain reaction using two synthetic 23 oligomers as primers and rat or hum a n genomic D N A as templates. Northern blot analysis demonstrated a low level of G T expression in untreated rat liver, but a marked increase of its m R N A 24 h after 3-methylcholanthrene-treatment (7-fold) and a moderate increase after treatment with phenobarbital (2-fold). Using the selective D N A probes for phenol GT it could be demonstrated that G T expression was markedly increased (2fold) in liver nodules produced by feeding N-nitrosomorpholine and in a rat hepatoma cell line (H41IE cells, 4-fold). A moderate expression of phenol GT could also be demonstrated in rat kidney (2-fold). Similar 280 bp D N A probes were obtained no matter whether rat or h u m a n genomic liver D N A were used as templates suggesting that no introns were present in this region. The two probes cross-hybridized under stringent conditions. The results indicate: (1) Expression of phenol GT is persistently increased in liver nodules. (2) The genes for rat and human phenol GT are closely related. Institute of Toxicology, University of Tfibingen, Wilhelmstrafe 56, D-7400 Tfibingen, F.R.G.

67 PHENOTYPIC HETEROGENEITY OF GLUCURONOSYL T R A N S F E R A S E - A L T E R E D R A T LIVER FOCI IN H E P A T O C A R C I N O GENESIS A.-B. Kobuscb

A f t e r N - n i t r o s o m o r p h o l i n e a d m i n i s t r a t i o n o f 3 d o s a g e s (20, 40 a n d 8 0 rag/l, in d r i n k i n g w a t e r f o r 7 w e e k s ) e n z y m e altered liver loci were analyzed histochemically and immun o h i s t o c h e m i c a l l y in W i s t a r r a t s a t w e e k 15, 23 a n d 31 o f t h e e x p e r i m e n t . C h a n g e s in t h e n u m b e r / c m 3 a n d v o l u m e t r i c f r a c t i o n (~) o f t h e s e f o c i w e r e f o u n d to be d o s e - a n d t i m e d e p e n d e n t . T h e aim w a s to s t u d y in d e t a i l t h e b e h a v i o u r o f g l u c u r o n o s y t t r a n s f e r a s e (GT) a l t e r e d l o c i . T h r e e p h e n o typic manifestations of GT-altered focal hepatocytes were observed: focal hepatocytes with increased enzyme level, GT(+), foci w i t h d e c r e a s e d e n z y m e l e v e l , G T ( - ) , a n d l o c i c o n s i s t i n g o f b o t h GT(+) a n d G T ( - ) h e p a t o c y t e s . F o r all of these foci a high correlation with 5 other enzyme-altered l o c i w a s o b s e r v e d . T h e s e e n z y m e s i n c l u d e d GSH t r a n s f e r a s e P, ~ - g l u t a m y l t r a n s p e p t i d a s e , P450IIE1, A T P a s e a n d g l u c o s e 6-phosphatase. GT(-) loci were predominant at early stages f o l l o w i n g low d o s a g e s . GT(+) a n d m i x e d l o c i i n c r e a s e d in a dose- and time-dependent manner. However, within each d o s a g e l e v e l t h e p e r c e n t a g e of GT(+) foci d i m i n i s h e d w i t h time while that of mixed loci increased. Futhermore, judged by their mean focal volume, mixed loci were by far the l a r g e s t . I n t e r e s t i n g l y , t h e d i a m e t e r size c l a s s o f G T a l t e r e d l o c i i n c r e a s e d w i t h t h e n u m b e r of o t h e r e n z y m e alterations per focus (phenotypic complexity level). The r e s u l t s s u g g e s t : (1) G T ( - ) a n d GT(+) l o c i r e p r e s e n t t w o different phenotypes while mixed loci may be later stages of GT(+) loci. (2) T h e n u m b e r o f a d d i t i o n a l p h e n o t y p i c a l t e r a t i o n s o b s e r v e d in G T - a l t e r e d l o c i s e e m s t o b e p o s i t i v e l y a s s o c i a t e d w i t h f o c a l size, i.e. w i t h t h e g r o w t h rate of focal hepatocytes. I n s t i t u t e of T o x i c o l o g y , U n i v e r s i t y o f T f i b i n g e n , W i l h e l m s t r a f e 56, D - 7 4 0 0 T f i b i n g e n , F.R.G.

68 D E T E R M I N A T I O N O F T H E L E N G T H OF T H E H I S T O L O G I C A L STAGES OF APOPTOSIS IN N O R M A L LIVER AND IN A L T E R E D H E P A T I C FOCI O F RATS W. Bursch,

S. Paffe,

a n d G. B a r t h e l

Apoptosis is a f o r m of cell d e a t h i n v o l v e d in the r e g u l a t i o n of cell n u m b e r in t i s s u e s ; it app e a r s to i n v o l v e a c t i v a t i o n of s p e c i f i c genes (e.g. o c c u r r e n c e of m R N A f o r t r a n s g l u t a m i n a s e ; "testosterOne repressed prostate message"). Chemicals may exert toxic effects through induction or i n h i b i t i o n of a p o p t o s i s . Q u a n t i t a t i v e determ i n a t i o n of cell loss t h r o u g h a p o p t o s i s in h i stological sections requires, in addition to c o u n t s of a p o p t o t i c cells, information on t h e d u r a t i o n of t h e h i s t o l o g i c a l l y v i s i b l e s t a g e s of a p o p t o s i s . A m e t h o d to d e t e r m i n e t h e d u r a t i o n of apoptosis in n o r m a l and p h e n o t y p i c a l l y altered t i s s u e of rat l i v e r is d e s c r i b e d : l i v e r h y p e r p l a s i a is i n d u c e d b y liver m i t o g e n s ; w i t h d r a w a l of the g r o w t h s t i m u l u s t r i g g e r s e l i m i n a t i o n of e x c e s s i v e c e l l s b y a p o p t o s i s . On t h e o t h e r hand, the i n i t i a t i o n of a p o p t o s i s c a n be b l o c k e d b y m i t o g e n s a n d t h e r e a f t e r , t h e t i m e c o u r s e of e l i m i n a t i o n of a p o p t o t i c cell r e s i d u e s f r o m t h e liver can be followed. The m e a n d u r a t i o n of the h i s t o l o g i c a l l y s t a g e s of a p o p t o s i s in n o r m a l liv e r w a s f o u n d to be a b o u t 3 hours. F u r t h e r m o r e , in p h e n o t y p i c a l l y a l t e r e d cell foci in r a t l i v e r the h i s t o l o g i c a l l y s t a g e s of a p o p t o s i s a p p e a r t o be as s h o r t as in n o r m a l liver. A s i m p l e f o r m u l a is g i v e n to c a l c u l a t e the cell loss rate by apoptosis. The method presented may provide data for q u a n t i t a t i v e c a n c e r r i s k a s s e s s m e n t f r o m m a t h e m a t i c a l m o d e l s of c a r c i n o g e n e s i s . I n s t i t u t fur T u m o r b i o l o g i e - K r e b s f o r s c h u n g B o r s c h k e g a s s e 8a, A - 1 0 9 0 W i e n

R18 69

71

CHANGED PATTERN OF LEUKOTRIENE C4 METABOLITES IN BILE OF ISOLATED PERFUSEDRAT LIVERS IN INTRAHEPATIC CHOLESTASIS H. Krell, G. Enderle and U. Delabar Drug-induced intrahepatic cholestasis in man is accompanied, to a variable extent, by hepatic inflammation. Since leukotrienes are potent inflammatory mediators which undergo enterohepatic circulation, we studied the b i l i a r y secretion of leukotriene C4 (LTC4) and LTC4 metaboiites under conditions of experimental intrahepatic cholestasis in rats. METHODS: Cholestasis was induced by pretreating rats with e-naphthylisothiocyanate (ANIT), ethionine (ETN), and estradiol valerate (EV). Bile secretion was studied in hemoglobin-free perfused livers (Krell e t a ] . , Hoppe-Seyler's Z. Physiol. Chem. 1984;365,1115-22) isolated from treated rats. 2H-labelled LTC4 was infused into the portal vein at a concentration of 2.5 nM. LTC4 metabol i t e s in bile were determined by HPLC separation (Denzlinger e t a ] . , J.Biol. Chem. 1986;33,15601-06). Permeability between perfusate and bile was assessed by sucrose clearance analyzing secretory compartmentation (Jaeschke et a l . , Biochem.J. 1987;241,635-40). RESULTS: In control l i vers, 80±20 % of infused LTC4 was secreted into bile. Pretreatment of rats with the cholestatic agents reduced bile flow and b i l i a r y secretion of LTC4. In control l i vers, LTD4 and LTE4 in bile amounted to 10.9±1.9 % and 1.3±0.4 %, resp., of total radioactivity. In all models of cholestasis, degradation was increased resulting in LTD4 in bile of 19.4±4.6 % (ANIT), 32.2±5.0 % (ETH), and 45.5±11.9 % (EV). The increase in LTD~ was correlated with both bile/perfusate ratio and b i l i a r y clearance of sucrose upon pretreatment with ETH or EV. In ANIT-induced cholestasis, in contrast, the relative amount of LTD4 remained constant at varying permeability. CONCLUSIONS: Hepatobil i a r y transport and metabolism of LTC4 is changed upon experimental cholestasis. The results are consistent with participation of paracellular reflux of leukotrienes from bile to perfusate resulting in intrahepatic circulation and further degradation of LTC4.

Effect of EGF and cyproterone acetate (CPA) on the DNAsynthesis of preneoplastic rat hepatocytes in vitro I. Neumann, D. Thierau and H. Greim During the stepwise transformation of a normal cell to a malignant cell there is a change in the control of proliferation. Already preneoplastic hepatocytes in altered hepatic foci exhibit a higher mitotic activity than the liver cells of the surrounding tissue. In order to elucidate the mechanistic basis of this differential growth behavior, in vitro studies are required. Thus, the stimulation of DNA-synthesis by EGF, rat serum and cyproterone acetate, a steroid hormone with tumor promoting activity, was examined in y-glutamyltranspeptidase (y-GTase)-positive and negative hepatocytes. Preneoplastic y-GTase-positive hepatocytes were induced in 2/3 partial hepatectomized Sprague Dawley rats by a single dose of diethylnitrosamine (30 mg/kg b.w.) and 0.1% phenobarbital in the drinking water for 6 months. The y-GTase-positive hepatocytes were enriched from the parent cell suspension by affinity binding to antiy-GTase antibody coated dishes (Lebsanft et al. Cancer Letters 29 r 29, 1985). Hepatocytes were kept in primary culture for 48 hrs and stimulated with the mitogens. Replicative DNA-synthesis was determined by liquid scintillation counting of isolated DNA and by autoradiography. DNA synthesis was stimulated in the parent cell suspension 2-, 3- and 4-fold by EGF (10 ng/ ml), EGF/ serum (i0 %) and EGF/CPA (5 uM), respectively. The labelling index was not significantly different in y-GTase-positive and negative hepatocytss when EGF or rat serum were used as mitogens. However, CPA in combination with EGF induced twice as many hepatocytes to enter S-phase in the preneoplastic cell fraction, which is compatible with the tumor promoting activity of CPA. Experiments are in progress to determine whether the mitogenic effect of CPA is mediated by receptor binding. GSF-Institut fur Toxikologie, D-8042 Neuherberg/M~nchen

Pharmakologisches I n s t i t u t , Wilhelmstr.56, D-7400 T~bingen

70 INDUCTION BY TUMOR PROMOTERS OF DNA SYNTHESIS IN PRIMARY CULTURES OF RAT AND HUMAN HEPATOCYTES. W. Parzefall, and E. Erber

72 ANALYSIS OF PLASMA MEMBRANE RECEPTORS AS A TOOL FOR THE DETERMINATION OF TUMOR PROMOTING PROPERTIESOF CHEMICALS. P. Cikryt and W. Muster

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

A large body of evidence suggests that liver tumor promoters are inducers of DNA synthesis in rat liver. We have shown previosly that cyproterone acetate (CPA), a potent liver mitogen in v i v % induced DNA synthesis and mitosis in rat hepatocyte primary cultures too. In the present study we have tested, whether or not other liver tumor promoters (alpha-hexachlorocyclohexane, (HCH); Pregnenolon-16a-carbonitril, (PCN); Nafenopin, (NAF); and Phenobarbital, (PB)) would induce DNA synthesis in rat and human hepatocytes. EGF was used as a positive reference compound. Cells were cultured on collagen gels in serum-free media for to 4 days. Analysis of DNA synthesis was done on H]-thymidine labeled cultures either by autoradiography or by determination of label incorporated into DNA. With rat hepatocytes it was found that HCH, PCN and NAF stimulated DNA synthesis in a dose dependent manner after a lag phase of approximately 24 hours. PB was the least potent of all compounds. EGF was consistently stimulatory in rat cells. The culture conditions developed for rat cells were then transferred to human hepatocyte cultures. EGF was also able to induce DNA synthesis here. However, the number of labeled cells was approximately by an order of magnitude lower than with rat hepatocytes. This might indicate that human liver cells are more strictly bound to the G^ state. CPA, HCH, NAF, PB, and RIF were also testUed for their growth inducing properties. In contrast to rat hepatocytes no increases in DNA synthesis could be found. These findings suggest that human hepatocytes are much less sensitive to liver growth stimuli and therefore may be less prone to their promoting actions in ~epatocarcinogenesis. Institut fur Tumorbiologie-Krebsforschung, Borschkegasee 8a, A-I090 Wien, Austria

A receptor-mediated mechanism has been suggested for a number of chemical tumor promoters. So far, we have studied the interaction of aromatic amines with different carcinogenic properties to the cytosotic aromatic hydrocarbon (Ah) receptor of rat liver and to the cytosolic estrogen receptor of rat liver and rat uterus. Receptors of the plasma membrane seem to play a key role in the process of tumor promotion. The objective of this study is to investigate the interaction of aromatic amines to the plasma membrane receptors of rat liver in vitro and in vivo. First, the methods for receptor analysis had to be established. We are focusing on protein kinase C (PKC) activity and its extracellular binding site for 12-O-tetradecanoyl-13-acetate, the so-called TPA receptor. In order to study the competitive binding affinity of our model compounds with phorbol-12,13-dibutyrate (PDBu) in vitro, we have tded a chromatographic method. The quarternary complex of the TPA receptor dissociates if applied to a HPLC gel filtration column and during sucrose density gradient centrifugation. PKC activity and the TPA receptor coelute as a sharp single peak in the absence of cofactors required for quarternary complex formation. Thus, a filter assay was established for the determination of the competitive binding affinity. The major problem in in vivo studies is the low amount both of PKC activity and TPA receptor in rat liver. In addition, these activities are masked in part by endogenous components which interfere with the determation of enzymatic activity as well as with the quantitation of the TPA receptor. Therefore, we have worked out a method which is based on the extraction of the receptor protein into cytosoL Subsequently, the endogenous cofactors are separated by gel filtration or ion exchange chromatography prior to the quantitation of the enzymatic activity and the receptor level. First results obtained with this new method will be discussed. Institute of Toxicology, University of W~rzburg, Versbacher Str. 9, D8700 W6rzburg, F.R.G. Supported by Deutsche Forschungsgemeinschaft (SFB 172).

R19

73 HEPATOPROTECTIVE ~'FZL~/? OF SEVERAL CALC!q3MANTAGONISTS IN RAT H~I~ATOCYTES I.Stauffert, H.Sippel, U.Steinmann Previous studies in our laboratory have shown, that a series of antiparasital diamidino cor~pounds, e.g. pentamidine, led to liver injuries in vivo and produced toxic effects in isolated rat hepatocytes. As several other hepatotoxins exert their deleterious action on the liver by increasing the cytosolic calcit~n-content (Review: Orrenius,Nicotera, 1987), we became interested whether the hepatotoxic side effects of the diamidino compounds are also due to a rise in cytosolic c a l c i ~ content. As parameter of the cytosolic calcium we measured the activity of the calcitEn-dependent phosphorylase a in isolated rat hepatocytes. In additional ~xperiments we tested if calcitc~Intagonists were able to lower the hepatotoxinstimulated phosphorylase a activity. The results show, that several diamidino compounds lead to a significant activation of phosphorylase a. The additional treatment with the calciumantagonists verapamil diltiazem and nifedipin in concentrations of 50-500 umol/l counteracted this effect and dependently decreased the phosphorylase a activity up to control levels. In correspondence, the calcium antagonists exerted a beneficial effect on cell viability: the cytotoxic action of the diamidino compounds was significantly lowered.

This work was supported by wilhelm-Sander-Stiftung, BRD, Orrenius S. and Nicotera P., Mechanisms and Models in Toxicology, Arch.Toxicol., Stppl. 11,1 I-I 9 (I987) Lehrsttthl fdr Toxikologie und Pharmakologie der Universit~t E r l a n g e n - N ' ~ g , Universit~tsstra5e 22 D-8520 Erlangen

74 INDUCTION OF HEPATIC MONOOXYGENASES BY 2,3,7,8TETRABROMODIBENZO-p-DIOXIN AND LIVER TISSUE CONCENTRATIONS IN RATS Tetsuji Nagao 1, Georg Golor 1, Harald Bittmann 2, Eckhard L6ser 3 The potency of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBrDD) to induce hepatic monooxygenases after a single s.c injection was studied in female rats. The substance was dissolved in a toluene/DMSO mixture 1 + 2 (application voL: 200 #l/kg bw). We tested six different doses between 3 and 3000 n g / k g body wt and measured E R O D (ethoxyresorufin-O-deethylase) activity in liver tissue seven days after injection. There was a good linear relationship between the monooxygenase activity and the doses lied. roups of rats were studied 1 to 78 days after a single dose of 600 ng TBrDD/kg body wt. The highest activity was measured 7 days after treaUnent (195 +_ 41 pmoles resonzfin/mg protein/ rain). Thereafter the extent of monooxygenase induction continually decreased: 39.5 -+ 4.2 pmoles resorufin/mg p r o t e i n / r a i n were measured on day 49 after injection. The extent and time dependancy of the induction very closely resembled results recently obtained in our laboratory under identical experimental conditions after a single injection of 300 ng 2,3,7,8.TCDD/kg body wt (Abraham et aL, 1988, Arch Toxico~ 62). If the difference in the molecular weight of the chemicals is taken into account, both halogenated dioxins reveal a similar enzyme inductive capacity. Liver tissue concentrations were determined in 18 samples. Highest concentrations were measured on day 3 after application (mean _+ SD: 5.7 -+ 1.0 ng/g; n = 3). On day 49 after application a concentration of 0.46 _+ 0.20 ng/g was determined. There was a gqod correlation between E R O D activity and liver concentrations (ff = 73%). Hepatic concentrations were very similar when compared with corresponding doses of TCDD. Studies supportml by grants from the Bandc~mini~teriumfiir Forschung trod Technologie to the Freie UnlvcrsitSt Berlin (07VDX01) and University of Tiibingen(9 and 020).

~p

1 Institutfar Taffkologi~ FUBedin, Garystr, 5, 1000Berlin 33 2 Institut f~r Organische Chemie Universitlit Tiibingen 3 Institutfiir ToMkologie, BAYER AG 146zppemd

75 TCDD-INDUC|BILITY OF SOME MONOOX~fGENASES IN LIVER MICROSOMES OF MARMOSET MONKEYS (Callithr~jacchus) Thomas Schulz-Schelga

2,3,7,8-Tetrachlorodibenzo-p-dloxJn (I"CDD) Is a very potent inducer of certain hepatic monooxyganases in several animal species, but the inductive potency of TCDD in non-human pdmatee Is still unkown. In this study, the activities of ethoxy-, benz~oxy-, pentoxyphenoxazone-Odeall
control a (N=4) 403 117 24 5

± ± + -+

97 33 7 I

167 ng TCDD (N=4) 558 4.99 53 17

± -+ + ±

44 232 15 6

300 rig TCDD (N=4) 535 1065 86 27

-+ + ± -+

65 128 14 4

a solvent only (toluene/DMSO; 0.1 mg/kg body wt) b pmo/es cytouhrome P450 x mg protein "1 o pmoles resorufln x mg protein "~ x min "1

EROD showed a ten-fold induction with the high dose. However, surprisingly also BROD and PROD showed a four- to five4old Increase in activity. The total P450 content, on the other hand, increased only slightly after TCDD treatment. The enzyme activities of ER~OD to BROD, EROD to PROD, and BROD to PROD coffelated very well (r= > 94%). No correlation was found between these enzymes and the overall cytochrome P450 content. Supported by grant Nr. 0318785A from the Bundesministertum f~r Forschung und Techno~ogis Institut for Toxiko~ogie und Embryopharmakologle, Freie Universlt~t Berlin, Garystr. 5, D-1000 Bedin 33

76 ASSESSMENT OF BIOLOGICAL ACTIVITIES OF MIXTURES OF POLYCHLORINATED DIBENZO-p-DIOXINS (PCDDs): COMPARISON B ~ E N DEFINED MIXToKES AND THEIR CONSTITOFd~TS. H.-P. Lipp, T. Wiesm~ller*, H. Hagenmaier*, and D. Schrenk

PCDDs r e p r e s e n t widespread environmental p o l l u t a n t s which are p r e s e n t as complex mixtures with widely d i f f e r i n g t o x i c i t y of t h e i r components. To a s s e s s the biological activity of mixtures, comparative studies were carried out with defined PCDD mixtures and pure constituents using primary rat hepatoeyte cultures and hepatoma H4IIE cells. Dose-response curves for the induction of 7-ethoxyresorufin O-deethylase by PCDDs were determined. With H4IIE cells, treatment with PCDDs led to optimal enzyme induction within 48 h. Similar maximal enzyme activities (efficacies) were obtained for all potent PCDDs. EC, ovalues of PCDDs were compared with the ECn0 of the most potent compound 2,3,7,8-C14DD (2,3,7,8-tetrachlorodibenzo-p-dioxin), and expressed as 2,3,7,8-C14DD equivalents (TEs). For 2,3,7,8-substituted PCDDs the following TEs were obtained: 2,3,7,8-C14DD: 1.00 1,2,3,7,8-C15DD: 0.18 1,2,3,4,7,8-CleDD: 0.18 1,2,3,7,8,9-C16DD: 0.06 1,2,3,6,7,8-CIeDD: 0.04 1,2,3,4,6,7,8-C17DD: 0.05 For CIoDD and PAHs lower ECho-values but also lower efficacies were obtained, making comparison of TEs difficult, TEs for defined mixtures containing 49 PCDDs

could be p r e d i c t e d from the sum of TEs for the 6 most potent 2,3,7,S-substituted congeners which accounted for only 13 - 20% of total PCDDs. These findings were substantiated by similar induction studies with primary hepatocyte cultures. The results suggest that biological activities of PCDD mixtures are mostly due to additive effects of their 2,3,7,S-substituted constituents. Institute of Toxicology, Wilhelmstr. 56 and *Institute of Organic Chemistry, Auf der Morgenstelle 18, University of T~bingen, D-7400 TQbingen, FRG.

R 20 77 PROMOTION OF RAT LIVER FOCI AND INDUCTION OF CYTOCHROME P-450-DEPENDENT MONOOXYGENASES BY 2,4,8-TRICHLORODIBENZOFURAN E. Deml, F. Kiefer and D. Oesterle The biological activity of 2,4,8-trichlorodibenzofuran (2,4,8-TCDF) was studied using two endpoints: a) the promotion of enzyme-altered, preneoplastic loci inititated by diethylnitrosamine (DEN) in livers of weanling female Sprague-Dawley rats and b) the induction of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), a marker for cytochrome P-450I activity, in livers of adult female SpragueDawley rats and in H4IIEC3 rat hepatoma cells. When animals were treated with 200 or 500 mg/kg 2,4,8-TCDF 5 x weekly over I0 weeks after a single application of i0 mg/kg DEN, the higher dose of 2,4,8-TCDF had a promoting effect on the appearance of preneoplastic foci. Thus number and total area of foci deficient in adenosine-5'-triphosphatase were significantly increased by a factor of 1.6. 2,4,8-TCDF induced AHH-activities in 9000 x g supernatants of liver 2-3-fold, when rats were treated with 100-1000 mg/kg/day for 5 days and monooxygenase activities determined after another 3 days. The amounts of 2,4,8-TCDF required for inducing AHH activity in H4IIEC3 cells were 7 orders of magnitude higher than those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The results indicate that 2,4,8-TCDF has a biological activity which is extremely low compared to that of 2,3,7,8-TCDD. GSF - Institut f[Ir Toxikologie, D-8042 Neuherberg (FRG)

79 SUPPRESSION OF IMMUNE RESPONSE BY 2,3,7,8-TCDD I N A POPLITEAL LYMPH NODE ASSAY (PLNA) I N RATS Maria Korte 1, Malgorzata Kubicka-Muranyi ", Ralf Stahlmann ~, Ernst GleiehmannL We used the PLNA in rats (Gleichmann, Arch Toxicol 63: 257, 1989) to investigate alterations of immunofunctions by TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin) in outbred Wistar rats. Three groups of rats were treated with single s.c. injections of 6, 60 and 600 ng TCDD/kg body wt. One control group received the vehicle (toluene + DMSO; 1 + 2) only. One week later 100/~1 of a suspension of human peripheral blood cells in buffer was injected subcutaneously as an antigen into one hind footpad of the animals. The contralateral side was left untreated to serve as an internal control. Another seven days later the lymphoproliferation was assessed by wei~,hing the popliteal lymph nodes and counting the number of cells m the lymph nodes. The quotient ("Index") of the treated and the eontralateral side was determined. The following results were obtained: Dose cell number index [mean ± SD (n)]

Vehicle 6 ng TCDD/.kg: 60 ng TCDD/kg: ooo ng TCDD•kg:

17.0-+10.1 1118/ 9.7 -+ 10.3 8.9 -+ 3.6 (12)* 6.6 -+ 3.2 (6)*

* = p < 0.01; t-test

No immune reaction was found after footpad injection of phosphate buffered saline in combination with TCDD (index: 0.96 to 1.03). We conclude that in the PLNA in rats TCDD reveals a suppression of immune reaction to antigens after a single dose of 60 ng/kg body wt or higher doses. Effects observed at lower doses were not significantly different from controls, because of the high variability of the data. Studies supported by a grant from the Bundesminlsterium fiir Forschung und Technologic (0765002 5) to the Freie Universit~itBerlin.

121nstitutfflr Toxikologi~ FU Bedin, Gatystr. 5, D-IO00Berlin 33 Medizinisches Insatut far Umwelthygiene, Heimich-Heine-Universitdt Dasseldorf, AuJ'm Hennekamp 56 D-4000 Dasseldorf

78 INCREASED COPPER CONCENTRATIONS IN RAT TISSUES AFTER ACUTE INTOXICATION WITH 2,3,7,8-TCDD B.Elsenhans,

W.Forth

and E.Richter

Recently, acutely toxic doses of the environmental pollutant 2,3,7,8-tetrachlorodibenzo-pd i o x i n (TCDD) h a v e b e e n reported to affect the h e p a t i c d i s t r i b u t i o n of e s s e n t i a l m e t a l s in t h e r a t (Wahba et al., J. B i o c h e m . T o x i c o l . 3, 121, 1988). H o w e v e r , t h e r e d u c e d f o o d i n t a k e b y T C D D was not taken into consideration. Therefore, metal concentrations were d e t e r m i n e d in d i f f e rent tissues at the end of a t o x i c i t y s t u d y w i t h T C D D o n rats. M a l e S p r a g u e - D a w l e y r a t s r e c e i v e d a s i n g l e i.p. i n j e c t i o n of c o r n o i l / a c e t o n e w i t h o r w i t h o u t T C D D (125 ~g/kg). C o n t r o l s a n d T C D D - t r e a t e d r a t s w e r e fed a d l i b i t u m ; additionally pair-fed controls received the a m o u n t of feed consumed one day earlier by their TCDD-treated partners. 21 d a y s a f t e r d o sing rats were killed and samples of liver, kidney and jejunum were t a k e n for t h e a n a l y s i s o f Ca, Cu, Fe, Mg, Mn, a n d Zn. A f t e r a c i d d i g e stion of the tissues metals were determined by atomic absorption spectrometry. The most outs t a n d i n g e f f e c t of T C D D treatment was an increase o f t h e c o p p e r l e v e l s in k i d n e y (4-fold, vs. p a i r - f e d controls) a n d l i v e r (> 2-fold, vs. pair-fed controls). Other metals were mainly a f f e c t e d b y t h e r e d u c e d f o o d i n t a k e only. S i n c e Cu is t h e o n l y m e t a l t h e h o m e o s t a s i s o f w h i c h d e p e n d s o n its b i l i a r y excretion, an impaired b i l i a r y e x c r e t i o n of C u b y T C D D is s u g g e s t e d as underlying mechanism. Walther Straub-Institut Toxikologie, NuBbaumstr.

f~r Pharmakologie und 26, D - 8 0 0 0 M ~ n c h e n 2

Supported by the Bayerisches\Staatsministerium f~r Landesentwicklung und Umweltfragen.

8O DOSE-RELATED EFFECT OF T C D D ON SPERMATOGENESIS IN RATS Jutta Hartmann 1, Gabriela M. Rune 2, Rail Krowke1 It is well known that chronic exposure to comparatively high doses of TCDD leads to functional suppression of male reproductive organs (Chahoud et aL, 1989, Arch Toxicol 63: 432). Our studies were aimed at finding out whether effects on sperm production and morphology of the testes may be induced by single doses of TCDD. Four groups of 4 male Wistar rats each were treated with a single subcutaneus injection of t4C-TCDD at a dose of 0.5, I, 3 and 5 gg/kg body wt, respectively. Testes were investigated by light- and electron microscopical means and the total number of sperm in testes and cauda epididymidis was counted after sacrificing the animals. Sperm count revealed a decrease (more than 50%) in the number of sperm in both organs after dosing with 5 and 3 ttg TCDD/kg body wt. Barely significant effects were observed after 1 gg/kg body wt. Morphological alterations after TCDD included lesions of the germinal epithelium, e.g. decreased intercellular contact between Sertoli and germ cells, the sloughing off of premature spermatids into the tubular lumen, and the appearence of necrotic germ cells, in particular pacliytene spermatoeytes. Sertoli cells often showed signs of damage and fragmentation. A decrease of smooth endoplasmic reticulum was perceptible in Leydig cells. No reduction in body weight, food consumptmn and testes weight was found with these doses. Summarizing, it seems probable from our results that TCDD leads to direct effects on spermatogenesis. Supported by a grant (07VDX 019) from the Bundesministerium fiir Forsehung und Technologie. llnstitut ~ Toxikologie und Embryopharmakologie, Freie Universetilt Berlih, Garystr. 5,1)-1000 Begin 33

Zlnsfitut ~ Anatomie, Freie Universit~ Begin, KSnigin-Luise-Str. 15, D-lOOOBerlin 33

R 21 83

81 NEURAL TUBE DEFECTS INDUCED IN RAT EMBRYOS IN VIVO BY EXPOSURE TO VALPROIC ACID (VPA) Frank Zappel, Stephan Klug

E F F E C T OF

GLUCOSE ON SURVIVAL OF MICE AFTER POISONING WITH ARSENIC TRIOXIDE (As203) F.X0 Reichl, L. Szinicz, and H. Kreppel

Valproic acid induced neural tube defects (.NTD) have been observed only in mice, up till now. However, using the whole-embryo culture technique NTD could also be induced in vitro in rat embryos in the presence of V I A (Lewandowski et aL, 198~ Arch Toxicol 58: 239-242). We performed/n vivo experiments with rats treated on a single day (day 10 of pregnancy) with different doses of VPA. Since equal doses in different animals leads to varying serum drug levels, a dose-response relationship could not be establisbed. Therefore, we compared the serum levels with the observed defects. The embryos were evaluated on day 11.5 (with a scheme identical to that used in the/n v/tro studies) and on day 21 (routine studies). Induction of NTD by VPA could be demonstrated on day 11.5 of pregnancy: VPA-serum embryos day of resorp, malform. NTD level(t~g/ml) (nJ evaluation rate rate 0-200 200-450 450-850

33 80 12

11.5 11.5 11.5

0-9% 0% 0-11% 0-100% 22-44% 20-100%

350-510 510-1120

35 ?

21 21

0-85% 100%

+ +

0-100% --

Carbohydrate (glucose and glycogen) depletion was reported to be a major problem in acute arsenic poisoning. In the present experiment the effectiveness of glucose substitution was investigated in mice after acute experimental poisoning with As203. Four groups, 10 mice each, received As203, 12.9 mg/kg, s.c.. The first group remained-without further treatment. 15 minutes after the As203 injection and then every 2 hours the second group received saline, the third 5% glucose, and the fourth 5% glucose + 0.12 IE insulin/kg i.p.. Groups 5 and 6, 5 mice each, received either saline or glucose only. The injection volume was 10 ~i/g mouse. Group 7 (5 mice), remained without any treatment. Immediately after death the livers were removed for the enzymatic determination of glucose and glycogen. The survival rate was 0/10 (group I), 1/10 (group 2), 6/10 (group 3), and 7/10 (group 4), the mean survival time was 12.4, 30.8, 40.7, and 43.6 hours, respectively. All mice not receiving As203 in groups 5-7 (controls) survived the 52 hours period. All mice which died showed a significant decrease in the liver glucose and glycogen content, compared to controls. In livers of survivors, the glucose and glycogen content was not different to the controls. The data support the assumption that carbohydrate depletion is an important factor in arsenic toxicity and its substitution should be considered in the treatment of arsenic poisoning.

When this VPA-induced effect was evaluated on day 21 of pre/~mcy no specific effects, such as NTD, could be detected, but a high resorption rate resulted. Apparently, defective rat embryos have a poor chance of surviving the NTD-lesion, in contrast to mouse embryos. Our data suggest that the apparent species difference is not the result of an inability of V I A to induce abnormal development (i.e. NTD) in rats, but is due to species differences with respect to the consequences of this lesion. Supported by grant Nr. 0318785A from the Bundesminlsterium fiir Forschung und Technologie. Institat fiir Toxikologie und Embryopharmakologie, Freie Universit~t Berlin, Garystr. 5, D-1000 Berlin 33

Walther Straub-Institut Toxikologie, NuBbaumstr.

82

84

ZEBRAFISH EMBRYOGENESIS UNDER THE INFLUENCE OF FORMAMIDE DERIVATIVES AND A L K Y L AMINES K. IKrenauer, G. Groth, and K. 3. Freundt

THE ACTION OF CADMIUM IN DRINKING WATER ON THE CONTENT OF METALLOTHIONEIN, ZINC AND COPPER IN THE LIVER, KIDNEYS AND TESTES OF RATS.

Isolated individual f e r t i l i z e d eggs from zebrafish (Brachydanio rerio) developed in vitro can be used to elucidate possible actions of hazardous agents on embryogenesis. This non-mammalian test system is easy to handle and allows one to obtain reproducible results within 4-6 days. In the present study, the following were the most important indicators of development at different stages during hatching that were checked: epiboly, yolk clot on primitive mouth (stage (st): 16), irregular muscle movement (st: 20), tail bud separation from the yolk (st: 20), heart beats: 200/rain (st: 24), hatehing of the embryo (st: 25), 54 somites (st: 25), delay of development. Normal development of each parameter (as %) was plotted against the concentrations of each agent applied in the incubation me;Jia (0.2 - 15 mg/ml)~ and the areas under the curves (AUC, 10 -~ x mg/ml) were calculated. The AUCs of dimethyiformamide (DMF) were compared to those of N-methylformamide (NMF) and formamide (FA). Epiboly, muscle movement, and hatching of the embryo were more disturbed by DMF than by NMF or FA. Changes of the other parameters (yolk clot on primitive mouth, tail bud separation from the yolk, heart beats: 200/min, 34 somites, delay of development) were more pronounced after application of NMF than of DMF or FA. In total, the order of activity was: NMF,$ DMF'~ FA. Methylamine (MA), dimethylamine (DMA), and diethyiamine (DEA), 0.1- 5 mg/ml incubation media (neutralized by HCI to pH 7.5), caused mortality of most of eggs by eytotoxie effects essentially at stage 2/4. Malformations were not observed. The order of activity was: D E A > M A > D M A ; this has been shown by the rates of e m b r y o n i c m o r t a l i t y : DEA - 60 % at 0.5 m g / m l , MA - 60 % a t 2 mg/ml, DMA - 40 % at 2 mg/ml ineubation media. The results show that the fish egg mode[ allows the researcher to easily differentiate types of embryogenesis lesions caused by ehemicals.

M. A. SAYEDand K.D. FRIEDBERG

Institute of Pharmacology and Toxicology, Fac. of Clin. Med., University of Heidelberg, Maybachstr. 3_4-16, D-6800 Mannheim 1

f~r Fharmakologie and 26, D-8000 M~nchen 2

We have developed a new method for the isolation of metallothloneln (MT) which gives a much higher purity than that obtainable with commercially available MT preparations. We use a polarographic method for the detection of MT which permits measurement to a limit of 33 ng/ml. This always permits the determination of entire MT, whilst the Cd-Haem-method provides lower values, depending on the degree of oxidation. In our experiments we provided male Sprague-Dawleyrats with 1, 10 and 100ppm cadmium (Cd2 + ) in their drinking water for 10, 30 and 90 days. We investigated the effects on body and organ weights, the MT level and on the distribution of zinc and copper in fiver, kidneys a~d testes. We found that the body weight was reduced only after 100ppm Cd -r during the first 3 days. The liver weight was reduced only at this dose, too. Similarly, a weight reduction of the kidneys could only be measured after administration of the highest dose for 10 days. The cadmium content rose both dose and time dependently in all the organs examined. The zinc content of the liver and kidneys behaved similarly. A fall in Zn content was measured only in the testes after 100ppm Cd2 + (10 and 30 days). The .copper content of the liver rose after 90 days in all dose groups. The kidneys showed a significantly increased copper content after 10 and 100ppm Cd2 + in all groups. In contrast, the level fell markedly in the testes after 10 days (10ppm) and after 30 days (1 and 10ppm) administration. The MT level was dose and time dependently increased in the liver and kidneys. In the testes a rise in the MT level was measured at all three Cd concentrations only after 30 days Cd treatment. It is notable that the MT value in~eased markedly in the testes of the control animals during a 90 day observation period. In the animals exposed to Cd 2 + there was further increase in content in the 100ppm group. lnstitut f~r Phanhakologieund Toxicologyder Fakul~t fir klinseheMedizinMannheim,Universit~t Heidelberg,Maybachstx.14-16,Mannhcim1.

R 22 85 A Cd-SATURATION ASSAY FOR Cu-CONTAINING M E T A L L O THIONEIN

D. Klein, R. Bartsch and K. H. Summer Due to the higher affinity of metallothionein (MT) to Cu than Cd, Cu-containing MT so far could not be determined with conventional Cdsaturation methods. This study describes the development of a Cd-saturation method to quantify Cu-containing MT in biological tissues. The principle of the rapid and easy to perform assay involves aeetonitrile to remove high molecular weight Cd-binding proteins, ammonium tetrathiomolybdate to bind Cu from MT and radiolabeled Cd to saturate the resulting apothionein. Excess of tetrathiomolybdate and its Cucomplexes are removed with the anion exchange resin DEAE-Sephacel, and excess Cd is bound to the cation exchange resin Chelex-100. The assay was capable of reliably measuring 14 ng MT and thus is suitable to determine basal MT levels even in biopsies or extrahepatic tissues. In combination with the recently developed Cd-Chelex assay (Bartsch et al., Arch. Toxicol., in press), the degree of the Cu-load in MT also can be assessed. A dose-dependent increase in the level and Cuload of MT could be demonstrated in Cu-incubated cultured human fibroblasts. In liver biopsies from patients with Wilson's disease and primary biliary cirrhosis, MT contents and Cu-loads in MT correlated with cytosolic Cu. In contrast, in livers of infants died from liver cirrhosis, probably caused by increased copper uptake via drinking water, MT-content was only little increased although Cu-levels were high. However, in these cases the protein was saturated with Cu. GSF-Institute of Toxicology, D-8042 Neuherberg, FRG

87 EFFECTS OF IRON OR DEFERRIOXAMINE ON CELL PROLIFERATION IN CULTUREDANIMAL AND HUMAN CELL LINES B. Schadwinkel, B. Steffen, G. Baretton* and C.-P. Siegers Iron is known to stimulate c e l l p r o l i f e r a t i o n in the hematopoetic system (Blood 64, l12a, 137a, 1984). No data, however, e x i s t concerning the influence of iron on e p i t h e l i a l or parenchymal c e l l p r o l i f e r a t i o n , in p a r t i c u l a r tumour c e l l growth. We t h e r e f o r e studied the influence of F e ( l l ) - and F e ( l l l ) - s a l t s as well as the iron-complexing agent deferrioxamine on d i f f e r e n t cultured established c e l l l i n e s . F e ( l l ) - s a l t s (I-I0 mg/l medium) i n h i b i t e d cell growth in a renal e p i t h e l i a l c e l l l i n e of pig (LLC-PKI), a human hepatoma c e l l l i n e (HepG2) and a human colon carcinoma c e l l l i n e (Caco-2). F e ( l l l ) - c h l o r i d e (up to 3 mg/l) stimulated cell growth only in the Caco-2 c e l l l i n e in a concentration-dependentRmanner. The i r o n - c h e l a t o r d e f e r r i examine ( D e s f e r a l ) markedly i n h i b i t e d c e l l growth in a l l three c e l l lines tested, maximum e f f e c t was seen with 150 pmol/l medium. The e f f e c t of iron seems to be dependent on the number of t r a n s f e r r i n - r e c e p t o r s on the cell surface, the s e l e c t i v e stimulation of c e l l p r o l i f e r a t i o n in the colon carcinoma c e l l l i n e Caco-2 may be t h e r e f o r e explained by an increased number of t r a n s f e r r i n receptors on these c e l l s , which is going to be i n v e s t i g a t e d . Iron-dependent tumour-cell p r o l i f e r a t i o n c o r r e l a t e s well with the cocarcinogenic e f f e c t s of an iron-enriched d i e t in a model of dimethylhydrazine-induced tumourigenesis in mice (Cancer Lett. 41, 251, 1988). I n s t i t u t e s of Toxicology and of Pathology*, U n i v e r s i t y of LUbeck, D-2400 LUbeck, FRG.

Medical

86

88

DITHIOCARBAMATE ANALOG N-(4-METHOXYBENZYL)-NDITHIOCARBOXY-D-GLUCAMI~TE, N E W E F F E C T I V E C A D M I U M ANTAGONIST V. Eybl, M. Koutensk~, JsKoutensk#, J.S~kora, V.Smol~kov~, M.M. Jones

IN VITRO TOXICITY SCREENING USING CULTURED SKELETAL MUSCLE CELLS M. GUlden and J. F i n g e r

In the experiments performed in male ICR mice the p r o t e c t i v e effect of a new d i t h i o c a r b a m a t e analog N-(4-methoxybenzyl)-N-dithiooarboxy-Dg l u c a m i n e (Me0BDCG) in acute e a d m i u m intoxieation was proved. The r e t e n t i o n and d i s t r i b u t i o n of Cd was then d e t e r m i n e d at 48th h after the single simultaneous ip a d m i n i s t r a t i o n of Me0BDCG w i t h ll5m CdCl~ iv (0.25mg Cd2+/kg). M e 0 B D C G was i n j e c t e d in- a dose c o r r e s p o n d i n g to chelator: Cd m o l a r ratio 10;l. The w h o l e body burden of Cd was d e c r e a s e d f r o m 82.2 to 72.4% of dose injected ( p < 0 . 0 1 ) . In a n o t h e r experiment M e 0 B D C G was a d m i n i s t e r e d 3 times w i t h i n 6 days (ist dose at 24th h after the Cd applieation). The dose c o r r e s p o n d e d to ehelatot: Cd m o l a r ratio 500:i. The w h o l e body burden of Cd (expressed as % of the amount of Cd r e t a i n e d at 24th h of the experiment) was decreased f r o m 98,3 to 3 4 ~ l % ( p ( 0 . 0 1 ) . ~ e mice w e r e saerified 48h after the last dose of a g e n t . . T h e amount of Cd in the liver, k i d n e y s and in the brain was also r e m a r k a b l y d e c r e a s e d in these experiments. The single peroral a d m i n i s t r a t i o n of this c h e l a t i n g agent did not affeet the whole body b u r d e n of cadmium. On a m o l a r dose basis, M e 0 B D C G was more effective than N-benz y l - N - d i t h i o e a r b o x y - D - g l u e a m i n e in r e m o v i n g c a d m i u m f r o m tissue deposits. D e p a r t m e n t of P h a r m a c o l o g y and CRL, Charles U n i v e r s i t y F a c u l t y of Medicine, CS- 301 66 Pilsen, ~SSR. + D e p a r t m e n t of Chemistry, V a n d e r b i l t U n i v e r s i ty, Nashville, TN, 37 235 U.S.A.

A t e s t system f o r in v i t r o t o x i c i t y screening has been d e v e l o p e d u s i n g p r i m a r y c u l t u r e s o f spontaneously contracting r a t s k e l e t a l muscle c e l l s grown i n 2 4 - w e l l t i s s u e c u l t u r e c l u s t e r s . More t h a n 30 model compounds f r o m v a r i o u s c h e m i cal c l a s s e s and w i t h d i f f e r e n t modes o f c e l l u l a r action (e.g. organic solvents, chlorophenols, detergents~ antibiotics, insecticides, mitochondrial poisons, neurotoxins) were t e s t e d . Concentration-dependent e f f e c t s were d e t e r m i n e d on t h r e e e n d p o i n t s : s p o n t a n e o u s c o n t r a c t i l i t y ( I ) and membrane i n t e g r i t y (2) a f t e r 1 and 24 h o u r s o f e x p o s u r e , and g l u c o s e u t i l i z a t i o n dur ~ i n 9 24 h o u r s ( 3 ) . Spontaneous contractility p r o v e d t o be a s e n s i tive parameter for measuring specific noncytot o x i c e f f e c t s on t h e e x c i t a b l e membrane as w e l l as g e n e r a l c y t o t o x i c i t y . The c o m b i n a t i o n o f different endpoints revealed distinct patterns of concentration-effect-relationships which were characteristic f o r some g r o u p s o f a g e n t s , i . e . chemicals acting selectively on e x c i t a b l e membranes, inhibitors or uncouplers of mitochondrial respiration and membrane s o l u b i l i z e r s . The i n v i t r o h a l f maximum e f f e c t i v e concentrat i o n s ( E C h o - v a l u e s ) measured w i t h t h e most s e n sitive e n d p o i n t v a r i e d between 0 , I m o l e / l and 0 , I n m o l e / l and c o r r e l a t e d rather well with lite r a t u r e d a t a on t h e a c u t e t o x i c doses (LD~nv a l u e s ) f o r r a t s or mice d e t e r m i n e d a f t e r ~v parenteral administration. Supported

by t h e BMFT (0318784 A)

Abteilung Toxikologie der Universit~t B r u n s w i k e r S t r . I 0 , D-2300 K i e l

Kiel,

R 23

89

91

VALIDATION PROJECT OF ALTERNATIVES TO THE DRAIZE EYE TEST IN WEST GERMANY: FIRST RESULTS H. Spielmann, I . Gerner~ S. Kalweit and R. Besoke

Genetically engineered cytochrome 1'450

Various methods have been developed to replace the Oraize r a b b i t eye t e s t f o r i r r i t a n c y t e s t i n g . As yet, p o t e n i t i a l l y a l t e r n a t i v e techniques have neither been generally accepted nor f u l l y validated. The neutral red c y t o t o x i c i ty t e s t (NR) according to Borenfreund and the embryonated hen's egg t e s t at the c h o r i o a l l a n t o i c membrane (HET/CAk4) according to Luepke are two premising methods f o r detecting potential irritants. Starting in June 1988 the Dept. of Research & Technology (BMFT) of the FRG i s , therefore, supporting a 2 1/2 year national i n t e r l a b o r a t o r y valiadal i o n study on the NR and the HET-CAM t e s t which is coordinated at the BGA. The aim o£ the study is to provide comparative data f o r the evaluation of the two tests with regard to t h e i r pred i c t i v e value f o r i r r i t a t i n g p o t e n t i a l of chemicals at the r a b b i t eye. The NR c y t o t o x i c i t y t e s t includes evaluat i o n of c y t o t o × i c i t y by the Kenacid Blue (KB) method and the HET-CAM t e s t w i l l be performed on hen's eggs on day 9 of incubation. 14 groups in t o x i c o l o g i c a l laboratories o£ the chemical industry, u n i v e r s i t i e s , the BGA and other research i n s t i t u t i o n s are studying chemicals From a v a r i ety o£ classes which are characterized by a broad spectrum of l o c a l l y i r r i t a t i n g properties. To f a c i l i t a t e management of the data and to reduce costs, PC-software was developed f o r both tests which allows storage of a l l data and s t a t i s t i c a l analysis on PC. Results on the following chemicals have so f a r been completed: butoxyethanol, dimethylsutfoxide, ethanol, NaC1, phenole, propandiol, p y r i d i n , sodiumdodecylsulfate, t r i e thanolemine, z i n c - p y r i d i n e t h i o n . Only zinc-pyridinethion, w h i c h is a paste with severely i r r i t a t i n g propeties in vivo, could net be tested in the HET/CAM-Test.

toxicological and pharmaceutical studies

Z e n t r a l s t e l l e zur Erfassung und Bewertung yon Ersatz- und Erg~nzungemethoden zu Tierversuchen (ZEBET), I n s t . f . Vet e r i ~ r m e d i z i n , BOA ( F e d . Health O f f i c e ) , B e r l i n , FRG.

expressing V79 derived cell lines for

Co Janssens, S. Dogra, M. Edigkaufer, H.R.Glatt, E. Molitor, S. Reichart, K. Platt, A. Seidel, F. Oesch und J. Dfihmer V79 Chinese hamster cells do not express cytochrome P450s key enzymes in metabolism of xenobiotics - but are perfectly

suited for mutagenicity studies due to their short generation time

of

12 hours, high cloning efficiency and suitable

test

gene (HPRT) on a single X-chromosome.

Cytochrome P450IA1, IA2 and lIB1 expressing V79 derived cell lines

were

generated

by

genetechnological

means

upon

genetransfer with recombinant SV40 expression vectors. Cell lines

were

metabolism efficient

validated studies.

in

These

analytical tool

in

mutagenictity-, cell

lines

will

cytotoxicitybe

a

new

and and

toxicological and pharmacological

studies.

Institut

fiir

Toxikologie,

Johannes

Gutenberg- Unversitiit

Mainz.

90

92

LIPOSOMES ARE SUITABLE CARRIERS FOR POLYCHLORINATED BIPHENYLS (PCB) IN CELL CULTURE SYSTEMS *J. Seedorf,2L. Potthoff,~M. Dubowy,3H.-J. Hapke,ZW. Leibold,tH. R~ssel-Sinn

R E D U C T I V E AND O X I D A T I V E M E T A B O L I S M OF 1,6- AND 1 , 3 - D I N I T R O P Y R E N E IN P E R M A N E N T CELL LINES

In order to investigate immunotoxicological effects of PCB congeneres we were looking for a potent solvent with minimal toxic properties on cultured cells. Organic solvents are c o m m o n l y used in comparable studies although their cytotoxic effects are well known. We tested lines of d i f f e r e n t cell-types for their tolerance of several organic solvents such as ethanol, butanol, acetone, hexane, toluol, dimethylsulfoxide, and isopropyl alcohol. The acceptable c o n c e n t r a t i o n s of these agents failed to solve sufficient amounts of PCB. Therefore we i n v e s t i g a t e d the properties of p h o s p h o l i p i d e - d e r i v e d liposomes as carrier for PCB. Their lipophilic compartments might qualify them as a shuttle for the e t r a o r d i n a r i l y lipophilic PCB. Besides the uptake of PCB into stable liposomes the influence of PCB-free liposomes on several cell lines were studied. Although different cell lines varied in their sensitivity to liposomes they tolerated much higher concentrations of liposomes than of organic solvents. W i t h i n the acceptable range of liposomes sufficient amounts of PCB can be integrated into the phospholipid-vesicles in order to expose cells to a reasonable c o n c e n t r a t i o n of PCB. By these criteria liposomes may become suitable carriers for PCB.

Previous studies have shown that m a m m a l i a n cell lines lacking d e t e c t a b l e cytochrome P450 activities (P450) are capable of r e d u c i n g 1,6-din i t r o p y r e n e (I,6-DNP) to organic soluble products (Hilper et al. [1988] 29 th Congress Europ. Soc. Toxicol, M~nchen, p.147). - The p r e s e n t results show that in cell lines containing P450 the total m e t a b o l i s m of 1,6-DNP is increased 2-3 fold after treatment with 0.4 nM 2,3,7,8-tetrac h l o r o d i b e n z o - p - d i o x i n (TCDD) for 24 h. Cell lines w h i c h were not, or only moderately, inducible for P450 a c t i v i t y did not show this increase. V i r t u a l l y all of the T C D D - i n d u c i b l e 1,6-DNP products were water soluble. Cell lines lacking P450, but c o n t a i n i n g g l u c u r o n o s y l and/or phenol sulfotransferases, also formed sizable amounts of water soluble products indicating that the p r e s u m e d r e d u c t i o n products of 1,6-DNP are extensively conjugated. - In contrast to 1,6-DNP, 1,3-DNP was m e t a b o l i z e d only in those cell lines w h i c h expressed P450. The products found in these cells were p r i m a r i l y water soluble. The apparent rates and inducibilities of 1,3-DNP m e t a b o l i s m were similar to those of the oxidative 1,6-DNP metabolism.

*Dept. of C h e m i s t r y , 2 I m m u n o l o g y Unit,JDept. of P h a r m a c o l o g y and Toxicology, V e t e r i n a r y School, 3000 Hannover

U. Hilper-Reuter, O. C u m p e l i k and F.J. Wiebel

The results suggest that in cultured cells 1,6-DNP is m e t a b o l i z e d by both oxidative and reductive pathways, w h e r e a s 1,3-DNP is metab o l i z e d n e a r l y e x c l u s i v e l y by oxidative pathway(s). GSF-Inst.

f.Toxikologie, D-8042 N e u h e r b e r g FRG

R 24 93 EVALUATIONOF XENOBIOTICACTIONONBIOTRANSFORNATIONIN PRIORY CULTUREDRATHEPATOCYTES G. Schepersand C. Aschmann The objective of this study was to evaluate the suitability of primary cultured rat hepatecj~cesfor screening the effects of xenobiotics on biotransfonnation pathways. As a relative broadmeasurefor this cellular function we investigated phase I (O-deethylation) and phase II (conjugation) metabolismof 7-ethoxycoumarin(7-EC) in intact cells. Experiments were started 24 h after plating. Biotransfonnation of 7-ECwas recorded by exposingthe menolayersfor 1 h to 200 ~ 7-EC and measuring the formation of 7-hydroxycoumarin(7-HC) by a fluorimetric assay. Free 7-HC was measureddirectly and total 7-HC after appropriate enzymatic hydrolysis of the sulfate and glucuronide conjugates. For detecting any direct interference of the test cmloouedswith the 7-EC~netabolism,they were addedto the cells together with the substrate, while delayedeffects of the chemicals on the monooxygenaseactivity were monitored following a prolonged exposureof the cells (up to 48 h). With respect to a direct modification of the drug metabolizing system different modesof action could be demonstratedfor somephenolic compounds (hexachlorophene,2,4~dinitrophenol, pentachlorephenol) and solvents (dimethylsulfoxide-[]g60,dimethylfonnamide-l]Mr). All these compounds led to a concentration-dependentinhibition of the O-deetylase activity. While the solvents did not affeht the subsequent7-HC-conjugation, the substituted phenols, however, inhibited conjugation morethan O-deethylation and showeddifferent influence on glucuronidation and sulfation. As to delayedeffects of chemicals on drug metabolism, enz3aneinduction was of prime interest. Therefore, the principal suitability of this test system was ascertainedby incubating the cells with the standard inducers phenobarbital (PB) and ~-naphtheflavone (8-NF). Following an exposure period of 48 h with PB (2 raM)or B-NF (30 ~) the O-deethy|ation of 7-ECwas increasedto 350 % and 690 %of the controls, respectively. Simultaneous incubation with cycloheximidepreventedthis effect, indicating inhibition of de novo synthesis of monno~genaseprotein. Enz~ne induction was also observedafter prolongedtreabmentof the cells with OvfiO, 13~F,and a technical preparation of pentachlorophenol.

95

SITE DIRECTED MUTAGENESISFOR SUBCLONINGTHE CYTOTOXIN GENE FROM PSEUDOMONASAERUGINOSAUSING THE POLYMERASE CHAIN REACTION (PCR) M.W. Struckmeier, G. O r l i k - E i s e l , H. Niemann*, F. Lutz .

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The cytotoxin of Pseudomonas aeruginosa is an acidic protein with cytolytJc a c t i v i t y against many kinds of eukaryotic c e l l s . The gene is already cloned and sequenced. The coding region should generate a protein of a mass of 31.6 kD, the cytotoxin isolated from bacteria through autolysis, however, is about 3 kD smaller. To investigate a potential p r o t e o l y t i c processing of the protein during autolysis we have expressed the cytotoxin gene in an E. c o l i expression vector by forced cloning in pTrc9gA. To generate a Ncol-compatible cloning s i t e , we amplified the gene with two oligonuc]eotides. One of them contained a 4-base mismatch to create a F o k l - r e s t r i c t i o n site g nucleotides upstream from the ATG-codon. Cleavage with Fokl thus produced'5'-CATG overhangs that could be cloned. PCR was performed f o r ~5 cycles f o r 2 min at 45 C (annealing), 3 min at 72~ C (polymerisation) and I min at 94~ C (denaturation). Six clones were analyzed. A processing step during autolysis similar to that observed in P. aeruginosa, couId not be detected in E. c o l l . Five clones produced a 30 kD protefn the c y t o l y t i c t o x i c i t y of which was activated by TPCK-trypsin. One clone produced larger amounts of a 30 kD protein than the other clones, but toxin from t h i s recombinant E. c o l i could not be a c t i vated by t r y p s i n . The c y t o l y t i c a c t i v i t y was examined in a human granulocyte swelling assay. I n s t i t u t ffir Pharmakologie & Toxikologie, Universit~t, 6300 GieBen, und *Bundesforschungsanstalt f o r Viruskrankheiten der Tiere, 7400 TObingen, FRG.

Abteilung Toxikologie der Universit~t Kiel, BrunswikerStr. I0, D-2300 Kie|

94 BINDING OF ABR1N AND RICIN TOXINS TO CELLS IS MEDIATED BY DIFFERENT CELL SURFACE COMPONENTS

96

K.- H. Jung

B.Marian*,

As previously reported, ribosome inactivating plant toxins from Abrus precatarius and Ricinus comrnunis have common mechanisms of protein synthesis inactivation, similar potencies when toxicities are evaluated in a cell-free system, but they differ considerably in their cell toxicities. Binding data obtained from studies with Jurkat cells (human T-cell line, CD5, CD8) reveal that abrin A, abrin B, abrin C, and ricin D have similar affinity constants. The number of binding sites, however, varies over a wide range. This may be the reason for the different cell toxicities. In displacement experiments, abrin A and ricin D displace each other, both toxins displace abrin B, and abrin C from their binding sites. Abrin C displaces abrin B, but neither abrin A nor ricin D. Abrin B is unable to displace any of these toxins. The results show that binding of abrin A, abrin B, and abrin C is mediated by different structures on the tell surface. Similarly, binding and uptake of abrin A and ricin D are mediated by different binding structures or conceivably by different subloci of identical binding sites. Abteilung Toxikologie, Med~iniseheHochschuleHannover, D - 3000 Hannover 61, FRG

MECHANISM OF SELECTIVE MITDGENESIS INDUCED IN PREMALIDNANT AND MALIGNANT HUMAN COLONOCYTES BY DIGLYCERIDES FOUND IN FECAL EXTRACTS S.Winawer

and

E.Friedman

Human c o l o n i c e p i t h e l i a l cells f r o m n o r m a l mueosa, p r e m a l i g n @ n t a d e n o m a s and m a l i g n a n t carc i n o m a s can be kept in p r i m a r y c u l t u r e for several days to w e e k s so that their r e s p o n s e s to m i t o g e n s can be o b s e r v e d . L o n g - c h a i n D i g l y c e r i d e s (LCDGe) c o n t a i n i n g oleie, p a l m i t i c and m y r i s t i c acid side c h a i n s are p r e s e n t in fecal e x t r a c t s and induce m i t o g e n e s i s in c u l t u r e s f r o m each of 13 a d e n o m a s and 2 of 4 c a r c i n o m a s , but in none of ? c u l t u r e s f r o m normal cells. P a r a l l e l to this a c t i v i t y p a t t e r n a p h o e p h o p r o rein m i g r a t i n g at 63 KD (p63) has b e e n i d e n tified in a d e n o m a - and c a r c i n o m a c e l l l y s a t e s , but not in n o r m a l cells. I m m u n o b l o t i n g w i t h an a n t i - p h o s p h o t y r o s i n e a n t i b o d y d e m o n s t r a t e d that the L D C G s d i m w r i stine and d i o l e i n i n d u c e d 3- to 6 - f o l d i n c r e a ses of t y r o s i n e - p h o s p h o r y l a t i o n of p63, Phosp h o r y l a t i o n was m a x i m a l a f t e r 0.5 min of t r e a t ment, but r e m a i n e d e l e v a t e d for at least 6 hours. P h o s p h o r y l a t i o n of p63 was also enhanced by t r e a t m e n t w i t h the p h o s p h a t a s e - i n h i b i t o r o r t h o v a n a d a t e and with s e v e r a l h o r m o n e s and g r o w t h - f a c t o r s , i n d i c a t i n g LCDGs u t i l i z e e s i g n a l pathway a l r e a d y i n use i n tumor c e l l s bv o t h e r mito~ens, but not present i n normal colonocytee. M e m o r i a l S l o a n - K e t t e r i n ~ C a n c e r Center, 1295 York Ave., New York, NY 10021, USA *Present address; Inst. of T u m o r b i o l o g y - C a n c e r Research, B o r s c h K e g . Ba~ A - I 0 9 0 Vienna, A u s t r i a

R 25

97

99

EFFECTS OF SUBCHRONIC FEEDING D E 0 X Y N I V A L E N O L ON INTESTINAL ABSORPTION FUNCTIONS IN MICE G. Hunder, K. SchUmann, G. Strugala , B. Fichtl

B I O M O N I T O R I N G OF AROMATIC AMINES - HEMOGLOBIN ADDUCTS OF D I A M I N O T O L U E N E S AND D I N I T R O T O L U E N E S I.Zwirner and H . - G . N e u m a n n

D e o x y n i v a l e n o l (DON; 3a,7o,15-trihydroxy-12, 13epoxytrichothec-9-ene-8-one) is a trichothecene m y c o t o x i n with high toxicity in man and animal. Most reports on chronic toxicity of DON have been focussed on feed refusal, reduced weight gain and feed efficiency. In the present experiments, we investigated the effects of DON on intestinal absorption of some nutrients in male mice after a 6-week feeding period. The animals received low levels of DON (0; 0,1 ppm; I ppm; 10 ppm) in the diet. The absorption of water, D-glucose, L-leucine, L-tryptophan, 5 - m e t h y l - t e t r a h y d r o f o l i c acid (SM-THF) and iron was measured in isolated luminally p e r f u s e d jejunal segments according to the method of Fisher and Parsons. Feed consumption was similar in all groups. Weight gain was reduced only at the highest exposure level (10 ppm DON). In this group a significant decrease was found in water and D-glucose transfer and, to a higher extent, in the transfer of 5M-THF. In addition, ,the gut c o n c e n t r a t i o n of 5M-TBF was markedly reduced. In the groups receiving 0,1 and I ppm DON the intestinal transfer of these compounds did not differ from that in controls. The absorption of L-leucine, L-tryptophan and iron remained unaffected in all groups. The findings presented here indicate that chronic ingestion of DON above I ppm in the diet may result in an impairment of transport mechanisms of the g a s t r o i n t e s t i n a l tract. Supported by Bayerisches S t a a t s m i n i s t e r i u m f@r Ern~hrung, Landwirtschaft und Forsten.

Most diaminotoluenes and dinitrotoluenes are mutagenic, but differ in their carcinogenic properties. 2,4-diaminotoluene (2,4-DAT) was classified as a carcinogen in rats, 2,6-DAT not. 2,6-Dinitrotoluene (2,6-DNT) on the other hand is considerably more carcinogenic than 2,4-DNT. If there is a common N - o x i d a t i o n product as the b i o l o g i c a l l y active precursor and if the differences in the biological effects have a pharmacokinetic basis, this should be reflected in the hemoglobin adducts which were studied after oral administration of the test compounds to female Wistar rats. Hydrolytical cleavage of the sulfinic acid amide adduct results in an amine function at the position where the reacting n i t r o s o - g r o u p was formed. With 2,4-DAT Hb-adducts could not be detected. The major cleavage product with 2,6-DAT was 2-Nacetylamino-6-aminotoluene, indicating that one amino-function was acetylated, the other N-oxidized. With 2,4-DNT and 2,6-DNT the reduction of one nitro-group leads to the p r e d o m i n a n t cleavage products 4 - n i t r o - 2 - a m i n o t o l u e n e and 2-nitro6-aminotoluene, respectively. In addition 2,4and to a larger extent 2,6-DAT were identified as cleavage products. A simple correlation between carcinogenicity and the b i o a v a i l a b i l i t y of a possible common N - o x i d a t i o n product could not be established, but the hemoglobin adducts reflect metabolic activation pathways and may be suitable for biomonitoring. The role of other pathways involving for instance the oxidation of the methyl group remains to be established. Institute of P h a r m a c o l o g y and T o x i c o l o g y of the University, Versbacherstr. 9, 8700 W@rzburg, FKG

Walther Straub-Institut fur Pharmakologie und Toxikologie, NuBbaumstr 26 8000 M@nchen 2 und ) Merckle AG, 7900 Ulm

98

100

CAN A T R O P I N E E N H A N C E O R G A N O P H O S P H A T E TOXICITY? S. de la M o t t e ............................................... Generally, e n h a n c e d t o l e r a n c e to a t r o p i n e (A) is a s s u m e d in o r g a n o p h o s p h a t e (OP) poisoning. H i g h doses of A are r e c o m m e n d e d for the t r e a t ment. R e c e n t l y c i r c u l a t o r y failure w a s r e p o r t e d a f t e r h i g h doses of A in OP p o i s o n i n g (LeBlanc et al, Clin Toxicol, 24:69, 1986). C i r c u l a t o r y and r e s p i r a t o r y p a r a m e t e r s were inv e s t i g a t e d in g u i n e a pigs a n e s t h e t i z e d w i t h u r e t h a n e (1.5-1.8 g/kg). 0, i, i0 or 50 m g / k g of A w e r e g i v e n iv 2 m i n u t e s after 32 u g / k g (2xLDS0) soman (S). R e s p i r a t o r y arrest o c c u r e d 3.9, 4.5, 4.0 or 3.1 m i n u t e s a f t e r S, and cardiac a r r e s t 2.8, 2.3, 3.6 or i.I m i n later. S u r v i v a l rates w e r e 0/6, 0/6, 3/7 or 3/7, resp. 4 m i n a f t e r S, b l o o d p r e s s u r e (BP) was 80%, 110%, 80% or 60% of the v a l u e s b e f o r e the experiment. A f t e r i, i0 or 50 m g / k g A alone (no S) the BP w a s I00%, 85% or 75% resp. No f u r t h e r i m p r o v e m e n t of r e s p i r a t o r y p a r a m e ters and survival was o b s e r v e d w i t h A, 50 mg/kg, c o m p a r e d to the i0 m g / k g g r o u p in S poisoning. The h i g h e s t A dose c a u s e d a m o r e p r o n o u n c e d d e c r e a s e in BP than in groups r e c e i v i n g a c o r r e s p o n d i n g dose of A or S alone. The dec r e a s e d intervall b e t w e e n r e s p i r a t o r y and cardiac a r r e s t m i g h t r e f l e c t some k i n d of impairm e n t of c a r d i a c f u n c t i o n b y the h i g h e s t A dose. O u r d a t a do not indicate e n h a n c e d t o l e r a n c e to a t r o p i n e t o x i c i t y in S poisoning. Doses for A t r e a t m e n t s h o u l d he e v a l u a t e d carefully, to avoid further impairment of vital functions.

NON-COVALENT AND COVALENT INTERACTIONS OF ESTROTHEIR METABOLITES WITH M I C R O T U B U L A R GENS AND PROTEINS E. Pfeiffer, R. Schnitzler, S. Endo, M. M e t z l e r

A k a d e m i e des S a n i t ~ t s - und G e s u n d h e i t s w e s e n s d e r Bundeswehr, I n s t i t u t f~r P h a r m a k o l o g i e und Toxikologie, I n g o l s t ~ d t e r Landstr. 98, D-8046 Garching-Hochbri~ck, FRG.

Various estrogens including d i e t h y l s t i l b e s t r o l (DES) and estradiol-178 (E2) have been shown to cause neoplastic t r a n s f o r m a t i o n of Syrian hamster embryo (SHE) cells i__nnvitrq. The only consistent genetic damage associated w i t h cell t r a n s f o r m a t i o n appears to be the induction of n e a r - d i p l o i d aneuploidy. In order to clarify the biochemical m e c h a n i s m of e s t r o g e n - i n d u c e d aneuploidy induction and to e v a l u a t e the role of p e r o x i d a t i v e e s t r o g e n metabolites, we have studied the inhibition of m i c r o t u b u l e a s s e m b l y in a cell-free system by several analogs of DES, e.g. 3,3'-DES, E,E- and Z,Z-dienestrol, indenestrol A and B, and indanestrol. The effect of these estrogens on m i c r o t u b u l e a s s e m b l y was also studied after p e r o x i d a s e - m e d i a t e d activation. Some steroidal estrogens and their catechol metabolites, e.g. 2-hydroxy-E2 were also included. The results show that some compounds (e.g. 3,3'DES and E,E-dienestrol) inhibit microtubule polymerization in the absence of p e r o x i d a t i v e metabolism, whereas others (e.g. indenestrol A) need m e t a b o l i c a c t i v a t i o n for the inhibition of m i c r o t u b u l e assembly. A n e u p l o i d y induction correlated better w i t h the requirement for peroxidative m e t a b o l i s m than with inhibition by the parent compounds, suggesting that a c t i v a t i o n to covalently binding m e t a b o l i t e s may be a prereq u i s i t e for a n e u p l o i d y induction by estrogens in SHE cells. Dept. of Food Chemistry and E n v i r o n m e n t a l Toxicology, U n i v e r s i t y of Kaiserslautern, P.O. Box 3049, D-6750 Kaiserslautern, Fed. Rep. Germany.

R 26 101 BINDING ALBUMIN

PRODUCTS

OF

ETHYLENE OXIDE WITH

HUMAN

SERUM

103 DNA ADDUCT FORMATION INDUCED BY ETHYLMETHANESULFONATE IN MOUSE EMBRYOS

Thomas Platzek*, Gerd Bochert U. F6st, H.M. Bolt It is a common phenomenon in pharmacology that serum albumin has a vehicle function for many lipophilic pharmaeeuties in human blood. Several binding sites in the protein molecule have been characterized. In contrast, the vehicle and target function of albumin for alkylating xenobiotics or their reactive metabolites has hardly been investigated in toxicology. Determination of albumin adducts is a new approach for biological monitoring of carcinogenic compounds like aliphatic epoxides, e.g. ethylene oxide. Human blood samples of non-exposed volunteers were centrifuged (400 g) to isolate the blood plasm. The plasm samples were mixed with 3 g hydroxyapatite suspended in I0 ml 0.01 mol phosphate buffer (pH 7.4) for 1 hour at 4 °C. Hydroxyapatite was then transfered into columns and the serum albumin eluted with phosphate buffer of 0.07, 0.Ii & 0.4 mol/l. The fractions containing albumin were dialysed towards distilled water (eut off: 8000 Da) and lyophilized. After acidic hydrolysis of the albumin samples, the released amino acids were dried and reacted with N-methyl-N-trimethylsilyltrifluoroaeetamide (MSTFA) to form the trimethylsilyl derivates for gas chromatographic separation and subsequent mass spectrometric analysis. We focussed our investigations on adduets of the Nterminal methionine in human albumin. N-hydroxyethyl methionine was also found in non-exposed humans. These findings support the hypothesis of an endogenous base level of ethylene oxide in humans. Our results confirm the observation by other authors of base levels of hydroxyethylated amino acids in human hemoglobin and are in concordance with the base levels of hydrexyethylated guanine in human lymphoeyte DNA previously found by us in pe[sons not occupationally exposed to ethylene oxide. Institut f~r Arbeitsphysiologie,Ardeystr 67,Dortmund,FRG

102 DNA MODIFICATIONS INDUCED BY PHOTOSENSITIZERS AND SINGLET OXYGEN ARE RECOGNIZED BY SPECIFIC REPAIR ENDONUCLEASES B. Epe and E. M~ller ..............................................

Specific repair endonucleases that recognize the characteristic base modifications induced by hydroxyl radicals ('OH), viz. 5,6-dihydrothymine derivatives, have been detected in all types of cells investigated so far. For modifications induced by singlet oxygen (iO2) or photosensitizers, however, which are - in part - chemically different from those induced by 'OH, no specific endonucleases have been described. We have analysed the recognition of DNA modifications induced by "OH, IO2 and photosensitizers in the presence of light by proteins from E. coli strains either overproducing or defective in endonuclease III, the major enzyme of this species to remove ~H-induced base modifications. The results indicate that102-induced modifications are not recognized by endonuclease III but by other endonucleases. Therefore, enzymes possibly specific fori~-induced DNA damage do exist. This suggests that this type of damage plays a role also under "natural" conditions. Both the enzymes involved and the chemical nature of the modifications recognized remain to be characterized. DNA modifications induced by photosensitizers (methylene blue, Rose Bengal) closel~ resemble those by~O2 with respect to the recognltion by the E. coli endonucleases and the relative number of single strand breaks and sites of base-loss generated. Supported by the DFG (SFB 172) Institute of Toxicology, University of W~rzburg, Versbacher Str. 9, D-8700 W~rzburg, FRG

In previous studies using methylating agents a correlation was found between the initial DNA adduct rate (O6-methylguanine) in the embryo and the teratogenic efficiency. This was shown by measuring DNA adduct rates in the teratogenic dose range which exhibited similar adduct rates at the equivalent teratogenic dose levels. A similar approach was performed using the ethylating agent ethylmethanesulfonate (EMS). In the teratogenic dose range (150 - 250 mg/kg body wt) the adduct rates of O6-ethylguanine were similar when compared to those which were obtained with inethylating agents. We conclude that a correlation between teratogenicity and adduct rate (O6-alkylguanine) exists for both methylating and ethylating agents. The no-observed-effect-level-dose (NOEL-dose) for teratogenicity was determined using a group of 28 litters with 322 fetuses and was established to be 100 mg/kg under the experimental conditions used. DNA adduct formation following doses at and below the NOELdose was measured. The lowest experimental dose was 45 mg EMS/kg body wt. Substantial DNA adduct rates in the embryos could be detected. Using the SAS NLIN-procedure an exponential function was fitted to the data. Function: Adduct rate (pmol O6-ethylguanine per pmol guanine) = -0.097 + 0.0000168 x dose EMS (mg/kg) E 2.54 We consider DNA adduct formation as a marker for an embryotoxic potential of EMS, especially at doses below the NOEL-dose. Studies supported by grants from the Deutsche Forschungsgemeinschaft given to the Sonderforschungsbereich 174. Institut fiir Toxikologie und Embryopharmakologie, Freie Universitfit Berlin, Garystr. 5, D-1000 Berlin 33; *pr¢sent address: Max yon Pettenkofer-Imtitut, Bundesgestmdheitsamt Berlin

104 QUANTITATIVEDETERMINATION OF N2,3-ETHENOGUANINE (EG) AND 7-(2'-OXOETHYL)GUANINE(OEG) IN RAT TISSUE DNAAFTER VINYL CHLORIDE (VC) EXPOSURE N. Fedtke*, J.A. Boucheron, and J.A. Swenberg Lactating female CD rats with lO-day-old pups were exposed to 600 ppm VC by inhalation for 5 days, 4 hrs per day. Groups of rats were sacrificed immediately and 3, 7, and 14 days after exposure. The formation and persistence of the DNA adducts EG and OEGwere measured in liver, lung, kidney, brain, and spleen of pups and mothers. OEG was determined by HPLC with fluorescence detection. Gas chromatography- negative ion chemical ionization mass spectrometry was used to quantitate a di-pentafluorobenzyl EG derivative by isotope dilution. The results, expressed as pmol adduct per ~mol unmodified base, indicate tissue differences in the formation of these adducts. The adduct concentrations in pup DNA were highest in the liver (OEG 162, EG 1.81), followed by kidney (OEG 29, EG 0.31), and lung (OEG20, EG 0.21). No adducts were detected in brain or spleen of the pups. The concentrations of adducts in the liver of the dams (OEG 43, EG 0.47) were about fourfold lower than the concentrations in pup liver. The ratio betweenOEG and EG was about 100:1 in all tissues immediately after exposure. In the liver of the preweanling rats, this ratio decreased to 14:1 one week after exposure, reflecting a greater persistence of EG. A half l l f e of 62 hrs was calculated for OEG, the estimated half l i f e of EG was greater than 30 days. In addition to the high persistence of EG, its high efficiency for causing base-pair mismatch in vitro suggests that this adduct may play an important role in VC-induced carcinogenesis. * Present Address: HUls AG, P.O. Box 1320, Toxikologie, 4370 Marl, Chemical Industry Institute of Toxicology, Biochemical Toxicology and Pathobiology, angle Park, NC 27709, USA.

Ps/BiologieFRG. Department of Research Tri-

R 27 105

107

METABOLISM AND DNA INTERACTION OF HYDROXYANTHRAQUINONES, PRESENT IN RUBIA TINCTORUM, L. B. PoKinsky, B. BlSmeke, J. Westendorf and H. Marquardt The roots of Rubla tlnctorum are used for the treatment of kidney and bladder stones. The active principle are hydroxyanthraquinone glycosides. Recently, we reported that some hydroxyanthraqulnones present in Rubla tlneto!um are genotoxic in a battery of short term tests. We now report on the metabolism and the DNA-interactlon of two such anthraquinone glycosides (alizarin and lucldin primveroside) and their corresponding aglycones (alizarin and lueidin) in the rat and in an in vitro system (S9mix). Alizarin, which is weakly mutagenic in Salmonella typh. strain TA 1537 and a weak inducer of UDS in primary rat hepatocytes, after oral application to rats is rapidly conjugated and excreted without alteration of the anthraquinone nucleus. However, if the prlmveroslde of alizarin is ingested, l-hydroxyanthraqulnone is excreted, indicating that a reductlve cleavage of the glycoside takes place. The same reductlve cleavage was observed when alizarin primveroslde was Incubated with S9-mlx. After oral application of lucidln (which was shown by us to be a potent genotoxic compound) three metabslites were observed in the urine of the rats. One of these metabolltes was identified as rubiadln (l,3-dlhydroxy-2methylanthraqulnone). The ingestion of lucldin prlmveroside to rats, too, resulted in the excretion of rubiadin, lucidln and two unidentified metabolltes. The reduction of lucldin to rubiadin was also demonstrated in vltro (Sg-mlx + NADPH). Incubation of lucldln, but not alizarin, with S9-mix and calf thymus DNA resulted in the formation of DNA adducts as demonstrated by the method of 32p-postlabeling. Covalent bondage of lucldin to rat liver DNA was also demonstrated after application of 14C-lucidln to rats. The data snpport our earlier findings on the genotoxic and possibly carcinogenlc potential of lucldln. Dept. of Toxicology, Hamburg University Medical School, Grlndelallee 117, D-2000 Hamburg 13, F.R.G.

&~LIMOR PROMOTING ACTIVITIES VARIOUS IN VITRO ASSAYS. C. ScDmutte and D. Welfle

106

108

ANTHRONES IN COMPARISON TO THEIR CORRESPONDING ANTHRAQUINONES ARE SEVERELY CYTOTOXIC BUT NOT GENOTOXIC. Marion Dominlak and Hildegard Marquardt,

THE THIOUREA METABOLITE FORMAMIDINE SULFIN~TE IS GENOTOXIC IN CULTURED MAMMALIAN CELLS K. Ziegler-Skylakakis and U. Andrae

Hydroxyanthraquinones and their glycosides are the active principle of many phytotherapeutics used as laxatives. It is well accepted that the laxative action is mediated by a micrsblal reduction of the anthraquinones to the corresponding anthrones. Because anthrones are llpophllic and highly reactive, the possibility exists that these compounds may have a genotoxlc potentlal. We, therefore, investigated the DNA binding capacity and the genotoxlc potential of four anthrones (Rheln-anthrone, anthralln, alse-emodln-anthron and chrysophannl-anthron) in a variety of short term tests (Salmonella mlcrosome assay, V79HGPRT-assay and UDS-inductlon in primary rat hepatocytee). Incubation of the anthrones with calf thymus DNA in yltro results in a tight binding which is not extractable by organic solvents. The anthroneswere 20-100fold more cytstoxic in bacteria or mammalian cells than their corresponding anthraqulnones. However, none of the investigated anthrones did induce mutations or DNA repair. These observations suggest that the conversion of anthraqulnonee to anthrones does not give rlse to genotoxlc moieties. However, the laxative effect of hydroxyanthraquinones may be related to the eytotoxic, i.e. intestinal cell wall-damaging effect, of their metabolically generated anthrones. Dept. of Toxicology, Hamburg University Medical School, Grindelallee 117, D-2000 Hamburg 13, F.R.G. Supported by a grant from Deutsche Forschungsgemelnschaft and Roggenbuckstiftung zur Krebshilfe, Hamburg.

OF HYDR0XY~HHAQUINOHES IN

Hydroxyanthraquinones (HA) are the a c t i v e p r i n c i p l e s of many c h r o n i c a l l y used p h y t o t h e r a p e u t i c drugs, !.~. laxatives. One of these compounds, danthron, is carcinogenic in rodents even though it is not genotoxic in a variety of short-term tests. Thus, the possibility exists that danthron and other HA may act as tumor promoters. We, therefore, investigated various HA for their tumor promoting activity in vitro: i.e., stimulation sf hepatocellular growth and promotion of transformation of C3H/M2 mouse flbroblasts. Danthron, chrysophanol, rhein and aloe-emodin were found to be active in these assays. These compounds carry hydroxygroups in the 1,8-posltions of the molecule which are also known to be essential for the laxative effect of HA, A further characteristic of many tumor promoters is the inhibitory effect on intercellular communication. To study this activity with HA, we established the

technique

of

lucifer

yellow

dye

injection.

With

danthron, however, - compared to the tumor promoting agent dieldrin - no change of dye transfer was observed in V79 cells or primary rat hepatocytes. Thus, it appears that the inhibition of cell communication is not involved in the promoting action of danthron on growth and transformation. Nevertheless, our data indicate that longterm-treatment with HA may result in tumor promotion. Further studies are, therefore, necessary to evaluate the carcinogenic risk of HA. Dept. of Toxicology, University Hamburg Medical School, Grlndelallee 117, D-2000 Hamburg 13, F.R.G. Supported by a grant from Deutsche Forschungsgemelnschaft and Roggenbuckstlftung zur Krebshilfe, Hamburg.

Thiourea (TU) is a carcinogen which causes thyroid tumours by a thyroid-specific non-genotoxic mechanism. However, there is evidence that TU can be carcinogenic in organs other than the thyroid, and we have recently shown that the compound is weakly genotoxic i n cultured rat hepatocytes and in V79 cells (Ziegler-Skylakakis etal., Arch. Toxicol. 58, 5, 1985). In the present study we have investigated whether the genotoxicity of TU in V79 cells might be due to a reactive oxygenated metabolite, formamidine sulfinate (FASA). FASA is formed from TU by the microsomal flavincontaining monooxygenase (FMO) via the intermediate fsrmamidine sulfenate which is rapidly reduced to TU in the presence of glutathisne (GSH) (Poulsen et al., Arch. Biochem. Biophys. 198, 78, 1979). Exposure of V79 cells to 5-20 mM TU for 3 h did not result in an induction of micronuclei but a 2-3-fold increase in the incidence of micronuclei was observed after exposure to 10-20 mM TU fsr 18 h. Treatment of the cells with 1.25-5 mM FASA for 3 h increased the frequency of micronuclei up to 12-fold. In cells pretreated with buthionine sulfoximine (BSO) for 18 h to deplete GSH, TU (5-10 mM) resulted in 2-2.5-fold increases already after 3 h. FASA also induced DNA repair synthesis and was weakly mutagenic. To determine whether V79 cells contain FMO activity we measured the capacity of the cells to metabolize the FMOsubstrates phorate and thiobenzamide. Considerable formation of phorate sulfoxide from phorate by postmitochondrial supernatant ($9) was observed. Sulfoxide formation could not be inhibited by prior boiling of the $9, suggesting that V79 cells can oxidize certain FMO substrates by nonenzymatic mechanisms. No measurable enzymatic activity towards phorate and thiobenzamide was detected. The results suggest that the genotoxicity of TU in V79 cells may be due to a slow chemical oxygenation of TU yielding the reactive metaholite FASA. In FMO-containing cells such as hepatocytes enzymatic formation of FASA may be the predominant mechanism underlying the genotoxicity of TU. GSF-Institut f. Toxikolgie,

D-8042 Neuherberg, FRG

R 28 109

111

8TEREOSELECTIVE REACTIVITY OF THE CHIRAL EPOXIDES WITH NUCLEIC ACIDS

TWO

ANTIPODES

OF

H. Peter, K. Golka, D. Wistuba , G. Snatzke ~. Marezynski Both diastereomers of chiral epoxides formed metabolically from their prochiral substituted ethylene precursors show different reactivity with chiral macromolecules (proteins, nucleic acids) in the organism. Enzymes involved in metabolism of epoxides such as glutathione transferases or epoxide hydrolases have stereoselctively different affinities for the two antipodes of chiral epoxides, so that finally one of the enantiomers is present in surplus. Therefore, the purpose of the study presented here was to investigate stereoselective differences in reactivity of chiral propylene oxide and styrene oxide towards nucleic acids. Different monobasic polynucleotides were incubated with either of the two chiral antipodes of propylene oxide or styrene oxide. After four hours of incubation in head space vials, the samples were shock frozen and lyophilized to remove the non-reacted surplus of the epoxides. The polynucleotides were dissolved in isotonic phosphate buffered saline solution, pH 7.4, and subjected to equilibrium dialysis, comparison of melting behavior, registration of circular dichroism and finally after enzymatic hydrolysis to HPLC separation. Thus the differences in nucleic acid binding between the diastereomers were quantified. The results indicate that stereoselective properties play an important role in genotoxicity of chiral epoxides.

Institut f. Arbeitsphysiologie, Ardeystr.67,Dortmund,FRG * Inst.f. Org. Chemie d. Univ. T~bingen,FRG ** Inst.f. Theoretische Chemie d. Univ. Bochum,FRG

SYSTEMIC

GENOTOXICITY OF METHYL BROMIDE AND METHYL IODIDE IN RATS B.Gansewendt, S.Deutschmann, K.Schr6der, E.Hallier ........................................................ The monohalogenated methanes methyl iodide and methyl bromide are widely used as alkylating agents in chemical industry. Furthermore, methyl bromide is used for sterilisation of soil and as a fumigant in greenhouses and mills. The eareinogenicity of both substances is at present under discussion. Methyl iodide has induced lung tumors and local sarcomas in animal experiments and methyl bromide is considered to cause carcinogenic lesions in the forestomach of rats. Due to limited experimental data, the carcinogenicity of these monohalogehated methanes has been evaluated differently by national and international regulatory organs. To clarify the genotoxic hazard of these chemicals, we investigated the reactions of methyl bromide and methyl iodide with DNA in vivo. Male and fem~¼e F-344 rats were exposed orally or by inhalation to C -labelled methyl bromide or methyl iodide. From liver, lung and stomach of the animals, DNA was isolated using phenol extraction. The DNA was either hydrolysed enzymatieally to deoxynucleosides or depurinated with hydrochloric acid. The samples were spiked with non-radioactive methylated guanines or deoxyguanosines as optical markers and separated by hplc. Radioactivity profiles during hple separation were determined. A comparison of the radioactive and optical hplc-elution profiles showed the formation of DNA-adducts. The formation of these adducts was confirmed by GC/MS analysis. The same distribution pattern of radioactivity after hplc-separation of hydrolysed DNA could be observed in all organs extracted for both methyl bromide and methyl iodide and for both ways of application. These results indicate systemic genotoxic effects of methyl bromide and methyl iodide in rats. Institut for Arbeitsphysiologie, Abteilung Toxikologie, Ardeystrasse 67, 4600 Dortmund, FRG

110

112

GENOTOXIC ARYLNITRENIUM IONS FROM ARYLAZIDES A. Dirr and D. Wild ....................................................

THE R O L E OF A L C O H O L D E H Y D R O G E N A S E IN T H E BIOACT I V A T I O N OF 1 , 3 - D I C H L O R O - 2 - P R O P A N O L . E. Eder, D. D e i n i n g e r and P. K n i e t s c h

Arylnitrenium

W e were r e c e n t l y a b l e to s h o w t h a t a l c o h o l d e h y d r o g e n a s e (ADH) is an i m p o r t a n t e n z y m e for the b i o a c t i v a t i o n of a l l y l i c a l c o h o l s . S o m e o t h e r r e a c t i v e a l c o h o l s , in p a r t i c u l a r the c a r c i n o g e n i c 1,3-dichloro-2-propanol, has g a i n e d i n c r e a s i n g i n t e r e s t in the l a s t few years due to its o c c u r r r e n c e in i n s t a n t s o u p s a n d s o u p spices. U n f o r t u n a t e l y the m e t a b o l i s m of t h i s c o m p o u n d is o n l y i n s u f f i c i e n t l y i n v e s t i g a t e d and somewhat sophisticated speculative activation mechanism via hypothetical epoxide formations are p o s t u l a t e d to e x p l a i n its c a r c i n o g e n i c a c t i v i t i e s t) . In order to i n v e s t i g a t e the r o l e of A D H in the b i o a c t i v a t i o n of 2 , 3 - d i c h l o r o p r o p a n o l we t e s t e d its m u t a g e n i c i t y in S . t y p h i m u r i u m T A I 0 0 in the p r e s e n c e and absence of yeast A D H / N A D , and f o u n d a f o u r f o l d h i g h e r m u t a g e n i c i t y w h e n A D H / N A D was present. We p r o p o s e the f o l l o w i n g b i o a c t i v a t i o n mechanism. ADH CICH2-CHOH-CH~CI ..... > CICHz- -CH2CI -H2 1,3-dichloroacetone is a s t r o n g m u t a g e n (30 000 r e v . / ~ m o l ) . The direct m u t a g e n i c i t y of 1 , 3 - d i c h l o r o p r o p a n o l e v i d e n t l y depends on b a c t e r i a l ADH. As no g e n o t o x i c i t y w a s f o u n d in the SOS C h r o m o t e s t w i t h E . c o l i PQ37 this b a c t e r i a s t r a i n seems n o t to possess A D H a c t i v i t y . We are p r e s e n t l y i n v e s t i g a t i n g w h e t h e r a d d i t i o n of A D H / N A D to the SOS C h r o m o t e s t s y s t e m leads to a p o s i t i v e result. I)A.R. J o n e s and G. F a k h o u r i (1979), X e n o b i o tica, 9, 595 Inst. of T o x i c o l o g y , U n i v e r s i t y of W ~ r z b u r g , V e r s b a c h e r Str.9, D - 8 7 0 0 W ~ r z b u r g

ions a r e e l e c t r o p h i l i c ,

DNA-binding,

and mutagenic species formed from arylamines in metab o l i c a l l y c o m p e t e n t cells. R e c e n t l y , w e h a v e s h o w n that they can also be produced non-enzymatically, by photolysis (A = 365 nm) of arylazides (Carcinogenesis 10, 335, 1989). T h i s s t r a i g h t f o r w a r d g e n e r a t i o n of n i t r e n i u m ions is u s e d for s t u d i e s of t h e i r D N A binding and mutagenic potencies. We report here on arylazides/arylnitrenium ions w i t h various aryl ring structures (benzene, n a p h t h a l e n e , q u i n o l i n e , diphenyl, f l u o r e n e , c h r y s e n e , pyrene, IQ etc.) a n d their mutagenic activity in Salmonella typhimurium TA98. No or l o w a c t i v i t i e s a r e f o u n d w i t h s e v e r a l s u b s t i tuted azidobenzenes; the activities increase w i t h the size of the a r y l r i n g a n d r e a c h m a x i m a l v a l u e s for the N-heterocyclic azido-imidazoquinoline compounds, a z i d o - I Q etc. In a d d i t i o n to r i n g s i z e a n d h e t e r o atoms, steric factors are important: 4-azidediphenyl is m u t a g e n i c , 2 - a z i d o d i p h e n y l is inactive. W e c o n clude that the extent of delocalization of the positive charge of the nitrenium ion governs its electrophilic and mutagenic potency. The structure-activity r e l a t i o n s h i p s f o u n d m a y a l s o b e r e l e v a n t for t h e arylamines. Supported by DFG, SFB 172. * Institute of Toxicology and Pharmacology, University of W~rzburg, Versbacher Str. 9, D - 8 7 0 0 W ~ r z b u r g , F.R.G.

R 29 113

115

M U T A G E N I C E F F E C T S OF C A R B O S U L F A N A N D F U R A T H I O C A R B IN T H E A M E S - T E S T A N D Y E A S T - A S S A Y D. W i e d e n m a n n , P a u l a S t e h r e r - S c h m i d , and H. U. W o l f

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 , Univ e r s i t y of Ulm, A l b e r t - E i n s t e i n - A l l e e ll/N-26, D-7900 Ulm/Donau, FRG

C H A R A C T E R I Z A T I O N O F THE C - H A - R A S G E N E IN N O R M A L AND CHEMICALLY TRANSFORMED SYRIAN HAMSTER EMBRYO FIBROBLASTS. R. Ebert, E. Reiss, G. R~llich, J. C. B a r r e t t ~, R. W i s e m a n ~, and D. Schiffmann ............................................... The S y r i a n h a m s t e r e m b r y o (SHE) cell transformation model s y s t e m has b e e n w i d e l y used to study the m u l t i s t e p p r o c e s s of c h e m i c a l l y ind u c e d n e o p l a s t i c t r a n s f o r m a t i o n . D N A s f r o m carc i n o g e n - i n d u c e d S y r i a n h a m s t e r cell l i n e s were s h o w n to i n d u c e t r a n s f o r m e d loci in N I H 3T3 c e l l s a f t e r t r a n s f e c t i o n , and an a l t e r e d p21 ras protein with a slower electrophoretic mobility was detected. Thus. a c t l v a t l o n of the H a - r a s g e n e v i a p o i n t m u t a t i o n m a y be one of the crucial events in the transformation of these cells. - We h a v e n o w c l o n e d the c - H a - r a s p r o t o o n c o g e n e u s i n g c D N A l i b r a r i e s and P C R (introns) One of the c D N A s r e v e a l e d an e l e m e n t ( i n t r o n D exon, IDX,) which represents an alternative t r a n s c r i p t of c - H a - r a s . It n e g a t i v e l y r e g u l a t e s the p21 ra5 e x p r e s s i o n . A p o i n t m u t a t i o n in IDX (coding for the 5 " - s p l i c i n g region) leads to decreased l e v e l s of a l t e r n a t i v e l y s p l i c e d Haras m R N A and r e s u l t s in an i n c r e a s e of normal H a - r a s mRNA, as has b e e n s h o w n w i t h h u m a n T 24 b l a d d e r c a r c i n o m a cells. S i n c e w e h a v e o b s e r v e d elevated H a - r a s m R N A l e v e l s in SHE cell lines neoplastically transformed by diethylstilbestrol, we are s e a r c h i n g n o w for IDX mutations. Moreover, we h a v e d e v e l o p e d a m e t h o d to s c r e e n m o r p h o l o g i c a l l y t r a n s f o r m e d SHE cell c l o n e s for the p r e s e n c e of H a - r a s m u t a t i o n s (PCR/sequencing). This a n a l y s i s of the e a r l i e s t a c c e s s i b l e s t a g e of cell t r a n s f o r m a t i o n (10-12 cell divisions) w i l l c o n t r i b u t e to r e s o l v e the question to w h i c h e x t e n t H a - r a s m u t a t i o n s are e a r l y / p r i mary events in chemical carcinogenesis. I n s t i t u t e of T o x i c o l o g y , V e r s b a c h e r Str. 9, 87 W ~ r z b u r g ; ~NIEHS, R e s e a r c h T r i a n g l e Park, U S A

114

116

D E T E C T I O N OF M U T A G E N I C A G E N T S BY D A R K M U T A N T S OF PHOTOBACTERIA H. B r u n n e r and Chr. F r a n k

TRANSFORMATION OF CARCINOGEN-INDUCED BY THE ACTIVATED c - E - r a s r ONCOGENE M. H6hne I and L. R. Schwarz z

The c a r b a m a t e i n s e c t i c i d e s c a r b o s u l f a n and fur a t h i o c a r b w e r e i n v e s t i g a t e d for m u t a g e n i c eff e c t s in the A m e s - t e s t u s i n g S a l m o n e l l a typhim u r i u m s t r a i n s T A 97, T A 98, T A 100, and T A 102, and in the y e a s t - a s s a y u s i n g g a c c h a r o m y s t r a i n s D7 and D 6 1 . M w i t h o u t ces cerevisiae and w i t h m e t a b o l i c a c t i v a t i o n b y rat l i v e r h o m o g e n a t e ("$9"), B o t h i n s e c t i c i d e s d i d not s h o w any m u t a g e n i c e f f e c t in the A m e s - t e s t w i t h o u t and w i t h m e t a b o l i c a c t i v a t i o n up to c o n c e n t r a t i o n s of 35.7 ~mol/l. In the y e a s t - a s s a y w i t h s t r a i n D7, c a r b o s u l f a n had no m u t a g e n i c a c t i v i t y w i t h o u t and w i t h m e t a b o l i c a c t i v a t i o n up to c o n c e n t r a t i o n s of 45.4 ~ m o l / l . H o w e v e r , f u r a t h i o c a r b s h o w e d mitotic gene conversion and r e v e r s e m u t a t i o n at c o n c e n t r a t i o n s of 38-47 ~ m o l / l w i t h o u t m e t a b o lic a c t i v a t i o n . W i t h m e t a b o l i c a c t i v a t i o n , this e f f e c t d i s a p p e a r e d . In S. c e r e v i s i a e D61.M, b o t h i n s e c t i c i d e s s h o w e d a p r o n o u n c e d i n c r e a s e of m i t o t i c a n e u p l o i d y at c o n c e n t r a t i o n s of 0 . 0 6 - 1 . 2 ~mol/l, w h i c h was 5 - f o l d (as c o m p a r e d to the c o n t r o l value) at 1.2 ~ m o l / l w i t h o u t m e t a b o l i c a c t i v a tion, and 1 7 - f o l d at the same c o n c e n t r a t i o n with metabolic activation. S i n c e the r e s u l t s o b t a i n e d so far s e e m to be of t o x i c o l o g i c a l s i g n i f i c a n c e , the i n v e s t i g a t i o n w i l l be c o n t i n u e d u s i n g test s y s t e m s w i t h other biological endpoints.

B a c t e r i a are w i d e l y u s e d b o t h in f u n d a m e n t a l s t u d i e s of the m e c h a n i s m s i n v o l v e d in the b i o l o g i c a l r e s p o n s e to D N A d a m a g e and in s h o r t term s c a n n i n g tests for p o t e n t i a l c a r c i n o g e n icity. A m o n g t h e s e tests d a r k m u t a n t s of l u m i n o u s b a c t e r i a h a v e a p p e a r e d to be v e r y s e n s i t i v e to d e t e c t a w i d e r a n g e of g e n o t o x i c agents. T h i s test c o n s i s t s in m e a s u r i n g the l u m i n e s c e n c e e m i t t e d b y the r e v e r t a n t l u m i n o u s w i l d type. The a p p e a r a n c e of t h e s e b r i g h t b a c t e r i a leads to an i n c r e a s e of the l u m i n e s c e n c e of the t r e a t e d c u l t u r e o v e r that of the c o n t r o l level d u r i n g g r o w t h e i t h e r in s o l i d or l i q u i d media. We h a v e i n v e s t i g a t e d the a c t i o n of s e v e r a l m u t a g e n i c agents, a m o n g t h e s e A f l a t o x i n BI, on c h e m i c a l l y i n d u c e d d a r k m u t a n t s of P h o t o b o t h in l i q u i d and bacterium phosphoreum s o l i d c u l t u r e media. The m u t a g e n i c a c t i o n led to an i n c r e a s e of l u m i n e s c e n c e b e t w e e n 110 % (1,25 ~g/ml) and 250 • (12,5 ~g/ml) as comp a r e d to the c o n t r o l level (i00 %). The e x p e r i m e n t s w e r e p e r f o r m e d b y u s i n g an a u t o m a t i c a l l y r u n n i n g 6 - c h a n n e l - l u m i n o m e t e r (Hamilton w i t h a r e a c t i o n v o l u m e of o n l y i ml. F u t u r e w o r k c o n s i s t s in f u r t h e r v a l i d a t i o n of the test, u s i n g o t h e r and m o r e s e n s i t i v e s t r a i n s , r e d u c t i o n of the test v o l u m e and an i n c r e a s e of the n u m b e r of s a m p l e s to be m e a s u r e d at the same time. 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 , U n i v e r s i t y of Ulm, A I b e r t - E i n s t e i n - A l l e e II/M-23, D-7900 Ulm/Donau, FRG

DIPLOID

HEPATOCYTES

Sequential treatment of rats with Diethylnitrosamine (DEN) and 2-Acetylaminofluorene (AAF) after partial hepatectomy leads to diploidity of the liver. These carcinogen-induced diploid hepatocytes are regarded as progenitor cells in the development of hepatocellular carcinoma. Until now it is not proven if these 'initiated' hepatocytes represent the target cell population in carcinogen or oncogene induced transformation. Diploid or polyploid hepatocytes were isolated from 2/3 hepatectomized rats treated by DEN (50mg/kg) and 0.02 % AAF (5 weeks) by means of centrifugal elutriation. Enriched cell populations (>85 % act. by FACS analysis) were transfected with retroviral vectors containing the human c-E-ras r or the N-myc gene in combination with a neomycin resistance marker by pulsed electroporation gene transfer. DNA uptake was optimized by transfeetion with CAT expression vectors and was similar in diploid and polyploid hepatocytes. However, only diploid hepatocytes could be transformed into continuously growing cell lines with hepatocyte morphology after transfection with c-Hras r or c- H-rasT/N-myc (transfection eff.: 0.07 G-418 resistant colonies/~g DMA x 106 cells). The established clonal cell lines contain the c-H-ras T gene in 2-10 copies/cell integrated, but lose the transfected gene after 20 pass. without changing the malignant phenotype. These data confirm that diploid hepatocytes are the target cell population of oncogene-induced transformation and that the 0ncogene product is possibly only involved in initiation but not in the maintenance of the transformed state of the hepatocyte. 11nstitut fGr Pharmakologie und Toxikologie, Universit~t G6ttingen, Robert-Koch-Str. 40, D-3400 GGttingen z Institut f~r Toxikologie, GSF, D-8042 Neuherberg

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INTERACTION OF RHODOPSIN WITH SMALL M r GUANINE NUCLEOTIDEBINDING PROTEINS (G PROTEINS) T. Wieland, P. Stannek and I. Ulibarri

EVIDENCE FOR AN AGONIST-FREE BUT ANTAGONIST-SENSITIVE ~-ADRENOCEPTOR ACTION IN TURKEY ERYTHROCYTE MEMBRANES K. GStze and K.H. Jakobs

Bovine rod outer segment (ROS) membranes contain four distinct small Mr G proteins (23 - 27 kDa) as detected by ligand blotting with [~-~2P]GTP. When ROS membranes were incubated with botulinum ADP-ribosyltransferase C3 (C3) and [32P]NAD, two proteins were labeled with Mr's of 22 24 kDa. Most interestingly, C3-induced [32P]ADP-ribosylation of these membrane proteins was inhibited by illumination of the membranes, in a manner similar to pertussis toxin-catalyzed [32P]ADP-ribosylation of ~-transducin. Inhibition of C3-mediated [32P]ADP-ribosylation by light was Mg2+-dependent. Light-induced inhibition of C3-catalyzed [32P]ADPribosylation of 22 - 24 kDa proteins was influenced by guanine nucleotides similarly to pertussis toxin-mediated [32P]ADP-ribosylation of ~-transducin in illuminated ROS membranes: GDP counteracted, whereas the poorly hydrolyzable GTP analogs, guanosine-5'-O-(3-thiotriphosphate) > guanosine5'-($,¥-imino)triphosphate, increased the light-induced inhibition of [~2P]ADP-ribosylation. Similar results as with the endogenous C3 substrates were obtained by measuring C3catalyzed [~2 P]ADP-ribosylation of recombinant human rho A protein and his 14valine mutant,, known C3 substrates, reconstituted with urea-treated ROS membranes. The results, thus, indicate that bovine ROS membranes contain in addition to the G protein transducin also small M r G proteins and that at least two of these proteins, being substrates of C3, apparently interact with rhodopsin in a lightdependent manner. The data, furthermore, illustrate an interaction of small M r G proteins with a membrane receptor (rhodopsin) in an "agonist" (light)-dependent manner and, thus, suggest that these proteins are somehow involved in light signal transduction.

In crude membranes of turkey erythrocytes, stimulation of adenylyl cyclase (AC) by GTP analogs, fluoride and the Badrenoceptor (BAR) agonist, isoproterenol (ISO) was studied. The hydrolysis-resistant GTP analogs, GTP[yS] and GppNHp, caused a time-dependent increase in AC activity. Addition of ISO (maximally effective at ~I ~M) diminished the lag phase of these agents. After prolonged preineubation (260 min at 30°C) with GTP[¥S] ±ISO, AC activities were almost identical and about 50-fold increased over basal activity. BAR antagonists decreased ISO + GTP[yS]-stimulated AC activity not only to the level seen with GTP[%S] alone but even to basal values. When added with the GTP analogs alone, the ~AR antagonists, propranolol and pindolol, completely prevented AC stimulation in a concentration-dependent and stereoselective manner. Pretreatment of the membranes with propranolol (I ~M) or adrenaline (0.1 pM), followed by washing, had no effect on AC stimulation by GTP[yS] and its inhibition by SAR antagonists, which data suggested that the membranes were not contaminated by endogenous catecholamines. On the other hand, when the membranes were pretreated with GTP[yS] ±ISO, subsequent addition of propranoloi had no effect on AC activity. AC stimulation by NaF (~10-fold at 10 mM) was not reduced by the SAR antagonists. The data indicate that in turkey erythrocyte membranes ~AR antagonists can stereoselectively prevent G s protein activation by GTP analogs, which requires prior dissociation of bound GDP, but not by fluoride, apparently even requiring G protein-bound GDP for activation. The findings, thus, suggest that in these membranes dissociation of G s proteinbound GDP is absolutely dependent on BAR action, which, however, does not exhibit an absolute agonist requirement and which can even be prevented by antagonist binding to the receptor.

Pharmakologisches Institut der Universit~t Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, F.R.G.

Pharmakologisches Institut der Universitgt Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, F.R.G.

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LIGHT-STIMULATED BINDING OF GTP[yS] IN BOVINE ROD OUTER SEGMENT (ROS) MEMBRANES: INHIBITION BY PERTUSSIS AND CHOLERA TOXIN C. Wieland

B-ADRENOCEPTOR BLOCKADE AND PSEHDOMONAS EXOTOXIN A PREVENT NORADRENALINE-INDUCED UP-REGULATION OF G i ~-SUBUNITS AND ADENYLYL CYCLASE DESENSITIZATION IN RAT HEART MUSCLE CELLS C.Reithmann and K.Werdan*

Binding of the labeled GTP analog [3sS]GTP[yS] and its regulation by light and the bacterial toxins, pertussis and cholera toxin, were studied in bovine ROS membranes. Binding of [3sS]GTP[yS] was stimulated by bright light compared to dim red light. This increase was due to an increase in both binding affinity (~3-fold) and binding capacity (~2-fold) and required Mg 2+ with a half-maximal and maximal effect at ~I and 10 ~M, respectively. Other guanine nucleotides competed for [3sS]GTP[yS] binding with the potency order, GTP >> GppNHp > GDP. While the apparent affinity of GDP was decreased by illumination, the apparent affinities of GTP and GppNHp were not. Treatment of the membranes with pertussis toxin caused a decrease in GTP[yS] binding capacity with no apparent change in affinity of the remaining available binding sites. Cholera toxin treatment of the membranes resulted in a decrease of the Vma x of the light-stimulated GTPase activity without change in the apparent Km value for GTP (~20 nM). Most interestingly, cholera toxin treatment of illuminated membranes caused a decrease in binding of [3SS] GTP[yS]. In contrast to pertussis toxin, cholera toxin reduced apparent GTP[yS] binding affinity (~I nM) without change in binding capacity. Apparent affinity for GTP (K I 3 nM) was also decreased. It is concluded (i) that binding of GTP[yS] to G proteins (mostly transducin) in bovine ROS membranes is receptor (rhodopsin)-regulated, (ii) does not require exogenous GDP and (iii) is inhibited by both pertussis toxin and cholera toxin treatment. While pertussis toxin apparently only prevents interaction of the affected G proteins with the receptor, cholera toxin-induced ADPribosylation of G proteins appears to result into two changes, a decrease in receptor-stimulated GTPase activity and a reduction in guanine nucleotide binding affinity.

In addition to the down-regulation of B1-adrenoceptors (~I-AR), noradrenaline (NA) (I ~M, 48 h) exposure of rat heart muscle cells leads to an about two-fold increase in the level of inhibitory G-protein ~-subunits and to a concomitant decrease in receptor-independent adenylyl cyclase (AC) stimulation by about 30 %. The present investigation was carried out to study whether the NA-induced increase in G i ~-subunits is due to the ~- or B-AR stimulatory effect of NA and whether it depends on protein synthesis. ~-AR blockade by I ~M timolol hut not ~I-AR blockade by I ~M prazosin completely prevented the NA-indueed BI-AR down-regulation and increase in Gi-protein ~-subunits. Similarly, the NA-mediated decrease in B-AR-dependent and -independent AC stimulation by isoproterenol and forskolin, respectively, were completely abolished by the B-AR but not by the ~ - A R blockade. Pseudomonas exotoxin A (PsExoA) (I ng/ml, 48 h) exposure of the cells led to an about 70 % ADP-ribosylation of elongation factor 2, thereby inhibiting protein synthesis. When added in combination with NA, PsExoA had no influence on NA-indueed BI-AR down-regulation but completely prevented NA-mediated up-regulation of G i ~-subunits. As a consequence, the NA-induced decrease in receptor-independent AC stimulation by forskolin was completely abolished. The decrease in B-AR-mediated AC stimulation by isoproterenol, while still present, was attenuated by 40 %. Conclusion: The NA-indueed increase in G i ~-subunits is B-AR-mediated and depends on de novo protein synthesis. By preventing Gi~-up-regulation PsExoA may attenuate catecholamine-induced desensitization and lead to an increased catecholamine cardiotoxicity. Pharmakologisches Institut der Universit~t Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, F.R.G. *Medizinische Klinik I der Universitgt MNnchen, Marchioninistr. 15, D-8000 MHnchen 70, F.R.G.

Pharmakologisches Institut der Universitgt Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, F.R.G.

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EFFECTS OF INSULIN AND ISOPROTERENOL ON THE PHOSPHORYLATION STATE OF GLUCOSE TRANSPORTERS IN THE ADIPOCYTE H.G. Joost

BIOCHEMICAL CHARACTERIZATION OF A SOLUBLE INOSITOL 1,3,4,5-TETRAKISPHOSPHATE 3-PHOSPHOMONOESTERASE FROM PIG BRAIN A. H6er, D. H~er, and E. Oberdisse

The glucose transport activity in adipocytes is controlled by insulin (stimulation) and catecholamines (inhibition). Since both hormones give rise to activation of protein kinases, the possibility has been investigated that glucose transport activity is modulated through changes in the phosphorylation of the transporter protein. Previously, we have reported (Joost et al., J. Biol. Chem. 262, 11261-11267 (1987)) that neither insulin nor isoproterenol altered the phosphorylation state of a transporter protein immtmoprecipitated with antiserum against the erythroeyte glucose transporter (GT1). However, the recent cloning of a glucose transporter (GT3) exclusively expressed in fat and muscle tissue prompted us to re-address the question with antiserum raised against the Cterminal tridekapeptide of GT3. Isolated adipocytes were equilibrated with [32p]phosphate (0.5 mCi/ml) for 90 rain, and were subsequently treated with or without insulin or isoproterenol for 30 men. The incubation was terminated by rapid centrifugation of cells through silicone oil. Immediately after centrifugation, the cells were frozen in a dry ice/methanol bath, collected from the top of the oil layer, and lyzed in buffer contninlng triton X-100 and phosphatase inhibitors. The lysates were centrifuged, and antiserum (1:200) was added to the supernatants. Immtmocomplexes were isolated with protein A sepharose, and were further separated by gel electrophoresis. From basal cells, the antiserum immunoprecipitated a phosphoprotein with an apparent molecular weight of 48 kDa. This protein was identified as the glucose transporter GT3 since (1) it corresponded with the transporter protein detected by Western blotting, (2) it was not precipitated by pre-immune serum, and (3) it was not precipitated by serum blocked with the C-terminal peptide of the transporter. Insulin failed to change the phosphorylation state of the immunoprecipitated GT3. In contrast, phosphate incorporation into transporters isolated from cells treated with isoproterenol subsequent to the incubation with insulin was moderately increased (25%). These data indicate that the stimulatory effect of insulin on glucose transport activity in adipocytes is unrelated to a change in the phosphorylation state of the tlssue-specific glucose transporter GT3. It cannot be excluded, however, that the inhibitory effect of isoproterenol is at least partially mediated by a phosphorylation of the glucose transporter GT3.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)PR), the intracellular messenger for Caz+ release, is gapid]y inactivated in stimulated cells by either dephosphorylation to inositol 1,4-bisphosphate or by phosphorylation to ino(Ins(1,3,4,5)Pa); the s i t o l 1,3,4,5-tetrakisphosphate latter may act in concert with Ins(1,4,5)P 3 td promote influx of Caz+ across the cell membrane or to r e f i l l Ins(l,4,5)P2-sensitive Caz÷ pools. Ins(l,3,4,5)P4, in turn, is metabolized to inositol 1,3,4-trisphosphate (Ins(1,3,4)P~). We recently showed that Ins(1,3,4,5)P4 is degraded to qns(1 4 5)P. by a soluble 3-phosphatase from plg braln. Now we present some propertles of th~s enzyme, using [5-SZP]Ins (1,3,4,5)P. as substrate. The 3-phosphatase, which was enriched b} ion exchange chromatography, showed a high a f f i n i t y for Ins(1,3,4,5)PA (Km = 400 nM); the i n i t i a l Vmx was 2 nmol/mg prot./min.- Th6 enzyme was inhibited by ~l~e two metabolites of Ins(1,3,4,5)P. degradation with comparable K1 values of about 2 . and "i • 75 pM for Ins(1,4,5)P 3 and Ins(1,3,4)P=, respectwely. There was no influence on enzyme a c t i v i { y by either Mg2÷ up to 30 mM or Ca2+ up to i mM. Commercially available inositol 1,4,5,6-tetrakisphosphate exhibited a marked inhibition on the 3-phosphatase (K~ = 500 nM). Significant inhibitory effects on enzyme a c t i v i t y were also found for GTP (100 ~ ) , UTP and CTP (half maximal at 10 ~ ) ; dUTP, dCTP and TTP were less effective (half maximal inhibition at i mM). The pathway described provides an additional source for Ins(1,4,5)P. generation. This possibility may be supported by the ~ecent description of a novel phosphoinos i t i d e (putatively phosphatidylinositol 3,4,5-trisphosphate). If this phospholipid is hydrolysed by a phospholipase C, Ins(1,3,4,5)P4 w i l l be generated, which is subsequently dephosphorylated to yield Ins(1,4,5)P 3. I n s t i t u t f{ir Pharmakologie, Freie Universit~t Berlin, Thielallee 69/73, D-IO00 Berlin 33.

Abteilung Pharmakologie and To~dkologie I, lustitutfiirPharmakologie trod Toxikologieder UulversithtG6tdngen,Robert-Koch-Str.40, D-3400 GSttingen, FRG.

122 INHIBITION OF VOLTAGE-DEPENDENT Ca2 + CURRENTS AND STIMULATION OF [o-32p]GTP AZIDOANILIDE-BINDING TO G-PROTEINS VIA o 2ADRENOCEPTORS IN THE INSULIN-SECRETINGCELL LINE, RINm5F A. Sehmidt, J. Hescheler, S. Offermanns, G.Sohultz, W. Rosenthal We attempted to elucidate mechanisms by which activated a2-adrenoce ptor,~ inhibit insulin secretion. Since insulin secretion requires activation of Caz+ currents we examined the effect of adrenaline on voltage-dependent Ca2 + currents of RINm5F cells. Whole-cell Ca2 + currents were determined by applying the patch-clamp technique (whole-cell configuration). Cells were kept under voltage-clamp conditions, and Caz + currents were evoked by depolarizing steps from -80 to 0 mV. The evoked currents were completely suppressed by the dihydropyridine, isradipine (1 +pM).Adrenaline (10 /JM, in the presence of 1/JM propranolol) inhibited Ca:' currents by 60 to 70 %. The inhibitory action of adrenaline was not observed in cells which had been treated for about three hours with an exotoxin of Bordetella pertussis, pertussis toxin (100 ng/ml) or in cells loaded with the GDP analogue, guanosine 5'-O-(2-thlodiphosphate) (500 pM). The findings indicate that a pertussis toxin-sensitive G-protein participates in the inhibition of Ca2+ currents by adrenaline. By probing membranes from RINm5F cells with antisera generated against synthetic peptides, several pertussis toxinsensitive G-proteins were identified, i.e. Gi2 with a 40 kDa a-subunit, another Gi subtype (possibly G 1) with a 41 kDa a-subunit, and two forms of Go with msubun ts of 39 and 40 kDa. Adrenaline (30 to 100/JM, in the presenee of 1 to 10 pM propranolol) and the a2-adrenoceptor agonist, clonidine (50/JM), stimulated incorporation of the photoreactive GTP analogue, [a32p]GTP azidoaniUde, into 39 and 40 kDa proteins eomigrating with the 40 kDa Gi2 o-subunit and the 39 and 40 kDa GO a-subunits. These data indicate that a2-adrenoceptors functionally couple to Gi-type G-proteins and to Go. The present data are consistent with the concept that G^ mediates between activated a2-adrenoceptors and voltage-dependent ~a 2 + channels of insulin-secreting cells. Taking into account the tissue disttibutlon of Go and data obtained with neuronal cells and with cells derived from the anterior pituitary (Rosenthal et al., Cold Spring Harbor Symposia on Quantitative Biology 53, 247-254, 1988; Schultz et al., Annu. Rev. Physiol. 52, 275-292, 1990), we suggest that Go couples Inhibitory receptors and voltage-dependent Ca2 + channels in neuronal, neuroandocrine and pituitary cells. Institut for Pharmakologie, Freie Universit~t Berlin, Thielallee 69/73, D-1000 Berlin 33

•

.

,

,

3

,

,

124 Effects of the peptide toxin Mastoparan in the human Thelper lymphocyte model Jurkat H. Sommermeyer G-proteins use a common mechanism for receptor-effector coupling. After GTP binding the G-protein is converted to an active state such that it can stimulate its effector system. Hydrolysis of bound GTP to GDP by the intrinsic GTPase activity of the G-protein terminates that activation. The agonist-receptor complex promotes both the dissociation of bound GDP and the subsequent binding of GTP, thereby increasing the time that the G-protein spends in the active state. It has been shown with purified G-proteins that Mastoparan, a toxin from wasp venom with the structure Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-LysLys-Ile-Leu-NH=, can mimic the role normally played by agonist-liganded receptors. The effect of Mastoparan was investigated in intact Jurkat cells (a human T-cell line). This cells can be stimulated via the antigen T-cell receptor complex (Ti/CD3) leading to enhanced phosphoinositide turnover• The cells also possess receptors coupled to the adenylate cyelase (prostaglandin Ez and ~-adrenergic receptors)• Both signalling pathways involve G-proteins. By use of Mastoparan to activate the G-proteins of this pathways we find neither an effect on basal or stimulated adenylate cyclase nor an effect on basal or stimulated phospholipase C. In higher concentration (50 pM) Mastoparan leads to influx of Ca 2+ by a detergent-like effect. This detergent effect of Mastoparan leads to cell death as can be revealed by an colorimetric viability assay (MTT-test)• We therefore conclude that activation of G-proteins (of the adenylate cyclase or the phospholipase C) by using Mastoparan in intact Jurkat cells is not possible. When Mastoparan is used in intact cells it has to be carefully controlled whether an observed effect is due to the lysis of the cells by the toxin• Div. of Molecular Pharmacology, Dept. of Pharmacology and Toxicology, Medical School Bannover, D-3000 Hannover 61, Federal Republic of Germany

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CHARACTERIZATION OF A MUTATION IN Gs~ DESIGNED TO ALTER THE REACTIVITY OF THE PROTEIN WITH PERTUSSIS TOXIN. M. Freissmuth* and A.G. Gilman +

PERTUSSISINTRACELLULAR PROSTAGLANDIN

A bovine cDNA which codes for the ~-subunit of Gs !Gs=) was altered using site-directed mutageneszs in an attempt to create a substrate for pertussis toxin-catalyzed ADP-ribosylation. The sequence near the carboxyterminus of Gs= -gln39°-tyr391-glu 39zwas changed to encode asp-cys-gly, which corresponds to the sequence found in all three forms of G~= and contains the cysteine residue modified by pertussis toxin. The mutant protein (Gs=PT) was expressed in E.coli and purified to homogeneity. In spite of the amino acid changes, Gs=PT is a poor substrate for pertussis toxin. As predicted from their similar kinetics of guanine nucleotide binding and hydrolysis, G PT and control Gs, are indistinguishable when {~eir activity is assayed with purified bovine brain adenylyl cyclase. However, when reconstituted into cyc- (Gs=-deficient) cell membranes, Gs=PT is three-fold more active in the GTP-bound form than control G s . This increased activity is due to accelerate~ GDPrelease in the presence of membranes suggesting that these provide an exchange factor, presumably a receptor, which interacts only with Gs=PT. By contrast with control Gs=, Gs=PT interacts poorly with B-adrenoceptors. These results show that a Gprotein ~-subunit containing a carboxyterminal cysteine does not necessarily serve as pertussis toxin-substrate and demonstrate the importance of the carboxyterminus of G s in determining the speclflty of its znteractlon with receptors.

Pertussis toxin (Ptx) has been shown to downregulate P2-purinoceptor-stimulated PGD 2 synthesis in rat astrocyte cultures (N.S.Arch. Pharm. 338,704) without affecting relative stimulatory capacities of cells (calculated as -fold increase of basal). It is shown here that Ptx also inhibited arachidonic acid (AA) release upon stimulation with ATP. Ptx inhibition was also observed when the phorbol ester TPA or the calcium ionophore A23187 were used. As shown previously, TPA and A23187 synergistically stimulate PGD 2 production and the purinoceptor is linked to both the phosphoinositoland the adenylate cyclass pathways. Ptx, therefore, likely interferes with some biosynthetic step commonly used as a result of all three stimuli. Treatment of the cells with Cholera toxin (Ctx) caused a generalized down-regulation of TPA-, or A23187-stimulated PGD 2 synthesis as observed with Ptx. In contrast, relative stimulatory capacities of cells were significantly inhibited upon ATP-stimulation. In the same way, ATPstimulated AA-release was markedly reduced, whereas TPA- or A23187-stimulated AA-release was unaffected. The specific inhibition of P purinoceptor mediated PGD 2 production by C~x may involve an additional antagonism at the site of adenylate cyclase. Institute of Pharmacology, University of Freiburg, Hermann-Herder-Str. 5, D-7800 Freiburg i.Br., F.R.G.

*Pharmakologisches Institut der Universit~t Wien, Wahringer StraSe 13 a, A-1090 Wien *Department of Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas 75235

126 GTP-SENSITIVITY OF SOLUBILIZED PGE 2 BINDING SITES FROM PORCINE GASTRIC MUCOSA IS LOST AFTER COVALENT CROSS L I N K I N G WITH DSP W. Schrammel

and M. Beinborn

High affinity 3H-prostaglandin (PG) E 2 binding sites are assumed to be coupled to a guanine nucleotide binding protein (G-protein) in porcine gastric mucosal cells (M. Beinborn et. al. , Gastroenterology 96 (5,2) A37 , 1989). We here investigated the sensitivity of 3H-PGE 2 binding to guanine nocleotids in solubilized plasma membranes before and after treatment with the cross-linking compound dithiobis(succinimidyl propionate) (DSP). Plasma membranes from porcine gastric mucosal homogenates were enriched by centrifugation methods and incubated with 5 nmol/l 3H-PGE2 . After solubilization with 20 mmol/l CHAPS, dissociation of bound 3H-PGE2 was enhanced about 70 % by 10 -4 mol/l GTP whereas other nucleotides (GDP, GMP, ATP, ADP) and PPi had almost no effect. When the membranes were incubated with the cross-linker DSP prior to solubilization, the effect of GTP disappeared. Treatment of the plasma membranes with pertussis toxin (1.8 ~g/ml) and cholera toxin (20 ~g/ml) for 30 min at 37 °C resulted in 32p-ADP-ribosylation of a 40 kDa- and a 45 kDa protein, respectively, as revealed by SDS-gel electrophoresis. We suppose from our data that 3H-PGE 2 binding sites in the plasma membranes of porcine gastric mucosa are closely connected and functionally coupled to a G-protein; this complex is solubilized intact with CHAPS. Abteilung Allgemeine Pharmakologie , Med. Hochschule Hannover, Konstanty-Gutschow-Str. 8 , D-3000 Hannover 61 Supported by DFG grant Se 151/16-1 and 2

AND CHOLERA SIGNALLING SYNTHESIS IN

P.J.Gebicke-Haerter,

TOXIN SYSTEMS ASTROCYTE

A.Schobert,

SENSITIVE LINKED TO CULTURES

G.Hertting

128 A SYNTHETIC LIPOPEPTIDE ACTIVATES HUMAN NEUTROPHILS THROUGH PERTUSSIS TOXIN-SENSITIVE AND -INSENSITIVE MECHANISMS R. Seifert 1, S. Hauschildt 2 and W. Bessler2

Upon exposure to the bacterial chemotactic peptide, N(fMet-LeuPhe), human neutrophils re]ease lysozyme and generate superoxide anions (02-). The synthetic lipoamino acid, Npalmitoyl-S-[2,3]-b~s(palmitoyloxy)-(2RS)-propyl-(R)-cysteine (Pam~Cys), which is derived from the N-terminus of bacterial qipoprotein, attached to (S)-seryl-(S)-lysyl(S)-lysyl-(S)-lysyl-(S)-lysine [Pam~Cys-Ser-(Lys)~], activated 02- formation and lysozyme ~elease in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, lipopolysaccharide and the lipopeptides, Pam~Cys-Ala-Gly, Pam~Cys-Ser-Gly, Pam3Cys-Ser, Pam~Cys-OMeand Pam~Cys-OH, did -not activate 02- formation. ~ertussis toxin prevented activation of 02- formation by fMet-Leu-Phe and partially inhibited the one by Pam3Cys-Ser-(Lys)v Lipopeptide-induced exocytosis was pertussis toxin-insensitive. 02- formation induced by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)~was enhanced by cytochalasin B, a phorbol ester and a diacylglycerol kinase inhibitor. Activators of adenylyl cyclase and removal of extracellular Caz+ differently inhibited On- formation induced by fMet-Leu-Phe and Pam3Cys-Ser-(Lys~~ PamRCys-Ser(Lys). synergistically enhanced fMet-Leu-Phe-in~uced OJ formation and primed neutrophlls to respond to the chemotactic peptide at non-stimulatory concentrations• Our data suggest that the signal transduction pathways activated by fMet-Leu-Phe and Pam~Cys-Ser-(Lys)~ are similar but not identical. In inflammatory processes, bacterial lipoproteins and chemotactic peptides may interact synergistically to activate 02- formation, leading to enhanced bactericidal activity.

formyl-L-methionyl-L-leucyl-L-phenylalanine

I I n s t i t u t f~r Pharmakologie, Freie Universit~t Berlin, Thielallee 69/73, D-tO00 Berlin 33, F.R.G. 21nstiut fQr Immunbiologie der Universit~t, Stefan-MeierStr. 8, D-7800 Freiburg, F.R.G.

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FORMYL PEPTIDE-STIMULATED FORMATION OF GTP [yS] AND SUBSEQUENT BINDING TO G PROTEINS IN MEMBRANES OF HL 60 CELLS J. Bremerich

A D P - R I B O S Y L A T I O N OF A C T I N I S O F O R M S BY B O T U L I N U M C2 T O X I N AND P E R F R I N G E N S I O T A T O X I N S. Mauss, C. C h a p o n n i e r * and G. G a b b i a n i *

Formation of the poorly hydrolyzable GTP analog GTP[yS] and its subsequent binding to G proteins was studied in membranes of differentiated HL 60 cells, using [S5S]ATP[¥S] as thiophosphate donator. Formation of [SsS]GTP[yS] was measured by TLC analysis of nucleotides after extraction with PCA and G protein binding by a filtration (GF/C) assay. In analyzing formation of [35S]GTP[7S] from [35S]ATP[¥S], it was found that the formation required the presence of GDP (maximal effect at ~I ~M) and was inhibited by high concentrations of UDP (~I00 ~M). Most important, the formyl peptide receptor agonist, fMet-Leu-Phe (fMLP), increased the GDP-dependent formation of [3sS]GTP[¥S]. In binding experiments, fMLP (EC50 ~30 aM) increased binding of radioactive material but only when GDP (maximal effect at ~1 ~M) or GTP were added together with [35S]ATP[yS], while other nucleotides (GDP[BS], GppNHp, GMP, ADP and IDP) were ineffective to support the agonist-induced stimulation. The presence of a NTP-regenerating system largely abolished the GDP-dependent binding, fMLP-stimulated binding required Mg2+ (~I00 ~M), while Ca 2+, Sr2+ and Ba 2+ were ineffective. Addition of exogenous GTP[yS] abolished formyl peptide-stimulated binding. Finally, control binding and particularly agoniststimulated binding were largely reduced in the presence of UDP (~I00 ~M). The data, thus, indicate that membranes of HL 60 cells contain (a) nucleoside diphesphokinase(s) catalyzing the formation of GTP[TS] from GDP and ATP[yS] and that the formed GTP[yS], then, binds to membrane G proteins. Most important, not only binding of GTP[¥S] t_o G proteins but apparently also its formation is facilitated by agonist binding to formyl peptide receptors, which findings provide evidence for an interaction of the agonist-receptorG protein complex with the nucleoside diphosphokinase(s). Pharmakologisches Institut der Universitgt Heidelberg, Ira Neuenheimer Feld 366, D-6900 Heidelberg, F.R.G.

C l o s t r i d i u m b o t u l i n u m C2 t o x i n and Clostridium p e r f r i n q e n s iota t o x i n b e l o n g to a n o v e l f a m i l y of ADP-ribosylating bacterial toxins which modify actin thereby inhibiting actin polymerization. The t o x i n ' s s u b s t r a t e is m o n o m e r i c G - a c t i n but not F-actin. A l t h o u g h b o t u l i n u m C2 toxin and p e r f r i n g e n s iota t o x i n m o d i f y actin in A r g - 1 7 7 , both toxins differ in their substrate specificity. We studied the substrate specificities of botulinum C2 t o x i n and p e r f r i n g e n s iota toxin by using five different actin preparations (skeletal m u s c l e ~-actin, cardiac muscle ~actin, s p l e e n c y t o p l a s m i c ~/y-actin, g i z z a r d ~actin and a o r t a actin). Whereas perfringens iota toxin ADP-ribosylated all actin p r e p a r a t i o n s tested, b o t u l i n u m C2 t o x i n did not modify skeletal muscle ~-actin or cardiac muscle ~-actin. Spleen cytoplasmic ~/~-actin and gizzard smooth muscle y-actin were s u b s t r a t e s of b o t u l i n u m C2 toxin. In the a o r t a actin preparation, smooth muscle y-actin but not s m o o t h m u s c l e ~ - a c t i n was ADP-ribosylated by b o t u l i n u m C2 toxin, w h i c h was d e m o n s t r a t e d by precipitation with a smooth muscle ~-actin s p e c i f i c m o n o c l o n a l antibody. The d a t a i n d i c a t e that in c o n t r a s t to C. p e r f r i n q e n s iota toxinr b o t u l i n u m C2 t o x i n ADP-ribosylates specifically c y t o p l a s m i c ~/y- and s m o o t h m u s c l e y-actin. 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 , D6300 GieSen, FRG, and *D@partment de Pathologie, Centre Medical U n i v e r s i t a i r e , 1211 G e n e v e 4, S w i t z e r l a n d

130

132

G-PROTEIN-ACTIVATION OF A SOLUBLE PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIP2)-SPECIFIC PHOSPHOLIPASE C IN HUMAN LEUKEMIA (HL 60) CELLS

DESTRUCTION

OF T H E A C T I N

BY BOTULINUM

C3 A D P - R I B O S Y L T R A N S F E R A S E

P. Gierschik, C. Hou, S. Gierschik, E. Strohmeyer, and M. Camps The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) was investigated using cytosolic fractions of myeloid differentiated human leukemia (HL 60) cells and sonicated phespholipid vesicles containing pH]PIP 2 as substrate. Soluble phospholipase C (PLC) activity was dependent on free Ca 2+ (ECs0 ~ 100 nM) and was markedly stimulated by the GTP analogs GTP[S] ~nd GppNHp. Halfmaximal and maximal (up to 5-fold) stimulation of PLC activity by GTP[S] occurred at I and 30 BM, respectively. Other nucleotides (GTP, GDP, GMP, GDP[S], ATP, ATP[S], UTP; 100 BM) did not affect PLC activity. GTP[S] stimulation of PLC was inhibited in the presence of excess GDP[S] or GDP. Analysis of [3H]inositol phosphates by anion exchange chromatography revealed [3H]inositol trisphosphate as the major product of GTP[S]-stimulated PLC activity. ~y-subunits purified from retinal transducin significantly enhanced basal as well as GTP[S]-stimulated PIP2-hydrolysis. In the absence of GTP[S], specific PLC activity was markedly reduced when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated PIP 2 hydrolysis were linear with time regardless whether studied at low or high protein concentration, these results suggest that (a) PLC is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating an ~-subunit of a heterotrimeric, large molecular weight G-protein present in HL 60 cytosol. Pharmakologisches Institut der Universit~t Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, F.R.G.

K. Aktories, and P. T r a u b *

I. Just,

CYTOSKELETON H. MUller,

INDUCED

W. W i e g e r s *

Botulinum C3 A D P - R i b o s y l t r a n s f e r a s e which is distinct from botulinum neurotoxins, modifies the G T P - b i n d i n g p r o t e i n s the and rac. So far the p h y s i o l o g i c a l f u n c t i o n s of t h e s e proteins are u n k n o w n . Here we s t u d i e d the e f f e c t s of C3 on FAO (rat hepatoma) and U 333 g l i o m a cells. T r e a t m e n t of FAO c e l l s w i t h C3 c a u s e d rounding up of cells in a time and concentration d e p e n d e n t manner. 70 to 90 % of the cells w e r e round a f t e r 48 h of i n c u b a t i o n w i t h 30 ~g/ml C3. Concomitantly, C3 A D P - r i b o s y l a t e d two proteins w i t h Mr of a b o u t 22 and 24 kDa which were localized in the m e m b r a n e and cytosol. ADP-ribosylation depended on the p r e s e n c e of divalent cations and/or guanine nucleotides. Rounding up of cells was paralleled by a d e s t r u c t i o n of the m i c r o f i l a m e n t n e t w o r k and an a l m o s t c o m p l e t e loss of a c t i n f i l a m e n t s s t a i n e d w i t h r h o d a m i n e - c o n j u g a t e d p h a l l o i d i n . W h i l e the intermediate filaments aggregated, the microtubules w e r e not a f f e c t e d b y C3. Similar e f f e c t s w e r e o b s e r v e d w i t h U 333 g l i o m a cells. Effects of C3 on m i c r o f i l a m e n t s occurred earlier than the a g g r e g a t i o n of intermediate filaments. The data suggest that ADPribosylating of GTP-binding proteins by C3 interferes with the r e g u l a t i o n of the actin cytoskeleton. Rudolf-Buchheim-Institut f~r Pharmakologie, F r a n k f u r t e r Str. 107, D - 6 3 0 0 G i e ~ e n and * M a x - P l a n c k - I n s t i t u t f~r Z e l l b i o l o g i e , Rosenhof, D-6802 Ladenburg

R 34 133

135

ISOLATION AND CHARACTERIZATION OF THREE SUBTYPES OF PROTEIN KINASE C FROM BOVINE AORTA. P. Klatt. C. Rudolph. and C. Sch~chtele Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC), an intracellular enzyme involved in second messenger-mediated signal transduction, has attracted much attention since its discovery in 1977. It is now known that PKC is not a single molecular entity, but a family of closely related isozymes. To date, at least 8 subtypes have been identified by recombinant DNA techniques, but only three forms, referred to as s (or III), B1/B2 (or II) and 7 (or I), have been separated by hydroxylapatite chromatography (e.g. from rat brain). Using this kind of chromatography, we have separated three subtypes of PKC from the cytosol fraction of bovine aorta. Forms I and II showed the well-known strong activation by Ca 2+ and phospholipids, whereas form III was only slightly stimulated by these enzyme activators. All three forms could, however, be detected by western blotting using PKC-specific monoclonal antibodies (Amersham, FRG). Peak II appeared to be the known s-form of PKC as revealed by western blotting with antibodies (Sakagaku Kogyo, Japan) that specifically detect the s-isozyme of PKC. These s-specific antibodies did not, however, interact with peaks I and III. Moreover, antibodies specific for the B- and 7-form of PKC did also not crossreact with any of the three forms. Several PKC inhibitors (e.g. staurosporine, polymyxin B, myricetin) were examined for their inhibitory effects against the three PKC subtypes and interestingly, showed differential inhibition of peak III relative to peaks I and II. G6decke Research Institute, 7800 Freiburg, FRG

PURIFICATIONOFA NECA BINDINGPROTEINPROMHUMANpLATELETS Thomas Fcin and Karl-Norbert Klotz N-Ethylearboxamido[SH]adenoslne ([3H]NECA) has been widely used as a radlollgand for eharaeterlzatlon of A2 adenosine receptors. This compound has been shown to bind also to non-receptor binding sites, which are present in human platelets in a 10fold higher density than A2reeeptors. These NECA-bindlng sites can be separated from A2 receptors by gelfiltration, ehitlng in a single peak. This suggested that a slnlge NECA-binding protein may exist which is about 10fold purified after gelfiltration. Additional 15-20fold purification was aehleved by ion-exchange chromatography on DEAE-Sepharese CL-6B. Binding activity eluted again as a single peak from the column. To further purify the oNECA-bindingprotein a hydroxyapatite column was used. Eleetrophoresis of the [q-I]NECA-binding peak from the hydroxyapatite column showed a band purified to near homogeneity with an apparent moleeular weight of 33,000. For pharmacological characterization of the NECA-binding protein, competition of adenosine derivatives for [31-I]NECA binding was tested. NECA was the compound with the highest affinity ( ~ 420 nM) followed by 2-ehloroadenesine (CIA, Ki 25,00nM), which was about 10 times more potent ~an adenosine. A series of N~-snbstituted adenosine derivatives including l~-phenyllsopropyladenosine (R-PIA), l~-eydopentyladenesine (CPA) and the respeetlve 2-ehloro derivatives exhibited Ki-valuas only in the high mieromolar range. Adenosine derivatives with substltuents in the 2.-position, whleh are selective for A2 receptors, are also only week inhibitors of [~I]NECA binding. Alkylxanthlnes as-the dassieal antagonists at adenosine receptors have virtually no affinity at the NECA-binding protein. The purification of this protein confirms the existence of a single protein in human platelets, which is responsible for the non-receptor binding of [3H]NECA. The pharmacological profile of this protein with a so far unknown function is distinct from A2 receptors. Pharmakologisehes Instltut der Universitit Heidelberg, Im Neuenhelmer Fold 366, D-6900 Heidelberg, FRG

134

136

NERVE GROWTH FACTOR - NONRESPONSIVE MUTANT PC12 CELL LINE - A T O O L T O S T U D Y SIGNAL T R A N S DUCTION MECHANISMS OF NERVE GROWTH FACTOR. P.Ehrhard, K. Cron and U.Otten

~ OCYTES

A

T h e r a t PC12 p h e o c h r o m o c y t o m a cell line h a s b e e n widely u s e d f o r t h e s t u d y of t h e mechanism of action of n e r v e g r o w t h f a c t o r ( N G F ) . PC12 cells r e s p o n d to NGF b y c e a s i n g cell division a n d a c q u i r i n g n e u r o n a l p r o p e r t i e s inc l u d i n g g r o w t h of n e u r i t e s . T h e r e is good e v i d e n c e t h a t NGF r e s p o n s e s a r e mediated b y specific h i g h a f f i n i t y r e c e p t o r s . To date, t h e mechanisms of N G F - m e d i a t e d s i g n a l transduction are only poorly understood. A m u t a n t PC12 cell line deficient b o t h in s h o r t - t e r m ( c - l o s p r o t o o n c o g e n e i n d u c t i o n , $6 k i n a s e activation) as well as l o n g - t e r m ( i n c r e a s e in choline a c e t y l t r a n s f e r a s e a c t i v i t y , g r o w t h of n e u r i t e s ) NGF r e s p o n s e s was examined f o r NGF r e c e p t o r e x p r e s s i o n u s i n g a 12SI-NGF c r o s s l i n k i n g J i m m u n o p r e c i p i t a t i o n a s s a y . We f o u n d t h a t t h e m u t a n t line e x p r e s s e s NGF r e c e p t o r molecules with a r e d u c e d molec u l a r m a s s as c o m p a r e d to p a r e n t PC12 cells ( 82 KDa versus 90KDa). P r e l i m i n a r y dissociation s t u d i e s i n d i c a t e d t h a t t h e m u t a n t PC12 ceil line p o s s e s s e s o n l y low a f f i n i t y ( K d ~ riM) NGF b i n d i n g s i t e s . F u r t h e r a n a l y s i s of t h e NGF r e c e p t o r in t h e m u t a n t PC12 cell line s h o u l d b e of g r e a t v a l u e in c h a r a c t e r i z i n g t h e signal t r a n s d u c t i o n of NGF r e c e p t o r s . D e p a r t m e n t of P h y s i o l o g y , U n i v e r s i t y lianum, V e s a l g a s s e 1, CH-4051, Basel.

of

Basel,

Vesa-

DEMONSTRATION ADENOSINE

OF RECEPTORS

ADENYLATE IN

CYCLASE-COUPLED GUINEA-PIG ATRIAL

Anke Wilken, Hoda Tawfik-Schlieper, Ulrich Schwabe

and

In atrial preparations adenosine exerts negative inotropic actions. The underlying mechanism has been proposed to be an increase in the potassium conductance. It is still subject to debate whether changes in adenylate cyclase activity contribute to the inhibitory actions of adenosine. Therefore, the effect of the adenosine derivatives 2-chloro-N6-cyclopentyladenosine (CCPA), R-N6-phenylisopropyladenosine (R-PIA), 5'-N-ethylcarboxamidoadenosine (NECA) and S-N6-phenylisopropyladenosine (S-PIA) on cAMP levels was examined in isolated guinea-pig atrial myocytes. Basal cAMP levels were reduced by the A 1 adenosine receptor-selective agonist CCPA. All agonists suppressed the isoprenalineinduced cAMP increase in a concentrationdependent manner. The rank order of potency wast CCPA (ICso 93 riM) > R-PIA (IC~o 309 nM) > NECA (ICs0 813 nM) >> S-PIA (~C50 26,300 nM), suggesting the involvement of A I receptors. The muscarinic receptor agonist carb~chol produced a complete suppression of the isoprenaline-induced cAMP increase (ICsn 398 ruM). The At-selective antagonist 8-cy~opentyl-l,3-dipropylxanthine (DPCPX) antagonized the effect of CCPA concentration-dependently with a K B value of 9.6 nM. It is concluded that adenosinA receptors in guinea-pig atrial myocytes, which are known to be coupled to K + channels, are also linked to adenylate cyclase and show the characteristics of the A 1 subtype. Pharmakologisches Institut der Universit~t Im Neuenheimer Fold 366, 6900 Heidelberg.

R 35

139

137 Pz-PURINOCEPTORS, BUT NOT AI-ADENOSINE (AR) RECEPTORS MEDIATE FORMATION OF INOSITOL-I,4,5-TRISPHOSPILATE (Ins-P3) AND ITS METABOLITES VIA A PERTUSSIS T O X I N - I N S E N S I T I V E PATHWAY IN THE RAT RENAL CORTEX. W. Sch~tz, C. Nanoff, and Elisabeth Tuisl AR is characterized by a variety of actions in the kidney (e.g. arteriolar constriction, decrease in renin release, decrease in norepinephrine release, stimulation of C l - s e c r e t i o n ) , but the cellular signalling mechanism mediating each particular effect is still unknown. Whereas activation of ~ - A R receptors was shown to stimulate cAMP formation, no coupling to the adenylyl cyclase system was demonstrated with the At-receptor (Freissmuth et al. NS A r c h Pharmacol 335:438,1987). As demonstrated in the present study, Az-receptor agonists affected neither basal nor norepinephrine- and angiotensin II-stimulated formation of Ins-P1, Ins-P2, and Ins-P 3 in slices of the rat renal cortex. In contrast, adenine nucleotides, agonists at the P2-purinoceptor, markedly stimulated inositol phosphate formation, with a rank order of potency consistent for an interaction . with the Pz -subtype Pretreatment of the rats wlth pertussls toxln caused a substantial reduction of functional .G - p r o t e i n , as indicated by the lack of [32p]NAD incorporatzon in a membrane preparation of the renal cortex, but the increase in inositol phosphate formation induced by n o r a d r e n a l i n e and by the P2-agonist adenylylimidodiphosphate [App(NH)p] was not significantly impaired. We conclude that P 2y- p.u r i n o c.e p t o r s are. present in the renal cortex, stlmulatlng formatlon of inositol phosphates via a pertussis toxin-insensitive pathway, which, however, was not used by renal A I - A R r e c e p t o r s as transmembrane signalling system. The physiological role of P2-purinoceptors in the kidney remains to be determined. .

y

.

i

•

.

Pharmakologisches Institut der Universit~t W ~ h r i n g e r Str. 13a, A-1090 Wien

°

MASTOPARANAFFECTS EICOSANOIDSYNTIIESISIN MACROPIIAGES

K. Weasel and V. Kaever Prostaglandin and leukotriene synthesis in m a c r o p h a g e s c a n be i n d u c e d b y d i f f e r e n t i n f l a m m a t o r y stimuli. T h e i n v o l v e m e n t of s e v e r a l signal transduction pathways s h o u l d b e e x a m i n e d to g e t f u r t h e r i n s i g h t in t h e r e g u l a t i o n a n d m o d u l a t i o n of e i c o s a n o i d s y n t h e s i s . Mastoparan, a peptide toxin from wasp venom, exhibits a range of biological effects in various cellular systems by mimicking the action of occupied receptors on guanine nucleotide binding proteins (G-proteins). To get informations about G-proteln involvement in the induction of elcosanoid release, we studied the influence of mastoparan on eicosanoid metabolism in murine macrophages. Masfoparan induced the release of arachidonic acid and in addition the subsequent synthesis of eicosanoids in this cellular system. This effect was accompanied by an enhanced activity of GTPase and phospholipase Az. However, while the induction of eicosanoid synthesis started at moderate concentrations of mastoparan (5 pM), higher concentrations (50 ~M) caused cell death. Taking into consideration the negative effect of mastoparan on cell v i a b i l i [ y , we conclude that it can be misleading to correlate the /'n vitro mastoparan action on G-proteins with effects o n complex systems in intact cells. institute of Molecular Pharmacology, Department of Pharmacology and Toxicology, Medical School Hannover, D3000 Hannover 61, FRG

Wien,

138

140

INTRACELLULAR ANALYSIS OF POSTSYNAPTIC ACTIONS OF SOME ADENOSINE AGONISTS AND ANTAGONISTS IN RAT H1PPOCAMPAL NEURONES

INHIBITION OF EICOSANOID RELEASE FROM PERITONEAL MACROPHAGES IN VITRO BY PETASIN AND ISO-PETASIN. D. Bickel, M.A. Popp*, J. Mollenhauer

Angela Ameri It is well known that adenosine and its agonists exert their depressant effects by interacting with the adenosine A1 receptor while there is no evidence for an electrophysiological effect mediated via the A2 receptor. To make a further attempt to determine A1 and A2 receptor-mediated changes in the electrical properties in neurone membranes, experiments were carried out with intracenular recordings from CA1 cells in isolated slices of the rat hippocampus using 5'-N-ethylcarboxamidoadenosine (NECA), L-phenylisopropyladenosine (PIA) and the A[ receptor antagonist 8-cydopentyl-l.3dipropyhanthine (DPCPX) administered by the superfusion medium. NECA (0.5-10)aM) caused a hyperpolarizalion (1-7 mV), a decrease (7-52%) in membrane resistance and an increase (40-220 %) in the threshold of action potentials. These effects were dose-dependent and antagonized by theophylline (150 pM). PIA produced NECA-like effects at low concentrations (0.1-1 ~aM), while at high concentrations (2.5 IJM) it caused either a hyperpolarizalion and a decrease in membrane resistance or a depolarization and an increase in resistance. Either type of effects was reduced by theophylline (150 IuM) administered in presence of PIA. DPCPX (0.1, 0.5 and I taM) produced depolarization (2-6 mV), increase in resistance (10-60 %) and generation of spontaneous impulse discharges. These effects remained unchanged even after I h washout. When NECA (5 taM) was administered in the presence of DPCPX, it caused further depolarization and increase in resistance (13-24 %). These effects were reversed into a hyperpolarization by theophylline (150 ~M). In contrast to NECA, the hyperpolarizing action of PIA was still present after administration with DPCPX, but it was substantially reduced. These data show that both NECA and PIA mainly bind to the At receptor and thus inhibit neurone activity. PIA exerts NECA-Iike effects at lower concentrations than NECA, indicating that it has a higher potency at A1 receptors. In certain conditions, NECA and PIA also appear to act on Pm receptors. Institut ffir Pharmakologie und Toxikologie der Universit/it des Saadandes, D-6650 Homburg/Saar

Petasin is an alcoholic sesquiterpen of the angelic acid extractable from Petasites hybridus (compositae). It has been shown, that petasin and iso-petasin have spasmolytic effects and the complete plant extract has ulceroand cytoprotective abilities. We investigated petasin and iso-petasin from various extraction procedures, starting from crude plant extract, on its abilities to inhibit prostaglandin E2 and leukotrien C4, D4, and E4 release from cultured mouse peritoneal macrophages activated by ionophore A23187. Peptidoleukotrien release was inhibited to a marked extent in a dosedependent manner by highly purified (>98%) petasin and isopetasin. ICS0 values of the extracts vary from 400 2~M for the crude extract to 2 /~M for petasin and 3 ~ M for isopetasin. Prostaglandin E2 release was almost unchanged in the concentration ranges tested so far. F r o m our data we conclude, that petacins may be specific leukotrien inhibitors. The investigation of the specific cellular sites of action is in progress. *Plantamed Arzneimittel GmbH, Kerschensteinerstr. 11-15, 8430 Neumarkt Institute of Pharmacology and Toxicology, University of Erlangen-Ndrnberg, Universit~tsstr. 22, D-8520 Erlangen, FRG.

R 36 141 ENDOTHELIUM-DEPENDENT RELAXATION OF BOVINE CORONARY ARTERIES BY PGD2 M. Braun

The vasomotor effects of PGD~ vary with species, the type and the tone of the vesCels (GILES H and LEFF P, Prostaglandins 35:277, 1988). This study investigates the influence of endotheIium on vasomotor effects of PGD9 on bovine coronary arteries (BCA). Isolated, helically cut strips of BCA were placed into an organ bath containing oxygenate~ (20% Op, 5% CO~, 75% Np) Krebs-Henseleit solution at 37~C. Cha~ges in ]ength w~re measured isotonically. Endothelial function was assessed by acetylcholine-induced relaxation (~50%) of precontracted (5-HT, I ~M) vessels. PGDo (0.01-I ~M) produced a concentration-dependent relaxation of the BCA at basal tone (n=4) and after precontraction with 5-HT (I ~M) (0.1~M: 61+7%, I~M: 102+5%; n=10) in vessels with intact endotheliffm. After removal of endothelium PGD9 exhibited a moderate contractile effect on BCA at ba~al tone and no significant change of vessel tone in precontracted vessels. A similar endothelium-dependent relaxation was observed for the metabolically stable PGD~-analogue ZK 110.841 ((5Z,13E)(9R,11R,15S)-9-chloro-35-cyclohexyl-11,15-dihydroxy16,17,18,19,20-pentanor-5,13-prostadienoic acid) (n=6). Preincubation of vessels with intact endothelium with indomethacin (3 ~M) did not antagonize the relaxing effects of PGDp (n=3). These data demonstrate an endothelium-dependent relaxation of bovine coronary arteries by PGD2. This action is not due to metabolic conversion of PGD~and seems not to be mediated by endothellal prostacycli#release. I n s t i t u t f~r Pharmakologie, Heinrich-Heine-UniversitBt DOsseldorf, Moorenstr. 5, D-4000 D~sseldorf I, FRG

143 THE PROSTACYCLIN ANALOGUE TAPROSTENE AND ACETYLSALICYLIC THROMBOLYSIS BY RECOMBINANT SINGLE-CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (SARUPLASE) IN RABBITS

ACID ENHANCE

J. Schneider The efficacy of fibrinolytics may be enhanced by antiaggregating agents due to inhibition of platelet apposition during lysis. A possible synergism of the ant±platelet prostacyclin analogue Taprostene and acetylsalicylic acid (ASA) and the fibrinolytic Saruplase have t h ~ been investigated in rabbits with pulmonary embolized "J-fibrin-labeled thrombi. Lysis was evaluated at 180 min af~ thromboembolization as decrease of the incorporated -'J-fibrin and as reduction of the total weight of the thrombi. In untreated rabbits the spontaneous lysis of the incorporated ---J-fibrin was 7 ± 1 % and the thrombus weight was reduced by 45 ± 2 % (mainly due to loss of B20). Saruplase (i0.0 - 46.4 Ug/kg'min i.v., 60 min) produced dose-dependent andl~gnificant lytic effects with a maximum decrease of the ---J-fibrin content by 41 ± 2 % and a thrombus weight reduction by 72 ± 2 %. Infusion of Taprostene alone (0.1 ~g/kg.min i.v., 180 min) ~ not modify the spontaneous lysis of the incorporated J-fibrin, but reduced the total thrombus weight by a significant higher percentage than in untreated controls. In combination with Saruplase Taprostenel~gnificantly enhanced the Saruplase-induced lysis of J-fibrin and the reduction of thrombus weight. Oral pretreatment (30 min before thromboembolization) with 50 mg/kg ASA, which did not exert a throm~9~ytic effect by itself, insignificantly augmented the ---J-fihrinolysis by Saruplase and significantly increased the Saruplase-induced reduction in thromhus weight. In conclusion, Taprostene and ASA enhanced the thrombolysis by Saruplase in a rabbit model of pulmonary thrombosis. T h i s effect may be ascribed to their antiaggregatory properties; an additional profibrinolytic effect of Taprostene must be considered. GrGnenthal GmbH, Center of Research, Zieglerztr. 6, 5100 Aachen

142

144

COMPARISON OF THE EFFECTS OF PROSTAGLANDINRELATED VASODILATORS ON PROTEIN PHOSPHORYLATION AND cAMP LEVELS IN INTACT HUMAN PLATELETS C. Friedrieh, M. Eigenthaler, P. Schanzenb~icher and U.Walter

E F F I C A C Y OF PGI2- ANALOGS IN V I T R O AND IN V I V O W. Dausch, H.Darius, G.G~rge, J.Meyer

Prostaglandin-related vasodilators such as prostacyclin (PG), the stable prostacyclin-analog iloprost and prostaglandin (PG-E 1) elevate cAMP-levels in t a r g e t tissues and inhithe dctivation of human platelets. Recently, our laboratory characterized and purified a vasod~lator-stimulated phosphoprotein (VASP) from human platelets~ and developed methods to_measure the extent of VASP phosphorylation in intact cellsZ. We have now compared the effects of PG-12, iloprost and PG-E 1 on cAMP-levels and VASP-phosphorylfition using intact ~vashed human platelets. PG-I9_ (5 pM) and PG-E 1 (10gM) rapidly raised the intracellular cAMP concentratio-n from 9 pM to 34 or 27 pM, respectively, and caused conversion of 80'/0 Q (55% with PG-E 1 ) of VASP from the dephospho- to the phosphoform. These effects were reversed upon removal of PG-I?~ or P G - E ! but were subsequently restored by addition of PG-I 9 or PG-E 1 to the platelets again. In contrast, iloprost ~ pM) rapidly increased the cAMP level to 70 ]lM and converted 80% of VASP to the phosphoform, but these effects were not reversible within 90 min after removal of iloprost from the p l a t e l e t suspension. Half-maximal effects on cAMP levels and VASP phosphorylation were observed with about 50 nM of PG-I 2, iloprost or PG-E 1. The results demonstrate significant differences betweefi these vasodilators with respect to their effects on extent and duration of VASP phosphorylation in intact human platelets. 1) M.Halbrfigge, U.Walter (1989) Eur.J.Biochem. 185,41-50 2) M.Halbrtigge, C.Friedrich et al. (1990) ].Biol.Chem., In press Medizinische Univ.-Klinik, Klinische Forschergruppe, JosefSchneider-Str. 2, D-8700 Wfirzburg, FRG.

Prostacyclin is a natural mediator with unique platelet antiaggregatory and vasodilator properties. We studied the antiaggregatory efficacy of epoprostenol (PGI2; EPO) with the chemically stable derivatives iloprost (ILO), taprostene (TAP) and ciprostene (CIP) in human platelets in vitro and compared antiaggregatory and vasodilatory effects of ILO and TAP in rats in vivo. All the compounds tested exerted a dose-dependent inhibition of platelet aggregation. ICbovalues for ADP-, collagenand PAFinduced platelet aggregation were 1.6±0.i, 1.2f0.4 and 0.2±0.1 nM when EPO was used as an antiaggregatory agent. ILO was almost equieffective with ICbo- values of 2.8±0.2, 0.6±0.i and 0.8±0.2 nM. In contrast, TAP and CIP were significantly less potent. When increasing doses of ILO and TAP were infused intravenously in anesthetized rats, diastolic blood pressure decreased from 59±11 to 38±4 n~mHg with ILO 1.6 ~g/kg/min and from 66±3 to 44±6 mmHg with TAP 8 ~g/kg/min, with systolic blood pressure being basically unaltered. Injection of collagen (80 ~g/kg) resulted in a transient drop in blood pressure and a decrease in platelet count by 42±ii %. When collagen was administered during drug infusion, alterations in blood pressure were markedly blunted and the decrease in platelet count was reduced to 19±5 % with ILO and to 38±12 % with TAP. There are marked differences in antiaggregatory potency of various prestacyclin analogs. The clinical usefulness of the compounds finally will depend on the ratio of antiaggregatory potency to other adverse drug effects. II.Med. Klinik, Jeh.-Gutenberg Univ., 6500 Mainz

R 37 147

145 DE NOVO SYNTHESIS OF THROMBOXANE-SYNTHASE DIFFERENTIATING HL-60 CELLS

IN

R. N ~ s i n g Monocyte

i n f i l t r a t i o n into i n f e c t e d tissues and morphologic and functional a l t e r n a t i o n s are k e y events in the i n f l a m m a t o r y process. Exposure of human promyelocytic l e u k e m i a cell line H L - 6 0 to tumor p r o m o t o r 12O-tetradecanoyl-phorbol-acetate (TPA) has b e e n assumed a model system for monocytic differentiation. After incubation w i t h 10 nM TPA these cells produced timeand concentration-dependent (basal, after a d d i t i o n of i0 ~M a r a c h i d o n i c acid or 20 ~M PGH2) h i g h e r amounts of TXB2 compared to c o n t r o l cells. Incubation with transcription-, translation-, or p r o t e i n k i n a s e C - i n h i b i t o r s abolished the s t i m u l a t i n g e f f e c t of TPA. By u s i n g s p e c i f i c a n t i b o d i e s to t h r o m b o x a n e s y n t h a s e in d i f f e r e n t techniques as ELISA, western blot and cell l a b e l l i n g we w e r e a b l e to d e m o n s t r a t e that an i n c r e a s e in TXB2 r e l e a s e from d i f f e r e n t i a t i n g H L - 6 0 c e l l s is a c c o m p a n i e d b y de novo s y n t h e s i s of thromboxane synthase. The mechanism for i n d u c t i o n of s y n t h e s i s of t h r o m b o x a n e s y n t h a s e by TPA is unclear, but there are strong indications for a close r e l a t i o n b e t w e e n the a c t i o n of p h o r b o l esters and the a c t i v i t y of p r o t e i n k i n a s e C. extensive

F a c u l t y of Biology, Konstanz

University

of Konstanz,

775

146 MEASUREMENT OF TRIENOIC ACID AND HUMAN PLASMA BY U. Hofmann a', E. Hiibel b

CHEMICAILY DIFFERENT THROMBOXANE A2/PROSTAGLANDIN H2 ANTAGONISTS AND AGONISTS SHOW DISTINCI PH D ~ C Y OF BINDING J. Theis, H.-G. Dellweq, R. GroB, E. Perzborn Bay U 3405 is a new TXA2/PGH2-antagonist which inhibits platelet aggregation and contraction of smooth muscle. The binding of [3H}Bay U 3405 to the TXA2/PGH2 receptor of human platelet n~mbranes was studied at different pH values and cc~pared with the binding of the antagonists SQ 29548 and J-PTA-OH and the agonists U 46419 end dYA2. The KD values of Bay U 3405, determined by equilibriu~ saturation studies, are 6.9 + 1 nmol/l and i.I + 0.05 rmol/l at pH 7.4 and 5.8, resp. B max is 6 ~mol/ mg protein in both cases indicating that affinity and not the number of binding sites is pH dependent. The increase in affinity results frcm a more than 10-fold higher association rate at pH 5.8 compared to pH 7.4. The rate of dissociation increases only 1.3-fold at pH 5.8. U 46619 and cTA2 also show significantly higher affinities at low pH. In contrast, J-PTA-OH exhibits a higher affinity at pH 7.4 and SQ 29548 shows no pH dependency. SQ 29548 and JPTA-OH have a second proton acceptor. This seems to be responsible for the altered pH-dependency. Bay U 3405: ( (3R)-3-(4-Fluorophenylsulfonamido)-l,2,3,4tetrahydro-9-carbazolepropanoic acid), SQ 29548 : (IS- ~, 2B (5Z) ,3B, 4 ~ ) )-7-(3( (2- (phenylamino) carbonyl) hydrazino) -7oxabicyclo(2.2.1 (hept-2-yl)-5-heptenoic acid) , J-PTA-OH: 9,11 -Dimethylmethano- 11,12 -methano-16 - (3-iodo-4 -hydroxyphengl)-13,14-dihydro-13-aza-15J.8-w-tetranor-TXA2, U 46619: 9,11-dideoxy-ll~,9~-epoxymethano-prostaglandin F2~, cTA2: (2B (Z),3~- (IE,3R*) -3- (3-hydroxy (l-octoenyl) -bicyclo (3. I. I) -hept- 2-yl-5-heptenoic acid Institutes of Pharmacology and Biochenlistry BAYER AG, Apratherweg 18 a, D-5600 Wuppertal 1

148 12(S)-HYDROXY-5,8,10-HEPTADECATHE 12-DEHYDRO METABOLITE IN GC/(NI-CI)MS L and U. Kuhlmann"

Studies on the human metabolism of infused thromboxane B 2 (TXB2) have shown that this eicosanoid is extensively metabolised. More than twenty metabolitus have been identified (Roberts LJ et al., J. Biol, Chem. 256:8384 (1981)). As the biological fate of infused TXB 2 might be different from its precursor, the enzymatieally formed ~FXAz, monitoring of more direct index metabolites would be desirable. Previous investigations of the molecular mechanism of thromboxane synthase strongly indicate that this enzyme converts cyclic endoperoxides stoiehiometrieally in equal amounts to TXA 2 and to a mixture of malondialdehyde (MDA) and 12(S)-hydroxy-5,g,10-heptadeeatrienoie acid (HHT) (Hammarstr6m S, Arch. Biochem. Biophys. 214:431 ( 1982 )). If H H T or its 12- dehydro metabolite 12- keto - 5,8, I 0heptadecatrienoie acid (KHT) were exclusively formed during TXA 2 biosynthesis, their plasma levels should be better suited to monitor the biosynthesis of the latter eicosanoid. Platelet rich plasma (PRP) was prepared from the blood of five healthy volunteers and was stimulated with arachidonic acid (AA) or collagen (C). In addition, non-stimulated platelet poor plasma (PPP) was obtained from blood which was collected under controlled, nonstimulating conditions (B-TG < 35 ng/ml, PF4 < 10ng/ml). The specific and sensitive quantification of the two metabolites in the plasma samples was carried out in the presence of the deuterated analogues (d6-HHT , d6-KHT), using gas chromatography/negativeion chemical ionization mass spectrometry. The amounts of H H T in PRP stimulated with AA or C ranged from 50 to 200 n g / m l and 50 to 130 ng/ml, respectively and were linearly correlated with simultaneous TXB~ formation. Besides, the 12-dehydro metabolite K H T was present m concentrations of 0.5 to 11 ng/ml. In contrast, low levels of H H T (10-20 pg/ml) were recorded in non-stimulated PPP and the amounts of K H T were below the detection limit of 10 pg/ml. K H T seems to be a sensitive parameter for platelet activation and might serve as an index parameter for TXA=-biosynthesis by blood platelets.

Supported by the Robert-Bosch-Foundation ~Dr. Margarete Fischer-Bosch-Institut ftir Klinische Pharmakologie, AuerbaehstraBe 112, D-7000 Stuttgart 50.- bRobert-BoschKrankenhaus, Auerbachstr. 110, D-7000 Stuttgart 50.

IIX)PROST, A POTENT FUNC'WIONAL ANTAGONIST THROMBOXANE A2 INDUCED VASOCONSTRICTION? G. SchrSder, G. Graichen, F. Fiske, D. Wehrmann

OF

Endogenous prostacyclin and thromboxaneA2 (TXA2) are generally considered to be functional antagonists concerning their effects on vascular smooth muscle and platelets. The functional antagonism of stable prostaeyclin analogues against TXA2 mediated vascular effects has recently been questioned. Since iloprost - in all investigations performed so far - has been shown to closely mimic the actions of PGI2 these data may be relevant for the general concept as outlined above. Therefore the influence of iloprost on U 46619 (stable TXA2 analogue) induced contraction of isolated rabbit mesenteric artery strips was tested. In addition, in vivo experiments were performed in conscious normoteusive Wistar rats. lloprost inhibited the U 46619 (2.5 x 10-7M) induced contraction in arter~ strips dosedependently and completely with an IC50 of 1.8 + 1.7 x 10" M. In Wistar rat bolus injection of U 46619 (8 izg/kg/min) increased blood pressure by 40mmHg approximately. Infusion of iloprost (0.1 ~g/kg/min) partly inhibited the U 46619 induced blood pressure increase and 0.3 ~.g/kg/min of iloprost completely blocked the U 46619 effect. The dose of iloprost did not lower basal blood pressure significantly. These results dearly show that iloprost is a potent functional antagonist of 1)U 46619 induced vascular constriction suggesting that iloprost may serve as a functional antagonist of endogenously released TXA2-mediated vasoconstriction. 2)The data support the original concept of a functional antagonism of PGI2 and TXA2. Research Laboratories of Schering AG, Cardiovascular Pharmacology, MiiUerstr. 170-178, D-1000 Berlin 65, West Germany

R 38 149

151

M~&LkaSchatz

METABOLISM DIHYDRO-OXO

D. T s i k a s Leukctriese ~ (LT~) is till mow the most potent inflammatory mediator produced by human calls. Neutrophils m2nthesize LT~ after stimulation with physiological (e.g. f~P, LT~, PAF) or n~physiological (e.g. Ca2+-ionc~aores) a~/sts. Two enzymes, phos~holipase ~ which liberates the arachidonic acid, and 5-1ipcx~enase are respc~ible for the generation of leukotrienes. H~an P ~ stimulated with physiological a~mts only produce small ~ao~ts of LT~ in contrast to Ca2+-i~hore stimulati0n. So neu~Is can, but will not, respond to receptor mediated stimulation. This phe~mer~ is known as '%T~-paradox". Our results show that also a mmbinat/on of two or more physiological agu~ists did not result in a significant increase in lenkotriene formation. Also, in a coinonbation of PMN with mcm~)cytes or platalets no definite enhancement could be detected. From 5-1ip~a~e~ise activty in R~super~tant it is }mown that the enm/me requires Ca2+ , AT? and fatty acid hydroper~ddes for maximal stimulation. 0 ~ question was therefore, whether one of these stimulatir~ factors is limiting for LTB4 synthesis in vivo. AT? seems not to be limiting, for the om~cantration is high enough in cells. Ca~ also ~oes not seem to be tbe missing factor, since human R ~ ere stinmlated by a physiol~/ical agm%ist and m~tracelluler a r a ~ c acid there is enon~ Ca~÷ for the activation of 5-1ipox~anase. Fatty acid ~ d e s are abeolutely necessary for l i ~ e m s e n . In su;ernafant, 5-1i;~x!a~mmse is inhibited by glutathione plus glutathione-peru~idase and can be reactivated by the additien of hydro~.rc~des (5- o¢ 15-HP~TE). Under physiological omditicms, e.g. f}KP-st/mulation, the addition of 10 ~M HP~TE only leads to a minimal stim~lati~ ( factor 1-2 ). These results indicate that the 5 - 1 i ~ o n e s e is not the rate limiting step in gram_ration of leukotrienes. So the msmmt of leukotriese m2ntheso depm~ls on ~spholipase ~ activity. ~/s mu"!~e also needs Ca~÷ for activation, but the level se~s to be much higher than for 5ll~enase.

OF LEUKOTRIENE PRODUCTS BY HUMAN

and

J.C.

B4 TO DIHYDRO MONOCYTES

AND

Fr~lich

Leukotriene B 4 (LTB4) is an i m p o r t a n t inflammatory mediator. In g r a n u l o c y t e s , LTB 4 is m e t a b o l i z e d to less b i o l o g i c a l l y active ~ - o x i d a t i o n products. The aim of this study was to i n v e s t i g a t e human m o n o c y t e m e t a b o l i s m of LTB4. The m e t a b o l i t e s w e r e s e p a r a t e d by R P - H P L C and i d e n t i f i e d by GC/MS. LTB 4 was c o n v e r t e d to two m a j o r m e t a b o l i t e s MI and MII less polar than LTB 4 w i t h an UV maximum at 232nm, suggesting that one of the t h r e e c o n j u g a t e d double bonds h a d been reduced. In the n e g a t i v e ion c h e m i c a l ionization mode (NICI) MI s h o w e d m a i n f r a g m e n t s i n c r e a s e d by two mass units c o m p a r e d to LTB4. The position of the conjugated double bonds was e s t a b l i s h e d by e l e c t r o n impact (EI) mass s p e c t r a and by splitting the remaining double bonds of MT with K M n O 4 / N a I O 4 and analysis of the p r o d u c t s by GC/MS. This resulted in the formation of a p r o d u c t with mass f r a g m e n t s due to 2 - h y d r e x y - h e x a n d i o i c acid indicating that the d o u b l e b o n d 10,11 was reduced. The EI mass s p e c t r u m of the p e n t a f l u o r o b e n z y l ester of MI was clearly d i f f e r e n t f r o m LTB 4 and s h o w e d fragment ions due to the c l e a v a g e b e t w e e n C1 to CI0 (m/z 435) and CII to C20 (m/z 213). These findings strongly suggest that MI is i d e n t i c a l w i t h 1 0 , 1 1 - d i h y d r o - L T B 4 . The lower p o l a r i t y of MII combined with the fact that MII may form a of the two methyloxime derivative suggest that o n e h y d r o x y g r o u p s is o x i d i z e d to an oxo group. NICI and El m a s s spectra of MII were clearly d i f f e r e n t from LTB 4 and MI. The EI s p e c t r u m of the m e t h y l e s t e r t r i m e t h y l s i l y l ether derivative of M I I showed fragments due to a d i h y d r o x y c o m p o u n d and to cleavage b e t w e e n C1 to C5 (m/z 203) and CII to C20 (m/z 225). This i n d i c a t e s that the oxo group in MII is e n o l i z a b l e and stays in the p o s i t i o n 12~ and s u g g e s t s that MII is 1 0 , 1 1 - d i h y d r o - 1 2 - o x o - L T B 4 . This study shows that human m o n o c y t e s c o n v e r t e LTB 4 to dihydro and d i h y d r o - o x o metabolites.

University of Konstanz, LS U11rich, Am eie~berg, D-7750 Kcmstanz, FRG D e p a r t m e n t of Clinical Pharmacology, Hannover M e d i c a l School, P.O. 610180, D-3000 Hannover 61, Germany (FRG)

150 REGULATION MONOCYTES J. Fauler

OF

LEUKOTRIENE

SYNTHESIS

IN

HUMAN

Receptor mediated stimulationof leukotrienesynthesisin human monocytesis modulated by complex mechanisms. Previouslywe have shown that pertussis toxin and cholera toxin inhibit fMLP stimulated LT84 synthesis suggestingthe involvement of G-proteins. The aim ot the present study was to further elucidate the mechanisms involved in receptor mediated leukotriene synthesis in monocytes. Supernatantsfrom stimulated human monocyteswere analyzed for leukotriene B4 synthesis by RP-HPLC UV-detection. The nonehydrolyzable analog of GTP, GTPyS stimulates the synthesis of LTB4 in saponin permeabilizedmonocytes. This GTPTS induced stimulation of LTB4was completely blocked by pertussis toxin (lOOng/ml) added 3 hours before GTPTS. Furthermore, NaF stimulated leukotriene synthesis in intact human monocytes. In additional experiments we investigated regulatory effects of protein synthesis on the receptor mediated mechanisms of leukotriene synthesis. Cycloheximide (10-6 tool/I) inhibited the fMLP induced leukotriene synthesis in a time dependent manner. After 6 hours oycloheximide completely inhibited the fMLP induced stimulation of leukotriene B4. Recently we have shown that the fMLP induced leukotdene synthesis entirely depends upon extracellular Ca++. In the present study we investigated whether potassium is involved in the mechanism of receptor mediated synthesis of leukotrienes. Hyperpolarization achieved by an extracellular concetration of 40 mmol/I K+ completely inhibited the tMLP induced LTB4 synthesis. The data of the present study further support the hypothesis that G-proteins are involved in the receptor mediated synthesis of leukotrienes by human monocytes. Furthermoreit is shownthat synthesisot proteins is involved in the signal transducing system and that synthesis of these proteins is rapidly regulated. In addition, receptor mediated stimulation of leukotrienes not only depends on extracellularCa++ but also on the polarizationstatus ot the cell as hyperpolertzation induced by augmentation of extraoellular potassium completely blocked fMLP induced leukotriene synthesis. Departmentof Clinical Pharmacology,HannoverMedicalSchool, P.O. Box 61 01 80, D-3000 Hannover61 W. Germany

152 IDENTIFICATION AND CHARACTERIZATION OF CYSTEINYL-LEUKOTRIENE FORMATION BY CLOTTING WHOLE HUMAN BLOOD Th. Simmer and W. Luck Biosynthesis of eicosanoids by whole human blood is a complex event but due to possible c e i l - c e l l i n t e r a c t i o n s c l o s e l y resembles in vivo conditions. C y s t e i n y l - l e u k o t r i e n e s (CYS-LT) possess potent b i o l o g i c a l a c t i v i t y which can lead to v a s o c o n s t r i c t i o n , increased vascular permeability and increased t-PA release. We have theref o r e investigated whether the process of c l o t t i n g might induce t h e i r biosynthesis in whole human blood incubated in v i t r o at 37oc. Spontaneous c l o t t i n g r e s u l ted in a time-dependent r i s e of immunoreactive CYS-LT in the serum reaching 538 • 9o and 1327 • 229 pg/ml at 6o and 12o min (n=7), r e s p e c t i v e l y . As assessed by bioassay, serum e x t r a c t s contained b i o l o g i c a l l y active material i d e n t i c a l to SRS-A. By combined reversed phase HPLC and radioimmunoassay the material was shown to consist of a mixture of CYS-LT. In a d d i t i o n , occurrence of ~ - o x i d a t i o n could be demonstrated. CYS-LT production was not affected by i n h i b i t i o n of the cyclooxygenase by indomethacin (2.8 ~M) nor by s t i m u l i such as the thromboxane agonist U 46619 (11~,9~-(epoxymethano)prostadienoic acid, Io uM) or PAF (I MM). S i m i l a r l y , the PAF antagonist WEB 2o86 (Br. J. Pharmacol. 91 1987 799, I or Io MM) did not modify CYS-LT production, suggesting t h a t i t occurs independent from^a possible PAF generation. In contrast, besides CaZ+-chelating anticoagulants, the f u n c t i o n a l l y unrelated heparin (2o IU/ml) i n h i b i t e d CYS-LT formation suggesting t h a t an event r e l a t e d to the I n t r i n s i c system of blood coagulat i o n might t r i g g e r CYS-LT production in whole human blood. Clotting-induced CYS-LT production may be of f u n c t i o n a l s i g n i f i c a n c e during thromboembolic events. Dept. of Pharmacology, Ruhr-University, s t r . 15o, D-463o Bochum, FRG

Universit~ts-

R 39 153 NONMITOCHONDRIAL CA2+/H+ ANTIPORT ACTIVITY IN SUBCELLULAR FRACTIONS FROM RAT LIVER H.-P. Bode

155 PROPERTIES OF SINGLE K + CHANNELS FROM THE BASOLATERAL MEMBRANE OF RABBIT COLON EPITHELIUM K. Turnheim, J. Costantin*, S.G.Schultz*

With a spectrophotometric method ATP-dependent v e s i c u l a r a c i d i f i c a t i o n in s u b c e l l u l a r f r a c t i o n s from r a t l i v e r was measured. The a c i d i f i c a t i o n in a crude microsomal f r a c t i o n (105000 x g p e l l e t ) , which could be i n h i b i t e d by N-ethylmaleimide but not by vanadate or oligomycin, was reversed upon addition of calcium. Equimolar amounts of strontium had a 48 +/- 29 % smaller e f f e c t . Manganese up to 10 mM did not influence the v e s i c u l a r a c i d i f i c a t i o n . Calcium t h e r e f o r e p r e f e r e n t i a l l y induced proton e f f l u x from i n t e r n a l l y a c i d i c vesicles in the microsomal f r a c t i o n . The observed a c i d i f i c a t i o n appears to be due to a vacuolar-type proton pump, as i t is not i n h i b i t e d by vanadate or oligomycin. These r e s u l t s suggest the existence o f a Ca2+/H+ a n t i p o r t system s i m i l a r to the one t h a t has been reported f o r the yeast c e l l vacuole (Ohsumi Y and Anraku Y, J. B i o l , Chem. 258: 5614-5617, 1983). F r a c t i o n a t i o n of the c e l l s into a mitochondrial, a crude lysosomal and a heavy microsomal f r a c t i o n revealed a c o r r e l a t i o n of Ca2+/H+ a n t i p o r t a c t i v i t y with the d i s t r i bution of the lysosomal markeb enzyme acid phosphatase among these f r a c t i o n s . No c o r r e l a t i o n with mitochondrial or endoplasmic reticulum marker enzymes was found. The described a n t i p o r t mechanism may t h e r e f o r e be associated with lysosomes or other organelles of the vacuolar apparatus of r a t l i v e r c e l l s .

Basolateral membrane vesicles isolated from surface epithelial cells of rabbit distal colon (H. Wiener, K. Turnheim, C.H. Van Os, J. Membr. Biol. ii0: 147-162, 1989) were fused with planar phospholipid bilayers, revealing spontaneous openings and closings • of highly cation-selective + channels o The permeabllity of the channels to K was, on average, 57 tzmes hlgher than to Na . Scorpzon (Leiurus quinquestriatus) venom, which contains charybdotoxin, blocked the channels from only one side of the membrane. This finding was used to establish the sidedness of the reconstituted channel, as the venom may be assumed to reach the cell membrane only from the extracellular solution. The open-state probability of the channels was increased by membrane depolarization. Decreasing free Ca Z+ in the solution on the cytoplasmic side of the membrane to approximately 2~.I ~M abolished channel activity, increasing ~ again to 1 ~M restored channel activity. Ba , quinidine, and trifluperazine inhibited channel activity. The effect of trifls~erazine may indicate that the interaction of Cawith the channel is mediated by calmodulin. The conductance of the single channel is high (340 pS with a K -gradient of 400/50 mM across the. membrane). The relatio~ of the szngle channel K conductance and the K activities on the two sides of the membrane suggests single-filing. These results demonstrate the presence of a Ca2+-activated "maxi" K--channel in the basolateral membrane of rabbit colonocytes.

I n s t i t u t f u r Pharmakologie und T o x i k o l o g i e , Karl von Frisch Stra6e, Lahnberge, D-3550 Marburg/L., FRG

Pharmakologisches Institut, Universit~t Wien, WMhringer Str. 13a, A-1090 Vienna, Austria * Department of Physiology and C e l l Biology, University of Texas Medical School, Houston, TX

154 SYNAPTOSOMAL OXYGEN CONSUMPTION AS VERATRIBIRE IHDUCED SODIDH IRFLUX

156 AN

INDICATOR

FOR

J. Urenlak. A. Belle, S. Khan, and F. Tegtmeler Exposure of synaptsosomes to veratridine results in transmembraneous influx of sodium (Na) and calcium (Ca). Veratrldine is thought to provoke a prolonged opening of Na-channels through which Ca-entry can proceed as well. In consequence of Na- and Ca-entry the activity of plasmalemmal ion pumps is enhanced stimulating which increases ATP consumption, thus mltoehondrial respiration. The present study was undertaken to delineate the respective contributions of veratrldlne induced Na and Ca inward movements to mitochondrial oxygen consumption in a rat synaptosomal preparation. Veratridine (i0 ~M/1) stimulated synaptosomal 02 consumption from 7.9 ± 0.4 nmol 02/min/mg under control conditions to 24.7 ± 4.9 umol 02/min/mg. Application of tetrodotoxln (5 ~M/I) inhibited the stlmulated 02 consumption by 86 ± 4 ~. Expectedly, blockade of the Na-pump by ouabaln (1 mM/1) led to a similar reduction of 02 consumption. Additional application of vanadate (i0 ~M/1) ylelded no further decrease. Induction of Ca-influx by either the Caionophore A23187 (i0 ~M/I) or the Ca-agonist Bay K 8644 (10 ~M/1) failed to augment synaptosomal 02 consumption. Moreover, the Na/Ca-exchange inhibitor amllorlde (200 pM/l) did not modify the respiratory response to veratridine indicating that the Na/Ca-exchange system is of minor relevance for veratridine's actions in this model. It can, therefore, be concluded that synaptosomal 02 consumption predominantly reflects increased Na- but not Ca-load, since the increased Na-load, e.g. by veratridlne, is the major energy demanding process whereas the accompanying Ca-load only minimally burdens energy consuming systems. In consequence, synaptosomes "Na-sensltive handled as above may be used as electrodes" JANSSEN RESEARCH FOUNDATION, D-4040 Neuss 21, FRG

DISTENSION-INDUCED SECRETION IN THE RAT COLON: MEDIATED BY PROSTAGLANDINS AND SUBMUCOSAL NEURONS M. Diener and W. Rummel Distension of the rat colon descendens in vitro by an hydrostatic gradient induced an increase in short-circuit current (Isc). In a preparation containing the plexus submucosus, the increase in Isc was biphasic with a half-time of about 200 s. In a preparation without the plexus submucosus time course was monophasic. The increase in Isc in the preparation with the plexus submucosus was inhibited by an inhibitor of phospholipase A2, quinacrine, and by indometacin, tetrodotoxin or atropine; each of them also abolished the second phase of the response. In contrast, in the preparation without the plexus submucosus only indometacin was effective in reducing the increase in Isc. In both preparations the response to distension was inhibited by scilliroside, by replacement of CI with gluconate, and by administration of furosemide or the chloride channel blocker, anthracene-9-carboxylic acid. The results indicate that distension induces chloride secretion by a release of prostaglandins, which act indirectly, i.e. mediated by the cholinergic neurons of the submucosal plexus, and directly at the epithelium. Institut for Pharmakologie und Toxikologie, Universitfit des Saarlandes, D-6650 Homburg/Saar, F.R.G. Supported by DFG, project Di 388/1-2

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E L E C T R O P H Y S I O L O G I C A L C H A R A C T E R I Z A T I O N OF THE TOXIC C O M P O N E N T FROM THE V E N O M OF THE G R E A T E R W E E V E R F I S H TRACHINUS DRACO. J. Beise, I. Chhatwal and F. Preyer

BUMETANID T R A N S P O R T P R O T E I N IS AN INTEGRAL MEMBRANE PROTEIN OF LIVER SINUSOIDAL PLASMAMEMBRANES A. Schenk, W. Honscha and E. Petzinger

Trachinus draco is a venomous fish which belongs to the piscine family Trachinidae. The venom extract isolated from the opercular and dorsal spines had a MLD of about 5 pg protein per g mouse and a total of about i0 000 MLD's were obtained from the venom apparatus of one fish. For e l e c t r o p h y s i o l o g i c a l experiments we had to use the crude venom, since the p u r i f i e d hemolytic protein component (92 kD, ECs0 ~ 3.5 ng/ml) looses quickly its activity in p h y s i o l o g i c a l solutions. The cytotoxic effect was studied on the n e r v e - m u s c l e - p r e p a r a t i o n of the M. triangularis sterni of mice. Within few minutes after application the venom (5-50 ug/ml) caused a massive quantal release of acetylcholine and a strong decrease of the resting potential followed by damages of muscle fibres and nerve terminals. The pore formation of the cytotoxic component was studied in ou~side-out patches from bovine adrenal chromaffin cells. About 1 minute after venom application (15 pg/ml) the formation of ion channels in the membrane could be recorded. Three to five subconductance states each of about 500 pS may be involved in the large transitions. The h e t e r o g e n e i t y in the pore conductances may be explained by the existence of pores composed of a different number of monomers. We conclude that the fish venom possesses cytotoxic and hemolytic activity which is related to a single component of the venom and which is most p r o b a b l y responsible for the clinical symptoms and the lethal activity at mice. Rudolf-Buchheim-Institut f~r Pharmakologie, Justus-Liebig-Universit~t, D-6300 Giessen, FRG.

158 EXPRESSION OF NaK-ATPase B-SUBUNIT OF TORPEDOCALIFORNIOA IN XENOPUSLAEVISOOCYTES RISES OUABAIN BINDING CAPACITY G. Schmalzing, S.KrSner, H.Omay, H. Appelhans & W. Schwarz The NaK-ATPase consists of a catalytic a-subunit with the binding site for cardiac glycosides and a smaller 8subunit. The B-subunit is essential for enzymatic a c t i v i t y and a subunit maturation (Zamofing et al. J. Membr. Biol. i04, 69-79, 1988). Expression of functional NaK-ATPase of Torpedo californica in oocytes has been shown to require mRNAs for both subunits. Injection of a-subunit-specific mRNA alone resulted in the expression of an inactive enzyme only (Noguchi et al. FEBSLett. 225, 27-32, 1987). Recently, we demonstrated that ouabain binding sites are exposed in Xenopus oocytes when inner membranes are rendered leaky by SDS (Schmalzing et al. Biochem. J. 260, 395-399, 1989). Taking advantage of this technique, we quantitated ouabain binding in oocytes injected with mRNA specific for either the a (mRNAa) or B subunit (mRNAa) of Torpedo NaK-ATPase. Cloned cDNAs of Torpedo californica NaK-ATPase were generously provided by Dr. M. Kawamura, Japan. Subunit-specific mRNAswere synthesized by in vitro transcription using Sal I-cleaved T=pSP65 or T~pSP65 as templates and SP6 polymerase. Oocytes were microinjected with mRNA(approx. 50 ng/cell) and cultivated for 2-3 days before being permeabilized with 10 NM digitonin. In mRNAa-injected oocytes, ouabain binding was not significantly different from noninjected controls. In mRNAa-injected cells, however, ouabain bindin9 determined in the absence and presence of 0.02% SDS was increased by 24 ± 3% (3 fmol/cell) and 36 ± 13% (12 fmol/cell), respectively (E ± SD, 3 experiments, p < 0.001). According to Scatchard analysis, the dissociation constant for ouabain was unchanged. We hypothesize that the NaK-ATPase 8-subunit of Torpedo californica expressed in oocytes associates with excess asubunit of endogenous NaK-ATPase, thereby increasing a subunits that are capable of ouabain binding. Max-Planck-Institut for Biophysik, 6000 Frankfurt 71

The elimination of loop diuretics in rat is achieved by both renal and hepatic pathways. Hepatic transport for b u m e t a n i d e ( 4 - ( 4 - a m i n o phenoxy)-3-butyl-amino-5-sulfamoylbenzoic acid anhydride) has been d e m o n s t r a t e d (Am. J. Physiol. 256:G78-G86, 1989). The kinetic data have revealed a high affinity and a low affin i t y component with K, =21 pM and K, =370 pM respectively. P h o t o a f f i n i t y labeling of hepatocytes and plasma membranes have suggested a localization of the transporter in the sinusoidal membrane. A more d e t a i l e d analysis was achieved by c o m p a r i n g p h o t o a f f i n i t y labeling of hepatocytes, sinusoidal plasma membranes and subfractions of the plasma membranes. The latter were associated membrane proteins and integral m e m b r a n e proteins which were separated by extraction of the membrane with the detergent Triton X-II4. The specific radioactive i n c o r p o r a t i o n of (3H)-bumetanide in integral membrane proteins was i0 times higher than in hepatocyte proteins. Specific labeling was neither found in associated m e m b r a n e proteins nor in the t r a n s p o r t - d e f i c i e n t hepatoma cell line AS-30 D. 2 D - g e l e l e c t r o p h o r e s i s of radiolabeled integral m e m b r a n e proteins r e v e a l e d a p p r o x i m a t e l y 30 proteins among which one single spot was radioactive. The m o l e c u l a r weight was 54 kDa with an apparent acidic pI of a p p r o x i m a t e l y 4,5. Institute of Pharmacology and Toxicology, Justus-Liebig-University, Frankfurter Str. 107, D-6300 GieBen

160 INTESTINAL

ABSORPTION OF B-LACTAN ANTIBIOTICS: TOPOLOGICAL SlllDIES OF THE INTESTINAL UPTAI(E SYSTEM FOR B-LACTANANTIBIOTICSAND SMALL PEPTIDES W. Kramer The intestinal uptake of orally active B-lactam antibiotics occurs by the H+-dependent transport system for small peptides in the brush border membrane of the entero cyte. A membrane protein of Mr 127 000 was identified as a component of this transporter in rabbit small intestine by photoaffinity labeling. Its involvement in transport was established by purification and reconstitution. For further characterization topological studies with brush border membrane vesicles (BBMV) were performed. Alkaline extraction with Na2CO3 and phase separation experiments with Triton X-I14 indicate that the 127 kDa protein is an integral membrane protein. In the absence and in the presence of 2-mercaptoethanol identical molecular weights were found demonstrating no coupling to other proteins by disulfide bonds. Twodimensional gel electrophoresis revealed a microheterogeneous protein with an isoelectric point (Pl) of 5-6. After solubilization with nonionic detergents the photelabeled 127 kDa protein could be completely precipitated with phytohemagglutinin (PHA); the uptake of cephalexin into BBMV and the photolabeling of the 127 kDa protein was not affected by PHA. Treatment of BBMVwith neuraminidase had no influence on the electrophoretic mobility of the 127 kDa protein, whereas after treatment of photolabeledBBMVwith endoglucosidase H a 115 kDa band and with endoglucosidase F a 115 and a 95 kDa peptide appeared. Incubation of photolabeled BBMV with trypsin decreased the Mr from 127 kDa to 50 kDa, whereas the uptake of cephalexin into BBMVwas not significantly altered by trypsin. Thus, the transport system responsible for the intestinal absorption of B-lactam antibiotics and small peptides is an integral microheterogeneous glycoprotein of Pl 5-6 and Mr 127 000. Hoechst Aktiengesellschaft, D-6230 Frankfurt am Main 80

R 41 161 A M~3VEL6EWEOf file I#,JLTifY2UG ~ESi$I~E TRAI~SPORIERFAWILY ISOLATED FROM NEUROBLASTOMAX 6LIOMA ~ELLS (NG-TRA) K.F. Becker, S. Reimer, M. Vanetti, M. Volm and V. H611~

163 RELEASE OF WATER SOLUBLE DRUGS FROM FROZEN AND THAWED PHOSPHOLIPID VESICLES L. Michaelis*, E. SchrSder, U. Weber and K.E. Wirth

A novel member of the multidrug resistance (MDR) transporter family was cloned from a mouse neuroblastoma x rat glioma hybrid (NGl08ccl5) cDNA l i b r a r y and termed NGTRA . Analysis of a partial sequence of about 2200 nucleotides of NG-TRA revealed a striking homology to that of the human MDRI gene: Up to 70 % of the amino acids were conserved between the two genes. Northern blot analysis of RNA of NG cells showed a single u~RNAspecies of 4.5 kb, when hybridized with NG-TRA cRNA, identical in size with that of MDRI. NG-TRAmRNAwas also found in the brain of rats. In situ hybridization experiments showed that NG-TRA is predominantly l o c a l i z e d within f i b e r tracts in the brain and over peripheral nerves. This indicates that oligodendrocytes and Schwann cells are major sites of NG-TRA biosynthesis in the nervous system. This distribution of NG-TRA is clearly different from that of MDRI in human brain which is present in endothel i a l cells only. On the other hand, both MDRI and NG-TRA B~RNA are expressed at sites with secretory/ excretory functions, such as kidney, l i v e r and adrenal gland. Cell lines derived from monkey kidney (CVI, COS) coexpress large amounts of both MI)RI and NG-TRA mRNAs. High levels of MDR mRNAwere also measured in murine sarcoma 180 cells rendered multidrug resistant with doxorubicin, whereas untreated cells contain very low levels of MDR mRNA. In contrast, the low concentrations of NG-TRA mRNA in these cells remained unchanged by the doxorubicin treatment. In conclusion, our findings show that NG-TRA is highly homologous to the MDRI gene. I t differs from MDRI by its response to certain drug treatments and by its localization in g l i a l cells. The characteristic distribution in the nervous system indicates that the putative NG-TRAtransporter plays a role in neuronal function. Physiologisches I n s t i t u t , University of Munich, Pettenkoferstr.; D-8000 MOnchen 2, FRG *German Cancer Research Institute, D-6300 Heidelberg, FRG

Above the lipid phasetransition temperature Tc the phospholipid fatty acy] chains are in a more fluid state and below in a quasi-crystalline array. Water soluble substances like carteolol HCl or methotrexate di-sodium salt (MTX) are released Bach faster from the phospholipid vesicles (liposomes) above the phasetransition temperature. We have used frozen and thawed large muitilamellar vesicles which were produced from either f u l l y hydrogenated soy lecithin (NC 95 H, Nattermann) or egg yolk phosphatidylcholine (PC) or a mixture of both. When e.g. about 20 mole% of PC are added to NC 95 H phasetransition temperature decreases from 54° to 30%. Frozen and thawed liposomes were produced from a thin l i p i d film by adding aqueous phase (carteolol MTX) f o l lowed by mechanical agitation for 10 min above phasetransition temperature. The suspension was then transferred to centrifuge tubes and sealed, frozen in liquid N9 and allowed to thaw in a water bath at 20°C. The l a t t e r two steps were repeated six times. Appearance of carteolol and MTX in the supernatants above and below phasetransition was analysed by high pressure liquid chromatography. Plasma protein binding was carried out by mixing liposomes with fresh human plasma and centrifuged for 16 h at 55 000 rpm (SW 55 Ti rotor). Our results may be summarized: I) Above l i p i d phasetransition temperature the tested water soluble drugs diffuse more rapidly into the surrounding aqueous compartmentand bind to plasma proteins Gcarteolol 2 %, MTX 48-52%). 2) By monitoring phasetransition to a physiologically relevant temperature drug release could be increased (above Tc) or again decreased (below Tc). I t is recommendedto test such systems in vivo. *Present address: Institute of Pharmaco]og/, University of Desseldorf, Moorenstr. 5, D-4000 DDsseldorf, FRG

162

164

REDUCED MITOXANTRONE CONTENTS IN A MITOXANTRONE-RESISTANT GASTRIC CARCINOMA CELL LINE UNRELATED TO THE MULTIDRUGRESISTANCE PHENOTYPE.

INTERACTION BETWEEN ORGANIC CATION AND ANION TRANSPORT AT THE BASOLATERAL MEMBRANE OF RABBIT P R O X I M A L TUBULE E. BRANDLE AND J. GREVEN

A.Reymann~* H.Arps,+ M.Dietel~ B.Schaefer + and C.Woermann* In the human gastric adenocarcinoma cell line EPG85-257 end in a subclone 185-fold resistant to mitoxantrone (DHAD, 5 day proliferation assay, IC~o nmol/l: 5 vs 923}, we measured DHAD uptake with a silicone oil filtration method (Reymann A et el, 1989, NSAP 340, Suppl.2: R 78). In contrast to typical multidrug-resistance (MDR, Bradley G e t el, 1988, BBA 948: 87), cells showed but slight cross resistance to anthracyclines (doxorubicin ll-fold, daunorubicin 3.5-fold) and no resistance at all to vinca alkaloids or eolchicine. In uptake studies (I umol/l DHAD in incubation medium, 30 min, n=6, SEM), DHAD-resistance did relate to decreased DHAD contents (pmol/mg protein, sensitive 206 + 21; resistant 116 + 8). This was due to a pronounced-reduction of the bound fraction of DHAD measured after CCI3COOM (10% w/v) precipitation of protein, DNA and RNA (pmol/mg protein, sensitive 128 t 16; resistant 78 + 15). Cytosolic DHAD (pmol/l, s~nsitive 19 ! i; resistant 16 ~ 2), as measured in the supernatant phase, was affected less. Indicative of diffusive influx, kinetics of DHAD (0.2 - I0 ~mol/l) uptake were linear in both cell lines. Low DHAD contents of resistant cells were not increased at 30°C, 25°C or 4°C. Light microscopy of resistant cells showed reduced DMAD uptake in the nucleus and formation of cytoplasmic vesic]es storing DMAD, which were lined by a hilayer membrane electron microscopically. Results indicate compartmentalization of cytoplasmatic DHAD in resistant cells and subsequent reduction of DHAD binding to vital cell structures as a possible mechanism of resistance. Whether vesicles mediate DHAD exocytosis from resistant cells remains to he investigated. (Supported by the DFG, SFB 232,C3 and Di 276/1-3) (*) Abteilung Allgemeine Pharmakologie, (+) Institut fur Patholoqie,Universit~ts-Krankenhaus Eppendorf, Universit~t HamDurg, Martinistra6a 52, D-2000 Hamburg 20, FRG (~) Institut f~r Patholoqie, Universit~t Kiel, Nichaelisstr. ii, D-2300 Kiel I, FRG

In a previous study we could demonstrate active transport of cimetidine across the basolateral membrane of rabbit proximal tubule. The transport system accepted only the positively charged eimetidine. Efflux-exchange studies provided evidence that cimetidine shares a common transport system with a number of organic bases. This lead us to conclude that cimetidine characterizes the transport system of organic cations. However, McKinney (Am. J. Physiol. 1981;10; F69 - F76) reported about an inhibition also by probenecid of cimetidine secretion in isolated perfused proximal tubules, suggesting an interaction between organic anion and cation transport in the kidney. The aim of the present study was to examine the interaction between organic anions and cations at the basolateral membrane of the isolated non perfused rabbit proximal tubule. S -segments of proximal tubules were incubated at 37.5°C with SHcimeti~ine (2"10- 7 mol/1) or 3H - P A H (4"10- 6 tool/I) and 14C-inulin (marker for the extracellular space) for 25 rain to achieve a steady-state. Afterwards, a non radioactive test substance was given to the bath and the change of the intraceUularly stored radioactivity was measured in time intervals of 5 min. The data given are the values obtained 25 rain after adding the test substance. Efflux-exchange of 3H-cimetidine: Probenecid at 5'10 -s mol/l decreased the intraceUular amount of eimetidine by 74%. At this concentration, furosemide and Na~SO4 had no effect. At a concentration of 10-s mol/l these substances, however, decreased cellular eimetidine ul~take by 67% (furosemide) and 43% (Na2SO4). EffIux-exchang e of JH..-PAH: At concentrations of 10 -s mol/l the organic bases cimetidine, N-methyl-nicotinamide and quinine decreased the intraeellular amount of PAH by 48%, 42% and 83%. Cimetidine and N-methyl-nieotinamide were also tested at a concentration of 5"10-s mol/l. At this concentration no effect was observed. Conclusion: The resuits indicate that the transport of organic cations across the basolateral membrane of rabbit proximal tubule can be inhibited by organic anions and vice versa. With the exception of probenecid this interaction, however, can only be demonstrated at high inhibitor concentrations (10 -3 mol/l).

Department of Pharmacology,RWTH Aachen, Wendlingweg, 51 Aachen

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CALCIUM OXALATE CRYSTAL FORMATION IN VITRO AND IN VIVO: EFFECTS OF PENTOSAN POLYSULFATE. H. Osswald*, G. Weinheimer

MICRO- AND mM Ca 2+ MODULATE DEVAPAMIL BINDING TO THE PURIFIED S K E L E T A L M U S C L E CaCB-RECEPTOR

The heparlnold pentosan pslysu]fate (PPS) was found to reduce stone recurrence in patients (Danielson et al., In: Urolithias{s, pp. I01-104, Plenum Press, New York, ig8g). With the present study we wanted to examine and quantitate in reproducible experimental cond[tlons the a b i l i t y of PPS to reduce calcium oxalate (CaOx) crystal formation in vitro and in viva. Methods: In vitro CaOx crystal formation was studied using the continuous flow crystallizer under steady state cend[tlons. OaOx crystals are formed in a r t i f i c i a l urine and were analyzed for size and density population by a Coulter counter. The [n vivo CaOx crystal formation was induced in rats by feeding a Vit B6 deficient diet and i % ethylenglycol as dr[nking water for 3-5 days. Energy dispersive X-ray microanalysis (EDX) of the kidney revealed that only CaOx crystals have been formed without contamination with phosphate or sulfate. PPS was adm[n[stered orally by a gastric tube, s.c. or by means of a subcutaneously implanted osmotic min{pump. Results: In the continuous flow crystalllzer PPS reduced at i0 NM the crystal growth rate from 0.39 + 0.02 to 0.26 + 0.02 gm/min while the nucleation rate ~as not affected. In the rat model o£ CaOx stone formation da{ly adm[nistradsn of i00 mg/kg p.o. PPS starting 2 days prior i n i t i a t i o n of the lithogenic diet showed 11ttle reduct{on of CaOx crystal formation whereas 30 mg/kg/2~h via the osmotic min~pump reduced CaOx formation by more than 80 %. Calcification of the kidney tissue expressed by peak to background ratio of the Ca signal after scanning the kidney section area with EDX was reduced from the control value of 0.g7 to o.ig by PPS. Conclusion: Pentasan polysulgate is an active inhibitor of CaOx crystal formation in vitro and in viva, thus csnfirming the concept of crystal growth inhibition by urinary polyanionic maeremolecules. The results provide an experimental basis for further search for exogenous inhlbitors that can substitute £o~ endogenous inhibitory macromolecules of crystal formation in the kidney.

T. Schneider,

~Pharmakologisches I n s t i t u t der Universit~t T~blngen, Wilhelmstr. 58, 7400 TUbingen, GBdecke Research I n s t i t u t e , Mooswaldallee l-g, 7800 £reiburg, F.R.G.

S. Regulla

The interaction of devapamil, a stereospecific analog of verapamil, with the purified receptor for calcium channel blockers (CaCB) was studied at 4 °C and 30 °C in the absence and presence of Ca 2+ . Devapamil was bound in a noncooperative manner at 4 °C in the absence of Ca 2+ with K d and Bma x values of 21.6 ± 9.3 nM and 1.41 ± 0.3~ nmol/mg (n = 5 preparations), respectively. The stoichiometry of 0.75 mol devapamil bound per mol ~l-subunit suggests that the ~aTsubunit has one binding site for devapamil. Ca ~-~ (i mM) increased the K d V a l u e to 106 nM without changing the number o f b i n d i n g sites. At 30 °C the devapamil.CaCB-reGeptor complex was instable in the absence of Ca 2~. It was stable at I ~ M aned s ~ mMv~2+ wi~h BKd values of 27 and 243 , r p cti y, an max values of 1.41 and 1.44 nmol bound per mg receptor protein. Almost identical K, values were obtained from the rate constants. ~he temperature and Ca 2+ affected the dihydropyridine binding site in a similar manner. Preincubation of the CaCB-receptor in the absence of Ca 2+ and devapamil at 30 °C but not at 4 °C resulted in an apparent loss of devapamil binding sites. The decrease was caused by a reduced affinity and was prevented by the addition of Ca 2+ and diltiazem during the preincubation at 30 °C. These results can be understood assuming two different Ca2+-binding sites interacting with devapamil binding. Institut f~r Physiol. Chemie, Medizin. Fakult~t, Universit~t des Saarlandes, D-6650 Homburg/Saar

168

166 PRIMARY STRUCTURE CHANNEL

S. Freundner,

OF A SMOOTH MUSCLE

Martin Biel, Peter Ruth, Veit Flockerzi

CALCIUM

IMMUNOPURIFICATION OF TEE DIHYDROPYRIDINE-SENSITIVE SKELETAL MUSCLE Ca2+-CHANNEL A. LUDWIG and W. NASTAINCZYK

Eva Bosse,

The cDNA of a L-type calcium channel (CaCh) el subunit was cloned from smooth muscle. The clone was obtained by crosshybridization of a random primed cDNA library with a skeletal muscle CaCh ~i subunit cDNA probe. The smooth muscle cDNA consists of 7374 nucleotides with an open reading frame encoding a sequence of 2166 amino acids. The calculated Mr is 242,498. The predicted amino acid sequence is much more homologous to the cardiac than to the skeletal CaCh ~i subunit. Like the skeletal and the cardiac proteins the smooth muscle ~i subunit contains four re ~peating units. Each repeat probably folds into six transmembrane helices. There are several potential phosphorylation sites for cAMP dependent protein kinase one of which is unique to the smooth muscle protein. These results suggest that slight variations of basic structural motifs apparently give rise to tissue specific Ltype CaChs. These variations might also contribute to differences in physiological regulation of native CaChs by hormones and by drugs. Institut f%r Physiologische Chemie, Medizinische Fakult~t, Universit~t des Saarlandes, D-6650 Homburg/Saar

The C a 2 + - c h a n n e l b l o c k e r (CaCB) r e c e p t o r purified from rabbit skeletal muscle contains five p o l y p e p t i d e s of 165 kDa (~i }, 135 kDa (~2), 55 kDa (B), 32 kDa (~) and 28 kOa ~) w h e n a n a l y z e d by S D S - P A G E u n d e r r e d u c i n g c o n d i t i o n s . Five monoclonal antibodies (mAb) have been produced a g a i n s t the ~ l - S U b u n i t (165 kDa) and one m A b a g a i n s t the B - s u b u n i t (55 kDa) of the C a C B receptor. An i m m u n o m a t r i x was prepared with an anti ~ l - S U b u n i t mAb and Affi Gel 15. This matrix was used to purify the CaCB-receptor from rabbit skeletal muscle membranes. Crude rabbit skeletal ~uscle m e m b r a n e ~ w e r e photoaffinity labeled with H-Azidopine or H-PN 200/110 and solubilized in d i g i t o n i n (1%). The s o l u b l e f r a c t i o n w e r e app l i e d to the a n t i ~ l - S U b u n i t m A b a g a r o s e gel. The c o l u m n w a s w a s h e d w i t h IM NaCI, pH 7.4 and the b o u n d C a C B - r e c e p t o r was eluted mit 2 M NaSCN, pH 7,4. The e l u a t e c o n t a i n e d all four s u b u n i t s of the C a C B - r e c e p t o r . A z i d o p i n e or PN 2 0 0 / 1 1 0 w e r e p r e s e n t in the ~ l - S U b u n i t . This s u g g e s t that all four s u b u n i t s can be p u r i f i e d on a c o l u m n c o n t a i n i n g a mAb a g a i n s t the O
R 43 169

171

PROPERTIES AND FUNCTIONAL SIGNIFICANCE OF TWO CALCIUM CHANNEL TYPES IN RAT PHEOCHROMOCYTOMA(PC12) CELLS H. Porzig, M. Usowicz and H. Reuter

DEPENDENCE OF GALLOPAMIL-INDUCED NEGATIVE INOTROPY ON EXTRACELLULAR K+: ROLE OF MEMBRANE POTENTIAL S. Herzig and Carola Pf¢iffer It has been observed that gallopamil and related calcium-antagonistic drugs exert their negative inotropic effect with a potency which depends on the extracellular potassium concentration ([K+]o). For instance, in Langendorff-peffnsed guinea-pig hearts stimulate~l at 3 Hz, the IC~o value for gallopamil was lowered• by 14-fold . , when [K +]o was raised from 2.7raM to 8.1raM. The mm of this study was to investigate the mechanism of this interaction.

Native PC12 c e l l s contain two populations of voltageactivated Ca channels that could be discriminated on the basis of t h e i r d i f f e r e n t electrophysiological and pharmacological properties: D i h y d r o p y r i d i n e ( D H P ) - s e n s i t i v e 6 channels (maximal DHP binding capacity 6.8±0.6 fmol/10 c e l l s ) were c l a s s i f i e d as L-type, w-conotoxin (CgTX)sensiti#e ones (maximal CgTX binding capacity 18.3+5.8 fmol/lO ~ c e l l s ) were c l a s s i f i e d as N-type. Nerve gTowth factor(NGF)-induced d i f f e r e n t i a t i o n of PC12 cells into cells with neuronal c h a r a c t e r i s t i c s was associated with a 2-3 f o l d increase in the density ofm-CgTX binding sites. The change in DHP binding was much less pronounced. These e f f e c t s were accompanied by a more than 5-fold increase in whole cell Ba currents through Ca channels. Almost all of this additional current flow could be blocked bym-CgTX. By contrast, the DHP-sensitive component of Ca channel currents was not s i g n i f i cantly enhanced during d i f f e r e n t i a t i o n • Induction of new functional channels may represent a specific action of NGF: Staurosporin, an i n h i b i t o r of NGF-mediated d i f f e r e n t i a t i o n , blocked the expression of additional m-CgTX binding s i t e s . In a NGF-insensitive PC12 mutant cell l i n e , ouabain-promoted d i f f e r e n t i a t i o n into a neuronal phenotype was not accompanied by an increase in functional Ca channels. Chronic block of L- and N-type Ca channels by verapamil andm-CgTX, respectively, during NGF treatment did not a f f e c t morphological d i f f e r e n t i a tion. Our results suggest that functional L- or N-type Ca channels are not o b l i g a t o r i l y required for neuronal d i f f e r e n t i a t i o n in PC12 c e l l s but may be involved in the regulation of neurotransmitter release. Pharmakologisches I n s t i t u t der Universit~t, FriedbUhls t r . 49, CH-3010 Bern, Switzerland

In guinea-pig papillary muscles (1 Hz), the influence of [K+], (2.7mM - 10.SmM) on resting membrane potential (RMP), action-potential duration (APD) and contractile force was assessed in the absence and presence of gallopamil (3xl0-¢M - 3xl0~M). When RMP was lowered from about -95mV at [K+]~ 2.7mM to about -70mV at [K+]~ 10.SmM, the effects of gallopamil were-markedly amplified under the-latter condition: for instance, A P D ~ was shortened by 20% in the presence of 3xl0"rM gallopamil w h e ~ [ K +] was high, whereas a tenfold higher gaUopamil concentration was still ineffective in low [K+]o. It is feasible that the decline of RMP accounts for the associated shift in the negative inotropic potency of gallopamil, since the inhibition of slow inward calcium currents is known to depend on the membrane potential. To test this hypothesis, single guinea-pig myocytes were voltage-clamped, using the patch clamp technique. Runs of stimuli (steps to +10mV, at 0.33Hz) were delivered from holding potentials of -70mY or -90mV, both in the absence and presence of gallopamil (3xl0-rM - 3xl0~M). L-type calcium current inhibition was exerted by nearly tenfold lower concentrations of gaUopamil when the holding potential was depolarized. Time courses of current inhibition by gallopamil could be described with single exponential functions. The time constants fitted to the time courses were plotted against the gallopamil concentration. These graphs suggest that both the association rate constant and the dissociation rate constant are affected by the holding potential. The half inhibitory gaUopamil concentrations calculated from these kinetic parameters agreed with those measured directly at equilibrium. It is concluded that the marked shift in the inotropic potency of gallopamil exerted by changing [K+],, is due, at least in large part, to the steep dependence on resting membrane potential of the gallopamil-induced inhibition of the calcium inward current. Department of Pharmacology, Univ. Kiel, Hospitalstr. 4, 23 Kiel, FRG.

170 ASSOCIATION OF THE MITOCHONDRIAL Ca 2+ ANTAGONIST BINDING SITES WITH AN INNER MITOCHONDRIAL MEMBRANE ANION CHANNEL

G.Zernig, I.Graziadei, T.Moshammer, D.Kandler, W.Peschina, N.Reider and The inner mitochonddal membrane contains specific Ca2 + antagonist binding sites which are unrelated to the L-type Caz+ channel &nd represent putative target structures for yet unexplained anti-ischemie Ca ;z+ antagonist effects (for a review see Zemig 1990, Trends Pharmacol Sci 11 (1), in press). Using mitochondrial swelling experiments (Gadid and Beevis 1985, Biochim Biophys Acta 853,187) we could demonstrate that Caz + antagonists from the 1,4-dihydropyridine- (DHP), phenylalkylamine- (PAA) and benzothiazepine (BTZ) classes specifically inhibited the activity of an inner mitochondrial membrane anion channel (IMAC) as did amiodarone, a known inhibitor of the IMAC (Beavls 1989, J Blol Chain 264,1508), displaying the following IC50 values (in pM; representative drugs): nitrendipine 20.2+5.9, nicardipine 8.4+0.4, (+)-iaradlplne 38.5+3.8, (-)-iaradipine 107_+27, niludipine 8.7+0.8, nifedipine 64.8+18.1; devapamil 17.6+3.1, verepamil 129_+37, gallopamil 69.5--.4.9; (+)-ols-diltlazem 61.3_+24.0, (-)-cis-diltiazem 58.2_+2.3; amiodarone 2-+0.3. Under the conditions of the swelling experiments, saturable (_+)]nitrendipine binding to mitochondrial membranes (KD , '2+2 -7 •0 r-M Bm 1.03_+0.37 nmol/mg protein) was inhibited with the following IC50 values i~n HM) by: nicardipine 2.1 _+1,0, (_+)-isradipine 6.6 _+1.5, (-)-isradipine 10.3+1.3; niludiplne 1.3+0.6, nifedipine 12.9+11.1; devapamll 43.6_+38.1, verapamil 14.7-+5.9, gallopamll 36.5-+17.0, (+)-cis-diltiazam 60.0_+49,0, (-)-cia-diltiazem 66.3_+17.8; amiodarone 0.8+0.2 (representative drugs). Linear regression of C50 values for inhibition of IMAC-induced swelling vs. plC.6n for (-+)H]nltrendipine binding Inhibition for all tested DHP's and ~Pniodarone yielded a correlation coefficient of 0.89 (n=11; plC50 IMAC=-O.37 + 0.95 x plC6n blndln,g). The correlation coefficient for amiodar~ne and all DHP-, PAA-, and-I~TZ Ca L* antagonists tested was 0.69 (n=17;plC50,1MAC = 1.32 + 0.65 x plC6n binding). Thes~ata strongly suggest the association of the mitochondrial Ca 2 + antagonist binding sites with the inner mitochonddal anion channel. Permeation of anigns through this channel could remove any restraint for mitochondrial Ca Z+ overload (Akerman and Nicholls 19~3, Rev Physiol Biochem Pharma£;ol 95,149). Thus, by inhibiting the IMAC, Ca_z+ antagonists could prevent Ca :~+ overload and ensuing structural and functional impairment of mitochondria in (iachemically) compromized tissue.

~

Inetitut for Biochemische Pharmekologie, P.Mayr-Str.1, A-6020 Innsbruck

172 CALCIUM UTILISATION IN RAT AND GUINEA-PIG J.G. Hugtenburg and J.J. Beckeringh~

PAPILLARY MUSCLES

The effect (PIE) of nifedipine (N), ryanodine (R), calcium (Ca), Bay k 8644 (Bk), caffeine (CAFF) and monensin (MO) on the developed force (DF) in rat and guinea-pig left ventricular papillary muscles (LVPMs) (paced at I Hz) was studied. In guinea-pig LVPMs N and R partially suppressed DF. Ca, Bk, CAFF and MO maximally increased the initial DF by 100, 210, 105 and 153%, respectively. In the presence of N (I wmol/l) or R (I ~mol/l) the positive inotropic effect (PIE) of Ca was not inhibited. N but not R reduced the PIE of Bk. Both N and R completely suppressed the PIE of CAFF. N slightly decreased the PIE of MO. R reduced the PIE of MO by about 70%. In rat LVPMa N (I ~mol/1) and R (I ~mol/l) almost completely suppressed DF. Ca and Bk maximally increased the initial DF by only 35 and 31%. CAFF and MO did not increase the DF. In N suppressed LVPMs the maximal response to Ca was increased by 95%. R did not influence the maximal PIE to Ca. N shifted the concentration-response curve to Bk to the right while R completely inhibited the PIE of Bk. In the presence of N or R a PIE of CAFF remained absent. In N suppressed LVPMs MO enhanced the DF by 54%. R did not induce an increase of DF by MO. The differential effect of Ca and Bk in the absence and presence of N and R on rat and guinea-pig LVPMs indicates that in rat LVPM the aarcoplasmic reticulum (SR) is mainly supplied with calcium via dihydropyridine (DHP) sensitive channels. Under control conditions the system operates at near maximum capacity. As shown by the ineffectiveness of R and the effect of MO in guinea-pig LVPM trigger calcium is also supplied from sources other than the DHP channel/SR system. The differential effects of Ca and drugs on DF in rat and guinea-pig LVPMs reflects the utilisation of differential sources of trigger calcium and the supply of calcium to these sources in the rat and guinea-pig myocardium. Division of Pharmacotherapy/Pharmacology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. *Fazantlaan 12, 2 2 6 1 B T Leidschendam, The Netherlands.

R 44 173

175

CARDIAC EFFECTS OF BAY K 8644, RYANODINE AND VARIOUS CALCILN ANTAGONISTS IN ISOLATED HEARTS FROM DIABETIC AND NORMAL RATS J.B. Heijnis and M-J. Mathy The effects of various drugs, differentially interacting with calcium metabolism were studied in Langendorff-perfused hearts of diabetic rats (DR), in comparison with hearts of age-matched control rats (CR). Diabetes was induced by i.v. injection of streptozotocin (50 mg/kg). At 45 days after STZ injection hearts (paced at 5 Hz) of both groups were used. The LVP (via an intravenbricular balloon) dPmax/dT and the coronary flow (CF) were determined. At 10-8 M Bay k 8644 induced a positive inotropic effect of 32% in hearts of CR, while in hearts of DR contractile force was reduced by 15%. At 3xI0 -6 M the same increase of contractile force was reached in CR and DR (290 vs 260%, n.s.). Bay k 8644 induced at 10-8 M a modest coronary vasoconstriction (15-20% of the initial CF) and a subsequent vasodilator activity from 2xIO-8 M and 2xIO-~ M, respectively in hearts from CR and DR. The same maximum increase of CF was reached in both groups (30%). Diltiazem (D) induced a leftward shift of the concentration-response curve for the inotropy in hearts of DR in comparison with those of CR (EC~0 1.8.10 -6 vs 10-s M). For verapamil (V) a moderate leftward shift was observed in the hearts of DR. The same response in both CR and ~R hearts was observed for nifedipine (N) and ryanodine (R). N, D and V induced an increase in CF (maximum 76-82% of the initial value). However, in'hearts of DR a stronger vasodilator activity was observed for N and D (105-128%). For R at 3xI0 -~ M a small decrease in CF was observed in CR and DR. These findings suggest a differential sensitivity of coronary vessels and an absence of consistent changes with respect to contractility in hearts from DR in comparison with hearts from CR for the various drugs studied.

Influence of endothelium on vascular activity of structurally different calcium antagonists in porcine isolated basilar arteries. G. Warner, G. Kojda and U. Fricke

Division of Pharmacotherapy, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Endothelial damage has been shown to decrease vascular activity of nitrendipine and some related 3-ester derivatives in porcine isolated arteries (Kojda et al., NSAP 340 (Suppl.): R 54, 1989). To study this interdependence further, we here report on the influence of endothelium on the action of calcium antagonists of rather different chemical structure: nisoldipine (NIS), gallopamil (GAL), flunarizine (FLU) and some calcium antagonists of the novel dihydronaphthyridine (DHN) type (Warner et al., NSAP 339 (Suppl.): R 47, 1989). In isolated porcine basilar artery rings bathed in oxygenated Krebs-HenseleitSolution (pH 7.4, 37°C) endothelial function was verified by substance P induced (3~mol/l) fading relaxations after precontraction with P G F ~ (l~mol/l). In control experiments these relaxations could he completely abolished by methylene blue (MB, 5~mol/l), the calcium antagonistic potency of NIS under these conditions being not different from that in artery segments mechanically denuded of endothelium. Following further precontraction with PGF2a (5~mol/l) or 5-hydroxytryptamine (5-HT,O.5 ~mol/l) the calcium antagonists were added cumulatively up to concentrations of 100pmol/l. In PGF2.-precontracted intact basilar arteries vasodilator potency (ICso) of DHNs (1.3-4.4 fold) and NIS (14.2 fold) was significantly (p< 0.05) enhanced over MB-treated vessels, GAL (1.2 fold) showing no such difference. Probably due to the 5-HT-induced release of endothelium derived relaxing factor (EDRF) this dependence was far more pronounced in 5-HT-precontracted vessels (DHNs = 13.5-28.2 fold, NI8 = 22.9 fold, GAL 4.3 fold). Opposite results were obtained ~ith FLU. This drug was about two times more potent in MB-treated vessels than in functionally intact basilar arteries. Thus, endothelium may influence vasorelaxation induced by selective (NIS, DHN, GAL) and non-selective calcium antagonists (FLU) rather differently. Inst. Pharmakol.Univ.E61n, Gleueler Str.24,'D-5000 K61n 41

174

176

EFFECTS OF NOP~RENALINE DEPLETION AND CALCIUM A N T A G O N I S M O N R E P E R F U S I O N A R R H Y T H M I A S IN CLOSED-CHEST RATS Michael Kirchengast

ENDOTHELIAL CYCLIC GMP-FORMATION AND -RELEASE R E S P O N S E T O A N P IS I N H I B I T E D B Y F E N D I L I N E . U. Pohl, I. W i n t e r , E. B a s s e n g e

IN

In c l o s e d - c h e s t , a n a e s t h e t i z e d r a t s r e p e r f u s i o n of t h e l e f t c o r o n a r y a r t e r y (LCA) a f t e r a 5 - m i n o c c l u s i o n a l w a y s led to v e n t r i c u l a r t a c h y a r r h y t h m i a (VT) a n d p e r i o d s of v e n t r i c u l a r fibr i l l a t i o n (VF), w h i c h , in m o s t a n i m a l s , s u b s i d e d s p o n t a n e o u s l y . To i n v e s t i g a t e a s p e c t s of t h e m e c h a n i s m of r e p e r f u s i o n a r r h y t h m i a s t h e i n f l u ~ of m y o c a r d i a l n o r a d r e n a l i n e d e p l e t i o n a n d of channel blockade were investigated. 4 groups of r a t s w e r e s t u d i e d , in w h i c h L C A o c c l u s i o n w a s c a r r i e d out v i a a p r e v i o u s l y i m p l a n t e d o c c l u d e r : c o n t r o l s , r a t s t r e a t e d w i t h r e s e r p i n e (5 m g / k g i.p.) 24 h b e f o r e o c c l u s i o n , a n d r a t s r e c e i v i n g 0.2 m g / k g ~ a l l o p a m i l i.v. 5 m i n b e f o r e o c c l u s i o n either with or without reserpine pretreatment. A l l c o n t r o l r a t s h a d V T a n d 9-Fs s t a r t i n g immediately after reperfusion. Gallopamil reduced V T t o 50% a n d V F to 20% of t h e rats. In the res e r p i n e g r o u p all r a t s h a d VT, 67% VF, this b e i n g not statistically different from controls. Addit i o n a l t r e a t m e n t of r e s e r p i n i z e d a n i m a l s w i t h gallopamil markedly reduced VT and totally prev e n t e d 9-F. It w a s s h o w n t h a t r e s e r p i n e t r e a t m e n t d e p l e t e d n o r a d r e n a l i n e s t o r e s to l e v e l s b e l o w 50 n g / g w . w . , w h e r e a s in c o n t r o l s 2000 - 3000 n g / g w.w. w e r e found. T h u s t o t a l d e p l e t i o n of m y o c a r d i a l n o r a d r e n a line stores did not prevent the occurrence or r e d u c e t h e s e v e r i t y of r e p e r f u s i o n a r r h y t h m i a in rats, w h i l e Ca ~ antagonism with gallopamil was effective.

R e c e n t l y a p o t e n t i a l r o l e of c a l m o d u l i n in the regulation of particulate guanylate cyclase (PGC) has b e e n r e p o r t e d . W e i n v e s t i g a t e d w h e t h e r c a l m o d u l i n a n t a g o n i s t s (CDA) i n t e r f e r e w i t h the f o r m a t i o n a n d / o r r e l e a s e of c G M P in e n d o t h e l i a l c e l l s (EC}, w h i c h e x h i b i t a h i g h c a l m o d u l i n content. c G M P - r e l e a s e ( r a d i o i m m u n o a s s a y ) f r o m cult u r e d (CEC; b o v i n e aorta) or n a t i v e (NEC; r a b b i t aorta) EC was d e t e r m i n e d u n d e r c o n t r o l c o n d i tions, and in the p r e s e n c e of a t r i a l n a t r i u r e t i c p e p t i d e (ANP: h A N P 9~-126 1 nM, 15 min) w i t h and w i t h o u t p r e i n c u b a t i o n (I0 min) of the c e l l s w i t h the C D A f e n d i l i n e (FEN; 1-30 ~M) or c a l m i d a z o lium (CAL; 3~M). Control cGMP c o n c e n t r a t i o n s (fmol/ml) a m o u n t e d to 49 ±12 ( s u p e r n a t a n t of CEC) and ii ±9 ( s u p e r f u s a t e f r o m NEC). A N P inc r e a s e d the cGMP c o n c e n t r a t i o n to 4280 ± 1 0 5 0 (CEC) and 712 ±158 (NEC) r e s p e c t i v e l y . P r e i n c u b a t i o n w i t h F E N d o s e - d e p e n d e n t l y a t t e n u a t e d the A N P - i n d u c e d r e l e a s e f r o m b o t h cell s y s t e m s up to 90 %, w h i l e CAL h a d no i n h i b i t i n g effect, F E N (3 ~M) a l s o r e d u c e d the A N P - s t i m u l a t e d cytosolic c G M P - c o n t e n t of C E C b y 32 ±2~. In c o n t r a s t , F E N did not r e d u c e the r e l e a s e of cAMP in r e s p o n s e to 1 D M i s o p r o t e r e n o l (593 ±0.8 vs. 675 ±0.7 fmol/ml) e x c l u d i n g an u n s p e c i f i c i n h i b i t o r y effect of F E N on p a r t i c u l a t e c y c l a s e s . It is conc l u d e d that the i n h i b i t o r y e f f e c t of F E N is due to an i n t e r f e r e n c e w i t h the PGC or one of its r e g u l a t o r s . 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 e f f e c t is d u e to the c a l m o d u l i n a n t a g o n i s tic p r o p e r t i e s of F E N s i n c e CAL d i d not act as an i n h i b i t o r .

D e p a r t m e n t of C a r d i o v a s c u l a r P h a r m a c o l o g y , AG, P.O. B o x 210805, 67 L u d w i g s h a f e n , F R G

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

Knoll

R 45 179

177 INHIBITION OF CELL P R O L I F E R A T I O N PYRIDINE DERIVATVES F. A b d a l l a h and K. G i e t z e n

BY D I H Y D R O -

It has been established in p r e v i o u s s t u d i e s that dihydropyridine c o m p o u n d s reveal a relatively w e a k p o t e n c y to i n h i b i t g r o w t h of tumor cells in vitro. B e c a u s e of the p r o f o u n d e f f e c t s of these a g e n t s on Ca 2+ c h a n n e l s and hence on the cardiovancular system, which occurs at considerable lower c o n c e n t r a t i o n s as c o m p a [ e d with the a n t i - p r o l i f e r a t i v e action, the use of these substances in anti-tumor therapy is prohibited. However, the fact that racemic d i h y d r o p y r i d i n e c o m p o u n d s may be s e p e r a t e d into enantiomers, one of which is in g e n e r a l far less p o t e n t on the c a r d i o v a s c u l a r system, o p e n s the attractive possibility to o b t a i n a novel class of therapeutics for the t r e a t m e n t of t u m o r s . Indeed, our e x p e r i m e n t s show t h a t B85935, which is the o p t i c a l a n t i p o d e of n i g u l d i pine and by far less p o t e n t on Ca 2+ c h a n n e l s , has the name p o t e n c y as n i g u l d i p i n e to i n h i b i t the growth of a g i v e n tumor cell line. This principle fact is also va'lid for the e n a n t i o mere sf n i t r e n d i p i n e and i s r a d i p i n e . H o w e v e r , the anti-proliferative potency sf B859-35 I C 5 0 = 0 . 2 ~M) on the m a m m a t u m o r cell line ZR-75 is about i00 times higher as c o m p a r e d with nitrendipine or i s r a d i p i n e . In a d d i t i o n , B 8 5 9 35 exhibits a certain selectivity with respect to the i n v e s t i g a t e d cell line in that f o l l o w i n g order of d e c r e a s i n g p o t e n c y has b e e n a s s e s s e d : ZR-75 > M C F - 7 > A-549 > Amnion. N e i t h e r isradipine or n i t r e n d i p i n e show any s e l e c t i v i t y for sne of the m e n t i o n e d cell lines, as is also the case for d o x o r u b i c i n e . D e p a r t m e n t of P h a r m a c o l g y & T o x i c o l o g y , U n i v e r sity of Ulm, O b e r e r E s e l s b e r g , D - 7 9 0 0 Ulm, FRG

178 INHIBITION

O F C a 2 ÷ - I N D E P E N D E N T L I P I D - M E T H Y L A T I O N BY

CALMODULIN ANTAGONISTS IN RINm5F CELL HOMOGENATES: QUANTITATIVELY DIFFERENT EFFECTS OF W7, TFP AND CGS 9343B H. S a f a y h i and M.I. Anazodo TFP:trifluoperazine w a s s h o w n to inhibit t h e incorporation of methyl groups i n t o e n d o g e n o u s Upids in rat i s l e t h o m o g e n a t e s s u g g e s t i n g a p a r t i c i p a t i o n of calmodulin (CAM) in lipid m e t h y l a t i o n [1]. Insulinoma cell h o m o g e n a t e s k a t a l y s e t h e t r a n s f e r of [31t]-methyl groups from [ 3 H - m e t h y l ] - S a d e n o s y l - L - m e t h i o n i n e (AdoMet) into e n d o g e n o u s lipids. In t h e p r e s e n c e of 5 mM Mg2. and 6pM AdoMet linear i n c o r p o ration of methyl groups w a s o b s e r v e d w i t h i n 10 rain at 0.25 to 1 mg protein/ml, and at 0.7 mg/ml w i t h i n 2 to 15 rain, r e s p e c t i v e l y . Methylation w a s almost c o m p l e t e l y a b o l i s h e d by 1 mM S - a d e n o s y l - h o m o c y s t e l n or by boiling o f t h e h o m o g e n a t e prior to i n c u b a t i o n . The addition of EGTA (5 mM) or Ca~÷ (0.1 to 100 pM) did not a f f e c t m e t h y l a t i o n . However, m e t h y l a t i o n w a s e f f e c t i v e l y i n h i b i t e d by t h e CaM a n t a g o n i s t s (CaM-A) of p h e n o t h i a z i n e - t y p e (25-1001JM TFP) and s u l f o n a m i d e - t y p e ( 2 5 - 1 0 0 tiM W7) by a b o u t 80 ~ . The n o v e l and more p o t e n t CaM-A CGS 9 3 4 3 B : 1 , 8 - d i h y d r o - 1 [1-((4-methyl-4H,6H-pyrrolo[l.2-a]-[4,l]benzoxazepin-4-yl) methyl) - 4 - p i p e r i d i n y l ] - 2 H - b e n z i m i d a z o l - 2 - one maleate (at c o n c e n t r a t i o n s which i n h i b i t e d d e p o l a r i z a t i o n - i n d u c e d i n c r e a s e in c y t o s o I i c Ca 2÷ and i n s u l i n r e l e a s e in i n t a c t insullnoma c e l l s as e f f e c t i v e l y as TFP and W7 [2]) d e c r e a s e d lipid m e t h y l a t i o n o n l y by 20 %. Our d a t a suggest: 1) Ca 2. (and t h e r e f o r e CaM) is n o t required for e n z y m a t i c m e t h y l t r a n f e r in i n s u l i n o m a cell h o m o g e nares. 2) The p o t e n t i n h i b i t i o n of methyl incorporation into lipids by W7 and TFP is p o t e n t i a l l y a CaS÷/CaM i n d e p e n d e n t , n o n s p e c i f i c s i d e e f f e c t of t h e s e t y p e s of CaM-A. [1] Kowluru et al. Arch Biochem Biophys (1985), 7 2 - 8 1 [2] S a f a y h i et al. N a u n y n - S Arch Pharmacol.(1989), 3 3 9 : 8 - 1 8 Lehrstuhl Pharmakologie, P h a r m a z e u t i s c h e s I n s t i t u t , A u f der Morgenstelle 8, D - 7 4 0 0 Tfibingen

COMPARISON OF FUNCTIONAL AND ELECTROPHYSIOLOGICAL EFFECTS OF DOBUTAMINE AND ISOPRENALINE IN NORMOXIC AND HYPOXIC GUINEA-PIG HEARTS K. G ~ t t l e r , O. R o s e n , R. P o d e h l r W. K l a u s

The a i m of the s t u d y w a s to e x p l o r e w h e t h e r dobutamine, an i n o t r o p i c a g e n t w i t h a m u l t i p l i c i t y of a c t i o n s at b o t h a l p h a and beta adrenoceptor sites, d i f f e r s d i s t i n c t l y in its d i r e c t cardiac effects f r o m the classical, nonselective beta a g o n i s t i s o p r e n a l i n e d u r i n g n o r m o x i a (pO2: 658 ± 8 mmEg) resp. h y p o x i a (pO2 : 12 ~ 1 mmHg). Experiments were carried out on guinea-pig papillary muscles with conventional microelectrode technique. In n o r m o x i a at equieffective concentrations with regard to the inotropic effect, isoprenaline (0.05 ~M) and dobutamine (i ~M) s h o w e d q u i t e i d e n t i c a l electrophysiological a c t i o n s on all p a r a m e t e r s tested, w h e r e a s in h y p o x i a of 120 m i n d u r a t i o n s t r i k i n g differences were e v i d e n t (drug t r e a t m e n t w a s s t a r t e d 30 min p r i o r h y p o x i a ) : H y p o x i a - i n d u c e d s h o r t e n i n g of the action p o t e n t i a l d u r a t i o n (91 ! 5 m s e c as compared to 209 + 4 m s e c in n o r m o x i a ) w a s enhanced under isoprenaline (70 + 6 msec), w h i l s t d o b u t a mine especially after longer lasting periods of hypoxia (> 90 min) r e v e a l e d a mitigated short e n i n g of the a c t i o n p o t e n t i a l d u r a t i o n (139 ! 5 msec a f t e r 120 m i n hypoxia). Additionally, it could be s h o w n t h a t d o b u t a m i n e w a s e f f e c t i v e in the same m a n n e r w h e n a d m i n i s t e r e d a f t e r the o n s e t of the h y p o x i c p e r i o d (70 - 120 min). C o n t r a r y to dobutamine isoprenaline induced a significant w o r s e n i n g of the m e c h a n i c a l f u n c t i o n as compared %o the c o n t r o l c o n d i t i o n . These results suggest tha£ in hypoxia dobutamine seems to be more appropriate than isoprenaline. Institut f~r Pharmakologie der K61n, G l e u e l e r s t r . 24, 5000 K 6 1 n

Universit~t 41 •

zu

180 REGULATION OF HUMANMYOCARDIAL~-ADRENOCEPTORSAND MUSCARINIC CHOLINOCEPTORSFOLLOWING CHRONICADMINISTRATION OF ~-ADRENOCEPTOR ANTAGONISTS. , N.M.Deighton, D.Borquez, and S.Motomura ) We have examined the effects of chronic ~-adrenoceptor (AR) antagonist treatment of patients with coronary artery disease on ~-AR and muscarinic (M) cholinoceptor number in right atria. In patients chronically treated with the ~-I AR antagonists atenolol, bisoprolol or metoprolol right a t r i a l ~-I AR number was significantly increased while right a t r i a l ~-2 AR numbers were unaffected. Concomitantly activation of right a t r i a l adenylate cyclase by 10 ~M GTP, 10 pM isoprenaline, and I ~M forskolin was enhanced. On.the other hand, on isolated e l e c t r i c a l l y driven (1.0 Hz, 37vC) right atria from ~-I AR antagonist treated patients the pD~ values for the positive inotropic effects of isoprenaline " and noradrenaline were only marginally higher than in nottreated atria, while that of the ~-2 AR agonist procaterol was significantly elevated (8.24 vs. 7.63). In contrast, in the same ~-I AR antagonist treated patients the number of right a t r i a l M-cholinoceptors was significantly reduced by about 25% and a significantly weaker inhibition of adenylate cyclase a c t i v i t y by the M-cholinoceptot agonist carbachol was observed. Concomitantly, the pD~ value for the negative inotropic effect of carbachol on isolated e l e c t r i c a l l y driven right atria was significantly decreased (6.93 vs. 7.23). I t is concluded that chronic ~-I AR antagonist treatment regulates ~-AR and M-cholinoceptors in the human heart in opposite directions: i t increases ~-I AR number, sensitizes ~-2 AR function and simultaneously desensitizes M-cholinoceptor function. *)Present address: Department of Pharmacology, Yamanashi Medical College, Tamaho, Yamanashi409-38, Japan Biochem. Forschungslabor, Med. Klinik & PoIiklinik, Universit~t Essen, D-4300 Essen, Fed. Rep. Germany N.M.D. is a recipient of a fellowship of the Deutsche Gesellschaft fur Herz- und Kreislaufforschung.

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183

BETA-ADEENOCEPTORS IN THE ISCHAEMIC AND REPERFUSED MYOCARDIUM OF THE RAT AND THE GUINEA PIG. R.van den Ende, H.D. Batink and P.A. van Zwieten.

TWO WEEKS INTERMITTENT DOBUTAMINE RESTORES CARDIAC FUNCTION AND INOTROPIC RESPONSIVENESS IN RATS WITH HEART EAILURE R.G. Sehoemaker, J.J.M. Debets

High concentrations of catecholamines, released during ischaemia may be expected to induce desensitization of cardiac adrenoceptors. However, variable results have been obtained in experimental models of ischaemia in vitro and in vivo. We established 6-adrenoceptor density, affinity and 61/62 ratios in rat and guinea-pig Langendorff hearts (LH), under normoxic and ischaemic circumstances. Neither ischaemia (30 or 60 min) nor post-ischaemic reperfusion caused any change in 6-adrenoceptor density, affinity or the 6~/62 ratio in the rat and guinea-pig LH. Perfusion of the rat LH with 10-5 M isoproterenol (15 min) caused a decrease in 6-adrenoceptor density, but no change in affinity or BI/62 ratio. In functional studies isoproterenol (3 x 10-11-3x10-s M) did not cause a rise in contractile force in rat LH which had been subjected to 30 min of global ischaemia and reperfusion, whereas in normoxic control hearts a concentration-response curve for isoproterenol could be established as usual with a pD2-value of 9.15 + 0.13. Bisoprolol (10-4 M) reduced contractile force of ~he rat LH by 35.5 ! 2.9% after 30 min of ischaemia and by 44.4 0.5% in nermoxic control preparations (means ~ s.e.m., n=4-6; p < 0.05). In conclusion, glcbal ischaemia did not change the density, affinity and Bi/62 ratios of cardiac 6adrenoceptors. However, the contractile response to isoproterenol was virtually abolished as a result of ischaemia. A mitigated negative inotropic response after ischaemia was found for the 61-blocker bisoprolol.

Division of Pharmacotherapy/Pharmacology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

In healthy rats, acute administration of dobutamine (dob) causes an increase in resting cardiac output (CO). Also maximal CO as obtained by rapid volume loading is increased. Coronary artery ligation in rats results in a long-term depression of resting and stimulated CO as well as a reduction of stimulated CO after acute dob administration. In the present study, we investigated the effects of intermittent dob therapy in coronary artery ligated rats. Three weeks after coronary artery llgation therapy (0.2 (low) and 1.0 mg/kg (high) i.p., twice daily) was initiated and continued for 2 weeks. Animals were implanted with an electromagnetic flowprobe on the ascending aorta to measure CO, and catheters in the abdominal aorta (for mean arterial pressure: MAP), thoracic vena cava (for central venous pressure), and abdominal vena cava (infusions). After 2 weeks of therapy, baseline and stimulated (infusion of 12 ml Ringer's solution in 60 s) hemodynamics were measured at least 90 min after the last injection. Two weeks intermittent dob neither affects MAP nor heart rate. Both baseline and stimulated CO were dose-dependently improved (baseline: infarct: 73±4; dob low: 81±5; dob high: 95±6 ml/min, stimulated: infarct: 105±5, dob low: 121±6; dob high: 144±8 ml/min). Two days later, while intermittent therapy was continued, effects of acute dob (60 ~g/kg.min iv) administration was measured on baseline and stimulated hemodynamics. Two weeks intermittent therapy preserved baseline responses to acute dob administration but enhanced CO response after stimulation (infarct: 128± 3; dob low: 175±6; dob hight 211±17 ml/min). Results indicate that 2 weeks dob therapy in conscious chronically in,farcted rats not only restores cardiac function but also the" positive inotropic response to acute dob. The study suggests that dob has a long-term inotropic effect, different from its acute inotropic effect. Dept. of Pharmacology, University of Li~burg, P.0. 616, 6200 MD Maastricht, The Netherlands

182 EFFECTS OF PHENYLEPHRINE, ISOPRENALINE AND SUCCINYLCHOLINE ON MYOCARDIAL INOSITOL PHOSPHATE CONTENT OF MALIGNANT HYPERTHERMIA SUSCEPTIBLE SWINE J. Seholz, N. Roewer, U. Rum, and J. Schulte am Esch

a-Adrenoceptor stimulation could induce malignant hyperthermia (MH) in swine. Many studies suggest that a defect of the sareoplasmic retieulum is involved in MH. Therefore the effects of the a-adrenoeeptor agonist phenylephriue (PHE) and for comparison the effects of the 6-adrenoeeptor agonist isoprenaline (ISO) and the trigger agent succinylcholine (SUC) on inositol-lipid-metabolism of MH susceptible (MHS) and healthy control swine (nMHS) were investigated. The experiments were performed on electrically driven (0.2 Hz) trabeeulae isolated from the right ventricles of the hearts of MHS and nMHS. After labelling with 3H-inositol for 6 h different inositol phosphates were determined by HPLC-analysis. Products measured were inositol-l-phosphate, inositol-l,4bisphosphate, inositol-l,3,4-trisphosphate, inositol-l,4,5-trisphosphate (1,4,5-IP3) and inositol-l,3,4,5-tetrakisphosphate. After stimulation with ISO (10 ~M) the inositol phosphate content did not increase and differ in MHS and nMHS. The same holds true for SUC (10-1000 #M). In contrast, all inositol phosphates increased after stimulation with PHE (10 #M) in MHS and nMHS but significantly more in MHS (246-393% of control) than in nMHS (168-300% of control), especially in 1,4,5-IP~. Since 1,4,5-IP~ has been shown to mobilize intraeellular calcium, it is concluded that an enhanced a-adrenergie response is involved in the development of MH. Abteilung fOr An~isthesiologie, Universit~its-Krankenhaus Eppendoff, Martinistrage 52, D-2000 Hamburg 20, FRG

Box

184 ~2TS OF ADenOSINE DI~/VATIVES ON PROTEIN PHOSPHORYIATION IN P~P/USED gJINEA-PIG HFAIqPS J. NeumannI, R. C. GuDta2 . A. M. Watanabe2 Al-adenosine receptors (ARs) are coupled in an ~ i t o ry way to adenylate cyclase (AC) activity whereas A2-ARs stimulate AC. Tne effects of the AI-AR agcrdst (-)-N~phenylisopropyladenosine (PIA, 1 ~a,ol/l) and the A2-AR agonist 5'-N-ethyl-carboxamidoadenosine (NECA, i ~mol/l) on rate of tension development (T), phospholamban phosphorylation (PL-P) and troponJ_n I phosphorylation (TnlP) were studied in intact myocardium in the presence of adenosine deaminase (1.5 u/ml). Isolated guinea-pig ventricles were perfused via the coronary arteries with 32pi (2mCi/heart). ~ereafter membrane vesicles and contractile proteins were isolated. Isoprenaline (Iso, i0 rmol/l, 60 s) increased T to 239 + 13.6 % of predrug value and PL-P frc~ 77 + 25 pmol 32p/rag protein (pP) to 431 + 66 pP and TnI-P from 96 + 28 pP to 247 _+ 39 pP (p< O.O5; n=6-7), silmlltaneous administration of Iso and PIA or NECA attenuated the increase in T to 143 + 9.5 % or 149 + 12 % of predrug value, respectively. ~he increases in protein ~ s p h o r y l a t i o n were significantly attenuated by simultaneous administz-ation of Iso and PIA or NECA (60 s ). Iso increased PL-P to 134 + 29 pP or 121 + 31 pP in the additional presence of PIA or NECA. Iso ~ TnI-P to 163 + 26 pP or 158 + 29 pP in the presence of PIA or NECA. Hence, both A 1 (PIA)- and A 2 (NECA)-AR agonists have similar effects on contractility and protein l~hosphorylation in the presence of Iso.- It is concluded that the negative inotropic effects of PIA and NECA are accc~panied by decreases in PL-P and TnI-P which might be due to activation of phos~%atase(s) and that ventricular ARs do not fit into the A 1 or A 2 classification but represent a special AIAR subtype. (Supported by the DFG. ) iAbteilung Allgemeine Pharmakologie, Universit~ts-Kranke/lhaus Eppendorf, [hliversit~t Hamburg, Martinistra~e 52, I)-20oo Ha~ourg 20, FRG; 2 D e ~ of Medicine and Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA

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187

EFFECTS OF CARBACHOL IN THE PRESENCE OF ISOPRENALINE ON CONTRACTILE RESPONSE AND cAMP AND cGMP CONTENT IN VENTRICULAR CARDIOMYOCYTES B. Stein and C. Schweiger

ACTION OF ANGIOTENSIN-I LATED RABBIT HEARTS R.R6sen and A.Korth

Carbachol (CARB) has been reported to antagonize the positive inotropic effect of catecholaraines in ventricular heart preparations. It is still a matter of controversy whether this effect of CARB is mediated by a decrease in cAMP content and/or due to an increase in cGMp content. Therefore we investigated the effects of CARB alone and in the presence of isoprenaline (ISO) on contractile response (extent of cell shortening in % of cell length, %CL) and cAMP and cGMP content (pmol/mg protein) in isolated ventricular myecytes from guineapigs. Myocytes were electrically stimulated at 1 Hz. CARB (10 )Imol/l) alone did not change CL or cAMP content, but increased cGMP content from 0.20 + 0.02 to 0.43 + 0.06 (n=10-11, p ~ 0.05) . ISO (0.01 ~inol/l) increased CL from 2.28 + 0.09 to 6.23 + 0.ii and cAMP content from 2.74 + 0,12 to 4.33 + 0.2~ (n=12-13, p a~ 0.05). ISO did not--change cGMP con[ent. In the presence of ISO CARB (I ~'nol/l) reduced CL by 61 % without influencing cAMP content. CALEB increased cGMP content from 0.23 + 0.04 to 0.37 + 0.05 (n=ll, p < 0.05). It is concluded that in isolated ventricular cardiomyocytes the negative inotropic effect mediated via m-cholinoceptors in the presence of ISO cannot easily be explained by a decrease in cAMP content. The increase in cGMP content might he involved in the negative inotropic effect of CANE. (Supported by the DFG.)

Abteilung Allgeraeine Pharmakologie, Universit~ts-Krankenhaus Eppendorf, Universit~t Hamburg, MartinistraBe 52, D-2000 Hamburg, FRG

AND CAPTOPRIL

IN

ISO-

The converting e n z y m e is t h o u g h t to b e a key e n z y m e of t h e v a s c u l a r endothelium. The aim of this s t u d y w a s to a n a l y s e t h e a c t v i t y of this e n z y m e s y s t e m in c o r o n a r y a r t e r i e s of an intact Langendorff-perfused rabbit heart. If t h e e n d o genous substrate angiotensin I was present, its inhibition by ACE-inhibitors like captopril should induce vasodilation. Captopril (10 -6 mol/l) did not significantly change the global coronary flow rate and the left ventricular pressure indicating t h e a b s e n c e of endogenous angiotensin-I. Therefore, the hearts were supplied with increasing concentrations of angiotensin-I without and with Captopril. In t h e a b s e n c e of C a p t o p r i l the coronary flow rate was decreased in a c o n c e n t r a t i o n - d e p e n d e n t manner u p to -25%, w i t h a n E C - 5 0 of 10-I°moi/i. Left ventricular pressure was concomitantly reduced up to - 1 5 ~ . By C a p t o p r i l this concentrationresponse curve was concentration-dependently shifted to t h e r i g h t as d e m o n s t r a t e d by the increase in t h e E C - 5 0 - v a l u e s (Captopril = 10 -7 m o l / l , E C - 5 0 = 5 x 10 -9 m o l / l ; Captopril = 10 -6 mol/l, E C - 5 0 = 1 , 5 x 10 -8 m o l / l ) . M o r e o v e r , the maximal flow restriction was diminished and at 10 -6 mol/l Captopril left ventricular pressure was not any longer different from control values. Thus, in isolated rabbit hearts the converting enzyme has a very high affinity to 'its s u b s t r a t e angiotensin-I, a n d m a y be effectively inhibited by Captopril. Institut f~r Pharmakologie der Universit~t zu K61n, Gleuelerstr. 24, 5 0 0 0 K ~ i n 41

186

188

POSITIVE INOTROPIC EFFECT OF CARBACHOL AND INOSITOL PHOSPHATE CONTENT IN GUINEA-PIG ATRIA AFTER PNETREATNENT WITH PERTUSSIS TOXIN C. Kohl and H. Scholz

ACUTE EFFECTS OF ACE INHIBITORS ON CENTRAL AND REGIONAL HEMODYNAMICS IN CONSCIOUS NORMOTENSIVE (WKY) AND INFARCTED

The m-cholinoceptor agonist carbachol (CARB) elicits a negative inotropic effect (NIE) in mammalian atria. Pretreatment with pertussis toxin (PTX) converts the NZE to a positive inotropic effect (PIE). We investigated the time course of the effects of CARB on force of contraction (FC) and phosphoinositide turnover in electrically driven left atria from guinea-pig hearts after pretreatment with PTX (180 ~.g/kg i.v.; 24 hrs). Inos~tol phosphates and phosphoinositides were labelled w i t h ~ H ] inositol (6 hrs) and separated with high performance liquid chromatography and thin layer chromatography respectively. The PIE of CARB (I0 Gmol/l) began within 2 min and was maximal within 15 min (158% of control). Inositol 1,4,5-trisphosphate (I,4,5-IP) rose within 1 3 min to 135% of control followed by an increase in inositol 1,3,4,5-tetrakisphosphate (IP~), inositol 1,3,4-trisphosphate, inositol 1,4-bisp~osphate and inositol 1-phosphate within 2 min (186%, 168%, 150% and 149% of control respectively). Both phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate decreased within 2 min to about 64% of control respectively.- It is concluded that the CARB-induced PIE is associated with an increase of the presumed second messengers 1,4,5-1P~ and IP A. Since the increase in 1,4,5-1P precedes the increase in FC it may initiate 3 . . the PIE. The increase in IP may be involved in main4 taining the PIE of CARB. (Supported by the DFG and the Claussen-Stiftung.) Abteilung Allgemeine Pharmakologie, Universit~ts-Krankenhaus Eppendorf, MartinistraBe 52, 2000 Hamburg 20, FRG

Recently, the existence of tissue renin-angiotensin systems besides a circulating renin-angiotensin system has been described. Tissue renin-angiotensin systems may be important in the regulation of hemodynamics in congestive heart failure. To investigate the role of tissue angiotensin II (ang II) in the regulation of central and regional hem0dynamics in heart failure, we studied in WKY and MI the acute effects of the ACE inhibitors captepril (CA), enalaprilate (EN) and lisinopril (LI) at doses which gave comparable inhibition of pressor responses to intravenous (i.v.) ang I. To induce myocardial infarction, a ligature was placed around the left coronary artery in WKY 5 weeks before measurements. The animals were instrumentated either with an electromagnetic flowprobe for measurement of cardiac output (CO) or with Doppler flowprobes for determination of regional blood flow distribution. Catheters were implanted for measurement of mean arterial pressure (MAP) and for i.v. injections of saline, CA (3 mg/kg)~ EN (0.I mg/kg) or LI (0.i mg/kg). The ACE inhibitors had no significant effects on CO and total peripheral resistance in WKY and HI. Although in WKY MAP was significantly reduced by EN (-8.3±1.i mmHg, n=8; p<0.05), no significant effects on MAP were obtained in MI. Renal blood flow was increased significantly by all 3 ACE inhibitors in MI (CA I0.I±2.0%, n=6; EN 10.3±2.4%, n=7); LI i0.8±3.6%, n=8), whereas in WKY only CA increased renal blood flow (12.5± 5.1%, n=7). Comparable results were seen for the decrease in renal resistance. Hindquarter and mesenteric flow were not affected. In summary, the ACE inhibitors have specific effects on renal blood fl0w in MI. Remarkably, a significant increase in renal flow is observed in HI at doses of ACE inhibitors which cause no decrease in MAP. This may suggest that formation of tissue ang II plays an important role in the regulation of renal flow in heart failure.

(HI) RATS H.J.M.G. Nelissen, J.F.M. Smits

Dept. of Pharmacology, University of Limburg, P.O. 616, 6200 MD Maastricht, The Netherlands

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189 CA~DIOy~SCULAR RESPONSESTO AMILORIDE ANALOGUESINHIBITING Na /Ca~ -EXCHANGE IN IS@~ATEDRAT TISSUES L.Brown, E.J.Cragoe, Jr ", S.Manley, and J.Bourke The positive inotropic and vasorelaxant responses+of amiloride analogues which selectively inhibit Na /Ca -exchange were measured in this study. The compoundstested were amiloride,+et~ylisopropylamiloride (EIPA, a potent antagonist of Na /H -exchange),+phenamil and 2',4'-dimethylbenzamil (DMB, bot~ potent Na channel antagonists with activity against Na /Ca -exchange) and 5-(N-4-chlorobenzyl)-+ 2'~}'-dimethylbenzamil (CBDMB, a potent antagonist of Na / CaZ~-exchange with reduced activity against Na~-channels). Cumulative concentration-response curves were measured in isolated right ventricular papillary muscles and thoracic aorta rings (intact or after mechanical removal of endothelium) from 12-16 week old female Wistar rats. A11 preparatiRns were bathed in Tyrode solution with 1.8 mM CaC19 at 35~C, gassed with 95% 09 and 5% C09. Papillary muscle~ were stimulated at I Hz while aortic ~reparations were contracted with I00 mM KCl;values are mean ± s.e.m. Phenamil, DMB and CBDMB increased the force of contraction of rat papillary muscles (neg. log EC5n: 4.77±0.06; 5.09±0.09; 4.97±0.17; maximal increase, 1.8±0.3 mN, 2.1±0.4 mN, 1.8± 0.3 mN; n=6 for each compound) comparedwith a maximal increase of 5.9±0.9 mN at 9mM CaCIg, while amiloride (300#M) and EIPA (30pM) gave small negative inotropic responses. A11 compoundscompletely relaxed endothelium-free thoracic aorta rings (neg. log EC~n: DMB, 5.30±O.06;EIPA, 4.99±0.11; CBDMB, 4.68~O.06;amilori~, 4.51±O.05;phenamil, 4.17±0.05; n=6 for each compound). Phenamil, CBDMBand DMB showed a shift to the l e f t of the concentration-response curves for relaxation of aortic rings in the presence of intact endothelium, while there was no endothelium-dependentshift of the concentration-response curves for EIPA o~ am~oride. These results i n d i c a t e that i n h i b i t i o n of Na /Ca~ -exchange leads to positive inotropic and endothelium-dependent vasorelaxant responses in isolated rat tissues. Department of Physiology and Pharmacology, University of Queensland, St. Lucia 4067, Australia; and *) 2211 Oak Terrace Dr., Lansdale, Pennsylvania 19446, USA.

191 Na/K.-ATPase AFFINITY OF STEROIDAL CARDIOTONICS AND THEIR POSITIVE INOTROPIC ACTION. Heinz Lallmann. Kay Peters and Joachim Pfeffer Experiments were performed to investigate the relationship between the affinities of some steroidal cardiotonics for the Na/K-ATPnse and their inotropic potencies. The affinities were determined by displacement of [aH]-ouabain from a membrane suspension of guinea pig heart ventricles. The inotropic effects were measured in electrically driven guinea pig left atria, exposed for 3h to the cardiotonic compounds. The compounds tested were classical cardiac glycosides (ouabain), genins (digitoxigenin, digitoxigenon), bis-guanylhydrazones of steroid-hormones (progesterone, hydrocortisone) and the newly synthesized hybride digitoxigenon-monoguanyl-hydrazone-nitrate. The inotropic effects induced by the concentrations occupying the Na/Ka) are summa: ized in the ATPase halfmaximaD IC~concentration pos. inotropy toxicity in % /-lydrocortisone1.8x10"SM 60 after 60 min. bisguanylhydrazone

T

Ouabain

1.3xl0-rM

70

after 90 min.

Digitoxigenln

4.6xl0-rM

70

after 120 rain.

Digitoxigenon

1.3xl0-rM

50

not toxic

Digitoxigenonguanyhhydrazone

1.5x10-TM= ICm

20

not toxic

3.0xl0-rM= 2xlC~

85

not toxic

Chlormadinonacetate- 3xl0-rM= ICr:e bisguanylhydrazone 1.5xl0-rM= 5xlCm

0

not toxic

80

not toxic

From the results the following conclusions are drawn: 1.) The affinities of steroidal eardiotonics for the Na/K-ATPase do not allow to predict inotropie potencies. 2.) Inspire of the replacement of a sugar moiety in Cs-f~-position by an equatorial, positively charged guanylhydrazone substituent the inotropic potency is maintained. This suggests that a great number of chemical variations of the genuine cardiac glycoside molecules are possible in the attempt to alter the pharmacological properties and shift them - with luck - into a favourable direction. Dept. of Pharmacology, Unlversitat Kiel, Hospitalstr.4, D-2300 Kiel

190

192

Na ÷ CONTROLS THE INOTROPIC EFFECTIVENESS OF D I F FERENT HOLDING POTENTIALS

M A G N E S I U M ENHANCES INOTROPISM AND ATTENUATES R I S E IN C O R O N A R Y T O N E C A U S E D B Y O U A B A I N (OUA) IN ISOLATED G U I N E A PIG H E A R T S . H.J.D6rin@, I.Schlicht, and V . H i l l e r Toxic concentrations of OUA are known to decrease myocardial contractile force and to induce coronary spasms. In the present study we investigated the influence of increasing extracellular Mg ÷÷ concentrations on myocardial contractility and coronary v a s c u l a r tone of O U A - p o i s o n e d isolated p e r f u s e d g u i n e a - p i g hearts. Coronary flow, isovolumetric LVP and p e r f u s i o n pressure (PP) were m e a s u r e d (KREBS solution with (mM) 2.5 Ca *÷ and 1.64 Mg+÷). As a measure of function the following curves were o b t a i n e d by means of computer controlled stepwise increases in PP (PP-range of 30 to 90 mmHg): 1 ) F R A N K - S T A R L I N G function curves (F-SFC) from d e v e l o p e d LVP, +dp/dtmax and -dp/dtmax, 2) coronary pressureflow curves (P-FC). Increasing Mg ÷÷ concentrations (0, 1.64, 3.28, 6.56, 16.4 mM) w i t h o u t OUA showed only slight elevations of F-SFC, but a strong increase in the slope of the P-FC's by 1904 at 16.4 mM Mg ÷÷ . OUA (1.4 x 10 -6 M) depressed F-SFC by 35 and 454 at 0 Mg ÷+ if LVP or +dp/dtmax were used for F-SFC construction. Moreover, -dp/dtmax d r o p p e d by 554. Increasing Mg ÷÷ concentrations g r a d u a l l y reversed this depressant effect so that finally a 35 to 65~ increase in F-SFC resulted, best seen in the -dp/dt~ax values. The M g + + - i n d u c e d increase of the slope of the P-FC's was less with OUA (max. 110%). Conclusion: Even in n o n - f a i l i n g hearts Mg ÷÷ detoxicates OUA resulting in more pronounced positive inotropic effects.

..................................................................................~.~.~.~_~..L...~.~..X~_~.~.~..._~.~.._~.~.~.~.~. If voltage-dependent Na-Ca exchange during diastole interferes with the subsequent contraction an effect of different resting membrane potentials on contraction should depend on extra- and intracelluler Na (Nao,i). This hypothesis was tested on myocytes from guinea-pig heart in the whole-cell clamp configuration. Contractions were elicited by depolarization to 0 mV at 0.5 Hz from different holding potentials. While a change of the holding potential was inotropically ineffective at Nao 140, Nal 5 mmol/l, with both the reduction of Nao to 70 mmol/l and increase of Nal by raising its concentration in the pipette or superfusion with ouabain at Neo 140 mmol/l a positive inotropic effect of depolarization of the resting membrane from -120 to -70 mV was observed. The Na dependence of the effect of the holding potential points to the control of Ca accumulation by Na-Ca exchange during diastole. When the m e m b r a n e is repolarized to the holding potential a transient inward tail current, carried in part by Ne-Ca exchange in the Ca extrusion mode, can be observed. The reported inotropic effect therefore could be due to reduced extrusion of that Ca which triggered contraction. The increase of the tail current in experimental situations with elevated intracellular Ca however excludes this suggestion. Therefore it is likely that the inotropic effect of depolarization of the resting m e m b r a n e originates from NaCa exchange which operates over the entire duration of the holding potential.

Institut fur P h a r m a k o l o g i e und Toxikologie der Technischen Universit~t M U n c h e n Biedersteinerstr.29, D-8000 M~ochen 40

Department of Physiology, U n i v e r s i t y of Freiburg, Hermann-Herder-Str. 7, D-7800 Freiburg

R 49 195

193 DUAL ,.~'.#~CI~ OF RESERPINE ON CARDIAC MYOFII~hILS. J.C. van Meel and W. Diederen

CALCIUM

SI~qSITIVITY

OF

Chronic treata~.nt with reserpine enhances inotropic responses to calcium in guinea pig papillary muscles. High doses of reserpine on the other hand are cardiotoxic. Sofar, no satisfying explanation has been presented to explain this phencmenon. We studied the effects of reserpine treatment on calcium sensitivity of chemically skinned right ventricular trabeculae. Guinea pigs (male, 500-600 g) were treated with reserpine (0.i mg/kg/day) during 7 days. Free running trabeculae were ch~mically skinned with Triton X-100 for 20-30 min at room temperature. Force-calcium curves were constructed at pH=7.0 at a ~ e length of 2.0-2.1 ~m. Fibers from reserpine treated animals were significantly more sensitive to calcium than those of control animals. Fibers from control animals elicited 3.3+3.9% tension at pCa=6.0 whereas fibers frc~ reserpine-treated animals elicited 13.3_+6.3% tension at pCa=6.0. Maximal tension development at pCA=4.25 was similar for both groups of fibers: 25.9+12.7 mN/~ for control animals and 28.8+10.0 mN/rma 2- for reserpin-treated animals. Acute administ/~tion of reserpine (10-6-10 `4 mol/l) to skinned fibers ooncentration-dependently reduced force. At 10 -4 mol/l, reserpine reduced tension at pCa=5.50 b y 85.5-+10.3% which was cc~oarable with a reduction of 47.0-+2.3% of maximal tension at pCa=4.25. In conclusion, acute treatment with high concentrations of reserpine decreases calcium sensitivity of cardiac myofibrils whereas chronic treatment with reserpine incalcium sensitivity of cardiac myofibrils. Dept. Pharmacology, Pharma Research, Dr. Karl Thomae GmbH, D-7950 Biberach i, FRG.

EFFECTS OF NOVEL Ca++SENSITIZING AND PHOSPHODIESTERASE (PDE) INHIBITING COMPOtrNDS ON C ~ I A C CON%~P~CTION AND HEI~XATION. M.J. Quiniou and Ph. I~houratate. The effects of five BRL compounds (I,II,III,IV,V) were compared with indolidan (IND) on isometric force of contraction (FC), relaxation time (RT) , myofibrillar Ca++dependent ATPase (MF-ATPase) and PDE activities. Chemical Structure: R1 R2 . R3 X R1 i -CH 3 -CH3 -CH 3 S ~IR2 R3 II - (CH2) 4 -CH 3 S O ~ %---- ~--X III - (CH2) 5-CH 3 S H / ~ - - ~ ~/ _ ~ N~_ ~ O~ IV -S-(CH2) 2-S-CH 3 S V -S- (CH2) 3-S-CH 3 S IND -CH 3 -CH 3 -H CH 2 The effects on FC and RT after cumulative addition of compounds were determined in electrically driven guinea-pig left atria. They were expressed as percent change, compared to the maximum effect of isoproterenol for FC and to the basal value for RT. The effects on MF-ATPase, at the concentration giving the maximum increase in FC (Cmax, ~M), were assessed on canine cardiac myofibrils by the shift of the pCa-MF-ATPase activity curve (npCa50) (Solaro and Ruegg, Circ. Res. 51:290, 1982). PDE inhibition (IC50, ~M) was assessed on canine cardiac sarcoplasmic reticulum k,gund-PDE (Kauffman et al., No1. Pharmacol. 30:609, 1986). (means, n =3-4)

I I I00 III 330 II 30 | IV 30 10 I IND 00

+75.1" +63.8* +58.3* +51.0" +32.7* +34.6*

- 5.4 +21.1 +39.7* +77.9* +54.2* - 5.4

+0.24* +0.35* +0.39* +0.57* +0.65* -0.04#

*: p < O . 0 5 (Student's paired t-test); #: studied at 200 ~M. The relative contribution of PDE inhibition and MF-ATPase Ca++sensitization to cardiotonic activity cannot be determined from these results. For compounds I to V, the Ca ++sensitization of MF-ATPase is correlated with the impairement of in vitro cardiac relaxation (r=0.883, P=0.048). That casts doubt on the value of inotropic agents possessing a myofibrillar Ca++sensitizing effect. Les Laboratoires Beecham, BP 45, 35760 Saint-Gr~goire, France

194

196

SENSITIZATION OF MYOFILAMENTS TO CA 2+ ACTIVATION AND PHOSPHODIESTERASE INHIBITION PRODUCE POSITIVE INOTROPY OF PIMOBENDAN IN THE HUMAN HEART M.B6hm, M.Wankerl, I.Morano*, J.C. R~egg*, R.Zimmermann and E.Erdmann In the failing human heart, there is a loss of myocardial ~-adrenoceptors with a concomitant reduction of the positive inotropic effect (PIE) of agents that increase the celluar cAMP levels. Therefore, additional Ca z* sensitizing properties on the contractile proteins could be useful to increase force of contraction (FOC) under these conditions. The effects of pimobendan (PIMO) and its demethylated metabolite UDCG 212 CL (UDCG) on FOC, on the activity of the cAMP-phosphodiesterase isoenzyms (PDE IIll) and on the Ca2+-force relationship of skinned myocardial fibers (SF) were measured in human myocardium. In nonfailing myocardium (NF), PIHO produced a PIE which was more pronounced than the PIE of PIMO in preparations from patients with moderate (NYHA II-III) or severe (NYHA IV) heart failure. UDCG alone failed to increase FOC in NYHA II-III or NYHA IV. In the presence of isoprenaline (0.03 pmol/l), the PIE of PIMO was more potent than with PIMO alone and a PIE of UDCG was observed . The inhibition of PDE III with UDCG (mean E C B 0 : 0 . 2 2 ~mol/l) was more potent than with PIMO (mean ECs0: 1.7 pmol/l). In SF, PIMO (I00 ~mol/l) shifted the Ca2÷-force relationship to the left by 0.15 pCa units. In contrast, UDCG (I00 pmol/l) desensitized SF by 0.Ii pCa units. It is concluded that both, inhibition of PDE III and sensitization of myofilaments to Ca 2+ are involved in the PIE of PIMO in the human heart. The failure of UDCG to increase FOC is likely due to a desensitiziation of the myofilaments to Ca z÷ because the PDE III was even more potently inhibited by UDCG than by PIMO.

EFFECTS OF IBMX, PIMOBENDAN AND SULMAZOLE CYCLASE ACTIVITY IN GUINEA-PIG HEARTS T. Dessauer, M. Nose and W. Schmitz

Hedizinische Klinik I der Universit~t MQnchen, Klinikum Gro~hadern, Marchioninistr. 15, D-S000 M~nchen 70, FRG * II.Physiologisches Institut der Universit~t Heidelberg.

0.32 1.1 1.3 0.35 0.26 0.65

ON ADENYLATE

The positive inotropie drugs 3-isobutyl-l-methylxanthine (IBMX) and sulmazole (SUL) have been shown to stimulate adenylate cyclase activity (ACA) in rat fat cell membranes (Parsons et al., Mol Pharmacol 33: 441, 1988; Mol Pharmacol 34: 37, 1988) To investigate whether this effect also occurs in cardiac preparations we studied the effects of IBMX, pimobendan (PIMO) and SUL on ACA in ventricular preparations from guinea-pig hearts with (PTX; n=8-9) and without (NPTX; n=8-9) pretreatment with pertussis toxin (180 ~g/kg i.v.; 24 hrs), after complete inhibition of phosphodiesterase activity with papaverine (300 ~nol/l). In NPTX pIMO (0.I-i00 ~mol/l) caused only a 23% increase of ACA. However, the effect of PIMO revealed no concentration dependency. IBMX increased ACA slightly at the high concentration of I00 ~mol/l (25% increase). In PTX, the increases of ACA by PIMO and IBMX were attenuated. SUL did not at all change ACA neither in PTX nor in NPTX. ~ the presence of the A -adenosine receptor 1 agonlst (-)-N -phenylisopropyladenosine (R-PIA) PIMO did not increase ACA neither in PTX nor in NPTX. IBMX caused a small increase of ACA in NPTX (21% over basal activity). No effects on ACA were found in the presence of R-PIA and SUL neither in NPTX nor in PTX. It is concluded that IBMX or PIMO stimulates cardiac ACA slightly at high concentrations only. Since the effects were attenuated by pretreatment with pertussis toxin, they are probably due to blockade of the G.-protein. It 1 is unlikely that these small effects contribute considerably to the positive inotropic action of these cardiotonic agents. (Supported by the DFG.) Abteilung Allgemeine Pharmakologie, Universit~ts-Krankenhaus Eppendorf, MartinistraBe 52, 2000 Hamburg 20, FRG

R 50 197 EFFECTS OF TRAPIDIL ON FORCE OF CONTRACTION, BEATING FREQUENCY AND PHOSPHODIESTERASE ACTIVITY IN GUINEA-PIG HEARTS Th. Bethke, H. Mehl, W. Meyer K. Thomas and H. Wenzlaff

199

Trapidil, a triazolopyrimidine with cardiotonic and vasodilating properties, has been shown to inhibit the cAMP phosphodiesterase activity from crude preparations of ventricular cardiac tissue. To further elucidate the mechanism of action of trapidil we investigated its effects on isometric force of contraction (FC, electrically driven papillary muscles, 1 Hz), beating frequency (BF, right auricles) and on phosphodiesterase (PDE) isoenzyme I-IV activities in guinea-pig hearts. For comparison theophylline, IBMX and milrinone were studied. Trapidil increased FC concentration-dependently (1000 3000 ~mol/l). The positive inotropic effect (PIE) was maximal at 3000 vmol/l with an increase in FC by 73.7 + 12.1% (n = 8). The EC.^u was 562.4 (204.5 - 1546.3) ~ o i / i . BF was increase~ maximally by 13% at 300 Bmol/l trapidil. Trapidil concentration-dependently (30 i0 000 Lumol/l) but nonselectively inhibited the activities of cAMP PDE I-IV (mean IC~0-values: 422.0, 227.4, 250.1 and 191.9 LLmol/I respectively). These effects were similar to those obtained with theophylline and IBMX. Compared to the ICsN-values of trapidil, theophylline was 1.7, IBMX 49 and miIrinone ll4fold more potent in inhibiting PDE III. In conclusion, trapidil is a nonselective PDE inhibitor like theophylline and IBMX. The inhibition of cAMPPDE III by trapidil, theophylline, IBMX or milrinone probably contributes to their PIE to a major extent. However, as the nonselective PDE inhibitors are positive inotropes, a selective inhibition of PDE III does not seem to be essential in increasing FC.

SODIUM PUMP INHIBITION AND POSITIVE INOTROPIC EFFECT OF SULMAZOLE IN GUINEA-PIG VENTRICULAR MYOCARDIUM R. Schmied, G.X. Wang, M. Korth .......................................................... Inhibition of Na+,K+-ATPase, inhibition of phoshodiesterase and increase in Ca z+ sensitivity of myofibrils have been proposed as mechanisms for the positive inotropic effect of the imidazopyridine sulmazole. In the present study, the contribution of Na*-K + pump inhibition to the positive inotropic effect of (±)-sulmazole and its stereoisomers was studied by means of Na+-sensitive microelectrodes. In the resting papillary muscle, (+)-sulmazole increased intracellular Na ÷ activity (at~a) within 15 to 20 min by 0.5+0.1 (n=3), 1.3+0.1 (n=7), 2.7+0.2 (n=6) and 4.9+0.5 mM (n=6) at 60, 1 0 0 7 300 and 1000-zM, respectivelyq (+)Sulmazole was more effective than the racemat, at,a was increased by 1.2+0.3, 2.1+0.3 and 4.0+0.2 mM at 60, I00 and 300 ~M, respectively In=2 for each-concentration). In the isometrically contracting papillary muscle (0.2 Hz), (+)- and (+)-sulmazole (600 and I000 ,M) produced a maximum positive inotropic effect which exceeded that of the cardioactive steroid dihydroouabain (DHO) by 8 and 21%, respectively. As an inotropic agent, (+)-sulmazole was almost twice as potent as the racemat. The maximum direct inotropic effect of (-)-sulmazole {I000 ~M) amounted to only 24% of the DH0 maximum and was, in contrast to the racemat and (+)-sulmazole, antagonized by 3 ~M carbachol.(-)-Sulmazole up to 300 BM did not affect at~a. DR0 increased al,a by 0.9+0.2 (n=3), 1.5+0.1 (n=7), 2.2+0.1 (n=2), 2.8+0.2 (n=4) and 4.1 mM (n=l) -at 30, 50, 80,-i00 and 120 ~-M, respectively. The increase in aIN, versus the positive inotropic effect of various concentrations of (±)-sulmazole, (+)-sulmazole and DHO could be fitted by linear regression (r=0.981). The results of the present study indicate that the rise in aINa due to Na+-K ÷ pump inhibition was the determinant mechanism of the positive inotropic effect of the ~midazopyridlnes (~)- and (+)-sulmazole. Inhibition of pho§phodiesterase was presumedly responsible for the inotropic effect of (-)-sulmazole.

Abteilung Allgemeine Pharmakologie, UniversitBtsKrankenhaus Eppendorf, Universit~t Hamburg, MartinistraBe 52, D-2000 Hamburg 20, FRG.

Institut f~r Pharmakologie und Univ. M~nchen, Biedersteinerstr. F.R.G.

198

20O BDF 9148: A NOVEL INOTROPIC AGENT THAT TRANSIENTLY PROLONGS CARDIAC ACTION POTENTIAL DURATION B.I. Armah, R. BrGckner, W. Stenzel

THE

cAMP

SPECIFIC ROLIPRAM INHIBITED PDE (ROI-PDE) IS IMPORTANT FOR THE INTRACELLULAR cAMP LEVEL IN MYOCYTES M. Klockow In contrast to cGMP inhibited PDE III (CGI-PDE), which is mostly bound to the plasma membrane, a large part of the Rolipram inhibited PDE (ROI-PDE) is firmly bound to the myofibrils of myocytes (NSAP 1989; 339: R 53). Specific inhibitors of ROI-PDE (Rolipram or Ro 20-1724") as well as CGI-PDE (CI 930**) increase the effect of isoprenaline on the cAMP level in isolated myocytes of rat (B) and guinea pig but exhibit only a small influence in the absence of isoprenaline. However, simnltaneous inhibition of both enzymes raises the intracellular cAMP even in the absence of isoprenaline (A). These results agree with the finding of C.D.Nicholson (NSAP 1989; 339: R 53) that only simultaneous inhibition of CGI-PDE and ROI-PDE display a positive inotropic activity on rat heart. ROI-PDE bound to myofibrils may have an important function (J.S. Hayes, K.L. Bnmton, J. Cycl. Nucleot. Res. 1982;8:1) in the heart cell. • 4-(3 -Butoxy-4-methoxybenzyl)-2-imidaznlidinone • *6-(4-Imidazolophenyl)-5-methyl-2,3,4,5-tetrahydropyridazin-3-on pmol cAMP .8

(A)

10•MCI

~mol cAMP 10 ~a2¢iCI 9 3 0 , 4+ Ro

930 + Ro 20-1724

3" ~ g ) ~ 1 7 2 4

.4 Ro 20-1724 buffer 0

CI 930 c~ buffer

1

3

10

0

.1

c1930 .3

1

V-M Pharmaceutical Research, E. Merck,D-6100 Darmstadt, FRO

3

10 vM

Toxikologie 29, D-H000

der Teehn. M~nchen 40,

Prolongation of inactivation of cardiac Na+channels has been described as the mechanism of action of naturally occuring and synthetic agents like DPI 201-106 (DPI) (Buggisch et al.: Eur. J. Pharmacol.(1985) 118, 303). Delay of inactivation of Na+channels results in prolongation of cardiac action potential duration (APD), and increases QT duration in the ECG. Thus DPI is characterized by prolongation of QT duration in man (R~egg, P.C. and E. N~esch: Hr. J. Clin. Pharmac. (1987) 24, 453). This report presents a new cardiotonic agent, BDF 9148, that transiently prolongs APD without affecting QT duration and QT (Bazett) in anesthetized animals. BDF 91~8, 4L3-(1-diphenylmethyl-azetidine-3-oxy)-2-hydroxy-propoxz7-1H-indol-2-carbenitril, is a chemical analogue to DPI, 4[3-(1-diphenylmethyl-piperazinyl)-2hydroxy-propox~-IH-indol-2-carbenitril), but pharmacologically different from the latter. In the guinea pig papillary muscle (stimulation at I Hz), BDF 9148 increased the force of contraction in a concentration-dependent fashion (0.1 -30 ~mol/l) with an IC50 of 0.6 Lunol/l. Treatment with propranolol, prazosln or earbacho! did not influence the inotropic effect, but tetrodotoxin com+ pletely abolished it, thus implying Na channel involvement. BDF 9148 did not influence Na+/K+ATPase activity, nor did it elevate intracellular cAMP content of myocardial tissue. Inotropic concentrations of BDF 9148 affected APD in a biphasic fashion; an initial prolongation was followed by a reversal of the prolongation, which was paralleled by a shortening of the isometric contraction curve. In contrast, DPI irreversibly prolonged APD and total contraction time. Dept. of Pharmacology, D-2000 Hamburg 20

BDP-Beiersdorf AG, Unnastr. 48

R51 201

203

POSITIVE INOTROPIC BUT NO CHRONOTROPIC EFFECT OF BDF 9148 IN GUINEA-PIG ATRIA. H. Brasch BDF 9148, systematic name 4[-l-Diphenylmethyl-azetidine3-oxy)-2-hydroxy-propoxy]-l-H-indole-2-earbonitrile, is structurally similar to the sodium chan~el activator DPI 201-106. In concentrations up to 3 x 15-~ mol/l, BDF 9148 did not change the frequency of spontaneously beating right atria (3~° C; 1.8 mmol~l Ca2+). In left atria and papillary muscles stimulated at a rate of I Hz, the drug increased the force of contraction three- to fourfold. From cumulativ~ concentration-response curves EC~n values of 1.32 x i0-I and 0.70 x i0-0 mol/l were calculated for atria and p~pillary muscles, respectively. With a single dose of I0-° mol/l BDF 9148,the inotropic effect in atria reached its maximum after 20 - 30 min and was accompanied by a slight lengthening of the time to peak force of the isometric contraction curve (from 53 + 1.7 to 64 + 2.0 ms) and a significant lengthening of th~relaxation--time (from 85 + 5.6 to 139 +7.4 ms). Simultaneously, the functional?efractory perTod, as measured by stimulation with twin pulses of equal strength, was increased from 120 + 7.3 to 197 + 14 ms. BDF 9148 accumulated in the atrium. After 30 mTn of incubation with 10- mol/l, the mean tissue concentration was 1.88 x I0-~ mol/kg, i.e. 18.8- fold the concentration in the organ bath. Repeated washing over a washout period of 4 h neither reduced the tissue concentration: nor the effect of the drug. Propranolol, 4 x I0-° mol/l, given 30 min before BDF 9148, did not modify the positive inotropic effect of the latter, but attenuated the accompanying increase of the relaxation time and the functional refractory period. All effects of BDF 9148 were promptly ^reversed by 3 x i0-n mol/l tetrodotoxin. 3 x i0-~ BDF 9148, a concentration that caused only a small inotropic effect by itself, shifted the concentration-response curve of ouabain in atria to the left, causing7a decrease of th~ EC~n of the glycoside from 3.21 x I0- to 2.00 x lOmoITl. These results are compatible with the suggestion that BDF 9148 exerts its positive inotropic effect by prolonging the open state of the sodium channel, and thus increasing the sodium influx during the action potential.

EFFECTS OF A NEW INDOL-2-CARBONITRILE, BDF 9148, AND ITS TWO ENANTIOMERS ON SODIUMCURRENT IN R A T H E A R T C E L L S P, Honerj~ger~ M. Dugas, and G. Wang

The effects of BDF 9148" (= azetidinoxy-for-piperazine analog of DPI 201-I06) and its enantiomers S-BDF and R-BDF on sodium current (INa) were studied in cultured late-fetal rat ventricular myoeytes using the whole-cell configuration of the patch-clamp technique (20°C; 70 mM (Na)o). During pulses from -I00 to -30 mV (50 ms; 0.2 Hz) BDF 9148 acted biphasieally on INa: at 1 and 3 ,M, peak INa showed little change but the rate of inactivation was slowed down; at I0 ,M, peak INa was reduced, and the rate of inactivation, slowed down by lower concentrations, was accelerated. S-BDF (I and 3 ~M) increased peak INa and slowed down the rate of inactivation, whereas R-BDF (I and 3 ~M) reduced peak INa with little effect on inactivation. The slowly inactivating INa (3 ~M BDF 9148) recorded during a l-s depolarization was described by the sum of two exponentials; a slow phase with time constant 0.47 s (n=3) accounted for 90 % of the decay. The current induced by 3 ~M DPI 201-106 decayed menoexponentially with time constant 0.40 s (n=2). Following prolongation of a pulse to 1 s in a train of 50-ms pulses (0.2 Hz), peak INa was transiently suppressed in the presence of 3 ~M BDF 9148, but remained irreversibly reduced with 3 ,M DPI 201-106.

Institut fur Pharmakologie der Medizinischen Universit~t zu L~beck, Ratzeburger Allee 160, D-2400 L~beck

In conclusion, BDF 9148 was found to resemble DPI 201-106 in many respects including biphasic action on INa, and slowing of inactivation and reduction of peak INa being due to the S- and R-enantiomer, respectively. We propose that the blocking kinetics of the R-enantiemers differ, however, in that R-DPI binds irreversibly to S-DPI-modified sodium channels, whereas R-BDF dissociates within seconds from those modified by S-BDF. (Supp. by DFG and Sandoz-Stiftung f. therap. Forschung.) *4-{3'-(l-benzhydryl-azetidin-3-oxy)-2'-hydroxypropoxy}IH-indol-2-carbonitrile Institut fHr Pharmakologie und Toxikologie der TU MHnchen Biedersteiner Str. 29, D-8000 M~nchen 40, F.R.G.

2O2

204

EFFECTS OF BDF 9148 ON SODIUM- AND CALCIUM-CURRENTS IN ISOLATED GUINEA-PIG XYOCYTES Thomas Pfeifer and Ursula Havens ..........................................................

POSITIVE IN(TfROPIC EFFECTS OF BDF 9148 IN ANESTHETIZED DOGS IS NOT ASSOCIAY~D WITH PROLONGATION OF Q-T INTERVAL D. Muster, A. Ramp

BDF 9148 (4-[3'-(l-benzhydryl-azetidin-3-oxy)-2'-hydroxypropoxy]-IH-indole-2-carbonitrile) differs from the positive inotropic agent DPI 201-106 (4-[3'-(l-benzhydrylpiperazinyl)-2'-hydroxy-propoxy]-IH-indole-2-carbonitrile) by an azetidin-3-oxy- instead of a piperazinyl-moiety. We have studied this new compound for its effects on excitation-contraction coupling in isolated ventricular myocytes with the whole-cell patch clamp technique. Action potential duration (APD) and cell-shortening of myocytes stimulated at a frequency of 0.2 Hz at 35°C were observed under control conditions and during 7 min of drug action. 1 ~M BDF 9148 prolonged APDzo from 188±21 to 201±17 ms and APD~0 from 387±31 to 409±36 ms. Contractility increased to 123~16 % of control-values within 7 min (n=5). In contrast, 10 ,M BDF 9148 shortened APUzo from 212±28 to 146±8 ms, but prolonged the APDgo from 337±11 to 401±49 ms. Contraction amplitude was reduced to 51±2 % of the control (n=5). - Membrane currents were studied at room temperature. In the voltage clamp mode, depolarizing pulses of 300 ms duration at a frequency of 0.2 gz were applied. With clamp steps from -80 to -40 mV a slowly inactivating inward current occurred, which was completely suppressed by tetrodotoxin (30 pM) and is therefore regarded as a modified sodium current (iNa). Control calcium currents (ica) measured with clamp pulses from -40 to 0 mV declined spontaneously by 13±6% (n=6) during a time period of 15 min. 1 ~M BDF 9148 added after a control period of 8 min further decreased ica. In conclusion, enhancement of APD and contractility by low concentrations of BDF 9148 are produced by modification of iNa, whereas high concentrations have an additional inhibitory effect on calcium currents, thereby shortening the APDzo and decreasing contractility. (DFG Ra222/6-2) .......................................................... Institut f~r Pharmakologie am Klinikum der Universit~tGHS-Essen, Hufelandstra~e 55, 4300 Essen 1

Prolongation of cardiac action potential duration is characterized by a lengthening of Q-T interval in the ECG in animals as in man. The Q-T interval of the ECG represents the duration of electrical activity in the ventricle. Many factors can shorten or lengthen the corrected Q-T interval (Q-T). The Q-T is shortened by digitalis and • c c hypercalclaemla, but lengthened by agents like amiedarone, quinidine, procainamide or the inotropic agent DPI 201-106 (DPI) (R~egg, P.C. et al: Br. J. Clin. Pharmac. (1987) 24, 453). While shortened Q-T is without serious consequences a long Q-T is associated with ventricular f.ibrlllatlon. . • ' Thus for posltlve c . . .lnotroplc . agents that affect the Na+ channel in similar fashion as DPI (4~3-(1diphenylmethyl-piperazinyl)-2-hydroxy-propox~7-1-H-indol2-carhonitril) the clinical value c a n b e limitedby lengthening of Q-T in man. We investigated the influence of BDF 9148 (4~-(1-diphenylmethyl-azetidine-3-oxy)-2hydroxy-propoxz7 IH-indol-2-carbonitril) on left-ventricular contraction, heart rate, total peripheral resistance (TPR) Q-T and Q-T in anesthetized dogs in comparison with DPI. Doses of ~.I, 0.3, I and 3 mg/kg i.v. gave eql/ieffective increases in LVdp/dt max, thus enabling an objective assessment of the other parameters. In contrast to DPI which lowered heart rate and TPR, BDF 9148 did not affect heart rate nor TPR. In regard to Q-T and Q-T there was a dramatic difference between DPI and BDF? While DPI dose-dependently prolonged Q-T and Q-Tcf BDF had no influence on Q-T nor on Q-T . The increase in cardiac contraction induced by BDF 914[ was accompanied by a marked increase of coronary perfusion. Thus BDF represents a greatly improved profile for a cardiotonic agent. Dept. of Pharmacology, BDF-Beiersdorf AG, Unnastr. 48 D-2000 Hamburg 20

R 52 205

207

EFFECTIVENESS OF Na+-CHANNEL ACTIVATORS IN FAILING HUMAN MYOCARDIUM R. Schwinger

COMPARISON OF THE ARRHYTHMOGENIC AND QUINDINE

Increasing degrees of heart failure are accompanied by progressive reductions of the effectiveness of cAMPdependent positive inotropic agents. Na+-channel activators mediate cAMP-independent positive inotropic effects (PIE) and would, therefore, be still effective in the failing human heart. We investigated the increase in force of contraction (FOC) elicited by BDF 9148 (BDF, 4(3'-(l-benzhydryl-azetidin-3-oxy) -2'-hydroxypropoxy)IH-indol-2-carbonitrile) and DPI 201-106 (DPI, 4-(3-(4diphenylmethylpiperazine-l-yl) -2-hydroxypropnxy)- IHindole-2-carhonitrile) in electrically driven (i Hz, 370C) auricular traheculae (AUT, aortocoronary bypass operation, n=12-15) and in papillary muscle strips from moderately (NYHA II-III, mitral valve replacement, n=712) and terminally (NYEA IV, heart transplantation, n=716) failing hearts. The PIE were studied with cumulative concentration-response curves (BDF, 0.01-100 pmol/l; DPI, 0.1-3 pmol/l). The PIE of Ca 2÷ (1.8-15 mmol/l) and isoprenaline ( I S O , 0.001-I pmol/l) were examined for comparison. AUT PAP: NYHA II-III NYHA IV max.PlE(mN) max.PlE{mN) max.PlE(mN) BDF +3.2±1.0 +1.6±0.2 p
In o r d e r to g a i n i n f o r m a t i o n a b o u t t h e p r o a r r h y t h m i c a c t i o n of L i d o c a l n e (LID) a n d q u i n i d i n e (QUI) r a b b i t h e a r t s , p e r ± u s e d a c c o r d i n g to t h e L a n g e n d o r f f - t e c h n i q u e , were t r e a t e d w i t h e i t h e r LID (2, 5, 10 Bmol/1) or QUI (1, 5, 8 lamol/1). T h e s e c o n c e n t r a t i o n s c o r r e s p o n d to low, middel a n d h i g h t h e r a p e u t i c p l a s m a l e v e l s . E p i c a r d i a l m a p p i n g (256 v e n t r i c u l a r A g C l - e l e c t r o d e s , 1ram s p a t i a l , 0.25 ms t e m p o r a l r e s o l u t i o n ) a n d v e e t o r a n a l y s l s of t h e e p i c a r d i a l a c t i v a t i o n p r o c e s s were u s e d to i n v e s t i g a t e t h e a c t i o n of QUI a n d LID. D i r e c t i o n a n d v e l o c i t y of t h e e p i c a r d l a l a c t i v a t i o n w e r e e x p r e s s e d in t e r m s of v e c t o r g e o m e t r y . H e a r t b e a t s under treatment and under control conditions were c o m p a r e d a n d t h e p e r c e n t a g e of s i m i l a r v e c t o r s ( d e v i a t i o n < 5*) c o m p u t e d . In a d d i t i o n , o r i g i n s of e p l c a r d i a l e x c i t a t i o n (=breakthroughpoints 'BTP') were determined and the p e r c e n t a g e of BTP w i t h u n c h a n g e d l o c a t i o n of two h e a r t beats was calculated. Furthermore epicardial potential d u r a t i o n ( R T - t i m e ) a n d t o t a l a c t i v a t i o n t i m e s (TAT) w e r e m e a s u r e d . O n l y t h e h i g h e s t c o n c e n t r a t i o n of LID led to a loss of v e c t o r s i m i l a r i t y w h i l e s i m i l a r i t y of BTP w a s o n l y s l i g h t l y r e d u c e d . R T - t i m e r e m a i n e d u n c h a n g e d , TAT w a s slightly prolonged and apparent epicardial conduction v e l o c i t i e s w e r e d e c r e a s e d b y a b o u t 20%. QUI s t r o n g l y r e d u c e d v e c t o r s i m i l a r i t y in all c o n c e n t r a t i o n s , b u t did n o t a f f e c t B T P - s i m i l a r i t y . The e f f e c t of QUI on TAT w a s c o n s i d e r a b l y g r e a t e r t h a n t h a t of LID, as w a s t h e d e c r e a s e of a p p a r e n t e p i c a r d i a l v e l o c i t i e s . In c o n t r a s t to LID, QUI s i g n i f i c a n t l y p r o l o n g e d R T - t i m e b y 9, 15 a n d 25 % r e s p e c t i v e l y . From t h e s e r e s u l t s t h e a u t h o r s c o n c l u d e , t h a t LID h a s a l o w e r a r r h y t h m o g e n i c r i s k in n o r m a l h e a r t s , as c o m p a r e d to QUI b e c a u s e of i t s s t r o n g e r disturbing i n f l u e n c e on e p i c a r d l a l a c t i v a t i o n .

A. Mfiller and

p vs~ NYNA IV A d e n o s i n e as w e l l a s c a r h a c h o l d i d n o t a f f e c t t h e PIE elicited by BDF or DPI but reduced the PIE of ISO. It is concluded that BDF and DPI increase cAMP-independently FOC in human myocardium. The PIEs of BDF and DPI were more pronounced in NYHA IV compared to NYHA II-TII. In the same hearts, the PIE of ISO was reduced in NYHA IV. Na*-channel activators keep their positive inotropic effectiveness in failing human hearts with reduced responsiveness to K-adrenoceptor agonists. Medizinische Klinik I der Universit~t Mfinchen, Klinikum Grosshadern, Narchioninistr. 15, D-8000 Nfinchen 70

BLOCK

INDUCED

J. W e i r i c h u n d H. A n t o n i ................................................. The f r e q u e n c y - d e p e n d e n t b l o c k a d e of fast sodium current induced by c l a s s - I - a n t i a r r h y t h m i c drugs (I-aa) s a t u r a t e s w i t h i n c r e a s i n g s t i m u l a t i o n frequencies (Weirich and Antoni NaunynSchmiedeberg's Arch Pharmacol 340:456, 1989). Thus, the s a t u r a t i o n b e h a v i o u r of b l o c k induced by 12 d i f f e r e n t I-aa was a n a n l y s e d and q u a n t i t a t i v e l y c o m p a r e d at an equipotent concentration (50~ r e d u c t i o n of the m a x i m a l u p s t r o k e v e l o c i t y of a c t i o n p o t e n t i a l s at 3.3 Hz). M a r k e d d i f f e r e n c e s in the saturation behaviour were found even b e t w e e n s u b s t a n c e s of the same s u b c l a s s (Ia bzw. Ic), as can be seen from the ranking with respect to the f r e q u e n c y at w h i c h h a l f - m a x i m a l s a t u r a t i o n occurs (Gr.= group, r = onset-rates (AP -1) of f r e q u e n c y - d e p e n d e n t b l o c k of each g r o u p at 3.3 Hz) : lidocaine(Ib), mexiletine(Ib), t o c a i n i d e (Ib).

Gr.2 (1.0-1.5Hz): q u i n i d i n e ( I a ) , l o r c a i n i d e ( I c ) , ( r = 0 . 0 2 - 0 . 0 7 A P -I) f l e c a i n i d e ( I c ) , e n c a i n i d e ( I c ) , procainamide(Ia). Gr.3 (0.2-0.3Hz): (r=0.02-0.1 AP-*)

S. Dhein

I n s t i t u t ±fir P h a r m a k o l o g i e d e r U n i v e r s i t i i t zu K61n, G l e u e l e r Str. 24, D - 5 0 0 0 KSln 41

208

206 SATURATION OF FREQUENCY-DEPENDENT BY CLASS-I-ANTIARRHYTHMIC DRUGS.

Gr.1 (2.1-2.7Hz): (r=0.3-0.6 AP -I)

RISK OF LIDOCAINE

pra~maline(Ic),nicainoprol(Ic), ethmozine(I?),disopyramide(Ia), propafenone(Ic)

From both, s a t u r a t i o n b e h a v i o u r and onset-rates of f r e q u e n c y - d e p e n d e n t block, d i f f e r e n t pro- and a n t i a r r y t h m i c p o t e n c y of these three groups may be inferred. Physiologisches Institut H e r m a n n - H e r d e r - S t r . 7, D - 7 8 0 0

der Universit~t, F r e i b u r g i. B r . , F R G

USE- AND VOLTAGE-DEPENDENCE OF LIGNOCAINE- AND QUINIDINE EFFECTS ON THE THRESHOLDS OF EXCITABILITY AND ACARRHYTHMIA Monika Adebahr, U. B6tel and A. Mescheder The excitation-threshold for rectangular pulses, the arrhythmia-threshold for alternating current and the force of contraction were determined in parallel in guinea-pig left atria paced at 3Hz. The concentration-response-curves were evaluated for lignocaine and qulnidine. The concentrations required for the elevation of the thresholds by 50% and for the reduction of the contractile force by 50% are indicated below.

quinidine fignocaine

ED m rect.pulse-excitation ac-arrhythmia 34~¢1 13~,t 2001~M 21pM

ICm contr.force -60~ 200~M

At 3Hz the membrane potential was lowered by raising the K+-concentration of the organ-bath from 2.7mM to 5.4raM, 8.1raM and 10.7raM. In another set of experiments the driving frequency was varied (IHz, 2Hz, 3Hz and 4Hz) at a constant K+-concentration of 2.7raM. Under drug-free conditions the arrhythmia-threshold was not altered by the K+-induced partial depolarization, whereas the excitationthreshold for rectangular pulses and the contractile force were decreased. The variation of the pacing frequency had no effect on the electrical parameters, the force of contraction was reduced at 1Hz. The effects of the drugs on the excitation-threshold were markedly augmented with increasing [K+]. The drug-effects on the arrhythmiathreshold were also elevated. With increased pacing rate, the drug-effects were similarly augmented, but to a lesser extent. In conclusion, both llgnocaine and quinldine depressed arrhythmia induced by alternating current with higher potency than the regular excitation induced by rectangular pulses. The use- and the voltagedependence of these actions were similar for the two drugs. Abt. Pbarmakologie, Universit~tt Kiel, Hospitalstr. 4, 2300 Kiel 1

R 53 209 COMPARISON OF THE EFFECTS OF RACEMIC SOTALOL AND OF THE (+) AND 6) ENANTIOMERS ON THE CARDIAC ELECTRICAL ACTMTY U. Stark, A. Lueger, S. Nagl and H.A. Tritthart Beta-blocking agents are very useful for the treatment of cardiac arrhythmias caused or exaggerated by sympathetic overdrive, e.g. sinus tachycardia, atrial and ventricular extrasystolie. The g-blocker sotalol is reported to prolong the action potential and the effective refractory period of the ventricular myocardinm, this effect is likely to be additionally effective against tachyarrhythmias.We evaluated the effects of (-+)-, of (+)- and of (-)-sotalol on the cardiac electrical activity in isolated Langendorff peffused guinea pig hearts using a new surface and stimulation ECG method (Stark et al.J Pharm Meth 21,195-209,1989). Drugs were applied in cumulatively increasing concentrations (0.1-100uM) for 15 mln periods. Following the application of the highest concentration the effective refractory periods (ERP) of each part of the conduction system as well as of the atrial and ventricular myocardium were evaluated by stepwise increasing pacing rate (ERPc) as well as by pacing with premature beats (ERPe). The AV-nodal conduction time was most prolonged by 100uM (-)-sotalol to 152-+14% (n=6,p<0.01) and less prolonged by (+)- and (-+)-sotalol to 114-+6% (n =6,p < 0.05) and 117_+4% (n =7,p< 0.05), respectively. The sinus rate was most reduced by the (+)enantiomer (to 63-+4%, n=6, p<0.01, 100uM) and was not significantly affected by (-)-sotaloi. The HV-interval was not influenced by either compound. During stimulation with increasing pacing rate ¥-ERPc and HisERPc were significantly prolonged by (-+)- and by (+)-sotalol, the latter beeing more effective in the His-bundle. (-)-Sotalol had only weak prolonging effects of borderline significance on V-ERPc and His-ERPc but bad an additional prolonging effect on the AV-ERPc (+ 18±5%,n=6,p<0.05). V-ERPe, evaluated by stimulation with premature beats, was prolonged by (-+)-sotalol by 30-+4% (n=7,p<0.01) and by (+)-sotalol by 28-+3% (n=6,p<0.01). These prolongations were significantly (p<0.01) more pronounced than that found with increasing pacing rate. (-)-Sotalol had no significant effect on refractoriness evaluated by premature beats, (-+)- and (+)-Sotalol significantly prolonged His-ERPe, AVERPe and also the ERPe of the sino-atrial conduction. These results show, that (-+)- and (+)-sotalol predominantly reduce sinus rate and prolong the ERPe of sino-atrial, AV-nodal and ventricular conduction and thus, reduce the likelyhood of extrasystolie. (-)-Sotalol has prominent inhibitory effects on the AV nodal activity only and is unlikely to be effective in the prevention of ventricular extrasystolie by prolongation of the refractoriness of the myocardium.

211 EFFECTS OF NICORANDIL, A NEW ANTIANGINAL AGENT, ON REPOLARIZING CURRENTSIN RAT VENTRICULAR MYOCYTES U. Borchard*, T. Weis, F. Berger, D. Hafner Enzymatically isolated rat v e n t r i c u l a r myocytes were analyzed with the whole-cell voltage-clamp technique. Myocytes had resting potentials of about -70 mV, which were not affected by 30 ~mol/l Nicorandil. Action pot e n t i a l duration, measured at r e p o l a r i s a t i o n potential of -50 mV (APD-50), was shortened to 76 + 4% of reference in=3) at 30 ~ o l / l Nicorandil. E f f e ~ s of Nicorand i l (3-300 ~mol/l) on inwardly r e c t i f y i n g i v l - c u r r e n t , transient outward current ( i t o ) and delayeC K-current (iv) were analysed, lv1-curren~ was activated by voltage st~ps from -60 to -rOD mV. I t ' s amplitude was reduced (30 ~ o I / I nicorandil) to 79 + 4 % of reference in=5). I . and i were triggered by-depolarizing the membrane f~Sm -80 ~ to + 40 mV. Membrane current at 10 ms a f t e r t e s t pulse onset, taken as a measure of i - -current, was increased to 159 + 33% in=6) by 30 ~mo~/el nicorandil. I n t e r e s t i n g l y , i n - c e l l s where no d i s t i n c t transient outward current was observed (control) a strong i , o current was induced by the drug. Membrane current at ~ s a f t e r t e s t pulse onset, taken as a measure of i Fcurrent, was also increased to 119 + 6% of referente in=6). In summary shortening of ~ n t r i c u l a r action p o t e n t i a l may be mainly due to an a c t i v a t i o n of i t o current. *Present address: I n s t i t u t e of Pharmacology, University of DOsseldorf, Moorenstr. 5, D-4000 DOsseldorf, FRG

Institute of Medical Physics and Biophysics, University Graz - Austria Supported by the Austrian Research Foundation (P/7141)

210

212

THE ANTIARRHYTHMIC AGENT E-4031 PROLONGS ACTION POTENTIALS IN GUINEA-PIG CARDIOMYOCYTES BY THE INHIBITION OF AN IONIC CURRENT DIFFERENT FROM THE DELAYED RECTIFIER (Ix). E. Wettwer .......................................................

EFFECTS OF C O + + DURING REPERFUSION AFTER CARDIOPLEGIC

The novel antiarrhythmic drug E-4031

(l-[2-(6-methyl-2-

pyridyl)ethyl]-4-(d-methyl-sulfonylaminobenzoyl)piperidine) prolongs the action potential duration (APD). This effect was attributed to an inhibition either of the delayed rectifier (I~) or of a different potassium conductance. In order to resolve this controversy we have studied the effect of E-4031 on outward membrane currents in whole cell voltage clamp experiments using isolated ventricular myocytes. At 37 °C, 0.i ~M E-4031 reversibly increased APPgo from 273±27 ms to 439±72 ms (n=5) within 5 min. For current recordings, the electrodes contained: (in mN) KCI 112; MgCI2 4; EGTA 10; HEPES 10; NazATP 4. Ic, was blocked with Cd 2÷ (0.i mM). Ix was measured as the steady-state outward current at the end of clamp pulses of 5s duration to +50 mV from a holding potential (Vh) of -40 mV at a rate of 1 min -~. We also analyzed the tail currents (It) upon repolarization to Vh. Within i0 min of regular stimulation IK and It showed run down between 15 and 25 % of the control. The monotonic decrease of I~ was not modified after addition of E-4031 (0.I ~M). However, It was reduced within 10 min by 62 % of the value before addition of E-4031 (u=4), suggesting that IK and It can he influenced differentially. Furthermore, in control experiments with clamp pulse duration varied from 20 to 4000 ms, I~ and It were not activated with a parallel time course as expected if they shared a common ionic pathway. This supports the conclusion that E-4031 affects APD by inhibition of an ionic current different from IK. ....................................................... Pharmakologisches Institut, Klinikum/GHS Essen, Hufelandstr. 55, D-4300 Essen i, FRG

CARDIAC ARREST G. Stark, H. M~ichler*, H. Udermann**, M. Decrinls, E. Pilger Cardioplegie cardiac arrest with a solution of high potassium concentrations is used to provide myocardial protection for cardiac surgical procedures. During the reperfusion period, an excessive influx of Ca-ions into the ceils occurs (calcium paradox) and, thus, being likely an adverse effect in addkion to the ischemia o~ the myocardium during cardioplegic arrest. In isolated beating rat hearts Co , an inorganic slow channel inhibitor, was shown to have a protective effect against the calcium paradox after a long+p+eriodof calcium free perfusion. We therefore investigated the effects of Co enriched St.Thomas Hospital cardioplegic solution and St.Thomas Hospital solution alone in isolated guinea pig hearts after cardiac arrest. During the experimental period the tissue calcium concentration, release of creatine kinase, contraction amplitude and EC~+parameters were evaluated continuously. Co (10uM) added to the perfusate, 10minutes before cardioplegic arrest induced a marked decrease in left ventriealar pressure by 13"+9% (p < 0.05,n = 10). During this period no el~c~yophysiologicalchanges were present. The hearts (n = 10 in the control and Co group) were arrested by the use of a St.Ths]mas Hospital solution at a temperature of 10°C containing either none or Co Tat a concentration of lmM. After 30minutes of cardioplegic arrest reperfusion was performed for lmin and no differences in tissue calcium concentrations of the hearts in the Co + +group (ll.3_+l.2umol/g) and in the control group (12.8-+1.9umol/g) could be detected. Also the creatine kinase release did not differ in both groups. The left ventrlcular contraction amplitude, which was comparable in both groups and about 71% of control values, recovered insignificantly earlier in the group with Co+ + present during cardioplegia (114_+27 seconds) than in the control group (133-+22 seconds). These findings may be the result of the inhibitory effect of Co + +on ATP breakdown during cardioplegia. During the reperfusion period in both groups sinus rhythm occured approximately within the same time span, in the control(15_+9seconds) and in the Co + +group (22-+15seconds). The recovery !.o+normal val~es+ on the AV-nodal conduction time was group. ++ prolonged in the Co Co exert inhibitory effects on the slow Ca channel activity and these effects and the reduction of left ventricular mechanical activity correspond to this. The prolonged recovery of AV-conduction after the cardioplegic arrest with the Co enriched St.Thomas Hospital cardioolegic solution may also be a result of the inhibition of slow channel activity. Co-~ +had no effect on the calcium tissue concentration in the early reperfusion period after cardioplegic arrest. Departments of Internal Medicine, surgery*, forensic medicine**, University of Graz, Auenbruggerplatz 15, A-8036 Graz Supported by the Austrian Research Foundation (P/7141)

R 54 213 E F F E C T S OF AZELASTINE ON CARDIAC ACTION POTENTIALS, N A + - I N F L U X , Ist, A N D C O N T R A C T I L I T Y . C . A c h e n b a c h , S. H a r t m a n n s , and P. S c h w e i k a r t The antiasthmatic/antiallergic agent azelastine with strong antihistaminic properties (Asta P h a r m a A.G., Frankfurt/M.) depresses cardiac activity by reducing upstroke velocity and p l a t e a u h e i g h t of a c t i o n potentials at d o s e s a b o v e 10 -7 mol/l. In addition, we m e a s u r e d atNa with Na electrodes (ETH 227) in s h e e p h e a r t P u r k i n j e fibres. R e s t i n g atNa d e c r e a s e d b y 40 % at h i g h a z e l a s t i n e doses. E x p o s i n g the fibres to p o t a s s i u m - f r e e solutions revealed a 30 r e d u c t i o n of (electrogenic) p l a t e a u Na-influx. The s l o w (Ca) i n w a r d c u r r e n t (ist) was m e a s u r e d in P u r k i n j e f i b r e s u s i n g a two-microelectrode v o l t a g e - c l a m p t e c h n i q u e and, for c o m p a r i s o n , in guinea pig ventricular myocytes using patch electrodes. 8 and 5 e x p e r i m e n t s , resp., w e r e p e r f o r m e d in e i t h e r tissue. Azelastine reduced ist by 50 % i d e n t i c a l l y at 3x10 -6 m o l / l in b o t h experimental models. R e d u c t i o n of c o n t r a c t i l e f o r c e in s h e e p v e n t r i cular m u s c l e b y a z e l a s t i n e t o t a l s approx. 60 % at 10 -~ m o l / l and is h a l f - m a x i m a l at a b o u t i0 -s mol/l. Muscle experiments with histamine show azelastine to be a m u c h s t r o n g e r a n t i h i s t a m i n e than a Ca-antagonist. H o w e v e r , it seems that azelastine counteracts the p o s i t i v e i n o t r o p i c e f f e c t of a d r e n a l i n e by blocking isl rather t h a n due to a s p e c i f i c a n t a g o n i s m .

Physiologisches Institut II der Universit~t Bonn, W i l h e l m s t r . 31, D - 5 3 0 0 B o n n i, F.R.G.

214 THE u-ADRENOCEPTOR BLOCKING AGENT NAFTOPIDIL ALSO INHIBITS CARDIAC CALCIUM CURRENTS. H.M. Nimmel Naftopidil is known to block at-adrenoceptors with a i00 times lower affinity than prazosin. Investigations in Z÷depolarized rat aortic strips suggested that naftopidil may have an additional vasorelaxation mechanism due to inhibition of Ca2+ entry. We have examined this hypothesis in various isolated preparations of the guinea-pig heart. The a-adrenoceptor antagonist prazosin and the Ca2+ channel blocker verapamil were studied for reference. In papillary muscles stimulated at 1 Hz, i0 ~M of either drug reduced the force of contraction by about 40% (naftopidil), 90% (verapamil), and < 10% (prazosin). The action potential duration and the refractory period were hardly affected by naftopidil, decreased by verapamil, and slightly increased by prazosin. The maximum rate of depolarization did not change with I0 pM of naftopidil, verapamil, or prazosin. In constant-flow bangendorff hearts, the drugs (10 pM) reduced the perfusion pressure, decreased the force of contraction, and slowed the spontaneous heart rate (order of potency: verapamil >> naftopidil > prazosin). In isolated ventricnlar cardiomyocytes, we studied the effects of naftopidil, verapamil, and prazosin, on the calcium current Ica by means of the whole cell patch clamp technique. The cells were depolarized from a holding potential of -40 mV to 0 mV at a rate of 0.5 Hz. Ics was concentration-dependently inhibited by verapamil (pDs-value: 7.0) as well as by naftopidil (pD~-value: 6.5). Even I0 BM of uaftopidil, however, reduced Ica to only 50% of the control value. Very high concentrations of prazosin (10 pM) also decreased Ica. In conclusion, our data are consistent with the hypothesis that naftopidil possesses weak calcium channel blocking properties. Pharmakologisches Institut, Universit~t-GH8 Essen, Bufelandstr. 55, D-4300 Essen I, FRG

215 EFFECTS OF BRETYLIUM ON MYOCARDIAL ATPASE PREPARATIONS N. Dzimiri and A.A. Almotrefi Bretylium is a class III antiarrhythmic agent which probably exerts its effects by interacting with potassium channels (Bacaner et al., Proc Natl Acad Sci 83:2223, 1986; Cook NS, Trends Pharmacol Sci 9:21, 1988; Sheldon et al., Clin Chem 35(5):748, 1989). Its effects on microsomal and mitochondrial ATPases were studied in guinea pig myocardial enzyme preparations. In microsomal preparations, bretylium inhibited the enzymatic hydrolysis of ATP by the Mg2+-dependent, Na+ and K+ activated ATPase system in a concentration dependent fashion qualitatively similar to that exhibited by ouabain, a specific ir~hibitor of this enzyme activity. However, the inhibitory potencies of bretylium were relatively weaker (ICs0 = 2.45 ± 0.17 mM; n=8) and occurred at higher concentration ranges (0.5 - 5 mM) than those of ouabain (range = 0.i - i000 ~M; ICs0 = 1.9 ± 0.2 ~M; n=7). In the presence of 2,5 ~M ouabain, the curve for hretylium exhibited a biphasic right-to-left shift which increased with concentration within its inhibitory active range and the reverse trends at lower concentrations. These combined effects remained nevertheless weaker than those of ouabain, requiring 1.28 ± 0.Ii raM of bretylittm to acquire half maximal inhibition of the enz)nne activity. In mitochondrial ATPase preparations the actions of bretylium were less marked, exhibiting inhibitory potencies only at concentrations above I0 mM. These results demonstrate interactions between bretylium and membrane bound ATPases. Furthermore, the biphasie nature of the concentration-response relationship for the combined drug effects on the mierosomal Na+-K+-ATPase activity seems to suggest a competitive type of interaction, probably involving the ouabain binding sites on the enzyme system. MBC-03, King Faisal Specialist Hospital & Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia

216 INFLUENCE OF HALOTHANE ON UPTAKE AND EFFECTS OF THIOPENTAL IN ISOLATED HEART PREPARATIONS OF RATS J . C h . I s e n b e r g , J . B a l d a u f , U. BOch*, P . A l t m a y e r * and H.P. BQch A f t e r i . v . t h i o p e n t a l (T) a p p l i c a t i o n T-concent r a t i o n in heart t i s s u e was much higher (+52 %) in r a t s anesthetized with 1.5 vol% halothane (H) than in c o n t r o l animals (BQch e t a l . , NaunynSchmiedeberg's Arch Pharmacol 337 N° 22, 1988). In order t o study t h a t pharmacokinetic i n t e r a c t i o n and i t s consequences on h e a r t f u n c t i o n Langendorff's h e a r t preparation as well as i s o l a t e d r i g h t and l e f t a t r i a o f r a t s were used. Tyrode-solution (gassed w i t h carbogen, pH 7.4) containing e i t h e r T (3-7 - I0 -s g/ml) or T and a d d i t i o n a l l y H ( 0 . 8 - 2 . 0 vol%) was used as perf u s i o n medium. I f steady s t a t e was reached the r a t i o o f T-concentrations ( h e a r t tissue/Tyrode) was about 3/1 independently o f the T-concentrat i o n used. In the presence o f H the T-uptake in heart t i s s u e was s i g n i f i c a n t l y increased. I n c o n t r a s t t o t h i s increased T-concentration o f heart t i s s u e the negative chronotropic a c t i o n o f T was reduced by simultaneously present H whereas the negative i n o t r o p i c a c t i o n remained unaff e c t e d . When using i s o l a t e d r i g h t and l e f t a t r i a , both, the negative chronotropic and inot r o p i c a c t i o n o f T were p o t e n t i a t e d by the presence o f H. I n s t i t u t f o 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 , I n s t i t u t f G r A n ~ s t h e s i e * , U n i v e r s i t ~ t des S a a r l a n d e s , D-6050 H o m b u r g / S a a r , FRG.

R 55 219 CARDIO~ON : THE ~ A N I N G OF IONIZED I N T R A ~ CAI~IIR AND s c o n m DURING CARD~OPLEGIA A. Morstadt, J. Preuner, C. de Beer , D. Birnbamn.

217 COMPONENTS OF ENERGY TURNOVER OF ISOLATED CARDIOMYOCYTES

H. Rose, S. Piipping Isolated ventricular myocytes of rat hearts were paced electrically and oxygen consumption as well as shortening was measured. The length-time intergral (I t ) was taken as a measure of the "work" performed by the contracting cells. I t was then correlated to the oxygen consumption per beat (VuO 2 }. Stepwise inhibition of the actin-myosin interaction by addition of 2,3-butanedione monoxime (BDM) was Characterized by a linear relationship between It and VbO 2. Extrapolation to the point of complete inhibition of the actinmyosin ATP-ase led to an oxygen consumption due only to the 4.+ + + cycling of ions (Ca ,Na ,K ), related to contraction. Basal oxygen consumption was 2t5 + 14 nl/mgpr* min and VbO 2 amounted to 0.722 nl/mgpr* beat (with 0.5 mM [Ca++]e), 22% of which was used for Ioncycling. This value increased to 42% when [Ca++]e was 1.8 raM. The influence of inotropic drugs on calcium cycling should be detectable with this new technique. (supported by DFG Ro 755 1-1) Institute of Physiology, Med. Fac. RWTH Aachen, Pauwelsstr. D-5100 Aachen, FRG

Perioperative ischemic damage of the myocar~um ultimately results in a sustained rise in cytosolic Ca ,which correlates with the subsequent loss of viability. Cardioprotection afforded by cold crystalloid cardioplegia(FRESENIUS), effective by reduction of sodium to 50 rmuol/l and partial removal of calczum' to 0.5 n~nol/l, was evaluated in. 22 P~T_, tients+undergoing mitral valve replacement. Ionlzed Ca and Na were determined in samples of coronary effluate during anaesthesia, cardioplegia and after weaning off cardiopulmonary bypass (CPB). Simultaneously myocardial biopsies were taken for analyzing total tissue calcium and sodium content. Reduction of coronary Na+ to 69 ±13 mmol/l during cardioplegia instantaneously followed the rate of perfusion. The intravascular Ca -concentration (0.68 ± 0.08 mmol/l) exceeded the values attainable (unspecified Ca-contamination of the cardioplegic solution(0.64 ±0.03 n~olCa/l), thereby reducing the efficacy of cardioplegia. Tissue calcium (Cat ) during induction of anaesthesia amounted to 2.2 ±0.6 ramol Cat/kg w.w. while intravascular Ca was 1.18 ± 0.06 ~nol/l. During cardioplegia a slight increase in tissue calcium (2.8+$1.2 mmolCa~/kg w.w.) took place,although intravascular Ca w a s reduced by 43%. After weaning off CPB tissue calcium increased by a factor of two up to 5.0 ±1.7 mlolCa~/kg w.w. (p<0.01). This cellular Ca,accumulation was prece~ded by an increase in tissue sodium from 32 ±3 to 51 ±9 /mnol Na~/kg cell (p<0.05). The applied cry~alloid cardiopl~gia inclompetely prevented cellular Ca -entry. The Na~-load imposed on the myocardial cell during cardiopleg~a may mediate Ca-influx by reactivating Na-Ca exchange on reperfusion. An alternative route of Ca-entry may be the postischemic impaired Caefflux via the ATP-dependent sarcolemmal Ca-pump. Dept. of Cardiovascular Surgery , Re~abilitationszentrum Sfidring 15, 7812 BAD KROZINGEN Dept. of Toxicology University of Kiel, Brunswiker Str. i0, 2300 Kiel 1

218 TRACER RE-UPTAKE INTERFERES WITH MEASUREMENT OF 45CALCIUM EFFLUX FROM GUINEA-PIG ATRIA Lutz Hein and Robert $¢ho¢h The determination of cellular Ca efflux from intact myocardial tissue preparations is complicated by diffusion-delays in the extracellular space. Experiments were performed to examine the influence of tracer re-uptake from the extracellular space on the washout of 45Ca from intact guinea-pig left atria. After labelling the exchangeable Ca pool of the atria with 4SCa for 60 rain, the washout of radioactivity into various tracer-free Tyrode solutions was studied for 300 rain. Only after 90 rain of washout, the ¢ C a efflux followed a single exponential course, corresponding to a constant fractional loss of ~Ca per rain from the muscle (rate coefficient k, eom.c0m-tmln-i). Under control conditions (1.8 mM Ca, 149 mM Na), the ~Ca efflux proceeded with a rate coefficient of k=7.74.10-°. This corresponded to a Ca exchange of 122umol.kg-Lmind. When the concentrations of Ca or Na in the washout Tyrode solution were reduced, the rate coefficient decreased significantly (s. table 1). During incubation of the atria in a "Ca-poor" medium (about 10 ?aM Ca), the probability for extruded 4SCa to be taken up again from the extracellular space is higher because of the low extracellular concentration o f non-radioactive Ca. U0on reduction of the extracellular Na concentration to 11 mM (sucrose as substitute), the atria developed a contracture and tissue Ca content increased by 100 ~umol. kg d . rain -1. In this case, the rate coefficient was decreased during washout both with 1.8 mM Ca and "Ca-lmor" Tyrode solution. Thus, the net Ca influx as induced by the low extracellular Na concentration decreased the apparent rate coefficient of 4sca effiux into the organ hath. When 5 mM EGTA was added as a Ca chelator to trap 4SCa having left the myocardial cells, the 45Ca effiux proceeded with a rate coefficient equivalent to the control condition. In conclusion, 45Ca efflux measurements using intact atria should be iute~l~reted with caution whenever re-uptake of ~ c a reduces the amount of Ca escaping from the extracellular space into the organ bath. Tab. 1: Rate coefficient ( ~ m . ~ m - Z . m i n d) of *sCa efflux from guineapig left atria (~-, n--4-6, *p,-0.025)

extracell~ar ENa+] e x u a ~ . . ~ 149 mM 11 mM

control 7.74.10 "z 5.54"10-8 *

~a ~+]

"Ca-pool' 5.68-10 -z * 4.53"10-3 *

Abt. Plaarmakologie, Universit/it Kid, HospltalstrA,

"Ca-free" 7.72.10 -~ 7.66.10 -3 D-2300 Kiel 1.

220 INFLUENCZ OF R 56865 ON ~ O H A N I O A L ~ND ACTION POTENTIAL ALTERATIONS INDUCED BY VERATRIDINE IN THE CUINEA-PI@ ISOLATED PAPIIJ.ARy NUSCLR D, w i l h e l m a n d C. M e u t e r R 56865 ( N - [ 1 - [ 4 - ( 4 - f l u o r o p h e n o x y ) b u t y l ] - p t p e r t d t n y l ] - N methyl-2-benzothiazolamtne) has been reported to prevent electrical and mechanical signs of cardiac glycoside toxicity in guinea-piE papillary muscle (Vollmer et al., E u r . J . P h a r m a e o l . 142, 137, 1987) s u g g e s t i n g a p o s s i b l e prevention of sodium and calcium gain. This hypothesis was t e s t e d by e x p o s i n g g u i n e a - p i g p a p i l l a r y m u s c l e s t o veratridine a compound t h a t p r o l o n g s t h e o p e n s t a t e o f t h e sodium c h a n n e l . I n t h e p r e s e n c e o f a t o x i c c o n c e n tration of veratridine ( 2 . 5 x 10 - 6 m o l / 1 ) , R 55865 (10 - 6 mol/l) abolished early afterdepolarlzatlons without affecting the profound prolongation of the action potential duration or the decline of the action potential amplitude. Concomitantly, veratridlne's positive inotroplc effect was attenuated and aftereontractlons were inhibited. In the presence of a subtoxie concentration of veratrldlne (8 x 10 -7 mol/l), R 56865 did not compromise the positive inotropic effect. In ease of pretreatment, R 56865 (10 -6 mol/l) prevented the occurrence of early afterdepolarlzatlons and attenuated the decrease in action potential amplitude exerted by the toxic concentration of veratrldlne, The prolongation of the action potential duration was not affected by R 56865. Mechanical signs of toxicity were Inblblted by pretreatment with R 56865 and the positive inotroplc response to veratrldine was maintained. In concluslon, R 56865 prevented and inhibited veratrldlne-lnduced early afterdepolarizatlons and aftercontractlons without affecting the prolonged action potential duration. It may he speculated that R 56865 interferes with intracellular sodium and calcium accumulation which is thought to be responsible for both veratrldlne- and ouabaln-lnduced afterdepolarlzatlons and aftercontractlons. JANSSEN RESEARCH FOUNDATION, D-4040 Neuss 21, FRG

R 56 221

223

EFFECTS OF R 5 6 8 6 5 AND R 59494 ON VERATRIDINE-INDUCED 45CA MOVEMENTS IN THE ISOLATED LEFT A T R I U M O F T H E RAT D. Wermelskirchen, U. Nebel and A. Wirth R 56865 (N-[l-[4-(4-fluorophenoxy)butyl]-piperidinyl]-Nmethyl-2-benzothlazolamine) protects against cardiac glycoside intoxication. In isolated left atria of the rat R 56865 diminished not only the ouabain intoxication-induced increase in intracellular Ca but also the increase in intracellular Na. To get further information on the mechanism of action of R 56865 and a structural analogue, R 59494 (N-methyl-N-[l-(4-phenoxy-butyl)-3pyrrolidinyl]-2-benzothiazolamine), we studied the intoxication of isolated left atria of the rat with veratridine (10-4 M), which increases the intracellular Na concentration. The accompanying 45Ca uptake was detected. Nifedlplne (10 -7 M) exhibited no effect on the veratridlne-induced 45Ca uptake. Verapamil (10-7-10 -5 M) diminished it maximally by 84~. However, the unstimulated 45Ca uptake was significantly suppressed by verapamil in the same concentration range. R 56865 (10 -810 -5 M) and R 59494 (10-9-10 -5 M) completely inhibited the veratrldine-induced 45Ca uptake without affecting the unstlmulated 45Ca uptake. Amilorlde (6 x 10 -3 M) exhibited no significant influence on the veratridineinduced 45Ca uptake. In summary, comparable to ouabain-indueed intoxication R 56865 and R 59494 protected against veratrldineinduced intoxication. Nifedipine failed to affect veratrldine-lnduced 45Ca uptake, indicating a minor involvement of L-type Ca channels. Verapamil diminished the veratrldine-induced 45Ca uptake, but the unstimulated 45Ca uptake was also suppressed. The lack of an effect of amilorlde contradicts a major contribution of the Na/Ca exchange to veratridine-induced 45Ca uptake. Since veratridine similar to ouabain increases intracellular Na and Ca, the antitoxic effects of R 56865 and R 59494 support the view, that these compounds protect against intracellular Na and Ca overload. JANSSEN RESEARCH FOUNDATION, D-4040 Neuss 21, FRG

CARDIOPROTECTIVE PROPERTIES OF SUBCHRONICAL LOW DOSE ACE INHIBITOR TREATMENT IN ISOLATED ISCHEMIC RAT HEARTS W. Linz, U. Albus, B.A. Sch61kens Cardioprotective effects have been shown in animals with myocardial ischemia and postisehemic reperfusion arrhythmias after single application of ACE inhibitors in an antihypertensive dose. To investigate whether this holds also true when an ACE inhibitor is given subchronically in a low dose without effect on blood pressure studies were performed in rats with two dose regimes. Rats were treated p.o. for two weeks with the ACE inhibitor ramipril 1. in the blood pressure lowering, high dose of 1 mg/kg/day or 2. with the low dose of 10 /lg/kg/day, which did not affect blood pressure. At the end of the 2 weeks treatment period animals were sacrificed, the hearts isolated and perfused according to the following procedure. After a 20 min control perfusion period we produced postischemic reperfusion injuries by setting an acute regional myocardial ischemia for 15 min followed by reperfusion for 30 min. High dose ramipril reduced duration and incidence of reperfusion induced ventricular fibrillations (VF) down to 2.0 -+ 0.6 rain (incidence 2/10) as compared to controls with 16.8 + 2.6 min (10/10) duration. Basal cardiodynamics as well as the metabolic status of the ischemic hearts from ramipril treated rats were improved, and release of lactate dehydrogenase and creatine kinase as well as lactate were decreased. Low dose ramipril showed comparable effects with a reduction in the duration of VF down to 4.5 + 1.2 rain and an incidence of 4/10. The cardiodynamic and metabolic changes were nearly identical to those seen in the group receiving the antihypertensive dose of the ACE inhibitor. The results suggest that a long lasting inhibition of the local ACE in the heart by subchronieal application of ramipril in a dose not affecting blood pressure is able to prevent postischemic reperfusion injuries. Hoechst AG, P.O.B. 80 03 20, 6230 Frankfurt 80, FR Germany

222

224

R 56865 POSTPONES ISCHEMIC CONTRACTURE AND POTENTLY INHIBITS POSTISCH~IIC REPERFUSION-INDUCED FIBRILLATIONS IN ~ GUINEA-PIG E, Scheufler and A. Mozes R 56865 (N-[l-[4-(4-fluorophenoxy)butyl]-plperidlnyl]-Nmethyl-2-benzothiazolamlne) potently inhibits ouabain induced toxicity. We investigated the antiischemic and antifibrillatory properties of the compound in guineapig Langendorff preparations perfused with constant flow. Left ventricular pressure (LVP) and perfusion pressure (PP) were monitored. R 56865 was given 45 mln before ischemia. Ischemia (45 min or 60 min) was elicited by stopping the perfusion flow or reducing it to 0.1 ml/min. During ischemia the onset of the ischemie contraeture was defined as the time, at which the diastolic pressure was raised by 2 mm Hg. After 60 min of reperfusion the occurrence of fibrillations was monitored. Ischemic contracture: 10 -7 M R 56865 significantly (P < 0.05) delayed the ischemic contracture from 24.7 to 33.2 min in stop flow Ischemla (45 min). The ischemic contracture was also significantly delayed by the same dose during 60 min of low flow ischemia (28.9 versus 37.9 mln). In both experimental groups LVP was slightly but not significantly reduced (P > 0.05). Antlfibrillatorv Droperties: R 56865 was given before ischemia (0.i ml/min low flow; 60 min). It did not siKnlficantly (P > 0.05) reduce LVP and PP from 10 -8 M to 10-7 M. The onset of the ischemic contraeture was not significantly delayed below 10 -7 M. However, the incidence of fibrillations at the end of 60 min of reperfuslon (83% in controls) was dose-dependently reduced to 0% in the concentration range of 10 -8 M to 10 -7 M R 56865. In conclusion, R 56865 delays the ischemic contracture at 10 -7 M. However, the compound was active between 10 -8 M and 10 -7 M in inhibiting the occurrence of fibrillations during reperfusion, without showing cardlodepressant effects. JANSSEN RESEARCH FOUNDATION, D-4040 Neuss 21, FR@

CARDIAC ELECTROPHYSIOLOGIC E F F E C T S OF T E D I S A M I L IN G U I N E A P I G P A P I L L A R Y M U S C L E S A.Fuchs, G.Buschmann, D.Ziegler, G.Varchmin, A. Farjam, U . G . K ~ h l The b r a d y c a r d i c K + - c h a n n e l b l o c k e r (Dukes a n d Morad, Am. J. P h y s i o l . 257, 1989 (in p r e s s ) ) t e d i s a m i l (T) h a s b e e n s h o w n to i n c r e a s e v e n t r i c u l a r a c t i o n p o t e n t i a l d u r a t i o n (APD) at l o w b u t to d e c r e a s e A P D at h i g h c o n c e n t r a t i o n s (Oexle et el., J. Mol. Cell. C a r d i o l . 19, Suppl. III, p.65, 1987; F u c h s et al., A r c h . P h a r m a c o l . 340, Suppl. If, R72, 1989). H e r e w e c o m p a r e electrophysiologic findings with T under normal and high K+ conditions and following application of T T X on g u i n e a p i g p a p i l l a r y m u s c l e . M e t h o d s : In i s o l a t e d p a p i l l a r y m u s c l e s (PM) the e f f e c t s of T on A P D a n d u p s t r o k e v e l o c i t y (Vma x) w e r e m e a s u r e d at n o r m a l (5.3 mM) or h i g h (22 mM) extracellular K+-concentration (nK +, hK ÷) or in the p r e s e n c e of 30 ~ M t e t r o d o t o x i n (TTX). R e s u l t s : In nK + T c a u s e d a b i p h a s i c c h a n g e of APD: a p r o l o n g a t i o n at 0 . 2 2 - 2 . 2 ~M, no c h a n g e at 4.6 ~ M a n d a s h o r t e n i n g at 10-22 ~M. Vma x w a s u n c h a n g e d up to 4.6 ~ M but r e d u c e d at 1 0 - 2 2 pM. U n d e r hK + or T T X T at 1-2.2 # M s h o w e d a m a r k e d p r o l o n g a t i o n of A P D w i t h o u t r e l e v a n t c h a n g e s of Vma x. A t hK + the r e d u c t i o n of A P D a n d Vma x by h i g h c o n c e n t r a t i o n s of T (10-22 ~M) w a s m a r k e d l y d i m i n i s h e d , w h e r e a s T T X h a d no i n f l u e n c e on the APD-shortening a n d Vs, x r e d u c t i o n by 22 ~M of T. C o n c l u s i o n : The r e s u l t s s u g g e s t t h a t T h a s c l a s s ill a n t i a r r h y t h m i c p r o p e r t i e s at l o w e r c o n c e n t r a t i o n s w h e r e a s at h i g h e r c o n c e n t r a t i o n s o t h e r effects including Na+-channel blockade become prominent. K a l i - C h e m i e P h a r m a GmbH, Herz-Kreislauf-Pharmakologie, D-3000 Hannover

Postfach

220,

R 57 225 AGONIST-INDUCED HUMAN NEUTROPHIL ACTIVATION IS INHIBITED BY DEFIBROTIDE IN VITRO P. Ney The po]ydeoxyribonuc]eotide d e f i b r o t i d e (DEF), affords protection against myocardial tissue damage. Since neut r o p h i l granulocytes (PMN) may aggravate the i n j u r y , the present study investigates the e f f e c t of DEF on PMN a c t i vation induced by f-Met-Leu-Phe (FMLP), p l a t e l e t activat i n g factor (PAF) and cal~imycin (A 23187) in v i t r o . Washed human PMN (5 x 10 /ml) were incubated f o r 10 min with DEF (I-1000 ~g/ml) and subsequently chal]enged by FMLP (30 nM), PAF (3 ~M) or calcimycin (I0 ~M). FMLP and calcimycin potently stimulated superoxide anion ( 0 ~ ) generation (22.2 + 1.8 and 15.2 + 3.6 nmoles O~-/SxID ~ PMN, respectively) aEd B-glucuronida-se re~ease (4~.4 + 8.2 and 34.3 + 4.9 ~g phenolphthalein/5 x 10 PMN, r e s p ~ t i v e l y ) . PAF ~ s a weaker a6ctivator of these reactions (7.8 + 0.9 nmoles 0~-/~ x 10 PMN and 16.5 + 2.3 wg phenolp-hthal e i n / 5 x~O PMN). Treatment of PMNwith DEF resulted in a dose-dependent i n h i b i t i o n of these reactions. At I mg/ ml DEF the 0~- generation stimulated by a l l agonists was inh i b i t e d b9 25-35% of control. DEF was a mere potent inhib i t o r of the stimulated B-glucuronidase release. The same concentration of DEF i n h i b i t e d the FMLP-, A 23187- and PAF-induced S-glucuronidase release by 67 ± 3%, 44 + 5% and 35 + 5% of control, respectively. In contrast, DEF-had no e f f ~ t on the agonist induced LTBa release an~+no major a l t e r a t i o n s in i n t r a c e l l u l a r cAMP l~vels or Ca" concent r a t i o n s were observed. These results demonstrate an i n h i b i t i o n of FMLP-induced superoxide anion generation and B-glucuronidase release from human PMN by DEF. The i n h i b i t o r y actions of DEF a f t e r PAF or calcimycin stimulation were only moderate. Any direct PMN-inhibiting action of DEF in combination with the previously demonstrated stimulation of vascular prostaglandin formation by DEF might be useful for the prevent i o n of PMN-induced tissue i n j u r y . Supported by the DFG (Schr 194/7-2) I n s t i t u t for Pharmakologie der Heinrich-Heine-Universit~t, Moorenstr. 5, D-4000 DOsseldorf I

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228 CHARACTERIZATION OF CARDIOVASCULAR EFFECTS OF SEROTONIN IN DOMESTIC PIGS P. METZENAUERAND R. DEDECKE

ANALYSIS OF =-ADRENOCEPTORS IN MICROVESSELS OF STRIATED MUSCLE OF THE SPONTANEOUSLY HYPERTENSIVE RAT M.M.J. Messing, H. van Essen, H.A.J. Struyker Boudier In this study we determined the distribution of =-i and =-2 adrenoceptors in the microcirculation of striated muscle in conscious spontaneously hypertensive rats (SHR). Male, 5 weeks old SHR (n=7) were implanted with a dorsal mlcrocirculatory chamber (TL Smith et al, Mierovasc Res 29, 360-370, 1985). Four weeks later, experiments were performed in conscious animals, using intravltal microscopy. Vldeo-recordings allowed later off-line analysis of diameter changes in the microvessels using a shearing monitor device. Arterioles were classified, according to their branching order as A1 (65-100 ~m) to A4 (15-25 ~m), as were the venules (VI: 125-200 ~m; V4:15-30 ~m). Mean arterial pressure (MAP) was registered from an intra-arterlal catheter. An intravenous catheter was used for drug administration. Agonists were given as cumulative infusions (phenylephrine (=-I): 1,3,10 ug/kg/min, azepexole (=-2): 30,100, 300 Bg/kg/min). Video-recordings were taken after establishment of a steady state. The antagonists were administered as bolus injections: prazosine (=-i): 0.I mg/kg, yohlmbine (=-2): i mg/kg. Phenylephrlne only constricted larger vessels (AI: 13±3%; A2: 13±4%; VI: 11±6%), whereas azepexole only caused constriction of the arterioles (At: 16±2~, A2: 12±3%, A3/A4: 12±3%). The antagonists caused differential dilatations. Prazosine increased the diameter of AI (17±2%), A2 (17±3%), VI (12±3%) and V2 (8±4%) vessels significantly. Yohimblne had a selectively vasodilator effect on the arterioles (AI: 12±5%, A2: 27±10%, A3/A4: 12±2%). From these data we conclude a predominant localization of =-i adrenoceptors on large (AI/A2 and VI/V2) vessels, whereas 6-2 adrenoceptors are distributed throughout the arteriolar but not the venular site of the mlcroeirculation of SRR striated muscle. Dept. of Pharmacology, University of Limburg, 616, 6200 MD Maastricht, The Netherlands

P.O.

Box

EFFECT OF a-FLUOROMETHYLHISTIDINE ON THE DEVELOPMENT OF HYPERTENSION IN SPONTANEOUSLY HYPERTENSIVE RATS. H. Prast Central histaminergic neurons seem to be implicated in the development of hypertension of spontaneously hypertensive rats (SHR). Elevated levels of histamine in hypothalamus and brainstem and changes in the activity of histidine decarboxylase in anterior hypothalamus and cortex have been found (Prast et al., this journal 338: 573,1988). Increased release of histamine in the posterior hypothalamus (Tuomisto et al., this journal 323:183,1983) and decrease in turnover of brain histamine (Oishi et al. Brain Res.343:180,1985) have also been reported. The effect of inhibition of histamine synthesis by ~-fluoromethylhistidine (a-FMH) on the development of hypertension in SHR was now investigated. Three or six week old SHR, Wistar Kyoto (WKY) and Sprague Dawley (SO) rats received dayly 75 mg/kg a-FMH for four weeks. The blood pressure was measured at least weekly by the tail cuff method. Treatment with a-FMH resulted in a substantial decrease of histidine decarboxylase activity and of histamine concentration in the brain. In six week old SHR, a-FMH did not influence the increase in blood pressure. However, in three week old SHE, the rise in blood presssure was delayed for I week. Subsequently, blood pressure increased to a level that was higher than that in untreated SHR. In WKY and SD aFMH h a d no influence on blood pressure. These results show that inhibition of histamine synthesis leads to a short-lasting delay in the development of hypertension. Probably, the effect of inhibition of histamine synthesis is subsequently superimposed by other neurotransmitters. Institut fur Pharmakodynamik und Toxikologie der Universit~t,Peter-Mayr-StraBe I,A-6020 Innsbruck,Austria

The e f f e c t of serotonin (5-hydroxytryptamine, 5-HT) is dose-dependent, but also varies with species. Our i n v e s t i g a t i o n is devoted to the effects of 5-HT and the characterization of d i f f e r e n t 5-HT-receptors in the cardiovascular system of pentobarbitone-anesthetized pigs. 5-HT-bolus-administration caused a dose-dependent biphasic e f f e c t on a r t e r i a l blood pressure: lower doses from 0.01 to 3 ~g/kg induced hypotension (max -35 mmHg) and higher doses from 10 to 100 #g/kg induced hypertension ( max +50 mmHg). In a l l tested doses tachycardia up to max +60 bpm was observed. There were no d i f f e r ences in the route of administration ( i . v . versus i . a . (aorta) versus i n t r a p u l m o n a r y - a r t e r i a l ) . Tachyphylactic reactions were absent in a l l experiments. The blood pressure e f f e c t induced by 30 ~g/kg 5-HT became inverted from an increase to a decrease, when selective 5-HT-2antagonists (for example ketanserin 0.I mg/kg i . v . ) were administered. Selective 5-HT-3-antagonists (for example ICS 205-930, 30 #g/kg i . v . ) d i d n ' t antagonize the hypertensive r e f l e x . The p o s i t i v e chronotropic response could not be i n h i b i t e d e i t h e r by 5-HT-2- or 5-HT-3-antagonists. Specific 5-HT-2-agonistic compounds like ~-methyl-5-HT induced an increase of blood pressure linked with tachycardia. Specific 5-HT-3-agonists l i k e phenylbiguanide always caused hypotension and bradycardia. In conclusion, 5-HT-2- and 5-HT-3-receptors are involved in the regulation of blood pressure response to 5-HT in pigs. The response of heart rate to serotonin seems to be unclear and has to be elucidate in f u r t h e r studies. ASTA Pharma AG, Department of Pharmacology, WeismUllerstr. 45, 6000 Frankfurt/Main, FRG

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5-HT1A-AGONISM IN HYPERTENSIVE DOGS DURING TREADMILL-EXERCISE. J . G . Grohs. S. Huber, G. F i s c h e r and G. R a b e r q e r F l e s i n o x a n i s known t o l o w e r s y m p a t h e t i c t o n e and hence a r t e r i a l b l o o d p r e s s u r e and h e a r t r a t e by stimulating 5-HTIA-receptors in the central n e r v o u s system in r a t s , c a t s and a n a e s t h e t i z e d dogs. Since f l e s i n o x a n f a i l e d in reducing a r t e r i a l blood pressure in conscious, normotensive dogs i t was o f interest to study its effects in hypertensive dogs. P r i o r t o experiments the dogs were c h r o n i c a l l y instrumented f o r measurement o f arterial blood pressure and h e a r t rate and h y p e r t e n s i o n was i n d u c e d by p e r i n e p h r i t i s . About six weeks l a t e r arterial blood pressure was m a r k e d l y i n c r e a s e d w h i l e h e a r t r a t e was unchanged. The e x p e r i m e n t s w i t h f l e s i n o x a n were p e r f o r m e d during the following period of constant hypertension. To evaluate the effects of f l e s i n o x a n on d i f f e r e n t l e v e l s o f sympathetic tone the dogs underwent grade d t r e a d m i l l - e x e r c i s e s . A f t e r a c o n t r o l t r e a d m i l l exercise f l e s i n o x a n was administered intravenously over a period o f f i v e minutes. 15 minutes l a t e r the post-drug exercise was performed, A dose o f 0 . 1 N g / k g f l e s i n o x a n led t o a decrease in s y s t o l i c and d i a s t o l i c a r t e r i a l blood pressure a t r e s t and during e x e r c i s e . A f t e r a d m i n i s t r a t i o n o f 0.2 #g/ks f l e s i n o x a n heart rate was i n c r e a s e d a t r e s t and d u r i n g e x e r c i s e b u t no further decrease in arterial blood pressure o c c u r e d . C o n s e q u e n t l y i t m i g h t be assumed t h a t this missing decrease in arterial blood pressure is sufficiently compensated by t h e b a r o r e c e p t o r reflex.

PHARMACOLOGICAL CHARACTERIZATION OF ZK 112.566, A DA2AGONIST/ot2-ANTAGONIST R. Beckmann, K.P. Gerbling, J.-D. Turner

Department of Pharmacology, University of Vienna, W ~ h r i n g e r s t r . 13a, A - I 0 9 0 V i e n n a , A u s t r i a .

ZK 112.566, a 3-(13-Amino-2,313-dihydro-2,6-dimethyl-8e~-ergolinyl)-l,1diethylurea, is an ergoline derivative, which was tested in different receptor binding assays with the following results (Ki-values): Receptor D2 D1 % % HT 5-HT2

Tissue pig striatum pig striatum rat forebrain bovine cortex bovine cortex rat forebrain rat forebrain

Ligand

Ki-value

3H-N-O437 3H-SCH23390 3H-prazosin 3H-paraamino-elouldin 3H-yoMmbin 3H-5-HT 3H-ketanserin

11.2 nM > 5000 nM 4000 nM 74 nM 6 nM 4200 nM > 1000 nM

ZK 112.566 inhibited constriction of rabbit ear artery induced by transmural ne~e stimulation concentration-dependently with an IC50-value of 8.2 x 10-' N. This effect could be reversed by the DA2-antagonist domperidone. A DAl-agonistic component of ZK 112.566 was excluded measuring renal blood flow in anaesthetized dogs. In isolated rabbit aorta p.ATvalue for the ~l-antagoulstie effect of ZK 112.566 against phenylephrme was 6.2. The ¢~Tantagonlstic effect was studied in isolated guinea pig atria, which were electricany stimulated during the refractory period. The PA2-value for the o%-antagonistic effect of ZK 112.566 against BHT 920 was 6.91. In isolated strips of the rabbit vena saphena contraction induced by BHT 920 was significantly inhibited by ZK 112.566. Concentration response curve of BHT 920 was shifted to the

right. Direct vasorelaxing effects of ZK 112.566 were excluded in potassium depolarized strips of rat aorta. In conclusion ZK 112.566 has been characterized in vitro as a potent dopamine receptor agonist selective for DA2-receptor~ with ~2" and minor ~l-antagonistic properties. Research Laboratories of Sobering D-1000 Berlin 65, West Germany

AG,

Mullerstr.

170-178,

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GABA B RECEPTOR-MEDIATED INHIBITION OF THE NEUROGENIC VASOPRESSOR RESPONSE IN THE PITHED RAT E. Schlicker and A. Kohlenbach The effects of GABA and related drugs on the vasopressor response induced by electrical stimulation (single pulse of 30 V and i ms) of the preganglionic sympathetic nerve fibres or by injection of noradrenaline 0.3 nmol/kg were studied in the pithed rat. The electrically induced increase in diastolic blood pressure (almost totally abolished by prazosin i0 plus rauwolscine 30~/mol/kg) was inhibited by GABA I0 and i00 ~umol/kg and by the GABA B receptor agonist R-(-)-baclofen i, i0 and i00 }/mcl/kg; the maximum extent of inhibition amounted to about 50 - 60 %. S-(+)-Baclofen and the GABA A receptor agonists muscimol and 3-aminopropane sulphonic acid (each up to 1001umol/kg ) did not affect the electrically induced vasopressor response. The dose-response curve of R-(-)baclofen was shifted to the right by the GABA B receptor antagonist 2-hydroxysaclofen 50 ~ m o l / k g by a factor of i0, but was not affected by the GABA A receptor antagonist bicuculline I0 mol/kg. 2-Hydroxysaclofen and bicuculline by hemselves did not modify the electrically induced vasopressor response. The increase in diastolic blood pressure induced by exogenous noradrenaline was not affected by the GABArelated drugs, which also had no (or very slight) effects of the basal diastolic blood pressure. It is concluded that GABA inhibits catecholamine release in the resistance vessels of the rat via GABA B receptors, probably located presynaptically on the postganglionic sympathetic nerve fibres.

CARDIO- AND HAEMODYNAMIC EFFECT OF ZKl12.566, A DA2AGONIST/~2 -ANTAGONIST B.-G. Scbalz, D. Kurth, C. Molitor, G. Kersten

~

Institut f%r Pharmakologie, Universitfit Reuterstra~e 2b, D-5300 Bonn 1

Bonn,

ZK 112.566, a 3-(13-Amlno-2,3~aydro-2,6-dimethyl-8c~-ergolinyl)-1,1diethylurea, has been characterized in vitro as a DA2 agoulst with some ~2and minor o,.1-antagoulstie properties. The following report inchides studies on the cardio- and haemodynamlc effect of ZK 112.566 in vlvo. ZK 112.566 given intravenously lowered blood pressure dose dependently in spontaneously hypertensive rats (SHR). Maximal reduction of blood pressure (PM) after 1 mg/kg bw was 47 % of preapplicatiou value without concomitant reflex tachycardia. The DAwantagoulst domperidone significantly inhibited the ZK 112.566 induced fail of blood pressure in SHR. Oral application of 3 mg/kg bw induced a long-lasting reduction of blood pressure up to 3 h p.appl. In post-DOCA-hypertenslverats ZK 112.566 was as potent as in SHR concerning dose-dependent blood pressure lowering effects. In conscious normotensive rats ZK 112.566 was significantly less effective concerning blood pressure reduction. The cardio- and haemodynamic profile of ZK 112.566 in nonnotensive sodium pentobarbital anaesthetized Wistar rats (1 mg/kg bw) is characterized by its sympatholytic effect with reduction of peripheral resistance and a moderate reduction of cardiac output mainly due to bradycardia. In sodium pentobarbital anaesthetized dogs ZK 112.566 lowered blood pressure without increasing heart rate and without affecting renal blood flow. In conclusion ZK 112.566 is a potent blood pressure lowering drug in models with elevated sympathetic tone. Inhibition of the cardio- and haemodynamlc effect with domperidone suggests that the described cardioand baemodynamie effects are due to the DA,2-receptor agonlsm of ZK 112.566. Research Laboratories of Sclaering AG, Cardiovascular Pharmacology, Miillerstr. 170-178, D-1000 Berlin 65, West Germany

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EFFECTS OF THE DA2-AGON1ST/o~2-ANTAGONISTZK 112.566 ON BLOOD FLOW DISTRIBUTION AND INTRAMUSCULAR PO2 IN THE RAT F. M. McDonald, B. Maaf3

VESSEL WALL PROPERTIES 0F THE CAROTID ARTERY IN HYPERTENSIVE PATIENTS TREATED WITH VERAPAMIL AND NEBIVOLOL L.M.A.B. Van Bortel, T. van Merode, F.A.M. Smeets, R.0.B. Bhhm, R.S. Reneman

The ergoline derivative ZKl12.566 [3-(13-amino-2, 3B-d~hydro-2,6dimethyl-8cz-ergolinyl)-l,l-diethylurea] is a DA2-agonist/~2-antagonist with antihypertensive properties. We have examlned the effects of this compound on blood flow distribution in conscious normoteusivc and spontaneously hypertensive (SH) rats using radioactive microspheres and ultrasounddoppler crythrocyte (RBC) flux. We have also investigated its effcets in anaesthetlsed normoteusive rats on intramuscular PO 2 (Sigma PO 2 Histo~:aph KIMOC with 200 ~m needle electrodes) during ischaemia (20 rain femoral artery occlusion) and reperfusion of the gastrocnemins muscle. In normotensive rats (n = 10/group), ZK 112.566 0.5 and i mg/kg i.v. dose-dependently increased cardiac output by 26 and *49 %. The most marked increase in regional blood flow was to (hlndlimb) skeletal muscle, by 43 and * 76 % respectively, 20 rain post-treatment. This increase in flow was reduced by approx. 50 % by the DA2-antagonist domperidone, 2 mg/kg i.v. ZK ]12.566 tended to decrease visceral blood flow. These findings arc supported by those in SH rats (11' = 6), in which 0.1 mg/kg ZK 112.566 increased RBC flux in the descending aorta by 18 % and in the femoral artery by 37 %. In anaesthedsed rats (n = 8/group), femoral artery occlusion reduced PO 2 in the gastroenemius muscle by 71%, from 15.8 + 1.3 to 4.5 :!: 0.7 mmHg, with subsequent recovery on reperfusion (to 15.7 + 1.3 mmHg). ZK 112.566 1.5 mg/kg i.v. reduced the change in PO 2 during isehaemia to *-50 %, from 14.9-+ 2.0 to 7.3 + 1.5mmHg; PO 2 during reperfusiun was also higher than in untreated rats (19.5 + 1.9 mmHg). These results indicate that ZK 112.566 causes relatively selective dilatation of skeletal muscle vascular beds in the rat. This results in improved tissue oxygenation, even in the pathophysiologic condition of acute arterial occlusion. • : p < 0.05, compared to solvent control.

Arterial wall distensibility has been found to be diminished not only in hypertensive patients, but already in borderline hypertensives. A better arterial compliance has been suggested to protect the hypertensive patient from atherosclerotlc complications. In the present study the effects of verapamil (V; a calcium-antagonist) and nebivoioi (N; a novel, 8-adrenoceptor blocker) on carotid artery distensibility (DC) and cross-sectional compliance (CC) were studied non-invasively in hypertensive patients with the use of a high resolution multi-gate pulsed Doppler system. This system also allows the assessment of the internal artery diameter. Arm blood pressure measurements (BP) were made by means of an automated device (Dinamap). After a 4 week wash-out period, 19 patients (aged 21-73 y) with essential hypertension entered a double-blind randomized placebo (P) controlled cross-over study. Each patient was given 120 mg V or P 3 times daily f~r 4 weeks. At the end of the V per~od~ DC was 13.1+1.0.10 /kPa and CC averaged 4.3±0.3.10-" m /kPa. These values were statistically slgnifi~antly larger (p<0.05) t~an those after P (10.7± l.l.lO-~/kPa and 3.4±0.4.10 " m-/kPa, reap.). Using the same protocol, 29 patients (aged 25-70 y) were given 5 mg N o{ P once daily for 4 weeks. After N, DC (12.1+1.1. 10-~/kPa) and CC (3.7±0.3.10-- mZ/kPa) were 3 signiflcantly higher ~p<~.05) than after P (10.2±1.1.10- /kPa and 3.1± 0.3.10-" m-/kPa, reap.). In both studies no significant differences in diameter and pulse pressure were found between P and V or N. V decreased BP from 153±4/95±2 mmHg to 147±3/91±2 mmflg. N decreased BP from 155±3/97±2 mmHg to 145±3/90~2 mmHg. This study shows that both V and N may influence DC and CC of the CCA favorably resulting in a better management of the systolic flo~ jet from the heart.

Research Laboratories of Sehering AG, Cardiovascular Pharmacology, Miillerstr. 170-178, D-1000 Berlin 65, West Germany

Depts. of Pharmacology, Biophysics, and Physiology, University of Limburg9 P.O. Box 616, 6200 MD gaastrlcht, The Netherlands

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REBIVOLOL DOES NOT INTERACT WITH THE PURIRERGIG S~uEOTPANSMISSION IN PITHED NORNOTENSIVE RATS J. Schneider. C. Fruh. and B. Wllffert is a potent and seleetlve Bl-adrenoeeptor Nehivolol blocking drug which lowers peripheral vascular resistance by an unknown mode of action (Van de Water et al., J. Cardiovasc. Pharmae. ii: 552, 1988). The aim of this study was to investigate nebivolol's interaction with the purlnergic neurotransmisslon in the vascular wall of the pithed normotensive rat. Electrical stimulation of the entire or local (Th5-L4) sympathetic outflow of the spinal cord was performed via the pithing rod after bilateral adrenalectomy and vagotomy. Neblvolol inhibited dose-dependently (10-7-10 -5 mol/kg, i.v.) the increase in heart rate (HR), but did not affect the increase in diastolic blood pressure (DBP) elicited by electrical stimulation of the entire sympathetic outflow of the spinal cord (time of stimulation: 25 s). Since there are indications that the purinerglc neurotransmission is more pronounced in short pulse-trains we studied the effect of nebivolol on the purlnerglc neurotransmlssion by using a shorter time of stimulation, i.e. 5 s. In order to prevent marked effects on heart rate we stimulated at the level of Th5-L4, thus providing a stimulation frequency dependent increase in DBP which was attenuated by =l- and ~2-adrenoceptor blockade. The remaining increase in DBP was not affected by nebivolol (10-6 mol/ kg, i.v.; -15 and -60 min). Desensitization of P2x-receptors with =,B-methylene ATP (2.65 mg/kg, i.v., as a cumulative dose) almost abolished the blood pressure response remaining after =I- and =2-adrenoceptor blockade. The i.v. administration of a single dose of a,Bmethylene ATP (0.01 mg/kg) produced a pressor response, which was not affected by pretreatment (-15 mln, i.v.) with nebivolol at a dose of 10-5 mol/kg. In conclusion, nebivolol neither affected the purinergic neurotransmission nor interacted with P2x-receptors in the vascular wall of the pithed normotensive rat. JANSSEN RESEARCH FOUNDATION, D-4040 Neuss 21, F.R.G.

CARDIOVAS(I/IAR EFFECTS OF THE INHIBITOR OF THE YHOSPHODIE~'I'~/~ASEIII, ADIBI~qDAN, IN COMPARISON TO NITROPRUSSIDE AND DO AMI IN CONSCIOUS DOGS A. Dorszewskl, B. Miiller-Bec~mmann, G. Sponer ~he aim of this study was to characterize the hemodynamic effects of adibendan (~M 14.478; 7,7-dimethyl-2-(4pyridyl)-6,7-dihydro-3H,5H pyrrolo [2,3-f] benzimidazol6-one), whether they ere due to both its cardial positive inotropic and its peripheral vasodilating properties or to a predominant vasodilation with concomitant reflex stimulation of the heart. Dose response curves were performed for adibe/ndan (A) by i.v. injections of incremental doses and for nitroprusside (N) and dobutamine (D) by i.v. infusions. The effects of A at a dose range from 0.01 - 0.03 Iag/kg resembled those of D (i.0 - 4.0 ~g/kg/min). Both drugs increased left ventricular dp/dt60 , cardiac output (CO) alld stroke volume (SV) significantly, while heart rate (HR) and blood pressure (BP) remained unchanged. The most obvious difference was a pronounced fall of left ventricular enddiastolic pressure (LVEDP) with A. With higher doses of D (8.0 - 20.0 Gg/kg/min) BP and LVEDP were only lowered slightly but HR rate increased alarkedly. Higher doses of of A (0.I 1.0 ~ag/kg) increased HR and decreased LVEDP, BP and SV to the same extend as N (0.5 - 12.5 ~g/kg/min), but in contrast to N A produced clear additional effects on dp/dt60 and CO. Out of these results it is concluded that both the peripheral vascdilating and the cardial positive inotropic action of adibendan contribute clearly to its general hemodynamic profile. Depar~t of Pharmacology, Boehringer M ~ e i m Sandhofer Str. 116, D-6800 Mannheim 31, FRG

(~nbH,

R 60 237 HELENALIN AND 11~,13-DIHYDROHELENALIN, TWO CONSTITUENTS FROM ARNICA MONTANA L., INHIBIT HUMANPLATELET FUNCTION VIA THIOL-DEPENDENT PATHWAYS W. LOsche*, U. T i l l * , H. Strobach, W. Leven**, G. Willuhn**, K. SchrOr This study investigates the effect on human p l a t e l e t function of two sesquiterpene lactones from Arnica montana L., helenalin (H) and lIK, 13-dihydrohelenalin (DH). The compounds were found to i n h i b i t collageninduced p l a t e l e t aggregation, thromboxane formation and 5-hydroxytryptamine secretion in a concentrationdependent manner at 3-300 ~M. In the presence of collagen, H- and DH-induced i n h i b i t i o n of p l a t e l e t aggregation was accompanied by a decrease in thromboxane formation. However, when arachidonic acid was used as a stimulus, thromboxane formation remained unaffected despite i n h i b i t i o n of p l a t e l e t aggregation. Both H and DH reduced the number of acid-soluble sulfhydry! groups in platelets, by up to 78%. This effect occurred at concentrations similar to those that inhibited p l a t e l e t aggregatory and secretory responses. Moreover, H- and DH-induced p l a t e l e t i n h i b i t i o n could be prevented by the t h i o l containing amino acid cysteine. I t is concluded that H and DH i n h i b i t p l a t e l e t function via interaction with p l a t e l e t sulfhydryl groups, probably associated with a reduced phospholipase A2 a c t i v i t y . Institut for Pharmakologie der Heinrich-HeineUniversit~t, Moorenstr. 5, 4 0 0 0 DOsseldorf, FRG; * I n s t i t u t for Pathobiochemie der Medizinischen Akademie Erfurt, NordhBuser Str. 74, 5010 Erfurt, GDR; * * I n s t i t u t for Pharmazeutische Biologie der Heinrich-HeineUniversit~t DOsseldorf, Universit~tsstr. I, 4000 DOsseldorf, FRG.

239 INHIBITION OF PROGRESSION OF A T H E R O S C L E R O S I S IN RABBITS BY DALTROBAN J. PILL*, O. Wolf, A. Schmelz, and J. Metz Daltroban, c h a r a c t e r i z e d as r e c e p t o r a n t a g o n i s t for t h r o m b o x a n e A 2 and endoperoxides (Stegm e i e r et al. N a u n y n - S c h m i e d e b e r g ' s A r c h Pharmacol (1986) 332 (Suppl.): R 36), r e d u c e d in rat hepatocyte cultures 14C-acetate i n c o r p o r a t i o n into cholesterol (CH)-ester in a d o s e - d e p e n d e n t manner. CH-ester a c c u m u l a t i o n as well as platelet a g g r e g a t i o n is involved in the d e v e l o p m e n t and p r o g r e s s i o n of atherosclerosis. We investig a t e d a t h e r o s c l e r o s i s in WNZ rabbits (n=24) fed a diet c o n t a i n i n g 0.5% CH: (A) for 42 d, n=6; (B) for 96 d, n=9 and (C) for 96 d, treated w i t h d a l t r o b a n I0 m g / k g / d from 42-96 d, n=9. CH levels in serum exceeded i000 mg/dl after 3 weeks and reached p e a k levels of about 1800 mg/dl in all animals. Total CH c o n t e n t of the aortic wall increased d r a s t i c a l l y in (B) but was d i m i n i s h e d about 30% in (C). Morphometric studies at the light m i c r o s c o p i c level were p e r f o r m e d on aortas divided into 20 segments. M e a s u r e m e n t s of outer and luminal perimeter and areas, covered surface of the aortic wall and area of plaque p r o t r u s i o n w e r e c a r r i e d out. The results of all segments summarized show a significant reduction (p
238

240

FENOFIBRATE AS A VASOPROTECTIVE AGENT IN ISOLATED TAIL ARTERIES OF RATS FED WITH ATHEROGENIC DIET H.I. Trzeeiak and B. Okopie~

-832.

Acute a n t i h y p e r t e n s i v e action of Cicletanine m e d i a t e d by muscarinic r e c e p t o r stimulation ~. Chunqt P. Rohmeiss t S. Rohmeiss C i c l e t a n i n e (Cic) is a novel a n t i h y p e r t e n s i v e belonging to the furopyridine class of drugs. It has been speculated that the blood p r e s s u r e (MAP) lowering effect of Cic is due to a stimulation of endogenous prostacyclin synthesis. In c h r o n i c a l l y instrumented conscious s p o n t a n e o u s l y h y p e r t e n s i v e rats intravenous (iv) infusions of Cic (125; 188; 250 mg/kg/h) led to a d o s e - d e p e n d e n t d e c r e a s e in MAP of m a x i m a l l y 36±2.7 mmHg. Equipotent iv depressor infusions of iloprost, a synthetic prostacyclin derivative, produced markedly different haemodynamic responses in cardiac output, renal, m e s e n t e r i c and hindlimb blood flow (pulsed Doppler) and splanchnic nerve activity. The MAP lowering effects of Cic were not influenced by iv p r e t r e a t m e n t with the cyclooxygenase inhibitor, indomethacin, but were abolished by the muscarinic receptor blocking agent, methylscopolamine. B e t w e e n the two Cic enantiomers, iv bolus injections of [-]Cic decreased MAP and heart rate significantly more than those of [+]Cic. Preliminary experiments with more specific MI, M2, M3 antagonists (Pirenzepine, A F - D X 116, Hexocyclium) suggest that Cic acts on all three muscarinic receptor subtypes. Our results provide no evidence for an involvement of prostaglandins but point to an important role of m u s c a r i n i c receptors for the acute MAP lowering effect of Cic.

Department of Pharmacology, Silesian Academy of Medicine Katowice, Poland.

Department Heidelberg,

The sensitivity to noradrenaline (NA) of isolated and perfused tail arteries prepared from Fats maintained on a%herogenic diet for one month was investigated. Fenofibrate was administered in a dose of i00 mg/day with a stomach caniula from the onset or the 15-th day of experiment. The data obtained as above were compared with the effect of NA on control arteries. The sensitivity of rat tail arteries was established according to Nicholas (1969). As far as arteries of rats fed with atherogenic diet is concerned that vasoconstrictor response was attenuated. When fenofihrate was administered from the onset of experiment it saved such responses in control arteries, while when administered from the 15-th day of experiment it had no such effect. We suggested that fenofibrate represents a class of vasoprotective agents and therefore may be effective in the management of arteriosclerosis. Nicholas T.E.: J. Pharm. Pharmae., 1969, 21, 826-

of Pharmacology, University INF 366, D-6900 Heidelberg, FRG.

of

R61 241

243

PHARMACOLOGICAL (~{~RACTERISATION OF t - P A PRODUCED IN NSCllN~CltY.A OOZE / ~ ' J . ~ SINGLE BOLUS INJECTION IN RABBITS U. Martin, S. Fischer, U. Eohnert, H. LiIl, R. Rudolph, A. S t e r n a n d K. Strein

SIMULATION AND TREATMENT OF INTERMITTENT CLAUDICATION IN DOGS WITH UNILATERAL LEG ISCHEMIA INDUCED BY INJECTION OF SEPHADEX AND ARTERIAL LIGATION E. B o h m , R. S c h w a l l , L. K l i n g

R e c c m ~ i n a n t t - P A w a s produced b y e x p r e s s i o n of t - P A in E. c o l i a n d subsequent renaturation (HM 06.021). In contrast to t - P A p r o d u c e d in eucaryotic cells (Alteplase, T h o m a e GatbH, Biberach, FI~G), HM 06.021 lacks carbohydrate side chains. ~ a i m of o u r studies w a s to investigate t h e ~ l y t i c p o t e n c y a n d pharmacokinetics of I ~ 06.021 a f t e r single i.v. bolus injection. In a rabbit model of jugular v e i n ~ i s ~M 06.021 or A l t e p l a s e w a s injected o v e r 1 m i n at 4 different dose levels (n = 6 / ~ ) . 1 h o u r a f t e r injection, the rate of ~ l y s i s w a s determined. Plasma concentrations of fibrinogen a n d plasminogen w e r e evaluated b e f o r e and 2 h o u r s a f t e r injection. Plasma samples b e f o r e and after injection of ~ M 06.021 or Alteplase w e r e assayed for t - P A activity b y a spectrophotcmetric m e t h o d w i t h Chromo-

~mR~. T h e analysis of t h e dose-respoDse curves showed that 256 k U / k ~ b w o f BM 06.021 add 623 kU/kg b w of A l t e p l a s e induced a rate of 45 % ~ l y s i s w h i c h corresponds to h a l f m a x i m u m effect in t h e model. Both plasminogen activ a t o r s caused a similar extent of fibrinogen-breakdown a n d plasminogen-constmption. A t equipotent doses (400 vs. 800 k U / k g bw) H M 06.021 demonstrated a 2-fold slower total p l a s m a clearance co,oared w i t h A l t e p l a s e (6.1 _+ 1.4 vs. 12.9 + 1.7 m l . m i n - l ' k g -I, m e a n _+ SD). In conclusion, H M 06.021 appears to h a v e improved pharmacokinetic p r o p e r t i e s w h i c h result in an increased thromb o l y t i c p o t e n c y after single bolus injection in a rabbit m o d e l of ve3xDus ~ i s . Furthermore, ~M 06.021 seems t o h a v e retained a relative fibrin-specificity like A I -

tepl~.

So far no dog model exists which is useful for the reproducible simulation of intermittent claudication on a treadmill. Aim of this study was to develop such a model and to assess whether it is appropriate for demonstrating the effect of hemodilution as an exampLe of a therapeutic approach. Method: In 12 mongrel dogs I mg/kg b.w. superfine Bephadex was injected into the femoral artery of the [eft hind leg. Subsequently this vessel and its side branches were ligated and extirpated, After a recovery period of I week, the walking capacity of the dogs was determined three times a week for 3 weeks on a treadmill at a constant velocity of 6 km/h. The waLking time (divided into 5 categories (t) of 3 min each; maximum: 15 min) and the degree of Limping (= L; divided into 5 categories) were taken as c r i t e r i a of the walking capacity. From t h i s values a score (S) was caLculated according to the equation: S = 5 x ( L - l ) + f . 6 dogs were treated by d a i l y infusion of 20 mt/kg b.w. HAES 10 % (Hydrox y e t h y l s t a r c h ) over a period of 3 weeks, the other 6 dogs served as a reference group. Results: ALl dogs easily reached the maximum score of 25 before the oper a t i o n . Afterwards the median of the score f o r a l l 12 dogs was reduced to 9 (range 1 to 15). The effect of the hemodilution is shown in the

foL towing table. Baler ....

Treatment eks)

Median q Seer

IInitial S | 9 . 5 | I

LJ

1

| 7.0 /

2

7.9

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Significance " n.s.

n.s. ....

,a o d i l o t i o n

Median I Significance Score -7.5 5.8

13.o| 15.8

|

" n.s.

oo.o5 p
These results show that chronic i n t e r m i t t e n t claudication in dogs can be induced by i n j e c t i o n of Sephadex and subsequent l i g a t i o n of the femoral a r t e r y and i t s side branches. Furthermore, the therapeutic e f f e c t of hemodilution beginning in the 2nd week of treatment could be demonstrated. ObviousLy the dog model is a useful p r e c l i n i c a t test f o r therapeut i c approaches for improving the walking capacity of patients with

intermittent claudication. D e p a r t m e n t of Pharmacology, Boehringer M a n n h e i m S a n d ~ o f e r Str. 116, D-6800 M a n n h e i m 31, F~G

galbH, Abteilung for Herz-/Kreislaufpharmakologie, Boehringer Mannheim, Sandhof e r Sir. 116, D-6800 Mannheim 31, FRG

242

244

THE PAr-ANTAGONISTS WEB 2086 AND WEB 2170 IMPROVE ENBOTOXIN-INDUCED DISTURBANCES IN THE MICROCIRCULATION IN RAT CREMASTER MUSCLE. B. Hergenr6der and Reich[ R.

N E U T R A L I S A T I O N OF H I R U D I N A N T I C O A G U L A N T A C T I O N BY A C T I V E - S I T E BLOCKED THROHBINS Ellen BrOggener, P, W a l s m a n n

Septic shock is a frequent complication of acute systemic infections and it is also known that the platelet activating factor (PAr) plays an important rote as a mediator of the septic shock (A. M. LEFER, Cicu[atory Shock 27:3-12, 1989). in this study the endotoxin-induced shock was used as an experimentaL model to test the effect of the s p e c i f i c Paf-antagonists WEB 2086 (3-[4-(2Chtorophenyl)-9-methyl-6Hthienor3,2- f ] [1,2,4] t r i a z o t o - [4,3-a] [1,4] -diazepin-2-yt]-1-(4-morphoLinyt)-l-propanone) and WEB 2!70 (8-(R,S)-6-(2Chlorophenyt)-8,9-dihydro 1-methyt-8-[(4-morpholinyl)carbonyi]-4a,7B-cyctopenta[4,5]thieno[3,2-f] [ 1 , 2 , 3 ] t r i a z o t o i 4 , 3 - a l [1,4]diazepine). In anesthetized rats the influence on systemic blood pressure, heart rate, and especially microc i r c u l a t o r y parameters such as vessel diameter in a r t e r i o l s , red cell v e l o c i t y and calculated microflow was evaluated in the cremaster muscle. the test procedure was divided into a preparatory control period, followed by bolus i n j e c t i o n of saline and the drugs WEB 2086 or WEB 2170 with 1 mg/kg i . v . , and endotoxin (E. coli 0111:B4) with 30 mg/kg i . v . and a f i n a l follow-up phase of 120 minutes. The endotoxin-induced shock showed a c h a r a c t e r i s t i c biphasic course. The f i r s t phase of about 30 minutes consisted of an increase in blood pressure accompanied by a vasoditation and an increased microcirculatory blood flow. i t was followed by a decrease in the s y s t o l i c and especia l l y in the d i a s t o l i c blood pressure, by a vasoconstriction to 77 % of the i n i t i a l value and a decrease in microflow to 30 % of the control value.

With the doses used quite clear but also differing therapeutic effects are obtained with the Paf-antagonists WEB 2086 and WEB 2170. Both WEB 2086 and WEB 2170 completely restore the s y s t o l i c blood pressure, whereas the decrease in d i a s t o l i c bLood pressure is affected s i g n i f i cantly only by WEB 2170. The heart rate shows no s i g n i f i c a n t d i f f e r e n ces. Only WEB 2170 antagonizes s i g n i f i c a n t l y the endotoxin-induced vasoconstriction and the reduced microftow. The anti-shock e f f e c t produced especially by WEB 2170 is based on i t s action on the m i c r o c i r c u l a t i o n . This publication is part of a doctoral thesis. Dept. of Pharmacology, Boehringer Ingelheim KG, D-6507 Ingetheim, FRG

H i r u d i n is a s p e c i f i c t i g h t - b i n d i n g t h r o m b i n inh i b i t o r , I t is i n t e n d e d for clinical use because of its strong anticoagulant and antithrombotic effects. Since bleeding is a dangerous complication of antithrombotic therapy a specific antidote would be a d v a n t a g e o u s that forms inactive complexes with hirudin. We p r e p a r e d active-site modified, enzymatically inactive thrembins by reaction of the enzyme with phenylmethyl~ulfenylfluoride (PMSF), diiseprepylfluorophosphate (DFP), amineethylbenzenesulfenylfluoride (AEBSF), 4-amidinophenylbenzoate and tosyllysylchlorcmethylketene (TLCK). These thrembinderivatives which are still able to bind hirudin in vitro, were administered to experimental animals in order to demonstrate the hirudin neutralieation in vivo. Rats received 1 mg h i r u d i n / k g subcutaneously. After 90 min a hirudin level of 0,5 ug/ml plasma w a s r e a c h e d , Curing the following 10 m i n t h e active-site blocked thrombine were infused at a dosage which corresponds to the hirudin binding capacity of 600 IU thrombin. After PMS-thrembin administration no m o r e h i r u d i n was d e t e c t a ble in plasma for a short period of time. After application of DIP-thrombin, AEBS-thrombin and benzoyl-thrombin, the hirudin level decreased to 40%, whereas only a weak effect was g a i n e d with TLCl<-thrombin. The decrease in the hirudin level after the infusion of the thrombin derivatives was followed by a rapid increase in the hirudin activity afterwards. The modification of thrombin by blocking its active centre without impairing the binding to hirudin may p r o v i d e e useful concept for the development of a hirudin antidote. Inetitut f~r Pharmakologie und Medizinischen Akademie Erfurt, Str 74, DDR-5OIO Erfurt

Toxikologie Nordh~user

der

R 62

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247

HORMONAL REGULATION OF THE L-TYPE CALCIUM CHANNEL CURRENT IN FRESHLY ISOLATED ADULT BOVINE TRACHEAL ~MOOTH MUSCLE CEL~S A n d r e a W e l l i n g ~, J o c h e n F e l b e l ~, K l a u s P e p e r 1 and Franz Hcfmann 2

~.T~--I-3 3 ~ TRANSI~ C ~ DIIATION •O THE RELEASE OF Et~F, NOT PGI2 , ~ RAT HEARTS. W Wienen, ABM Mauz

i. F r e s h l y isolated adult bovine tracheal smooth muscle cells were voltage clamped by the tight-seal whole-cell technique. 2. D e p o l a r i z i n g v o l t a g e s t e p s f r o m a h o l d i n g p o t e n t i a l of -60 m Y t o 0 m V e l i c i t e d a transient negative current carried by calc i u m (L-type). It w a s r e v e r s i b l y b l o c k e d by Ni-ions, Nitrendipine and increased by the calcium channel agonist. 3. T h e c a l c i u m p e a k c u r r e n t (Ica) was inc r e a s e d 2-3 fold b y i s o p r o t e r e n o l (EC50: 1 t o 6 nM). T h e i s o p r e t e r e n o l e f f e c t w a s m e diated by the ~-receptor. 4. I n t e r n a l d i a l y s i s of t h e c e l l w i t h c A M P (100 ~M) o r c A M P - k i n a s e h a d n o e f f e c t on b a s a l or i s o p r o t e r e n o l s t i m u l a t e d Ica. 5. C a r b a c h o l i n h i b i t e d t h e i s o p r o t e r e n o l s t i mulated I Conclusion: ~ t y p e c a l c i u m c u r r e n t is r e g u l a t e d b y t h e E - r e c e p t o r in f r e s h l y i s o l a t e d b o vine tracheal smooth muscle. Apparently, the regulation does not involve cAMP or cAMP-kinase. l: P h y s i o l o g i s c h e s I n s t i t u t 2: F.R. M e d i z i n i s c h e B i o o h e m i e M e d i z i n i s c h e Fakult&t, U n i v e r s i t ~ t landes, D - 6 6 5 0 Ho~Lburg-Saar

des

Saar-

IS DUE

The effect of endothelin-i (ET-I) on coronary flc~ and its underlying m e c / ~ m n ( s ) were investigated in isolated, constant-flow perfused rat hearts. Administration of ET-I (I*IO~8M) for 12 min induced an instantaneous and transient (i rain) decrease in perfusion pressure frc~ about 85 to 45 ~m~g, followed by a progressive rise to about 140 m~Hg. This was accompanied by a pronounced increase in the venous release of 6-keto-PGFlm (PG) frc~ 1.26 pg/~i~ (i rain before EI~-I) to 6.2, 23.1 and 12.7 pg/min resp. at i, 5 and i0 rain after administration of ET-I. In hearts, pretreated with ind~methacin {3,I0-6M), the release of PG was completely abolished, but coronary response to ET-I was not significantly different from that of control hearts with respect to initial coron@zydilation and later constriction. Bradykinin (i*i0"I0 to I*Io-9M; bolus injection) induced a concentration- and endothelitma-dependent decrease in coronazy perfusion pressure (to about max. -30%), which was virtually abolished after treatment of these hearts with methylene blue (MB). In accordance to this, the initial transient decrease in perfusion pressure after infusion of ET-I was also abolished after pretreatment with MB. In contrast to these results, the coronary-dilating effect of sodium nitroprusside (I*10-7M; bolus injection) was not altered after the addition of MB. It is concluded from thence experiments that the initial and transient coronarydilating effect of ET-I is predominantly mediated through the spontaneous release of EDRF in rat hearts. In addition, Er-i is a very potent stimulant for the cardiovascular de-novo synthesis of PGI2 . However, the initial ET-induced coronary-dilation is not related to the release of this endogenous vasodilator nor is the coronary-coD~icting effect of h~f-i attenuated by PGI2 under the experimental conditions tested. Department of Pharmacology, Pharma Research, Dr. Karl ~33~mae GmbH, D-7950 Biberach i, FRG

246

248

ELECTRICAL AND MECHANICAL EFFECTS OF ENDOTHELIN IN PULMONARY AND CORONARY VASCULAR MUSCLE G. Haeusler and J.-E. de Peyer

K+-CHANNEL OPENING CONTRIBUTES TO THE RELAXANT A C T I V I T Y O F F O R S K O L I N IN V A S C U L A R S M O O T H M U S C L E S E. Klaus, H. E n g l e r t , M. H r o p o t , H. M e t z g e r , H. Tilly, a n d G. W i e m e r

The experiments were carried out on strips of pig coronary artery (PCA) and of rabbit aorta (RA) and main pulmonary artery (RMPA). Mechanical tension was measured isometrically and changes of membrane potential with intra~ellular7 glass micro-electrodes. Over the range of 3-10 to 10 M porcine endothelin (endothelin-1, ET-1) produced concentration-dependent contractions which were slow in onset. As compared to noradrenaline, the maximum vasoconstrictor effect of ET-I was for the RA and RMPA 30% and 50%, respectively. In the PCA maximum contractions to acetylcholine and ET-1 were similar. Removal of the endothelium augmented the contractions in response to ET-I in the RA, but had no major effect in RMPA and PCA. ET-I depolarized in a concentration-dependent manner the membrane of the vascular smooth muscle cells of the PCA and the RMPA. The resting membrane potential of the muscle cells of PCA varied around a mean value of -55mV; maximally effective concentrations (I0- M) of ET-I reduced it to -42mV. Respective depolarization was less in the RMPA, namely from -58mV to -52mV. Nifedipine and nitrendipine did only igcompletely relax vascular strips contracted by ET-I (i0- M). By contrast, EMD 52 692 (6-cyano-2,2-dimethyl-4-[oxo-l,2-dihydropyridine-lyl]-2H-l-benzopyran), a potent potassium channel activator, completely antagonized ET-l-induced vasoconstriction. The inhibitory effect of END 52 692 was similar irrespective of whether contraction was produced by ET-1 or noradrenaline. Although vasoconstriction in response to ET-1 is, at least in part, dependent on calcium entry from the extracellular space, it does not seem that this occurs exclusively through dihydropyridine-sensitive calcium channels. However, depolarization by ET-1 and relaxation by EMD 52 692 suggests that smooth muscle contraction by ET-I may involve a potential-linked mechanism.

B e s i d e s a c t i v a t i o n of a d e n y l a t e c y c l a s e h y p e r p o l a r i z a t i o n of t h e cell m e m b r a n e b y f o r s k o l i n (FO) has been observed in several tissues including heart, smooth muscles and neurons. However, the underlying mechanism remains e l u s i v e and has b e e n a t t r i b u t e d t o a s t i m u l a t i o n of t h e N a + / K + - A T P a s e , a c t i v a t i o n of K + - c u r r e n t s o r t o a s t i m u l a t i o n of e l e c t r o g e n i c C a + + - p u m p S o T h e p r e s e n t s t u d y s h o w s t h a t g l i b e n c l a m i d e (GL) previously shown to inhibit vascular ATPdependent K+-channels interferes with FOs r e l a x a n t a c t i v i t y in v e s s e l s . FO (0.4 a n d 1 ~M) relaxed KCl-precontracted isolated thoracic a o r t i c s t r i p s of g u i n e a pigs. G L (0.5 a n d 5 ~M) d e c r e a s e d r e l a x a n t a c t i v i t y of FO t o 76 a n d 63 % of p r e t r e a t m e n t v a l u e s , r e s p e c t i v e l y . FO (0.i i0 ~M) h y p e r p o l a r i z e d c o n c e n t r a t i o n - d e p e n d e n t l y (up to 20 mY) t h e cell m e m b r a n e p o t e n t i a l of rabbit pulmonary artery which could be abolished b y G L (i - i0 ~M). FO (I~M) a l m o s t c o m p l e t e l y inhibited contractions in i s o l a t e d g u i n e a p i g a o r t a t h o r a c i c a p r e c o n t r a c t e d w i t h 25 m M KCl, whereas no activity was found in vessels precontracted with 50 mM KCI. FO induced stimulation of adenylate cyclase was not a f f e c t e d b y G L (I00 ~M). O u r r e s u l t s i n d i c a t e t h a t FO i n d u c e s h y p e r p o l a r i z a t i o n 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 v i a o p e n i n g of G L - s e n s i t i v e K+-channels. This hyperpolarization contributes t o FOs r e l a x a n t activity. In t h i s r e g a r d o t h e r a g e n t s k n o w n to e x e r t r e l a x a n t a c t i v i t y m a i n l y b y an i n c r e a s e of c - A M P w i l l b e a s s e s s e d .

Pharmaceutical Research Department, E. Merck, Frankfurter Strasse 250, D-61O0 Darmstadt, FRG

H o e c h s t AG, M a i n 80

Postfach

800320,

D-6230

Frankfurt

am

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GLIBENCLAMIDE DOES NOT INHIBIT ENDOTHELIUM-MEDIATED 86RB° EFFLUX FROMRAT AORTAAND PIG CORONARY. U. Quast and Y. Baumlin

Spiperone-~ced relaxation of pig coronary artery is probably mediated by ATP release G.J. Molderin~s

Stimulation of the endothelzum by agonzsts llke acetylcholine (ACh} or substance P (SP) leads to the release of factor(s) which relax and hyperpolarzze vascular smooth muscle. In the arterla cerebrz media of the rabbit thls hyperpolarzzatlon is inhlblted by glibenclamlde (I0 NM), suggesting that it ls due to an opening of ATP-sensztzve K ~ channels (KA, p) (Standen et al., Selence 245, 177 (1989)). Since K ^ ~ in all tissues examined thus far is Rb ~ permeable, we have examined the gllbenclamlde sensitivity of 86Rb ~ efflux stimulated by ACh from the rat aorta or SP from the plg coronary artery.

The effects of spiperone on vascular smooth muscle were determined in rings of the pig coronary artery contracted with the thrcmlboxane analogue U 46619 (9,11-dideoxy-ll~ 9 o ~ -epoxy-methano-prostaglandin F2~. Spiperone (1-30 _~i/i) induced a complete, endothelium-dependent relaxaItlon ~ at higher concentrations (30-300 ~ o i / i ) an endothellL~n-independent contraction of the artery . Suramin (300 ~mo!/l) and reactive blue II (I00 ~mDl/l) antagonized the relaxing effect, whereas metltepine (0.i-I0 ~nol/l), spiroxatrine (i p~ol/l), propranolol (4)/tool/l), idazoxan (3 ~nol/l), flupenthixol (i 2mnol/l), atropine (i ~a~ol/l) and 8-phenyltheophylline (I ]amml/l) did not. Nucleotide pyrophosphatase (0.25 U/m/) shifted the concentration-response curve for spiperone to the left. The er~othelium-independent contraction was reduced by surarain (300 pmol/l) and 8-phenyltheophylline (I ~nol/l) but not by metitepine (i0 ~m~i/l). - In arterial strips of porcine coronary artery incubated with [3H]adenosine (0.2 ~m~i/l) and subsequently superfused with physiological salt solution containing dipyridamol (30 ~m~I/l) the effect of spjn p~rone on tritium efflux (which is assumed to reflect [ H]ATP release) was investigated . Spiperone (i0 ~m~)i/l) evoked an increase in tritium overflow above basal efflux from arteries with intact endothelium but considerably less from those in which endothelium was destroyed. We suggest on the basis of the present data that spiperone at the concentrations investigated induces a release of ATP from the pig coronary artery. ATP, in turn, probably activates endothelial P2-purinoceptors, leading to the release of an endothelium-derived relaxing factor with subsequent relaxation of vascular smooth muscle.

Rings were loaded with 86Rb* and the 86Rb ÷ efflux determined (Quast, Br. J. Pharmac. 91, 569 (1987)). Basal rates (k, in 10-a/mln) were 9 ± l'(aorta) and 3.5 ± 0.3 (coronary). Stimulation with ACh (aorta) or SP (coronary) led to concentratlon-dependent, transient (% min I increases in k. These effects required an intact endothelium. Stimulation for 5 mzn by ACh (i0 ~M, aorta) or SP (30 nM, coronary} gave peak increases in k by 31 ± 4~ and 61 ± 5 ~. A second challenge after a recovery perlod of 50 mln gave a significantly smaller response (75~ or 60~ of the first response in aorta or coronary, respectively). Pretreatment with glibenclamide (3 or I0 HM) for 20 mln before the second challenge did not inhibit the increase in k but abolished the increase in 86Rb ÷ efflux Induced by cromakalzm (0.3 and

3 ~M). It Is concluded that in rat aorta and pig coronary artery cromakallm and the endothelzum-derlved hyperpolarzzzng factor (EDHF) act on different K ~ channels. The results provlde no support for the view that in these two (large) vessels EDHF-induced hyperpolarlzation is mainly carried by glibenclamlde-sensitive (KA,m) channels.

Institute of Pharmacology and Toxicology, University of Bonn, Reuterstr. 2b, D-5300 Bonn i, Fed.Rep. Germany

Sandoz AG, Praklinische Forsohung, CH-4002 Basel.

250

252

COMPARISON BETWEEN ISOBUTYL I S O B U T Y L NITRITE: T O L E R A N C E OF A N D V E N O U S STRIPS P R E I N C U B A T E D TRINITRATE. T. Zimmermann,

M. L e i t o l d

NITRATE AND RABBIT AORTIC WITH GLYCERYL

and R.A.

Yeates

This s t u d y i n c l u d e s the first s y s t e m a t i c comp a r i s o n of an o r g a n i c n i t r a t e w i t h the corresponding organic nitrite isobutyl nitrate and i s o b u t y l nitrite. The s p a s m o l y t i c a c t i v i t y of the n i t r i t e on i s o l a t e d rabbit a o r t i c and venous strips was s t r o n g e r than the a c t i v i t y of the nitrate. In v i t r o t o l e r a n c e to g l y c e r y l trinitrate greatly weakened the a c t i v i t y of i s o b u t y l nitrate, but had m u c h less e f f e c t on i s o b u t y l nitrite. P o s s i b l e r e a s o n s for these differences are d i s c u s s e d . The p a p e r demons t r a t e s the m u l t i f a c t o r i a l n a t u r e of t o l e r a n c e development in aortic and venous smooth muscle. Dept. of Pharmacology, Pfizer/Heinrich Mack Nachf. Chem.-pharm. Fabrik, P.O.Box 2064, D-7918 Illertissen,FRG

NO GENERATIONBY ENDOTHELIUM-DEPENDENTVASODILATORS IS IMPAIRED AFTERMYOCARDIAL ISCHEMIA AND REPERFUSION I. Woditsch This study compares the NO release by bradykinin (BK), substance P (SP) and g l y c e r o l t r i n i t r a t e (GTN) with coronary vasomotor responses before and a f t e r ischemia + reperfusion (MI + RPF). Langendorff hearts of rabbits (n = 8) were perfused at constant flow and subjected to 2 h of low-flow MI and subsequent RPF f o r 0.5 h. BK (0.05 i~M), SP (0.05 ~M) and GTN (40 ~M) were infused f o r two periods of 3 min each, ie. before and a f t e r MI + RPF. Coronary perfusion pressure (CPP) was continously monitored and NO was measured online in the coronary effluent by the oxyhemoglobintechnique: before MI I after MI + RPF Decrease in CPP Decrease in CPP Agent [mmHg] [%] NO [nM] I [mn~Hg] [%] NO [nM] BK SP GTN

16 +3 14~3 22~3

34 +5 25 +5 357-4 34~5 397-4 43~7

24 +5 30~6 35T6

33 +6 12 +3* 43~7 19¥3" 43+-'/ 35T-4

The basal CPP rose from 40 + 5 mmHg beforeMI to 66 + 11 mmHg after MI + RPF. SP an~ BK induced NO generation-was significantly(*) diminished after MI + RPF, while NO generation from GTN and coronary vasodilation by all substances were unaffected. BK and SP induced PGI~ release (6keto-PGF~L) remained unchanged after MI + R~. These data suggest 6C impairment of endothelial NO generation in reperfused ischemic hearts which might contribute to the increased basal CPP but does not affect BK, SP and GTN induced vasodilation. Institut for Pharmakologie, Heinrich-Heine Universit~t, Moorenstr. 5, D-4000 Dfisseldorf, FRG

R 64 253 REGULATION OF SOLUBLE GUANYLATE CYCLASE HUMAN PLATELETS BY CARBONMONOXIDE ANIONS SUPEROXIDE K.-U. Schmidt

FROM AND

Human platelet guanylate cyclase activity w a s studied with respect to the function of its heme-containing regulatory subunit. The partially purified enzyme could be stimulated by carbonmonoxide (CO) and sodium nitroprusside (SNP) in a dose dependent manner. Superoxide anions generated by the xanthine/xanthine oxidase system were strongly inhibitory in the enriched preparation as well as in the CO stimulated platelet supernatant (ECs0 = 0.i mU/ml). Unlike CO and SNP the effect of superoxide cannot be mediated through the heme-regulatory subunit, since heme-free and nonstimulable guanylate cyclase w a s inhibited to the same extent as the heme containing enzyme and al,l attempts to obtain a spectral shift in the presence of xanthine/ xanthine oxidase were unsuccessful. No effect of superoxide was seen in the presence of Mn 2+ ions which correlated with their dismutase activity. Superoxide turned out to be a potent and reversible inhibitor of soluble guanylate cyclase which, together with Endothelium Derived Relaxing Factor (EDRF), recently identified as NO, could form a physiologically relevant effector system. Faculty of Biology, University of Konstanz, Universit~tsstra~e I0, 7750 Konstanz, F.R.G.

255 RADIOLABELING OF MUSCARINIC RECEPTORS OF BOVINE AORTIC ENDOTHELIAL CELLS F. Brunner It was previously shown that fresh helical strips of bovine coronary artery with intact endothelium relax to low concentrations of acetylcholine while higher concentrations cause vessels with and without endothelium to contract. Since these findings could reflect muscarinic receptors (mAChR) on endothelial cells, the nature of muscarinic binding sites on endothelial cells of bovine aorta was investigated by labeling with [3H]N-methyl-quinuclidinyl benzilate ([3H]NMeQNB). Muscarinic binding sites on membranes derived from endothelial cells by mechanical removal with a scalpel resembled mAChR in that, a) the binding of [3H]NMeQNB was reasonably specific (80 % - 50 % of total at 0.1 - 3 nM NMeQNB); b) saturable (KD ~ 0.4 nM, Bmax ~ 15 fmol/mg microsomal protein); c) highly stereospecific as determined with dexetimide (KI ~ 0.6 nM) and levetimide (KI ~ 3 #M); d) the binding of 1-2 nM [3H]NMeQNB was inhibited by acetylcholine and several muscarinic antagonists with potencies similar to those in other tissues; e) 5"-guanylylimidodiphosphate (100 #M) shifted the acetylcholine competition curve to the right by a factor of < 2. - Binding of [3H]NMeQNB to mechanicallyderived intact endothelial cells was likewise specific, saturable and stereospecific and yielded similar binding parameters as the membrane homogenates. They contain ~ 1000 mAChR per cell. However, cultured endothelial cells (3 passages) that had originally been harvested by enzymatic digestion, showed no specific binding. These results for the first time demonstrate that endothelial cells do contain muscarinic receptors, albeit in comparatively very low number. These receptors may directly mediate the synthesis and release of endothelium-derived relaxing factor. Institut f~r Pharmakodynamik und Toxikologie, Universit&t Graz, Universitfitsplatz 2, A-8010 Graz (Austria)

254 MAMMALIAN SMOOTH MUSCLE CONTAINS TWO IMMUNOLOGICALLY DISTINCT cGMP-DEPENDENT PROTEINKINASES Alexandra Keilbach, Wernet

Wolfgang Landgraf,

Wolfgang

Two distinct cDNA's (I~ and I@) for cGMP-kinase have been identified and cloned from a bovine trachea library. The derived amino acid sequence suggested that the two putative isoenzymes differ only in the aminoterminus (amino acid 189 of I~), whereas the rest of the protein (amino acid 90-671) is identical. Peptide antisera were raised in rabbits against sequences which are present only in the putative I~enzyme. The antisera crossreacted in immunoblots of tracheal extracts with a 75 kDa peptide which was indistinguishable from pure cGMP-kinase. DEAE-cellulose chromatography yielded two peaks of cGMP-binding fractions eluting at about 90 and 175 mM NaCI. Both peaks contained cGMPkinase as demonstrated with a pclyclonal antiserum which recognizes the common sequences of the I~ and I~ isozymes. However, the peptide antisera reacted only with the second peak. Further experiments showed that the second peak is present in vascular and bronchial smooth muscle. These results suggest that smooth muscle contains two closely related isozymes of cGMP-kinase. It will be of interest to see if the expression of isozyme I~ is regulated by a prolonged change in smooth muscle tone. Institut f~r Physiologische Chemie, Medizinische Fakult~t, Universit~t des Saarlandes, D-6650 Hemburg/Saar

256 SODIUM FLUORIDE INCREASES Ca 2+ INFLUX, cGMP FORMATION AND EDRF BIOSYNTHESIS IN ENDOTHELIAL CELLS. W.F. Graier and W.R. Kukovetz Since the endothelial cytosolic free Ca 2+- concentration ([Ca2+]i) is discussed as a possible signal for EDRF-production, the mechanism of Ca2+-influx regulation in endothelial cells (ECs) becomes important. Sodium fluoride was used to investigate the possible involvement of G-proteins in the regulation of porcine aortic endothelial Ca 2+- channels. Sodium fluoride increased [Ca2+]i with an ECso value of about 5 mM from 62 + 3.8 nmol to 395 + 26 nmol. This effect strictly depended on the presence of extracellular Ca2+, indicating an enhanced influx of extracellular Ca2+ rather than a release of Ca 2+ from intracellular stores. In the presence of the AI3+ chelator deferoxamine the effect of sodium fluoride was abolished which confirms the current hypothesis that only [AIF4]- but not F- stimulates G-proteins. Pertussis toxin, which is known to catalyze the ADP-ribosylation of some G-proteins had no effect on sodium fluoride induced Ca2+ increase.Thus and the findings that sodium fluoride did not increase cAMP-levels in ECs indicates that neither Gi nor Gs seems to be involved in this mechanism of Ca2+- channel activation. Similar to its effect on [Ca2+]i sodium fluoride also increased endothelial cGMPlevels which are described as a biochemical marker for EDRF formation. This effect also strictly depended on the presence of extracellular Ca 2+ and could be potentiated by addition of AICI3. In agreement to these results sodium fluoride also induced an endothelium dependent relaxation in coronary artery. Thus, similar to the activation of receptor operated Ca2+channels, direct stimulation of a G-protein by sodium fluoride results in an increase of [Ca2+]i and in the formation of EDRF. Institut f0r Pharmakodynamik u Toxikologie, Universit~tsplatz 2, A-8010 Graz, Austria

R 65 257 OXIDIZED LOW DENSITY LIPOPROTEIN (LDL-ox) INHIBITS STIMULATION OF SOLUBLE GUANYLATE C Y C L A S E BY E D R F (NO). *K. S c h m i d t a n d + G.M. K o s t n e r S i n c e h y p e r c h o l e s t e r o l e m i a is a s s o c i a t e d w i t h i m p a i r e d e n d o t h e l i u m d e p e n d e n t r e l a x a t i o n , we i n v e s t i g a t e d t h e effect of LDL-ox, a l i p o p r o t e i n w i t h a t h e r i o g e n i c p r o p e r t i e s , on c u l t u r e d p o r c i n e aortic e n d o t h e l i a l cells. I n c u b a t i o n w i t h b r a d y k i n i n , A T P or t h e c a l c i u m i o n o p h o r e A 23187 p r o d u c e d a n e n h a n c e d f o r m a t i o n of E D R F r e s u l t i n g i n i n c r e a s e d e n d o t h e l i a l cGMP-levels. P r e t r e a t m e n t (15 m i n ) w i t h LDL-ox d o s e - d e p e n d e n t l y d i m i n i s h e d t h e effect of E D R F on e n d o t h e l i a l c G M P - l e v e l s w i t h a n ICGo of a b o u t 100 [tg cholesterol/ml. A s i m i l a r i n h i b i t o r y effect of LDL-ox w a s obs e r v e d w h e n e n d o t h e l i a l cells were s t i m u l a t e d w i t h s o d i u m n i t r o p r u s s i d e w h i c h i n d i c a t e s a n i n t e r a c t i o n b e t w e e n LDLox a n d g u a n y l a t e cyclase r a t h e r t h a n a n i n h i b i t i o n of E D R F formation. To prove w h e t h e r t h i s i n a c t i v a t i o n only occurs in e n d o t h e l i a l cells, we i n v e s t i g a t e d t h e effect of L D L - o x o n p a r t i a l l y purified soluble guanylate cyclase from bovine thrombocytes. W h i l e LDL-ox i t s e l f i n c r e a s e d b a s a l g u a n y l a t e cyclase activity 3 to 4-fold, t h e s t i m u l a t o r y effects of NO r e l e a s i n g c o m p o u n d s ( s u c h a s n i t r o p r u s s i d e , S - n i t r o s o g l u t a t h i o n e or n i t r i c oxide itself) were i n h i b i t e d w i t h a n ICGo of a b o u t 100 I~g cholesterol/ml. I n c o n t r a s t to LDL-ox, n a t i v e L D L w a s i n a c t i v e u p to 300 ~tg cholesterol/ml. T h e q u e s t i o n w h e t h e r LDL-ox directly i n a c t i v a t e s E D R F or w h e t h e r LDL-ox reacts w i t h t h e g u a n y l a t e c y c l a s e r e s u l t i n g in a d i m i n i s h e d r e s p o n s e to NO is c u r r e n t l y i n v e s t i g a t e d . N e v e r t h e l e s s , i n d e p e n d e n t o f t h e e x a c t m e c h a n i s m of i n a c t i v a t i o n , t h e s e data could explain, why in arteriosclerosis the dilatory r e s p o n s e of blood v e s s e l s to E D R F is i m p a i r e d . * I n s t i t u t fiir P h a r m a k o d y n a m i k u n d T o x i k o l o g i e bzw. + I n s t i t u t fiir M e d i z i n i s c h e B i o c h e m i e , U n i v e r s i t ~ t G r a z , A-8010, A u s t r i a .

258 NITRIC O X I D E - G E N E R A T I N G ADP-RIBOSYLTRANSFERASE

AGENTS ACTIVATE A CYTOSOLIC 13. Briine, and E.G. Lapetina •

Agents t h a t are able to release nitric oxide (NO), like s o dium nitroprusside (SNP) or 9-morpholinosydnonimine (commonly known as SIN-l), are known vasodilators and inhibitors of platelet activation. It is thought t h a t t h e s e effects are mediated by the spontaneous release of nitric oxide and the s u b s e q u e n t activation of soluble guanylate cyclase. We have found t h a t SNP (5-200pM) and SIN-1 (20-1000 pg/ml) activate an endogenous ADP-ribosyltransferase t h a t ADP-ribosylates a soluble 39-kDa protein. This enzymatic activity causes a mono-ADP-ribosylation of the 89 kDa protein, since digestion of the electroeluted protein from SDSgets with snake venom phosphodiesterase releases mainly 5 ' AMP, which is identified by comigration with a standard of AMP on polyethyleneimine TLC-plates. In addition to platelet cytosol, the enzymatic activity was also present in the 100000 x g s u p e r n a t a n t of rat brain, heart, intestine, liver and lung, although basal and stimulated activity varied b e t ween the t i s s u e s tested. Dithiothreitel, reduced glutathione, or cysteine was needed in order to observe the stimulatory effect. Hemoglobin which binds nitric oxide, inhibits the s t i mulatory effect of SNP and SIN-1 dose dependently. The activation of the endogenous ADP-ribosyltransferase by the NO-releasing agents is not related to the stimulation of soluble g u a n y l a t e eyclase and the production of cyclic GMP, because cyclic GMF itself. Dibutyryl cyclic GMP, and 8 bromo-eyclie GMP are ineffective at stimulating ADP-ribosylation of the 89-kDa protein. These studies demonstrate and effect of nitric oxide r e leasing compounds t h a t is totally independent of their known action on g u a n y l a t e cyclase. D e p a r t m e n t of Biology, U n i v e r s i t y of K o n s t a n z , U n i v e r s i t ~ i t s s t r . 8 - 1 0 , 7 7 5 0 K o n s t a n z , W e s t - G e r m a n y a n d "The B u r r o u g h s Wellcome R e s e a r c h L a b o r a t o r i e s , 8 0 3 0 C o r n w a l l i s Road, RTP, N.C. 2 7 7 0 9 , U.S.A.

259 THE ENDOTHELIAL SYNTHESIS OF NITRIC OXIDE AND PROSTACYCLIN IS MEDIATED VIA INDEPENDENT PATHWAYSAND INHIBITED BY NEUTROPHILS H. Schr~der The calcium ionophore calcimycin (A23187) is known to stimulate the formation of prostacyclin (PGIg) and n i t r i c oxide (NO) in endothelial c e i l s . The ~resent study investigates the question i f a decrease in NO generation results in a compensatory increase in PGIp production and vice versa. Endothelial cGMP stimulatioh was used as a biochemical marker for NO formation and 6oxo-PGF1~ as an index metabolite for PGI2. In argininedepletedhorcine aortic endothelial c e l l s , L-arginine (2 mM) enantioselectively potentiated calcimycin-induced cGMP formation whereas PGI9 ~ormation was not affected. The L-arginine antagonist:N%monomethyl-L-arginine ( I 100 ~M) induced a 65-90% decrease in the 55-fold cGMP stimulation by 3 ~M calcimycin but had no effect on PGI9 formation under the same condition. In the presence of indomethacin (10 pM), the 37-fold PGI9 stimulation by 3 pM calcimycin was completely abolished, whereas cGMP stimulation remained unaltered. Upon co-incubation with washed human neutrophils, calcimycin-induced PGI9 and cGMP stimulation in endothelial cells was reduced ~y 50% and 88% of control, respectively. I t is concluded that the endothelial synthesis of NO and PGI2 is mediated via independent pathways. The i n h i b i t i o n of both PGI9 and NO ,formation by activated neutrophils may be invd'Ived in neutrophil-induced changes in vascular tone and permeability.

Supported by the DFG (Schr 194/7-2) Institut for Pharmakologieder Heinrich-Heine-Universit~t, D-40OOD~sseldorf, Moorenstr. 5

260 L-ARGININE GENERATED IN CULTURED ENDOTHELIAL CELLS IS USED FOR EDRF FORMATION M. Hecker, J.A. Mitchell, E. Angg~d and J.R. Vane ..................................................................................... Endothelium-derived relaxing factor (EDRF) has been identified as nitric oxide and is derived from the guanidino group of L-arginine (LArg). The L-Arg concentration in bovine cultured aortic endothelial cells (BAEC) is about 100 I.tM as determined by reversed phase HPLC/fluorescence detection analysis. These cells lose about 90-95% of their intracellular L-Arg when grown in an L-Arg free culture medium for 24 h but retain their ability to release EDRF when stimulated with ADP. In contrast to BAEC grown in normal medium, this EDRF release was substantially increased over 4-6 consecutive challenges, whereas basal EDRF release remained unaffected. Subsequent doses of ADP did not result in a further increase (n=9) and in 3 out of 5 experiments infusions of L-Arg (50 ~M) could no longer potentiate EDRF release. The intracellular L-Arg concentration of these cells increased 1.4 fold, whereas all other amino acids measured fell considerably. An even more pronounced rise in L-Arg (5.1 fold) occurred when the cells were perfused for 90 min in the absence of ADP (n=3). In separate experiments, BAEC depleted of L-Arg were incubated for 60 min in Krebs' buffer at 37°C. Under these conditions, L-Arg levels were markedly elevated (17.9 fold; n=6), whereas they increased only 3 fold in the presence of ADP (10 gM), indicating that transfer from the culture medium somehow stimulates the generation of L-Arg which is subsequently utilized for EDRF formation. In summary, our data show BAEC generate L-Arg from an intracellular source and that the level of L-Arg is rate-limiting for EDRF synthesis. Thus, they support the notion that L-Arg is the endogenous substrate for EDRF synthesis. The origin of the newly generated L-Arg and the control of its mobilization will be discussed. The William Harvey Research Institute is supported by a grant from Glaxo Research Group, Ltd. The William Harvey Research Institute, St. Bartholomew's Hospital Medical College, Charterhouse Square, London EC 1M 6BQ, U.K.

R 66 261 C A L C I U M D E P E N D E N C Y OF E N D O T H E L I A L CYTOSOLIC N I T R I C OXIDE SYNTHESIS IS M O D U L A T E D BY CALMODULIN A. M61sch, R. Busse Release of nitric oxide (NO) from endothelial cells c r i t i c a l l y depends on a transmembrane calcium influx into the cells. Therefore, we studied w h e t h e r the free cytosolic calcium c o n c e n t r a t i o n d i r e c t l y affects the activity of the N O - f o r m i n g enzyme(s) p r e s e n t in the cytosol from freshly h a r v e s t e d porcine aortic endothelial cells. In the p r e s e n c e of i m M L-arginine, 0.i mM N A D P H and 0.1 m M EGTA NO accumulated in the gas-phase covering the endothelial cytosol as d e t e c t e d by chem i l u m i n e s c e n c e with ozone. In the same incubates cytosol activated a p u r i f i e d soluble guanylate cyclase (GC) 5 ± 0.5-fold. C a l c i u m chloride increased this activation further by 136 ± 15 % in a c o n c e n t r a t i o n - d e p e n d e n t fashion (ECs0 0.3 ~M free calcium). The c a l c i u m - d e p e n d e n t NO synthesis (as q u a n t i f i e d by activation of GC) was p o t e n t l y inhibited (ICs0 1 ~M) by the calmodulin antagonists m e l i t t i n and calcineurin, but not by calmidazolium. This inhibition was overcome by a d d i t i o n of porcine brain calmodulin. We conclude that the c a l c i u m - d e p e n d e n c y of endothelial cytosolic NO synthesis provides the molecular basis for the a g o n i s t - i n d u c e d formation of NO in intact cells, w h e r e a s the c a l c i u m - i n d e p e n d e n t NO synthesis reflects the basal NO formation. Furthermore calc i u m - c a l m o d u l i n may be tightly a s s o c i a t e d with the r a t e - l i m i t i n g enzyme in the oxidative Larginine pathway, thereby p r o v i d i n g the calciums e n s i t i v i t y of endothelial NO synthesis. Institute of A p p l i e d Physiology, U n i v e r s i t y Freiburg, Hermann-Herderstr. 7, D-7800 Freiburg, F R G

263 EDRF SYNTHASE IN N1E-115 NEUROBLASTOMA CELLS IS A CALCIUM- AND CALMODULIN-REGULATED ENZYME U. F6rstermann*$, L.D. Gorsky$, K. Ishii*, and M. HeUer*~

N1E-115 cells produce a substance with the pharmacological profile of endothelium-derived relaxing factor (EDRF) (Ishii et al., J Appl Cardiol 4: 1989, in press; F6rstermann et al., Naunyn-Schmiedeberg's Arch Pharmacol 340; 1989, in press). In the present study, increases in cyclic GMP levels in rat fetal hmg flbroblasts (RFL-6 cells) were used to assay EDRF. Intact N1E-115 cells suspended in Locke's solution released a material that markedly enhanced cyclic GMP levels in RFL-6 cells. The synthesis of this substance could be stimulated with the receptor agonist neurotensin (10 ~tM) or by adding the EDRF substrate L-arginine (100 ktM). In Ca2÷-free Locke's solution containing 0.2 mM EGTA (ethyleneglycol-bis-[13-aminoethyl ether] N,N,N',N'tetraacetic acid), stimulation of EDRF production by both neurotensin and L-arginine was abolished. EDRF synthase activity was localized in the cytosol of N1E-115 cells. The enzyme was Ca2+-sensitive, the major increase in activity occurring between 100 nM and 500 nM Ca 2+. The Ca2+-sensitive enzyme was inhibited about 80% by No-nitro-L-arginine (10 [.tM) or NG-monomethyl-L-arginine (100 I.tM). The effect of EDRF was completely abolished by hemoglobin (10 ~tM) or methylene blue (10 gM). The synthesis of EDRF was also inhibited by the following calmodulin antagonists (with the approximate ICs0 values given in parentheses): calmidazolium (I0 IxM), trifluoperazine (I0 ~tM), fendiline (80 ~tM), W-7 (N-[6-aminohexyl]-5-chloro-l-naphthalenesulphonamide, 120 [.tM) and compound 48/80 (3 ~g/ml). In further experiments, N1E-115 cytosol (about 25 mg of protein) was applied to a D13-52 anion exchange column in potassium phosphate buffer (KPi, 20 mM, pH 7.4). After washing the column with KP i buffer, EDRF synthase was eluted with 0.1 M KC1. The retrieved enzyme was inactive, but its activity could be fully restored by adding exogenous calmodulin. The ECs0 of calmodulin in restoring enzyme activity was about 2 units/ml. It is concluded that hormonal stimulation of N1E-115 neuroblastoma cells leads to the influx of extracellular Ca 2+ which in turn activates a cytosolic Ca2÷- and calmodulin-sensitive enzyme that synthesizes EDRF from L-arginine or a related material. *Dept. of Pharmacol., Northwestern Univ. Medical School, Chicago, IL 60611, and SAbbott Laboratories, Abbott Park, ]1, 60064, U.S.A.

262

264

METHYLENE BLUE INHIBITS THE SNP MEDIATED SUPPRESSION OF AGONIST INDUCED CALCIUM RISE IN P L A T E L E T S B Y T H E P R O D O C T I O N OF S U P E R O X I D E IONS. J. U t z a n d V. U l l r i c h

CHARACTERIZATION AND PURIFICATION OF A RAT BRAIN E N Z Y M E ( S Y S T E M ) WHICH C A T A L Y Z E S THE FORMATION OF AN EDRF-LIKE FACTOR FROM L-ARGININE H.H.H.W. Schmidt',2, J.S. Pollock1, and F. Murad~,2

Human platelets were loaded with the Ca ~+sensitive fluorescent dye FURA2/AM and the Ca +*signal o b t a i n e d after stimulation w i t h arachidonic acid (AA) was registrated. P r e i n c u b a t i n g the platelets 1 min with sodium n i t r o p r u s s i d e (SNP), which liberates NO and elevates cGMP levels via s t i m u l a t i o n of guanylate cyclase, inhibited the AA induced C a + + - i n c r e a s e in a dose d e p e n d e n t manner. This h a p p e n d in the presence and absence of extracellular calcium. If m e t h y l e n e blue (MB), an inhibitor of soluble guanylate cyclase was added prior to the addition of SNP, the s u p p r e s s i o n of the AA induced Ca++-increase was nearly completely abolished. Simultaneous addition of superoxide dismutase a n d MB and subsequent addition of SNP however fully r e s t o r e d the inhibition by SNP. Catalase alone or in combination with ME had no effect on the Ca *+ rise with and w i t h o u t added SNP. These results confirm the proposal of M a r t i n et al. [i] obtained from their work w i t h rabbit aorta, that MB not only inhibits guanylate cyclase itself, but also interacts d i r e c t l y with the endothelial d e r i v e d relaxing factor which has been identified as NO. M o r e o v e r the direct inhibition of human p l a t e l e t soluble guanylate cyclase by euperoxide might r e p r e s e n t a third m e c h a n i s m of action of MB.

Endothelial cells have been shown to synthesize a factor (EDRF) which induces vasorelaxation via activation of vascular smooth muscle soluble guanylyl cyclase and a subsequent increase of the intracellular cyclic GMP concentration. The formation and release of an EDRF-like activity has further been demonstrated in other tissues, e.g. rat brain. EDRF is also a potent activator of soluble guanylyl cyclase in intact cultured rat fetal lung fibroblasts (RFL-6), which have been used as a sensitive detector system for EDRF and EDRF-like activity. Here, we describe the characterization and partial purification of an enzyme(system) which catalyzes the formation of an EDRF-like factor from L-arginine in rat brain cytosol (100,000 x g supernatant) using RFL-6 cells as the detector system. Hemoglobin interfered with this assay. The removal of hemoglobin from the rat brain cytosol was achieved by phosphocellulose chromatography. Subsequent anion exchange chromatography (Q Sepharose FF) of the phosphocellulose pool resulted in an almost complete loss of enzyme activity which was concentration-dependently reconstituted by addition of calmodulin (apparent Kr, 1-2 U/ml). Binding of the enzyme activity to Q Sepharose FF and the elution characteristics at pH 7.4 were dependent on the type of buffer system used, i.e. Tris/HCI or potassium phosphate. Under the latter conditions, stepwise elution with KCI resulted in two fractions which when combined in the absence of exogenous calmodulin partially reconstituted the enzyme activity applied to the column. One of these fractions was replaceable by calmodulin. Further purification was obtained using S Sepharose FF and dye affinity columns. The partially purified enzyme (system) was dependent on NADPH, L-arginine, Ca2. and calmodulin, bound to calmodulin-agarose and was inhibited by the L-arginine analogs, Na-monomethyI-L-arginine and NG-nitro-L-arginine.

[I] Martin, W., Villani, G.M., Desingaro, J. and R.F. Furchgott: J. Pharsacol. Exp. Ther. 232: 708-716, 1984.

Fakult~t f~r Bio!ogie, U n i v e r s i t y of Konstanz, U n i v e r s i t ~ t s s t r a S e 10-12, D 7750 Konstanz, FRG.

1Abbott Laboratories D-47R AP9A, Abbott Park, Illinois 60064, USA, and 2Department of Pharmacology, Northwestern University, Chicago, Illinois 60611, USA.

R 67 267

265 ANALYSIS OF BRAIN ANGIOTENSINOGEN USING TRANSGENIC ANIMALS J.Mullins, B.Bunnemann, K. Fuxe

EXPRESSION

Angiotensinogen is widely expressed within the brain, being particularly abundant in the hypothalamus. As a prerequisite to the elucidation of the functions of angiotensinogen expression in different brain regions it is essential to define the cis-acting DNA sequences within the angiotensinogen gene which are responsible for expression in each region. Clonston et.aL (EMBO J. 8, 3337-3343, 1989) have constructed transgenic mice possessing a mouse angiotensinogen minigene and demonstrated that DNA sequences neccessary for tissue-specific expression of the angiotensinogen gene reside within 0.75kb 5' of the transcription start site and 2.0kb 3' of the polyadenylation site. We have extended this approach by introducing the rat angiotensinogen gene into the mouse germline. The use of a homologous, but distinct gene, permits the localisation of expression within the brain by in-situ hybridisation with a rat-specific cRNA probe which does not cross-hybridise with the endogenous angiotensinogen mRNA. Preliminary results indicate that the transgene is expressed within specific brain areas including the thalamus (paraventrieular thalamie nucleus, mediodorsal thalamie nucleus and the other thalamic midline nuclei), the hypothalamus (paraand periventricular hypothalamic nuclei, supraehiasmatic nucleus and arcuate nucleus), the superior and inferior collicull, the central periaqueduetal gray with the dorsal raphe nucleus and the Purkinje cell layer and the granular layer of the cerebellar cortex. Lower levels were present in the septurn (medial septal nucleus and the nuclei of the diagonal band), the mammillary body and the hippocampal formation however not confined to any layer. These studies will facilitate more detailed analysis using deletion constructs with the aim of identifying those DNA sequences required for expression within specific regions. Dept. Pharmacology, Univ. of Heidelberg, INF 366, D-6900 Heidelberg, FRG, and Karolinska Institute, Stockholm, Sweden.

TISSUEANGIOTENSINOGENAND THE ROLE OF THE CARBOHYDRATE CONTENTFOR THE RENINKINETIC. U.Hilganfeldt and T.Muley ............................................... In recent years evidence has accumulated indicating the existence of a tissue renin-angiotenslnsystem.The actual conception of this systemsuggeststhat both renin and plasma anglote~inogan (Ao) are diffusinginto the tissue where the processingof angiotensintakes place. In addition it has been found that ranin and Ao mRNA are expressed and secreted in tissues other than kidney and liver, respectively. We have comparedAo from perfused rat heart and hind limb with plasma Ao. Tissue Ao displays a smaller molecular weight and a broad band in the SDS disc eleetropboresis.This observationand the data obtained by isoeleetric focussingof tissueAo suggeststhat the carbohydratecontent is partially degraded. Therefore, the role of the carbohydrate content of Ao for the kinetic with renin was investigated. Purified angiotensinogeawas treated with ueuraminidase, Oglyean-peptidehydrolase and peptid-N-glycosidaseF, respectively.The amount of cleaved neuraminleadd was followedby a colorimetrie assay.The deglyeosylafioa was e~ramined by SDS eleetropboresis mad isoeleetde focusing. After neursmin;dasetreatment a 2-fold increase in Vmax could be measured. Complete cleavage of the glyean residues of Ao by peptid-N-glyeosidaseF showeda 3-fold decrease in kM. These data suggestthat in the tissue the carbohydrate content of angintensinogen can be enzynmtieally degraded. This would improve the processing velocity of angiotemin by renin. Department of Pharraacology,Universityof Heidelberg, Im NeueulaeimerFeld 366, D-6900 HEIDELBERG

266

268

CHARACTERIZATION OF TRANSGENIC RATS WITH HYPERTENSION D. Ganten, J. Peters, M. Lee, Yi Zhao, F. Zimmermann, S. Bachmann

CONTRIBUTION OF ENDOGENOUS KININS TO THE ACUTE ANTIHYPERTENSIVE EFFECTS OF A CONVERTING ENZYME INHIBITOR, RAMIPRIL: A STUDY IN KININ-DEFICIENT BROWN NORWAY RATS. L. Danckwart and Th. Unger

Transgenic techniques allow precise and systematic testing of gene functions. They also allow the establishment of novel and specific animal models for pathophysiological and pharmacological investigations. Primary hypertension is considered a polygenic inherited disorder but to date the nature of the genes involved is unknown. We have now established for the first time TG rats by introducing the mouse renin gene (Ren2u) into the genome of the rat using transgenic techniques. Three independent lines of transgenie rats have been established and those animals which possess the mouse gene exhibit extreme hypertension (systolic pressure 180-260 mmHg). The segregation of the hypertensive phenotype with the presence of the transgene, in independent lines, indicates that expression of the mouse renin gene is responsible for the hypertension. Interestingly, these rats do not have abnormal levels of active renin, angiotensin I or angiotensin II in their plasma and therefore over-expression of renin in the kidney does not account for the phenotype, plasma prorenin on the other hand is high. These animals are a possible model for normal or low renin hypertension and high tissue renin. We are presently determining the underlying cause of their hypertension. Additionally, these TG rats provide an attractive new experimental model for pharmacological and pathophysiological cardiovascular studies and will be suitable to study other genes involved in hereditary cardiovascular disorders in the future.

The contribution of endogenous kinins to the acute antihypertensive actions of the converting enzyme (CE) inhibitor ramipril was investigated in kinin-deficient Brown Norway rats (BN), and in Brown Norway Hannover (BN-HA) and Wistar rats as controls. In BN, urinary bradykinin (BK) excretion was measurable but extremely low when compared to control strains. The depressor responses to intraarterial (i.a.) BK injections were not different between BN and BN-HA, and were potentiated by intravenous (i.v°) ramipril (60pg) and attenuated by i.a. i~fusi~n~of theTBK antagonist, B4146 (D-Arg-(Hyp ,Thi '~,D-Phe')bradykinin), (40~g/kg/min) to a similar extent in both strains. In renal hypertensive (two-kidney-one-clip) BN, the blood pressure reductions to i.v. bolus injections of ramipril (100ug) were significantly reduced, both in extent and duration, when compared with hypertensive BN-HA and Wistar rats. I.a. infusion of B4146 (40~g/ kg/min) attenuated the depressor response to ramipril in Wistar rats and BN-HA, but had no effect in BN. In contrast, all three groups showed similar depressor responses to i.e. infusions of the angiotensin II-receptor antagonist saralasin. These responses were not influenced by the BK antagonist. Our data, showing that the depressor response to ramipril was linked to the integrity of the endogenous kallikrein kinin system, support the hypothesis that kinins are important for the acute antihypertensive actions of CE inhibitors.

German Institute for High Blood Pressure Research and Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, FRG

Dept. of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, FRG.

R 68 269

271

EFFECTS OF BRADYKININ ANTAGONISTS ON SYMPATHETIC OUTFLOW OF 8HR AFTER INHIBITION OF THE CONVERTING ENZYME. P. Dominiak, M. Simon and A. Blbchl It is suggested that inhibition of the converting enzyme (CE) is linked to accumulated bradykinin (BK). Benetos et al. 1986 (Hypertension 8:1089) could demonstrate that the hypotensive effect of a CE inhibitor was blunted when the BK antagonist B4146 was administered to rats. Because we observed an increase in sympathetic outflow after administration of BK to rats, particularly if the CE is inhibited it was of interest to study the role of BK antagonists on the sympathetic outflow after inhibition of the CE.Male spontaneously hypertensive rats (SHR), weighing about 240g were orally treated by garage with ramipril (img/kg/d) for 14 days, controls received water at the same volume. SHR were pithed by standard method and the sympathetic outflow was induced by stimulating the thoraco-lumbar spinal cord (ims, 50mA, 0.5Hz) for 3 min. The stimulation was repeated during the infusion of the BK-l-antagonist Des-Arg-Leu^-BK (5~g/kg/min) or the BK-2-antagonist B4146 (0.17 and Im~/kg /min). In another group of SHE, ramipril was administered acutely (O.img/kg i.v.) and the stimulation and the BK-antagonists were applied as described above. As parameters for the sympathetic outflow circulating noradrenaline (NA) and adrenaline (A) were deter'mined at the end of each stimulation period by using HPLC and ELCD.Infusion of the BK-l-antagonist did not significantly change NA and A after chronic or acute administration of ramipril. However, the BK-2-antagonist caused a dose dependent increase in NA (about 40%) and in A (100%) in the ramipril treated group when compared to controls. The effects of the BK-2-antagonist on sympathetic outflow in chronically treated SHR were less pronounced. In control SHR, only an increase in A was observed after infusion of img/kg/min of the BK-2-antagonist.From our results we conclude that the antihypotem sire effect of the BK-2-antagonist in rats treated with a CE inhibitor is partly due to the significantly increased sympathetic outflow. Physiol. Inst., Uai MUnchen, Pettenkoferstr.12, 8Minchen 2

DIFFERENT MECHANISMS FOR THE CENTRAL ANGIOTENSIN II-INDUCED PRESSOR RESPONSE WITH AND WITHOUT D R I N K I N G . R. Retti@, P. B r ~ n d l i r and U. S a u e r Intracerebroventricular (icy) angiotensin II (ANG II) c a u s e s a p o t e n t p r e s s e r response. The u n d e r l y i n g m e c h a n i s m s are n o t c o m p l e t e l y u n d e r s t o o d and m a y be i n f l u e n c e d by A N G I I - i n d u c e d drinking behavior. In c h r o n i c a l l y i n s t r u m e n t e d a d u l t m a l e W i s t a r rats (n=19) i0 ng A N G II icv e l i c i t e d an i n c r e a s e in m e a n a r t e r i a l p r e s s u r e of 18~2 m m H g w h i c h was f o l l o w e d by a s i m i l a r p r e s s e r p e a k (17+3 mmHg) I0 m i n l a t e r w h e n w a t e r w a s o f f e r e d and--the a n i m a l s drank. The f i r s t p r e s s e r p e a k was a s s o c i a t e d w i t h a s i g n i f i c a n t i n c r e a s e in v a s c u l a r m e s e n t e r i c r e s i s t a n c e (MR, pulsed Doppler) and a decrease in directly measured efferent splanchnic nerve activity (SpNA). D u r i n g the s e c o n d p r e s s e r p e a k M R increased initially followed by a long lasting decrease; S p N A i n c r e a s e d . P r e t r e a t m e n t w i t h P r a z o sin (i0 ~ g / k g / m i n iv) d i d n o t s i g n i f i c a n t l y alter any o f the p a r a m e t e r s a s s o c i a t e d w i t h the f i r s t p r e s s e r p e a k b u t a t t e n u a t e d the s e c o n d p r e s s e r p e a k a n d the a s s o c i a t e d i n i t i a l i n c r e a s e in MR. In c o n t r a s t , p r e t r e a t m e n t w i t h the v a s c u lar vasopressin (AVP) receptor antagonist d(CH~)5Tyr(Me)AVP (I0 ~ g / k g iv) a t t e n u a t e d the firs£ p r e s s e r p e a k and the a s s o c i a t e d i n c r e a s e in MR, w h i l e it d i d n o t s i g n i f i c a n t l y a l t e r a n y of the p a r a m e t e r s a s s o c i a t e d w i t h the s e c o n d p r e s s e r peak. We c o n c l u d e t h a t the c e n t r a l A N G II-induced presser response may preferentially involve AVP release and mesenteric vasoconstrict i o n on the one h a n d or s y m p a t h e t i c a c t i v a t i o n a s s o c i a t e d w i t h m e s e n t e r i e v a s o d i l a t i o n o n the o t h e r hand, depending on w h e t h e r or n o t the a n i m a l s e n g a g e in d r i n k i n g b e h a v i o r . 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 Neuenheimer Feld 366, 6900 Heidelberg, FRG

270

272

CAPTOPRIL INHIBITS THE BRAIN RENIN ANGIOTENSIN S Y S T E M (RAS) I N D E P E N D E N T L Y OF C O N V E R T I N G E N Z Y M E P__,Gohlke a n d M. R i t z a l A s t i m u l a t e d b r a i n R A S m a y c o n t r i b u t e to some t y p e s of g e n e t i c h y p e r t e n s i o n . We t e s t e d the h y p o t h e s i s t h a t s u l f h y d r y l (SH) g r o u p c o n t a i n i n g c o n v e r t i n g e n z y m e i n h i b i t o r s (CEI) e x e r t a c t i o n s on the brain RAS independently of their converting enzyme (CE) inhibiting properties. The SH g r o u p c o n t a i n i n g CEI, C a p t o p r i l (CAP), was i n j e c t e d i n t r a c e r e b r o v e n t r i c u l a r l y (icy) in c o n s c i o u s rats a n d c o m p a r e d to the n o n SH g r o u p c o n t a i n i n g CEI, r a m i p r i l . F o l l o w i n g CAP at 150 and 500 ug the presser responses to icv angiotensins I and II (ANG I, A N G II) w e r e b l o c k e d for as long as 1 w k (150pg) to 3 wks (500~g). The d r i n k i n g r e s p o n s e to A N G II w a s a l s o i n h i b i t e d b u t o n l y for 24 hrs. The p r e s s e r responses to icy injections of another neuropeptide, s u b s t a n c e P, w e r e n o t a f f e c t e d . The non C E - i n h i b i t i n g s t e r e o i s o m e r of CAP, SQ 14543, and the SH g r o u p c o n t a i n i n g c o m p o u n d , g l u t a t h i o n , p r o d u c e d s i m i l a r e f f e c t s as CAP. In contrast, ramipril (100~g icy) o n l y i n h i b i t e d the c e n t r a l A N G I e f f e c t s for less t h a n 24 hrs. O u r d a t a r e v e a l t h a t CAP p r o d u c e s a l o n g - l a s t i n g i n h i b i t i o n of the b r a i n RAS t h a t is p r o b a b l y r e l a t e d to the SH g r o u p a n d i n d e p e n d e n t of CE. S i n c e CEI can g a i n a c c e s s to the b r a i n a c c o r d i n g to t h e i r l i p o p h i l i c i t y (Gohlke et al, JPET, 249: 609-616, 1989) this e f f e c t m a y c o n t r i b u t e to CAP's persistent antihypertensive actions.

EFFECTS OF CONVERTING ENZYME INHIBITORS ON CARDIAC HYPERTROPHY AND THE TISSUE RENINANGIOTENSIN SYSTEM (RAS) Keuneke C, Yacullo R, Sugiura M, Mall G.

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 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, D - 6 9 0 0 Heidelberg, FRG

Therapeutic actions of converting enzyme inhibitors (CEI) can be independent of the inhibition of the plasma RAS and are possibly mediated through the tissue RAS. In order to study the regulation of the RAS within different tissues we treated male SHRSP for 28 days with CEI and a peripheral vasodilatator (n:10 rats each group) 1. Control, 2. Captopril (50 mg/kg), 3. Lisinopril (10 mg/kg), 4. Cilazapril (10 mg/kg), 5. Hydralazine (30 mg/kg). This treatment lead to a reduction of systolic blood pressure from 200 mmHg to 140 mmHg in all groups. The heart- to bodyweight ratio was significantly reduced versus control in all CEI treated animals while Hydralazine had no effect on this parameter. Renin mRNA in the kidney showed a significant increase following CEI treatment from 6.71_+0.06 pg/pg total RNA (Control), to 1.64_+0.14 pg/pg RNA (Captopril), to 11.2+0.42 pg//zg RNA (Lisiaopril), to 5.2+0.44 pg/pg RNA (Cilazapril) while Hydralazine treatment (0.77_+0.07 pg//lg RNA) did not change renin mRNA. Plasma renin activity was changed in parallel. Liquid-hybridisation-assay and northern blotting revealed specific regulation of the angiotensinogen gene in the hypothalamus and in the adrenal gland, Lisinopril, Cilazapril and Hydralazine but not Captopril resulted in a 17-16% reduction of angiotensinogen gene expression in the hypothalamus. Cilazapril treatment stimulated significantly 3.3 times adrenal renin mRNA from 84.7_+10.3 pg/gg RNA to 261.2_+64.1 pg/pg RNA. Our data indicate that CEI interact specifically with the tissue RAS and these local actions may contribute to their therapeutic antihypertensive and cardiac effects. German Institute for High Blood Pressure Research and Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg, FRG.

R 69 273 EFFECTS OF THE NEW ANGIOTENSIN CONVERTING ENZYME (ACE) INHIBITOR PERINDOPRIL ON STRUCTURAL AND MECHANICAL VASCULAR PROPERTIES IN EXPERIMENTAL HYPERTENSION E. Scalbert*, M. Devissaguet and H. Langenbahn Perindopril is a potent and long-lasting ACE inhibitor whose antihypertensive properties have been extensively demonstrated in hypertensive animals and humans (reviewed in: J. Hypertension 6, suppl. 3, 1988). Based on the key role of the arterial wall in the genesis and/or the maintenance of hypertension, the effects of perindopril on the structural and mechanical vascular properties have been thoroughly studied. Levy et al. (Circ. Res. 63:227, 1988) showed that chronic treatment with perindopril completely reversed the aortic media hypertrophy and the decrease of arterial compliance observed in the renovascular hypertensive rat. Christensen et al. (J. Hypertension 7:83, 1989) reported similar benefits in mesenteric resistance vessels of the spontaneously hypertensive rat. The effect of perindopril treatment was much more potent than that of other antihypertensive therapies. This has to be related to the potent ACE inhibition obtained with the drug in the arterial wall (Unger et al., J. Cardiovasc. Pharmacol. 8:276, 1986; Jackson et al., J. Pharmacol. Exp. Ther. 245:950, 1988) and emphasizes the potential role of angiotensin II as a vascular growth modulator. Such effects of perindopril could be relevant not only from a mechanistic (Harrap et al., Clin. Exp. Pharmacol. and Physiol. 13:753, 1986), but also from a pathogenetic point of view in relation to the vascular complications of hypertension, namely the decrease of the vascular reserve, e. g. coronary reserve (Gosse et al., Clin. Exp. Hypertension A9:1899, 1987). * Present address: IRIS Company and Development, 22 rue Gamier, 92200 Neuilly-sur-Seine, France

275 EFFECTS OF OCTREOTIDE (SMS 201-995, SANDOSTATIN~), PIRENZEPINE, RANITIDINE AND OMEPRAZOLE ON BETHANECHOL-STIMULATED PEPSINOGEN SECRETION IN THE ISOLATED PERFUSED MOUSE STOMACH Th. Buhl. D. Eggenschwyler Pepsin is thought to be an important aggressive factor in the pathogenesis of peptic ulcers. In several animal models it could be clearly demonstrated that acid alone does not produce ulcerations; in all cases pepsin was required to cause severe lesions in the stomach or duodenum (1,2). Therefore we set up an in vitro model for measuring pepsinogen secretion independently from concomitant changes in aod secretion using the isolated perfused mouse stomach. In this model, the muscariuic agonist bethanechol stimulated pepsinogen secretion with an ECso value of 9.1x106 M. The stimulation of pepsinogen secretion by bethanechol (3x10-s M) could he completely inhibited by pirenzepine at high concentrations (10-s M). Octreotide (SMS) also significantly inhibited pepsinogen secretion at concentrations as low as 104 M. The H 2 antagonist ranitidine and the proton pump inhibitor omeprazole were unable to reduce bethanechol-stimulated pepsmogen secretion at concentrations which are maximally effective upon acid secretion in this same model. Basal pepsinogen secretion was even enhanced by omeprazole (10-4 M). In conclusion our results provide evidence that SMS and pirenzepine ~uscarinic sumulated pepsinogen secretion, whereas ranitidine and omeprazole have no effect or even stimulate the secretion of pepsinogen in vitro. Thus SMS and pirenzepine, in contrast to ranitidine and omeprazole, lower peptic activity in gastric juice not only by inhibiting gastric acid secretion but also by decreasing total pepsinogen output. (1) Alphin RS, Vokac VA, Gregory RL, Bolton PM, Tawes JW (1977) Gastroenterology 73:495-500 (2) Joffe SN, Roberts NB, Taylor WH, Baron,JH (1980) Dig Dis Sci 25:837-841 Preclinical Research SANDOZ AG, CH-4002 Basel

274 EFFECT OF ACE I NHIBITORS ON EXPERIMENTAL DIABETES IN NOI~VIOTENSIME AND HYPERTENSIVE RATS S.W.Gumulka, I.Wisniewska E f f e c t o f a n g i o t e n s i n e c o n v e r t i n g enzyme /ACE/ inhibitors

on e x p e r i m e n t a l d i a b e t e s produced by

s t r e p t o z o t o c i n was i n v e s t i g a t e d in normotensive /~KY/ and s p o n t a n e o u s l y h y p e r t e n s i v e r a t s /SHR/. Streptozotocln i.v.

(30 and 6Omg/kg) markedly

i n c r e a s e d glucose l e v e l s in both blood and u r i n e as w e l l as decreased the b l o o d c o n c e n t r a t i o n o f insulin

. The maximal s u r v i v a l

tlme was d o s e - d e -

pendent and amounted t o 4 and 2 weeks r e s p e c t i v e l y . No changes in b l o o d p r e s s u r e were n o t e d . Captopril

( 8 . 0 or 4.0mg/kg 3 t . i , d . ,

p . o . ) but not

enalapril

(6.O o r 3.Omg/kg 3 t . i . d . ,

p.o.)

longed the s u r v i v a l urine

pro-

time, decreased blood and

l e v e l s o f glucose and increased b l o o d con-

c e n t r a t i o n of

insulin

in b o t h : SHR and WRY s t r e -

p t o z o t o c i n t r e a t e d a n i m a l s . C a p t o p r i l decreased b l o o d glucose in c o n t r o l animals but d i d not fluence

the blood i n s u l i n

.

Department o f Pharmacodyna~nics Medical Academy o t Warsaw, Krak.Przedm.26/28,

in-

276 ELEVATION OF INTRAGASTRIC PH BY THE NOVEL H+/K+ATPase INHIBITOR BY 1023/SK&F 96022 IS NOT COUNTERACTED BY EXOGENOUS STIMULATIONOF ACID SECRETIONIN THE FISTULA DOG. S. Postius .

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BY I023/SK&F 96022, a substituted benzimidazole, has been shown to potently inhibit gastric acid secretion in rat, dog and man (Gastroenterol. 96, 5 (Suppl.) A 397 and A 473, 1989). I t is known (J. Chem. Soc. Chem. Commun., 125-127, 1986) that this compound has to be activated in an acidic environment to produce the active principle. We therefore suggested that exogenous stimulation of acid secretion might enhance rather than attenuate the antisecretory a c t i v i t y of this class of antiulcer drugs. The aim of the present study was to compare the histamine Hp-antagonist cimetidine and BY 1023/SK&F 96022 with re@ect to their effects on intragastric pH elevation. The drugs were administered orally to gastric f i s t u l a dogs either during submaximal stimulation by continuous s.c. pentagastrin infusion (6 #g x kg-1 x h-l) or without exogenous stimulation. The results clearly indicate that the pH elevating effect of cimetidine lasts considerably shorter under pentagastrin infusion than in unstimulated dogs. Nevertheless, a similar potency with respect to maximum increase in pH was observedunder the two experimental conditions. By contrast, BY 1023/SK&F 96022 elevated intragastric pH for a comparable time both during pentagastrin infusion and i~] ~nstimulated animals, These results suggest that the H'/K--ATPase inhibitor BY I023/SK&F 96022 may be advantageous under hyperacidic intragastric conditions as found in patients with Zollinger Ellinson syndrome and duodenal ulcer or under stress and during the early night. Clinical studies with BY 1023/SK&F 96022 are underway.

00-92? Warsaw, Poland BYK GULDENPharmaceuticals, D-7750 Konstanz

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R 70 277 MECHANISM OF GASTRIC ANTISECRETORY EFFECT OF CLOFIBRATE W. Beil The phenoxyisobutyrate derivatives ciprofibrate, bezafibrate, and clofibrate exhibit hypolipidaemic activity in experimental animals and in man. In addition to this primary pharmacological effect the drugs show at high dose level (100-500 mg/kg) antisecretory activity (Eason et al. Scand J Gastroenterol 23, 1063, 1988). The nature of this antisecretory activity is not understood. In order to define the mechanism of how phenoxyisobutyrate derivatives reduce acid secretion we have studied the effects of clofihrate on acid production i~ i~olated and enriched g$insa-pig parietal celSs, on H /K -ATPase activity and on H /K -ATPase mediated H uptake in intact gastric membrane vesicles. Results: In isolated parietal cells clofibrate caused a concentration-dependent inhibition of HCI production (IC50:47 ~mol/l), as measured by "~C-aminopyrine uptake, durlng histamine and dibutyryl-cAMP stimulationI T~e type of inhibition was of a non-competitive nature. H-/K ATPase activity in purified gastric membrane vesicles was only weakly affected by the drug ~395 inhibition at I m~ol /i). In contrast, the effect on H-/K -ATPase mediated uptake was more pronounced. I0 and ~0 ~mol/l clofibrste reduced the extra- intravegicular H gradient to 80 and 50% £f ~ontrol value, respectively. The action of the drug on H-/K--ATPase mediated H uptake+was not affected by mercaptanes or by increasing the K concentration in the medium. Furthermore it was found that elofibrate enhanced the proton efflux rate from vesicles in which the ATPase reaction was stopped at the steady-state level of proton uptake by addition of EDTA. Conclusions: Clofibrate inhibits gastric acid secretion by a direct effect on the parietal cell. The site of action is the secretory membrane. From the observed type of interaction found in the vesicle preparation we suggest that the drug acts as a protonophor. Abteilung Allgemeine Pharmakologie, Medizinische Hochschule Hannover, D-3000 Hannover 61

279 BISMUTH ( I l l ) SALTS AND CARBENOXOLONEARE POTENT INHIBITORS OF LYSO-PAF TRANSACETYLASE IN VITRO F. v° Bruchhausen and M. Rochel Since PAF has been found to be an ulcerogenic mediator (Rosam et a l . , Nature 319, 54, 1986), we studied the influence of most usual ulceroprotective or ulcer healing agents on its last biosynthetic step. We used a microsomal preparation of ovine intestinal lymph nodes as source of the enzyme lyso-PAF-transacetylase (EC 2.3.1.67; 1-Oalkyl-2-1yso-sn-glycero 3-phosphocholine:acetyl-CoA-acetyltransferase). B i ( I I I ) salts (as subcitrate) in concentrations of 2 x 10-o M halfmaximally inhibit the enzyme. Carbenoxolone and 18~-glycerrhetic acid inhibit with ICso values of 0.12 and O.OB mM, respectively. The inhibition was non-competitive to both substrates acetyl-CoA and lyso-PAF. 18~-glycerrhetic acid and other 3~-hydroxy steroids (methandriol, pregnenolone, cholesterol~ were without any effect at concentrations up to 10- M. Sb(III) salts had a slight, Sb(V) salts no inhibitory effect. Kisoprostol inhibits at concentrations (ICRn 0.15 mM) which a~e far beyond gastroprotective dosage. Other agents such as cimetidin, r a n i t i d i n , famotidin, unmetabolized omeprazol, pirencepin, proglumide, methoclopramide and A l ( I I I ) salts up to mM concentrations were ineffective. The inhibitions of this enzyme by some non-steroid antiinflammatory drugs (v. Bruchhausen, 1989, Arch. Pharm. 322:776), flavonoids (Nakos & v. Bruchhausen, 1989, Arch. Pharmacol. 340:R61) and gold compounds (Rayamajhi & v. Bruchhausen, 1989, Arch. Pharmacol. 340:R84) indicate that the last step in the biosynthesis of the inflammatory mediator PAF could be impaired by some agents. Here we show the influence of some gastrotberapeutic agents on the same step. In case of bismuth salts its action is understood by k i l l i n g effects on Campylobacterpylori besides some metabolic actions. The involvement of reduced PAF biosynthesis as mechanism of action must be considered. I n s t i t u t f~r Pharmakologie, Freie Universit~t Berlin, Thielal]ee 69/73, D-IO00 Berlin 33

278

280

IBMX P O T E N T I A T E S EFFECTS O F HISTAMINE ON GLYCOPROTEIN AND PROTEIN SYNTHESIS OF ISOLATED PIG GASTRIC MUCOSAL CELLS N.-K. Heim, A. Oestmann and K.-Fr. Sewing

BASE-LINE CONDUCTANCE OF G U I N E A - P I G D U O D E N A L MUCOSA AS A DETERMINANT OF SUSCEPTIBILITY TO ACID DAMAGE H.J. Maeherey

Histamine enhances protein and glycoprotein synthesis of isolated (pronase/collagenase) and enriched (counterflow centrifugation) pig gastric non-parietal cellsz as measured by the incorporation of [3H~L-leucine ([3H]Leu) and N~acetyl_[14C]D_glucosamine ([1C]GIcNAc) respectively into cellular acid-insoluble material [Heim et al.; unpublished data]. To evaluate a role of cyclic AMP in this process we investigated now the influence of 3-isobutyll-methylxanthine (IBMX), an inhibitor of cyclic AMP phosphodiestersses, on the concentration-dependence of the histamine, forskolin (receptor independent activator of the adenylste cyclase, positive control) and 12-O-tetradecanoylphorbol-13 acetate (TPA, protein kinase C activator, negative control) stimulated protein and glycoprorein synthesis of cells incubated with the tracers for 20 h at 37 ° C in Dulbecco's modified Eagle's Medium. In the presence of IBMX 30 ~mol/l concentration-response curves for forskolin and histamine were shifted to the left and EC50values (indicated in nmol/l) lowered as follows

Serosal HCO a decreases electrical conductance (Gt) of guinea-pig duodenal mucosa by prostaglandin-dependent actions, probably on the paracellular pathway (Macherey and Petersen, Gastroenterology, in press). This effect enables the mucosa to withstand an acid lumen (pH 2.0) for a prolonged period before G~ starts to rise, indicating progressing tissue injury (Macherey and Petersen, Naunyn-Schmiedebergs Arch. Pharmacol. 338: R 53, 1988). Here we have investigated segmental heterogeneity of duodenal mucosa in base-line G t and its response to luminal acidification. 5 cm pieces of duodenal mucusa were divided into 4 segments, which were stripped and mounted in Ussing chambers filled with HCOs-free Ringer's. Tissues were continuously short-circuited. 60 min after mounting, pooled segments 1 + 2 (proximal end) exposed a Gt (18,4 -+ 0,8 mS/era ~) significantly lower than pooled segments 3 + 4 (22,9 -+ 1,6 mS/cm 2) taken from the same animals (n=10 for each segment). In a further set of experiments, the luminal bath was acidified to a pH of 2.0 and the eontraluminal acidification determined by clamping the pH at 7.4 (pH-stat titration). Within a few minutes, both G~ and rates of contraluminal acidification commenced progressive increases. Re-arranging mucosae according to their rates of acidification yielded two groups (n=7 each) of which the high rise group exhibited also higher base-line G t ( AGt = 7,2 mS/cm ~) and larger increases in G t over 100 rain (by 41.2 -+ 4,8 versus 26,5 +- 3,6 mS/cmZ). The mean segment number was 3 in the high-rise group compared to 2 in the low-rise group. As a conclusion, a low base-line Gt (spontaneous or furnished by serosal HCO z) conditions the tissue for a better resistance against acid-induced damage; in the absence of HCO s, this mechanism provides for a better protection of proximal segments.

Incorporation

Forskolin + IBMX

[3H]Leu [14C]GIcNAc

2000 430

250 50

Histamine + IBMX 540 <100 2000 120

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whereas response to TPA was not potentiated. Since histamine has also been shown to enhance cyclic AMP levels of gastric non-parietal cells (sewing et al.; Life Sciences 1985, 12, 1097-1106), these results suggest a "second messenger" function for cyclic AMp in the stimulation of protein and glycoprotein synthesis by histamine. Abteilung Allgemeine Pharmakologie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Str. 8, D-3000 Hannover 61, FRG. Supported by BMFT grant no. 03 8507/5.

Supported by D F G grant Pe 280/2-1 Institut fiir Pharmakologie, RWTH Aachen, D-5100 Aachen, F R G

R 71 281

283

EFFECTS OF ACETYLSALICYLIC ACID ON ELECTRICAL CONDUCTANCE OF G U I N E A - P I G A N T R A L AND D U O D E N A L MUCOSA IN VITRO P. Wurm and J.M. Winterhager

EFFECTS OF CYCLIC AMP AND A CL C H A N N E L BLOCKING A G E N T ON SECRETORY HCO 3 TRANSPORT BY RABBIT G A L L B L A D D E R EPITHELIAL CELLS K . - U . Petersen

In guinea-pig duodenal mueosa (DM) in vitro, serosal HCO 3 reduces tissue conductance (Gt) and delays the Gt rise that is produced by lowering luminal pH (Macherey and Petersen, Gastroenterology, in press; Naunyn-Schmiedebergs Arch. Pharmacol. 338:253, 1988). These effects, which are considered indicative of tissue integrity and protection require the availability of prostaglandius, but are unrelated to HCO s secretion. Here we have investigated a) whether HCO 3 has a similar role in antrum mucosa (AM), and b) effects of acetylsalicylic acid (ASA) on G~ of AM and DM, in absence and presence of HCO s. Isolated mueosae were mounted in Ussing chambers and continuously shortcircuited. In AM bathed in HCOa-free solution, spontaneous G t was about 20 mS/cm 2. Serosal, not mucosal addition of HCO 3 (20 raM) caused G t decreases by 2-3 mS/em z, indicating a similar HCO s effect as in DM. In the absence of HCO a, reducing luminal pH to 2.0 by adding HCI led, within 60 rain, to G t increases by 9_+4 and 11_+4 mS/era 2 in AM (n=5) and DM (n=8), respectively. Luminal ASA (10 -2 M), when added together with HCI, enhanced ZkG~ to 23_+8 mS/cm ~ in AM (n=5) and 20_+7 mS/em 2 in DM (n=8). This effect was largely diminished by serosal HCO 3. The presence of serosal ASA had no influence on HCIinduced rise in G t. Luminal addition of ASA at neutral pH (7.4) evoked, within 60 rain, G t decreases by 5-6 mS/em 2 in AM and DM. The drop in G t was reduced by serosal HCO s to = 1.5 mS/cm z in both tissues. Effects of serosal ASA at neutral pH were moderate to negligible. In conclusion, HCO s has comparable effects on G t in AM and DM. Tissue damage by ASA, in terms of G t increases, requires acid pH at the side of ASA addition. Serosal HCO 3 decelerates G~ increases upon addition of HC1 or HCI + ASA. • Supported by DFG grant Pe 280/2-1

Secretory HCO 3 transport by rabbit gallbladder epithelial cells has been investigated in vitro by means of pH-stat (HCO a fluxes) and intracellular mieroeleetrode techniques (membrane voltages and relative resistances). Previously we have formulated a mechanism by which HCO s secretion (Jam) is accomplished b y sequential Na,HCO s co-transport at the basolateral and CI/HCO a exchange at the luminal cell membrane (Petersen et al., Pfliigers Arch. 408: R34, 1987). The latter notion was supported by the finding that bilateral removal of CI reduced and only luminal addition of C1 stimulated Jsrn" Luminal, not serosal addition of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 10-4 M), a blocker of electroneutral and eleetrogenie HCO s secretion in guinea-pig gallbladder (Petersen et al., NaunynSchmiedebergs Arch. Pharmacol. 337: R47, 1988) inhibited Jsm (from 2.8 + 0.2 to 1.2 ± 0.3 /~mol/cm2h; n=4). NPPB must act on transceltular J~m' as it was inefficient in the absence of Na, a condition which confines Jam to the paraeellular route. It cannot act by inhibiting anion channels because transepithelial voltage and current were not changed and the apical membrane is devoid of sizable anion conductance. Hence NPPB most likely inhibits CI/HCO s exchange. 8 - B r - c A M P (10 -3 M, luminal bath) increased Jam by 0.8 -+ 0.1 #mol/cm2h (n=7). This stimulation cannot be due to opening of luminal membrane anion channels as luminal membrane voltage (- 67 _+ 3 mV) and relative resistance ( R J R b ; 3.5 ± 1.7) remained unchanged (n=4). Moreover, the typical membrane response to luminal C1 removal (slow hyperpolarization) was only in a fe~w instances converted into a small, transient depolarization. Hence basolateral Na,HCO s cotransport remains a more likely target of cAMP in stimulation of Jam" Supported by DFG grant Pe 280/2-1

Institut for Pharmakologie, RWTH Aachen, D-5100 Aachen, F R G

Institut for Pharmakologie, RWTH Aachen, D-510Q Aachen, F R G

282

284 KAPPA OPIOID RECEPTORSCONTROLREFLEX PERISTALSIS IN THE GUINEA PIG ISOLATED ILEUM W. Kromer The longitudinal muscle myenteric plexus preparation of the guinea pig ileum is widely regarded as a #-type preparation, although R opioid receptors have also been demonstrated in this tissue. Electrically-induced longitudinal muscle contractions and distension-induced reflex peristalsis (circular muscle contractions) represent different functions and no information is available on the opioid receptor type(s) involved in the l a t t e r . The function of endogenous opioids can be unmasked by specific receptor blockade which enhances peristalsis in v i t r o (see Kromer, Pharmacol. Rev. 40, 121-162, 1988). The present study compared the effects of the non-select i v e opioid antagonist naloxone with those of 3 receptor type-selective antagonists: binaltorphimine (~-selecrive), CTP-NH~ (#-selective), and ICI 174,864 (6-selective) (see To'rtella, TIPS ~, 366-372, 1988). Concentrations were between 0.1 and I,O00 nmol/l. The biological a c t i v i t y of the antagonist samples was confirmed on the electrically-stimulated guinea pig ileum and mouse vas deferens against receptor type-selective agonists. Naloxone at concentrations of O.I and I #mol/l increased the frequency of p e r i s t a l t i c waves within the f i r s t 15 min interval, and thereafter in a declining fashion, by 68 and 88%, respectively. Binaltorphimine behavedsimil a r l y . I t was one order of magnitude more potent than naloxone but a l i t t l e less effective. Its maximumeffect was 57% enhancement at 10 nmol/l. Both CTP-NH9 and ICI 174,864 were completely ineffective (both ~1 #mol/l; n=10). I t is concluded that, in contrast to the widespread assumption that # opioid receptors are responsible for opioid inhibition of gut m o t i l i t y , ~ receptors are used to this end by intestinal opioids under the present conditions. Byk Gulden Pharmaceuticals, D-7750 Konstanz, FRG

EFFECTS OF TWO CHLORIDE CHANNEL BLOCKERS ON CI TRANSPORT IN ISOLATED MUCOSA OF G U I N E A - P I G DISTAL COLON G. Sprakties Guinea-pig colonic mucosa responds to high intracellular concentrations of cAMP with electrogenic C1 secretion. Here we have tested the ability of two CI channel blockers, NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) and MPTC ( 4 - N - m e t h y l - N - p h e n y l a m i n o t h i o p h e n e - 3 - c a r boxylic acid), to inhibit this transport. Isolated mucosa was mounted in Ussing chambers in HCO3-free solutions for determination of shortcircuit current ( Is)c ~ secretory (J 8 m ) and absorptive ( J , ) C1 fluxes. NPPB caused a concentration-dependent (range 3 , 10-6 M) reduction of spontaneous tissue conductance (G~) and I . Only for Ise, mueosal addition was somewhat more effective than serosal one. Subsequent stimulation of I8c by PGE 1 (10 -8 M, serosal side), was accordingly reduced. In tracer flux experiments (n=13-14) PGE. elevated both unidirectional C1 fluxes ( Z k J r = 0.8 + 0.4 /zmol/cm~h; /kJsm= 5.6 -+ 0.7 ~mol/cm2h), the increase in Jnet exceeding the concurrent rise in lae (= 3.2 _+ 0.2 /.~mol/cm~h). Thus PGE 1 seems to stimulate not only electrogenie chloride secretion but also paraeellular CI flow. Mueosal addition of NPPB (3"10 -~ M) reduced I , JmB and J,m by 2.5 ± 0.2 , 8.2 + 1.1 and 9,3 ± 1.4 /~mol/cm2h, respectively. Corresponding values with serosal addition were 2.1 ± 0.2 , 2.7 -+ 1.1 and 7.0 ± 1.1 /~mol/cm2h. Hence both effects of PGE 1, stimulation of C1 secretion and C1 paracellular flow, are reverted by NPPB which lowers CI permeation even below control values without PGE r MPTC (3'10 -4 M) reduced spontaneous G t and I witli no particular side preference and diminished the effect of PGE1, added subsequently. As with NPPB, inhibitions of Jmm and J were more pronounced than that of Iae, suggesting reductions of both transeellular and paracellular CI flow induced by PGE r In conclusion, the C1 channel blockers seem to act on CI channels in the cell membranes as well as on junctional structures controlling paraeellular CI flow. Supported by DFG grant Pe 280/2-1 Institut for Pharmakologie, RWTH Aachen, D-5100 Aachen, F R G

R 72 285

287

THE EFFECT OF SEVERAL SPASMOLYTIC DRUGS ON ISOLATED GUINEAPIG DUCTUS CHOLEDOCHUS. Martina Moormann and Martin Pfaffendoff Spasmolytie drugs are widely used in therapy of billary and renal eollcs. Beside a symptomatic treatment with analgesics, the usual therapy attempts to relax the smooth muscle by N-butylscopolamine. It may, however; be worthwhile to investigate other spasmolyfic drugs possessing different mode of action and no cholinolytie activity. We therefore compared the smooth muscle relaxing efficacy of N-butylscopolarnine with that of papaverlne and the calcium-antagonists nifedipine and gallopamil. The experiments were performed using the isolated guinea-pig ductus chotedochus. The duetus was suspended in warm oxygenated Tyrede-solution after mounting a catheter in the proximal end and closing the distal opening by ligation. The catheter was connected to a pressure transducen The rnusenlar activity was recorded as an increase of the intraluminal pressure. BeC12 and eathachol were used to stimulate the preparation. Concentmtlen-reSlm~So-relatiunships were established and the ECs0 values were found to be 0.35 (:1:0.06) pM and 532 (:h 106) IIM for carbachol and BaC12, respectively. As standard stimuli nearly maximal effective concentrations of earbadaol and BaCI2 were used (3 pM and 5000 }1M respeetively). Cumulative concentratlon-response eurves were determined for each spasrnolylic drug with both stimulants. Cathachol (3/aM) BaCl2 (5000 pM)

P o t a s s i u m channel b l o c k i n g p o t e n c y of tolbutamide in c e l l - a t t a c h e d m e m b r a n e patches from m o u s e pancreatic B-cells at 37 ° Christina Schwanstecher, Corinna Heipel and U. Panten

B~x(%)

Bes0¢~M)

(%)

Bcs0~lVO

N-butylseopolamin~ 95 ~ 2.4) 10.37 (~ 0.04) 23 0: 2.9) 30.0 0: 4.8) papaverine 91 ~ 8.5) 6.4 (t: 1.70) 93 ~ 9.0) 15.7 ~ 5.4) galtepamil 94 ~. 2.2) 0.12 0: 0.04) 99 (+_'3.1) 0.09 (+. 0.02) nifedipine 79 (+_ 5.6) 0.03 (.% 0.01) 96 (~ 3.9) 0.03 ~ 0.004) According to the pharmacological profile of the stimulants a distlnctiun of eholinolytics and 'directly' acting spasmolytles shouM be possible. Indeed, NImtytscopolamine was found to be effective in reducing the carbachol induced contraction but was less effective in relaxing BeC12-stimulaled preparations. Papaverine, galloparull and nlfedlpine on the other hand, revealed the same efficacy concerning carbaehol and BaCI2 induced pressure iucrease. The calcium-antagonists used were much more potent then papaverine and N-butylscopolamine. We therefore conclude that calclum-antagonists are highly effective drugs in reducing an increased muscular tone of guinea-pig dnctos elroleduchns. The effect is independent of whether it is generated by high parasympathetic activity or by a 'direct' muscular stimulation. Abt. Pharmakologin, Universlt~t Kid, Hospitalstrasse 4, D-2300 Kiel I

Tolbutamide initiates insulin release by blocking ATP-dependent K~channels in the plasma m e m b r a n e of pancreatic B-cells. At room temperature tolbutamide has been shown to be half m a x i m a l l y effective at 4.1~M or 4.2~M in the whole-cell (0.3-1mM ATP in the patch pipette) or the inside-out configuration (imM ADP at the cytoplasmic face of the membrane), r e s p e c t i v e l y .The aim of the present study was to determine the K÷channel b l o c k i n g p o t e n c y of tolbutamide at more physiological conditions in the cell-attached c o n f i g u r a t i o n at 37 ° . Cultured pancreatic B-cells from NMRI-mice were used. At 370 the A T P - s e n s i t i v e K÷cur rent had an amplitude of 6 p A at the resting potential and a conductance of 88 pS (pipette potential b e t w e e n -60 and +80 mV). The current r e v e r s e d at a pipette potential of -69 mV and showed a typical inward rectification. T o l b u t a m i d e b l o c k e d these currents half m a x i m a l l y at 2.2~M. Thus, the K÷channel b l o c k i n g potency of tolbutamide in intact B-cells compares well to its p o t e n c y in the whole-cell or the insideout c o n f i g u r a t i o n of the p a t c h - c l a m p technique. This finding supports the view that ,sulfonylureas stimulate B-cell function solely by b l o c k i n g the A T P - d e p e n d e n t K'channel. Institut f~r P h a r m a k o l o g i e und Toxikologie, R o b e r t - K o c h Str.40, D-34 G S t t i n g e n

286

288

FUNCTIONAL DIFFERENTIATION OF TWO FORMS O F INSULIN RECEPTORS WITH T H E RECEPTOR ANTAGONIST B29,B29'SUBEROYL-INSULIN M. Welland

HYDROLYZABLE NUCLEOTIDES INHIBIT M I D E B I N D I N G IN P A N C R E A T I C I S L E T S M . S c h w a n s t e c h e r and Iris Rietze

In mature rat adipocytes the covalently dimerized insulin derivative B29,B29'suberoyLinsulin is a partial agonist of the insulin receptor tyroslne kinase activity (Joost et al., Biochem. Pharmacol. 38, 2269-2277 (1989)). In the present study, we compared the effects of thns partial receptor agonist in mature rat adipocytes with those in 3T3-L1 cells in an attempt to differentiate diverse effects of insulin. 3T3-L1 fibroblasts grown to confluence were differentiated by treatment with dexamethasone, isobutyl-methylxanthine and insulin. In these cells, insulin receptor binding ( lzs I-insulin binding to 3T3-L1 cells), glucose transport activity (2-deoxyglucose uptake rate), DNA synthesis ([SH]thymidine incorporation into total DNA), and receptor tyrosine kinase activity (autophosphorylation of partially purified insulin receptor) were assayed. B29,B29'-suberoyl-insulin fully inhibited specific insulin binding to 3T3-L1 cells with about the same affinity as insulin itself. In contrast, the dimer only partially (25% of the maximal response) mimicked insulin's effect on glucose transport activity and DNA synthesis. In the presence of half-maximally stimulating concentrations of insulin, B29,B29'-suberoyl-iusulin inhibited insulinstimulated glucose transport, DNA-synthesis and receptor kinase activity in a concentration dependent manner. Further, the dimer inhibited insulinstimulated DNA synthesis and glucose transport by a shift of the concentration response curve to higher insulin concentrations, indicating a competitive type of inhibition. In striking contrast, the dimer stimulated glucose transport (initial 3-O-methylglucosu as well as 2-deoxyglucose uptake rates) in rat adipocytes to the same extent as insulin did. Parther, the combination of B29,B29'-suberoylinsulin and insulin stimulated glucose transport in a purely additive fashion over the entire range of insulin's concentration-response curve. Thus, the covalently dimerized insulin derivative is a pure agonist of i~sulin-stimulated glucose transport in rat adipocytes, but an insulin receptor antagonist with intrinsic activity in 3T3-L1 cells. It is concluded that B29,B29'-suberoyl-insulin differentiates two types of insulin receptors present in 3T3-LI adipocytes and in the mature rat adipocyte.

Evidence was p r e s e n t e d recently that sulfon y l u r e a - i n d u c e d closing of the A T P - s e n s i t i v e K+channel requires p h o s p h o r y l a t i o n of the drug r e c e p t o r and/or channel. Therefore we investigated whether binding of glibenclamide to its receptor is altered by the presence of both Mg 2~ and ATP or related compounds. In microsomes obtained from mouse pancreatic islets ATP and ADP inhibited binding of SHglibenclamide. These effects were not observed in the presence of the nonhydrolyzable analogues adenosine 5 ' ( ~ , F - i m i - d o t r i phosphate) (AMP-PNP) or ~ , ~ - m e t h y l e n e - a d e n o sine 5'-diphosphate (AMF-CP), in the presence of AMP or in the absence of Mg z÷ . In the presence of Mg z~ , however, SH-glibenolamide binding was also reduced by GTP, GDP, UTP and CTP. This p a t t e r n of inhibitory effects of nucleotides was still observed after an additional wash of the microsomes. At a c o n c e n t r a t i o n of ImM adenosine 5'-0-(3thiotriphosphate) (ATP~S) was as effective as ATP. The submaximal inhibition of binding of S H - g l i b e n c l a m i d e induced by lower concentrations of A T P F S was not altered by cyclic AMP, cyclic GMP or calmodulin. The data suggest that the affinity of the sulfonylurea receptor is modulated by protein p h o s p h o r y l a t i o n induced by a kinase which could not be identified so far.

Abtellung Pharmakologle und Toxikologie I, Iustitut fiir Pharmakologie and Toxikologie der Unlversit~it G6ttingen, Robert-Koch-Str. 40, D-3400 Gbttingen, FRG.

GLIBENCLA-

Institut f~r P h a r m a k o l o g i e und Toxikologie, Robert-Koch-Str.40, D-34 G 6 t t i n g e n

R 73 289 I S THE F A I L U R E OF GLUCOSE TO PROMOTE I N S U L I N S E CRETION OF F E T A L LANGERHANS ISLETS C A U S E D BY

INADEQUATE D E P O L A R I Z A T I O N ? M.A. Wahl and R.G. W A L D M E R

291 [~II]IDAZO~

BINDS TO mz~ABRENOCEPTORS AS WELL AS INIDAZOLINE RECEPTIVE SITES IN XICROSOMES FROg RINmLF INSULINOMA CELLS

Annette Fetal rat i s l e t s are k n o w n to i n a d e q u a t e l y secret i n s u l i n a f t e r g l u c o s e s t i m u l a t i o n . The f i r s t steps in the cascade of events, w h i c h fin a l l y lead to exocytosis are c l o s u r e of the pot a s s i u m channel, d e p o l a r i z a t i o n and o p e n i n g of the v o l t a g e d e p e n d e n t Ca z+ channel. In collagename i s o l a t e d i s l e t s of a d u l t rats elevation of the g l u c o s e c o n c e n t r a t i o n f r o m 3 to 5.6 m M or t o l b u t a m i d e (i00 pg/ml in the p r e s e n c e of 3 m M glucose) causes the w e l l k n o w n i n h i b i t i o n of aSRb + efflux and increase of Ca 2+ n e t uptake. In islets of fetal rats (21 days p.c.) increase of the g l u c o s e c o n c e n t r a t i o n from 0 to 3 m M also p r o d u c e d i n h i b i t i o n of eaRL÷ e f f l u x but n o further effect was o b s e r v e d when g l u c o s e was raised u p to 5.6 mM. T o l b u t a m i d e i n h i b i t e d "SRb ÷ e f f l u x in the absence of g l u c o s e but no e f f e c t of tolb u t a m i d e was o b s e r v e d in the p r e s e n c e of 3 m M glucose. N e i t h e r g l u c o s e (3 mM; 16.7 raM) nor t o l b u t a m i d e (100 ~g/ml) s t i m u l a t e d net u p t a k e of calcium. Our d a t a suggest that in islets of fetuses the failure of insulin secretory response to g l u c o s e or t o l b u t a m i d e s t i m u l a t i o n is due to n o n a d e q u a t e o p e n i n g of the voltage sensitive c a l c i u m channel. Since "SRb • e f f l u x is m a x i m a l l y i n h i b i t e d a l r e a d y at 3 m M glucose, in the u t e r i ne life at m a t e r n a l b l o o d sugar levels fetal p a n c r e a t i c B-cells are r e f r a c t o r y to s t i m u l a t i o n of insulin s e c r e t i o n via d e p o l a r i z a t i o n .

D e p a r t m e n t of Pharmacology, Institute of Pharmaceutical Sciences, E b e r h a r d - K a r l s U n i v e r s t i y T~bingen, Auf der M o r g e n s t e l l e 8, D-7400 T ~ b i n g e n

Schulz

and A. H a s s e l b l a t t

We h a v e shown p r e v i o u s l y that a2-adrenoceptor antagon i s t s e q u i p p e d w i t h an i m i d a z o l i n e - m o i e t y e n h a n c e i n s u l i n r e l e a s e from mouse p a n c r e a t i c i s l e t s . This property r e s i d e s i n t h e i m i d a z o l i n e s t r u c t u r e and d o e s n o t r e s u l t from a 2 - a d r e n o c e p t o r b l o c k a d e as az-antagonists of different structure failed to stimulate insulin release. We now i n v e s t i g a t e d w h e t h e r an i m i d a z o l i n e r e c e p t o r s i t e does e x i s t i n t h e c e l l membrane o f i n s u l i n p r o d u c i n g cells from where t h e s i g n a l for additional insulin originates. [ 3 H ] I d a z o x a n ([°If]IDA), a r a d i o l i q a n d of affinity for both, the az-adrenoceptor as w e l l as "imidazoline-preferring binding sites", was employed i n b i n d i n g s t u d i e s and RINm5F i n s u l i n o m a c e l l s were u s e d as a model system. The following findings suggest that [oil]IDA binds to a heterogenous population of receptors (probable the az-adrenoceptor and an imidazoline binding site): i. Competition of [SH]IDA by unlabeled idazoxan revealed a biphasic curve with a high (ICnoh=7.6 nmol/l) and a low (ICso,=0.4 ~mol/l) affinity site. 2. Competition of [oil]IDA by rauwolseine affected almost exclusively the high affinity binding site (ICLoh=0.1 nmol/l) and only 55% of total radioligand binding. 3. The imidazoline antazoline which exhibits negligible effects on ~m-adrenoceptors competes only for the low affinity binding site (IC~o.=l.9 ~mol/l). 4. Imidazoline compounds with known affinity to ai-adrenoeeptors compete for both binding sites (phentolamine ICsoh=5.9 nmol/l, IC5o,=75.6 ,mol/l; clonidine IC5oh=2,3 nmol/l, IC~oi=9.4 ]amol/l).

Institut f~r Pharmakologie und Toxikologie, Universit~t G6ttingen, Robert-Koch-Str. 40, D-3400 G6ttingen. Supported by DFG, SFB "Organprotektion"

290

292

INHIBITION OF CAMP RELEASE FROM RIN M5F - CELLS BY PHORBOL ESTER TPA. H.P.T. Ammon a n d S.E. S c h u l z e

IMMUNOADSORPTION OF THE TISSUE-SPECIFIC, INSULINRESPONSIVE GLUCOSE TRANSPORTER FROM ADIPOCYTES A. Schiirmann

Phorbol esters by activating protein kinase C are known e i t h e r to e n h a n c e or to i n h i b i t a g o n i s t - s t i m u l a t e d c A M P - p r o d u c t i o n b y i n t a c t c e l l s or m e m b r a n e p r e p a r a t i o n s . T h e s e a c t i o n s m a y be m e d i a t e d b y e f f e c t s on t h e h o r m o n e r e c e p t o r , Gs- or G l - p r o t e i n or t h e c a t a l y t i c s u b u n i t 11,2]. In c r u d e m e m b r a n e f r a c t i o n s of RIN m5F-cells basal adenylate cyclase activity and stimulat i o n b y f o r s k o l i n ( 1 0 0 ]aM) a n d NaF (10 mM) w a s s i g n i ficantly augmented after 5 minutes pretreatment with TPA (100 nM): w i t h o u t TPA TPA basal 2024-62.4 3384-98.1 forskolin 4144-56.8 711 + 8 5 . 7 NaF 5794-127 9214-149 [pmoles cAMP/rag p r o t e i n ' m i n ] , means_+SEM, n = 4 . In c o n t r a s t , t h e r e l e a s e o f cAMP from i n t a c t RIN m S F c e l l s i n d u c e d b y f o r s k o l i n (100 pM), g l u c a g o n (5 lig/ml) a n d i s o p r e n a l i n e ( 1 0 0 pM) w a s s i g n i f i c a n t l y d i m i n i s h e d b y TPA (100 nM): w i t h o u t TPA TPA basal 3.3+0.2 3.0-+0.05 forskolin 1944-17.5 86+5.8 glucagon 10.84-1.1 6.74-1.8 isoprenaline 4.94-0.3 8.84-0.05 [fmoles cAMP/pg p r o t e i n ] , means-+SEM, n = 4 . TPA did n o t a f f e c t f o r s k o l i n - m e d i a t e d i n c r e a s e of cAMPc o n t e n t of RIN m 5 F - c e l l s . l t is s u g g e s t e d t h a t TPA a f f e c t s t h e c A M P - s y s t e m o f RIN m 5 F - c e l l s a t d i f f e r e n t locations: first at the adenylate cyclase and second t h r o u g h i n h i b i t i o n of t h e r e l e a s e m e c h a n i s m . [1] J o h n s o n e t el. J . C y c l . N u c l e o t . P h o s p h . R e s . ( 1 9 8 6 ) 11,3:199-215 [2] Y o s h i m a s a et el. N a t u r e ( 1 9 8 7 ) , 3 2 7 : 6 7 - 7 0 Lehrstuhl Pharmakologie, Pharmazeutisches A u f d e r M o r g e n s t e l l e 8, D - 7 4 0 0 T f i b i n g e n

Institut

Glucose transport activity in adipose and muscle tissue is controlled by insulin through a translocation of glucose transporters from an intraceUular pool to the plasma membrane. These intracellular transporters are located in microsomal, probably Golgi-assodated vesicles which can be separated from plasma membranes by differential centrifugation. It is as yet unknown, whether the vesicles comprising the glucose transporter contain additional proteins, and, if so, whether these proteins are also translocated in response to insulin. In order to approach this question, we have further purified the Golgl-assodated vesicles which contain the glucose transporter. Specific antiserum was raised against the C-terminal tridekapeptide of the adipocyte/mnscle tissue-speciflc glucose transporter (GT3), and was characterized by Western blotting and immanoprecipitation of glucose transporters from adipocytes, r-Globulins were isolated from the serum by adsorption to protein A sepharose and eluted by a low-pH wash. The r-globulins were covalently linked to either tresyl- or CNBractivated agarose. The excess of reactive groups was deactivated with ethanolamine. After exhaustive washing, the affinity adsorbens was incubated with low-density microsomes prepared from adipocytes and solubilized in triton X-100, washed again thoroughly, and the bound proteins were eluted with electrophoresis sample buffer. Western blotting of the eluates indicated that the affinity adsorbens specifically retained the glucose transporter. As judged from both specificity and yield of the adsorption, the CNBr-linked affinity adsorbens appeared superior to the tresyl-linked material. Further, glucose transporters from intact microsomes were retained by the CNBr-linked immunoadsorbens, indicating that the adsorbens can be used to isolate the vesicles containing the glucose transporter. Thus, the results provide an approach to isolate and identify the proteins assodated with the translocated glucose transporter in insulin-sensitive cells. Abteilung Pharmakologie and Toxikologie I, Institut fiir Pharmakologie and Toxikologie der Universit~it GSttingen, Robert-Koch-str. 40, D-3400 Gtttingen, FRG.

R 74 293 EFFECTS OF CHOLECYSTOKININ (CCK) RECEPTOR AGONISTS AND -ANTAGONISTS ON GLUCOSE PRODUCTION, -UTILIZATION, PLASMA INSULIN AND -GLUCOSE IN RATS C. Zoll, E.J. Verspohl CCK increases insulin secretion in vitro and in vivo. The aim of this study was to ~n~estigate the effect of CCK on glucose metabolism. The primed constant D[3- H] glucose infusion technique in anesthetized rats was used. Plasma glucose and plasma radioactivity were measured and the rates of glucose production and -utilization were calculated by employing the equations of Steele et al. in the derivative form using a modified single compartment model in the non-steady-state. Plasma insulin and glucose levels were determined at 5-10 minute intervals for 2 hours. By validating the system employing glucagon, a hormone with well known effects on glucose metabolism, there was a transient increase in glucose production and a decline of -utilization followed by a sustained increase in glucose utilization. The i.v. application of CCK in various doses (0.5 , 2 , 10 nmoles/kg b.w.) resulted in a concentration dependent increase in plasma insulin. This insulin secretion was inhibited by the CCK receptor antagonist L-364,718. At plasma glucose levels of 6 mM, achieved by infusion of 100 mg glucose/kg*h, CCK (2 nmol/kg) increased glucose production and decreased glucose utilization transiently followed by eounterregulations. When plasma glucose levels were increased to 9 mM by infusion of 500 mg glucese/kg*h CCK (2 nmol/kg) resulted in a sustained increase in glucose utilization whereas glucose production remained unchanged. These results indicate that cholecystokinin may have regulatory functions in glucose metabolism: whereas it increases glucose production at basal glucose levels CCK rises glucose utilization causing a decrease of plasma glucose concentrations if they are enhanced. Department of Pharmacology Institute of Pharmaceutical Sciences Eberhard Karls University Tiibingen Auf des Morgenstelle 8 7400 Tiibingen

294

To investigate a novel model of inflammation, we studied peptone as an inflammatory stimulus and non steroidal anti-inflammatory drugs (NSAIDs) on edema formation, cell migration and cartilage degradation in the rat air pouch. Subcutaneous a i r pouches were created in female SPF rats by inflation of 20 ml s t e r i l e air. Rats had rabbit cartilage implanted on day 8 and received either 10 ml of 3% peptone (PEP) in saline or vehicle (SAL) only (every other day), followed by daily injections of different doses of diclofenac (DIC), piroxicam (PIR) or placebo into the air pouch. On day 15, the rats were killed, a i r pouch f l u i d (APF) volume and cell number recorded and uronic acid in cartilage quantitated. Furthermore, chemotaxis of neutrophils was measured by using the under-agarose method of Nelson ( j . Immunol. 115:1650) with rat zymosan-activated serum as the chemoattractant and MEM as the cont r o l . SAL-treated rats showed a significantly lower APF volume and less cells than PEP-treated air pouches. On the other hand, chemotaxis of neutrophils derived from inflamed air pouches was significantly lower compared to neutrophils obtained from blood. However, there was no difference in uronic acid content of implanted cartilage after either treatment. NSAIDs dose-dependently decreased the APF volume after PEP application but had no effects on cell number. However, DIC administered in vivo caused inhibition of chemotaxis when tested ex vivo (PIR not tested). The major side-effects of NSAIDs were intestinal perforations which were more pronounced with PEP co-admioistration than with SAL only. Thus, NSAIDs act as antiphlogistic drugs on the inflamed ~ir pouch but do not decrease cell accumulation• supported by DAAD; present address: I n s t i t u t ffir Pharmakologie, FU Berlin, Thielallee 6g/73, D-IO00 Berlin 33 Dept. of Rheumatology, Royal North Shore Hospital, University of Sydney, St. Leonards, N.S.W. 2065, Australia

296

HAEMOSTATIC SYSTEM IN MALE RATS: INFLUENCE TESTECTOMY AND TESTOSTERONE SUBSTITUTION G. Paul, B. Miiller, B. Baldus

OF

Males are at a higher risk of cardiovascular events linked to arterial thrombosis (i.e. myocardial infarction) than premenopansal women. We investigated whether testosterone representing the main determinant of the male sexual endocrinologic status might influence the haemostatic system in a way favouring thrombosis. Three groups of adult (-> 3 months) male Wistar rats (10anlmalR each) were formed: 1 contxols (intact males) 2 testectomy 3 testectomy followed by daily s.c. injection of testosterone propinnate (TP; 1 mg/kg) starting 3L0days after testectomy Haemostatic parameters were measured 38 days after testectomy (28 days after start of rip treatment in group 3). No difference between the groups was noted with respect to coagulation parameters (prothrombin time, partial prothrombin time) and plasma fibdnolytic potential (fibrin plate test). Platelet aggregation, however, assessed as fall of platelet count in heparinized whole blood upon addition of ADP (5 x 10"7 M), was reduced by 56.5 % in testectomized vs. control animals (cz _<0.05) and fully restored to control values with TP treatment. In a thrombosis model where thrombus size is knowa to depend from both platelet function and activation of coagulation (combined thermal and mechanical damage of jugular veins, assessment of thrombus size 3 hours later by measurement of Hb content in damaged vs. intact vessel segments) the largest thrombi were encountered in controls. Thrombus size in testectomized animals was 59.5 % of control values ((z < 0.05) while TP treatment resulted in only nonsignifieantly smaller thrombi (79.7 % of controls; (z _> 0.05). Decreased platelet aggregability/fosmation of smaller thrombi after testectomy and reversion of these effects by testosterone replacement indicate a link between the male endocrinological status and a higher liability for platelet dependent thrombotic events. Research Laboratories of Schering D-1000 Berlin 65, West Germany

295 THE SUBCUTANEOUS RAT AIR POUCH AS A MODEL TO EVALUATE ANTI-INFLAMMSTORY DRUGS B. Nuernberg", G.E. Freeman, and P.M. Brooks

AG,

Mitller str.

170-178,

The distribution and tissue. M. Kurowski

of

NSAIDs in

synovial fluid

The distribution of nonsteroidal antiinflammatory drugs between plasma and synovial fluids follows physiological and biochemical-pharmacological rules. Under conditions of destructed, pathophysiologically alterated capillary membranes an accelerated equilibration can be observed. At a constant functional environment of the synovial capillaries the half-life and the PKa-values correlate with synovial drugs accumulation at steady-state. Among the drugs, which have been studied in patients with rheumatoid arthritis, the ratio of AUC-values (synovial fluids/plasma) was 0.77 (highest value) for piroxicam and 0.22 (lowest value) for tiaprofenic acid. A different behaviour could also be observed regarding concentrations in synovial tissue. With oxaprozim, a new npropionic acid derivative, tissue concentrations were considerably higher than plasma levels. In contrast the tissue levels of tiaprofenic acid reached less than 10% of the corresponding plasma values. Since the synovial tissue can be regarded as the therapeutical target for antiinflammatory treatment, the different tissue affinities of various NSAIDs should be investigated more carefully under clinical conditions. *Institut fur Pharmakologie und Toxikologie, Universit~t Erlangen N~rnberg, Universit~tsstraBe 22, D-8520 Erlangen, FRG

R 75 297 SOME METABOLIC CHIRAL INVERSION OF IN CLINICALLY USED ARYLPROPIONIC ACIDS DIFFERENT SPECIES S. M e n z e l - S o g l o w e k and G. Geisslinger 2-Arylpropionic acids like ketoprofen and ibuprofen are clinically used in their racemic forms. The anti-inflsmmatory activity, however, appears to reside in the S(+)-enantiomers. A unique characteristic of the m e t a b o l i s m of this class of drugs is inversion at the asymmetrical C-atom. This inversion seems to be unidirectional in m a m m a l i a n species. The present study was done to examine w h e t h e r metabolic chiral inversion of k e t o p r o f e n and ibuprofen is enantioselective and whether enantioselectivity is species-dependent. Stereospecific HPLC assays were developed to determine simultaneously the plasma concentrations of the enantiomers using different chiral columns. Metabolic chiral inversion was investigated after ivadministration of I0 m g / k g BW of the pure R(-}-enantiomers of ketoprofen and ibuprofen to various species i.e. rat, gerbil, guinea-pig and man. Plasma concentrations of the R(-)and m e t a b o l i c a l l y formed S(+)-enantiomers were quantified and p h a r m a c o k i n e t i c parameters calculated. Our data demonstrate, that the extent of m e t a b o l i c inversion is substanceas well as species-dependent. D e p a r t m e n t of Pharmacology and Toxicology, U n i v e r s i t y of Erlangen-N61rnberg Universititsstr. 22, D-8520 Erlangen, FRG

298 GASTROINTESTINAL ULCERATIONS INDUCED BY ANTIINFLAMMATORY DRUGS IN RATS: PHYSICOCHEMICAL, BIOCHEMICAL, PHARMACOKINETIC FACTORS INVOLVED W.S. Beck and K. Brune Gastric and intestinal side-effects represent the most common adverse drug effects of non-steroidal anti-inflammatory drugs (NSAIDs). Aspirin, diclofenac, diflunisal, ibuprufen and indometacin were investigated both in vitro and in vivo. Their inhibitory potency at the cyclo-oxygenase was determined in macrophage cultures. Their acute gastric and intestinal toxicity was assessed in fed or fasted rats alter oral or intravenous administration. The amounts of these drugs excreted in bile were studied in rats with biliary fistulas and were quantified by HPLC. Firstly, we found that the gastric damage occurred only in lasted animals, and is strongly correlated with the dose given and the drug's solubility at pH 2. Neither the drug's eyclo-oxygenase inhibitory potency nor the degree of its biliary excretion contribute significantly to acute gastric toxicity. Secondly, ulcerations of the small intestine occurred in fed animals only. The degree of damage is strongly correlated to the amount of unchanged or conjugated drug excreted in bile and eyclooxygenase inhibitory potency. The absolute dose and drug solubility arc not relevant factors in the development of intestinal ulcerations, it is concluded that, in the rat, acute gastric and intestinal toxicity of nonsteroidal anti-inflammatory drugs are due to different mechanism. Whereas gastric toxicity is strongly influenced by the amount of drug dissolved under the pH conditions in the stomach, intestinal toxicity appears to depend on biliary excretion and enterohepatic circulation of a drug as well as on its potency as an inhibitor of prostaglandin synthesis. Department of Pharmacology and Toxicology, University of ErlangesNueruberg, Universitaetsstrasse 22, D-8520 Erlangen, FRG

299 THE EFFECT OF DERMAL APPLICATION OF A GLUCOCORTICOID ON THE RM3/I + MACROPHAGE IN THE BLOOD G. Zwadlo-Klarwasser, S. Bent and W. Schmutzler Previous studies revealed that i.v. injection of a glucocorticoid (prednylidene) causes an induction of the antiinflammatory macrophage subtype RM 3/1 in the peripheral blood of man (ZwadloKlarwasser et.al.,Int.Arch.Appl.Allerg. Immunol. in press). Its proportion among blood monocytes increased from a basic level of 3% to about 80% positive cells within 24h. The present study was designed to investigate the effect of topical application of glucocorticoids on the RM 3/1 + subtype in the blood. Healthy probands treated a skin area of 200 cm 2 with a creme containing 0,1% fluprednidene for periods up to ii days. The daily administered dose amounted to about 6 mg. Before and at different times during application blood was taken, mononuclear cells were isolated and analyzed for RM 3/1 expression on cytospin preparations with an indirect immunoperoxidase technique. After 1 day no effect could be observed. On day 4 a weak increase in RM 3/1 + cells occurred in half of the probands. On day 7 and ii no significant changes could be found. These results indicate that topical application of fluprednidene to a limited area of healthy skin did net cause a comparable effect to a single i.v. injected dose of prednylidene on the RM 3/1 + macrophage subpopulation in the blood. institute of Pharmacology, RWTH Aachen Wendlingweg, D-5100 Aachen, FRG.

300 INFLUENCE OF CORTICOSTEROID TREATMENT ON CELL MOBILIZATION IN PATIENTS WITH SYSTEMIC LUPUS ERYTHENATOSUS (SLE) UNDER PSYCHOLOGICAL STRESS N. Rueckemann, J. Berth, R. Ferstl Characteristic changes of immunoregulatory cells due to physical or psychological stress in patients with SLE and in healthy subjects were described. Aim of the present study was to investigate the correlation between the attenuated cell mobilization in SLE patients and the changes in plasma hormone concentrations as well as influence of treatment with corticosteroids. 14 patients with SLE (57 + 8 yrs., X + SEN; 11 under prednisone 4-10 mg/die), 14 sexand age matched healthy subjects and 10 further patients under corticosteroid treatment without SLE were studied. The stress model consisted of different reproducable neuropsychological tests and lasted 2 hours. In healthy subjects a significant decrease in the lymphocyte count, of the Tlymphocytes, of the Th-lymphecytes and of the Th/Tsc-ratio (p< 0.05) was found. An increase of the B- and Tsc-lymphosytes was also seen in this group. Patients on corticoateraid treatment without SLE showed no difference in the degree of cell mobilization compared with healthy subjects. In SLE-patients, however, no significant changes of these parameters were detected. In the three groups of subjects investigated the stress tests caused a significant rise in plasma adrenaline and noradrenaline levels. The height of this increase was comparable in all three groups. The plasma cortiaol levels after the 2 hours lasting stress test remained unchanged compared with the baseline values. In conclusion a reduced cell mobilization following a psychological stress test was seen in SLE patients, while the increase of catecholamines was comparable in all groups. This attenuated cell mobilization to cstechelamine stimuli is not altered by a low dose certicosteroid treatment in patients with systemic lupus erythematosus. Present address: I. Medizinische Klinik, ChristianAlbrechts-Universit~t, Schittenhelmstr. 12, 2300 Kiel

R 76 301 A L T E R A T I O N S IN T H E P E R C E N T A G E O F L Y M P H O C Y T E S U B T Y P E S IN P E R I P H E R I A L B L O O D O F M A R M O S E T M O N KEYS AFTER EXPOSURE TO 2,3,7,8-TETRACHLORO. D I B E N Z O - P - D I O X I N IN VIVO A N D IN VITRO R e i n h a r d Neubert, Ursula Jakob-Miiller, H a n s Helge*, Diether Neubert

osure of experimental animals to very high doses of 2,3,7,8T F •(TCDD) e been reported to intertrachlorodibenzo-p-dio~n fere with man] variables of the immune system, but the significance has

of these findings with respect to a risk assessment for man is questionable. We have studied the effect of very small single doses of TCDD on l?eripherial blood lymphocytes of the non-human primate Callithrix jacchus (marmoset). Classification of the lymphocyte subtypes was performed with monoclonal antibodies (MAN) and a FACSean (Becton-Dickinson). Following a single s.c. dose of 10 ng TCDD/kg body wt the percentage of CD4* cells (especially those of the subtype CD4+/ CDw29 +) was found to be reduced (R. Neubert et al., Braz J Med Biol Res, Dez 1989). Due to the persistance of TCDD within the body such an effect could still be demonstrated more than 10 weeks after the single dose of 10 ng TCDD/kg body wt. Our data provide evidence for' defined immunological changes occurring in peripherial lymphocytes of non-human primates after exposure to TCDD, and they may form the basis for special clinical studies on human populations at risk exposed to PCDDs/PCDFs. Although the effects observed/n vivo may be secondary (to effects on lymphatic organs), additional studies performed by us indicate that TCDD also may exert a direct effect/n v/tro on poke weed mitogen-stimulated lymphocytes from marmosets or man at the extremely low concentration of l x 10 13M. Supported by grant Nr. 0765002 from the Bundesministerium fiir Forschung und Technologic. • ' " * and Instltut " fiir Toxikologle " " und Embryopharma k oKinderkhnik logic, Freie Universit~t Berlin, Garystrasse 5, D-1000 Berlin 33

302 TBE REGULATION OF SIALYLTRANSFERASE ON THE MEMBRANE OF HUMAN LYMPNOCYTES W.Gielen, E. Hoermeyer, U. Scholl

On the membrane of human lymphocytes we can demonstrate an interesting interaction between a galactophilic lectin and a membrane-bound sialyltransferase. Both receptors are located in spatial proximity but on different structures of the membrane, for the solubilized enzyme doesn't show any lectin property. This galactophilic lectin seems to regulate the access to the active center of the sialyltransferase. Both receptors exclusively recognize galactose, offered as mono-, di- or oligosaccharides and as a sugar molecule in terminal position of the carbohydrate chain of a glycoprotein. Only after the lectin has been saturated completely, the enzyme can perform the transfer of the sialic acid from the activated sugar nucleotide to the corresponding galactose acceptor. The cell to cell sialylation still becomes m o r e difficult by the circumstances that the availability of the enzyme is additionally hindered by the glycoproteins of the lymphocyte membrane and especially by the sialic acid, the anionic sugar moiety bound peripherally on these membrane glycoconjugates. Previous exposure of the lymphocytes to neuraminidase significantly enhances the cell sialylation, and the presence of galactose induces an additional effect, possibly by increasing the substrate concentration in the enzyme area. For the rosette formation with asialo-erythrocytes both receptors of the lymphocyte membrane must be unoccupied. The blocking of one receptor, by galactose or by sialyltransferase antibody respectively, prevents the rosette formation according to the degree of the highly specific binding at the receptor sites.

Institute of Pharmacology, University of Cologne, Gleueler Str. 24, 5000 Cologne 41, FRG

303 INFLUENCE OF NAAGA (N-ACETYL-ASPARTYL-GLUTAMICACID) ON THE IN VITRO MEDIATORRELEASE OF HUMANCELLS I. Andresen, J. Ring*, D. Rehn, G. Hennings NAAGA is a new active compound which produces a mast cell s t a b i l i z i n g effect, prevents the degranulation and i n h i b i t s the spontaneous release of histamine induced by allergen and compound 48/80. There is also evidence for i n h i b i t i o n of the anaphylactic release of leukotrienes (LTC~, LTD. and LTE4) from the sensitized lung tissue and t h e I~E mediated contractions of tracheal strips obtained from the sensitized guinea pigs. The aim of this study was to investigate the effect of NAAGA (200 mg/ml) on histamine, leukotriene B. and serotenin release of peripheral blood c e l l s from healthy subjects and patients with a l l e r g i e s (histamine release of basophil granulocytes, leukotriene synthesis of monocytes and neutrophil granulocy~es and serotonin release of thrombocytes) induced by anti-lgE, allergens (bee and wasp venom) and thrombin. NAAGA i n h i b i t e d s i g n i f i c a n t l y the serotonin release induced by thrombin in a l l concentrations ( I : I 0 , I:I00, |:fOOD) with p
804 EFFECT OF AZELASTINE ON CHEMILUMINESCENCE OF HUMAN GRANULOCYTES J. SCHMIDT, B. KAUFMANN, and I. Szelenyi Measurement of emitted photones by means of lucigeninand 7-Dimethylamino-naphthalene-l,2-dicarbonic-acidhydracide (DMNH)- chemiluminescence (CL) enables to evaluate the respiratory burst of polymorphonuclear leukocytes (PMNL). Lucigenin detects very sensitively and specifically superoxid anion, whereas DMNH-CL represents more the myeloperoxidase-activities. Phorbolmyristate acetate (PMA) as well as opsonized zymosan induce both a lucigenin- and a DMNH-dependentCL in PMNL. Azelastine, an o r a l l y e f f e c t i v e antiasthmatic/antia l l e r g i c drug, inhibited the PMA-induced chemiluminescence in a dose dependent manner. IC~nvalues amount to 2.56 #M and 2.61 /~M for lueigenin- a ~ DMNH-CL, resp e c t i v e l y . In contrast, no i n h i b i t o r y a c t i v i t y was found a f t e r zymosan-induction. Therefore we conclude, azelastine acts specifically on the generation of oxygenderived radicals and is not simply a scavenger of oxygen-derived radicals. Considering the observation, that lucigenin- and DMNH-CLare affected in the same manner, we conclude, the myeloperoxidase-system is not influenced by azelastine. Comparing the effect of azelastine with the effect of protein kinase C (PKC) inhibitor staurosporin, we got the the same inhibitionpattern of PMNL-CL. Therefore we suggest, a possible target of azelastine is probably PKC. ASTA Pharma AG, Department of Pharmacology, Weism~llerstr. 45, 6000 Frankfurt/Main, FRG

R 77 305

307

THE CYTOKINE ACTIVATED O X Y G E N RADICAL GENERATING SYSTEM IN H U M A N GLOMERULAR MESANGIAL CELLS A N D SKIN FIBROBLASTS ÷

PHARMACOLOGICAL INTERVENTION AGAINST TNF-a-INDUCED HEPATITIS IN D - G A L A C T O S AMINE-SENSITIZED MICE

H.H. Radeke and B. Meier* We have recently demonstrated t h a t human skin fibreblasts (HSF) and mesangial cells (HMC) release Oz- (4.5 nmol/106 HSF/hr and 3.2 nmol/10~HMC/hr) into medium upon stimulation with physiological concentrations of IL1 - a and TNF-a. The effect of t h e s e cytokines was rapid in onset (0 - 10 min for I L - l - a , 10 - 40 rain for TNF-a) and continued for up to 5 hrs in contrast to the o x i d a t ive burst of neutrophils or macrophages. The addition of cyanide, azide, rotenone, allopurinol or x a n t h i n e had no effect t h u s excluding radical generating systems like mitochondrta and the x a n t h i n e / x a n t h i n e oxidase. Added NAD(P)H increased 02release by both cell types s e v e r a l - f o l d , and diphenyl-iodinium, a specific, covalently binding inhibitor of the e s s e n t i a l flavoprotein in the NADPH-oxidase complex inhibited the radical production in a dose dependent way. These r e s u l t s indicate the presence of a "phagocytic NADPH-oxidase". Moreover, employing low temperature difference spectometry (M. Baggiolini, Bern) the presence of cytochrome b-245 was shown in HMC. At p r e s e n t we are using both of t h e s e cell s y s t e m s to the pathway leading to NADPH-oxidase illucidate activation. The calcium ionophore A2~lsv was the most potent activator (46 nmol O2-/106HSF/hr) and was inhibited by TMB-8 dose dependently and by Quin 2 AM, whereas chelating of extracellular calcium with EGTA, EDTA or DETAPAC had no effect. PMA, FMLP, LTB4 had only marginal activities in either HMC or HSF (0.3 - 0.88 nmol Oz-/106cells/hr). Diacylglycerol and staurosporine, C3b, lipopolysaccharides and Lipid A or other cytokines like IL-6 and IFN-gamma had no direct effect. These first observations indicate a protein kinase C independent, intracellular calcium s e n s i t i v e mechanism of NADPH-oxidase activation in the non-pagocytic cells, HSF and HMC. This pathway may also explain our previous observations t h a t I L - l - a and TNF-cx stimulated rapid Ozgeneration in HSF and HMC. Dpt. Molecular Pharmacology, Medical School, " Chemical Institut, Veterinary School, Hannover, FRG. ÷ SFB 244

306 RECEPTOR-RELATED ASPECTS OF OF TUMOR NECROSIS FACTOR AND

Gisa Tiegs A d m i n i s t r a t i o n of e i t h e r 1 0 # g / k g s a l m o n e l l a a b o r t u s equi l i p o p o l y s a c c h a r i d e (LPS) i.p. or 15/zg/kg r e c o m b i n a n t m u r i n e t u m o r n e c r o s i s f a c t o r a ( T N F a ) i.v. to Da l a c t o s a m i n e ( G a l N ) - s e n s i t i z e d m a l e N M R I a l b i n o mice d to h e p a t i c injury within 8 hrs as a s s e s s e d by p l a s m a t r a n s a m i n a s e activities. In this m o d e l , l e u k o t r i e n e s y n t h e s i s i n h i b i t o r s or r e c e p t o r a n t a g o n i s t s as well as s u p e r o x i d e d i s m u t a s e or a l l o p u r i n o l p r o t e c t e d a g a i n s t G a l N / L P S b u t not against GalN/TNFa. We concluded that leukotrienes as well as reactive oxygen s p e c i e s a r e likely to be involved in the L P S - i n d u c e d T N F a - r e l e a s e by m a c r o p h a g e s (1). W e now r e p o r t t h a t p r e t r e a t m e n t with e i t h e r 0 . 5 m g / k g colchicine i.v., or 1 0 m g / k g c y t o c h a l a s i n B i.p., or 5 0 m g / k g cyclosporin A i.v. 15 hrs a n d 1 h b e f o r e t h e h e p a t o t o x l c c h a l l e n g e p r o t e c t e d a g a i n s t G a l N / L P S as well as a g a i n s t GalN/TNFa. E v i d e n c e was o b t a i n e d that neither r e t r e a t m e n t a f f e c t e d t h e r e l e a s e of t h e m a c r o p h a g e erived h e p a t o t o x i c m e d i a t o r s i n c l u d i n g T N F a . T h i s e x p e r i m e n t s s u g g e s t t h a t this p r o t e c t i o n m i g h t be d u e to an i n t e r f e r e n c e with t h e T N F a r e c e p t o r .

~

(1) G . T i e g s , M . W o l t e r , a n d A . W e n d e l . B i o c h e m . P h a r m a c . 38,627-631,1989. B i o c h e m i c a l P h a r m a c o l o g y , F a c u l t y of Biology, U n i v e r s i t y Of K o n s t a n z , D-7750 K o n s t a n z , F R G .

308 THE MODE OF ACTION ITS M O D U L A T I O N .

H. Holtmann, C. Brakebusch, H. Engelmann*, M. K6nig, R. Klocke, Y. Nophar*, K. Resch, and D. Wallach* K n o w l e d g e of the m e c h a n i s m s i n v o l v e d in and c o n t r o l l i n g the action of the cytokine tumor necrosis factor (TNF) is still limited. TNF binds to s p e c i f i c sites on the cell surface, h o w e v e r the s i g n a l l i n g p a t h w a y initiated thereby is a m a t t e r of debate, and the p o s s i b i l i t y of an i n t r a c e l l u l a r f u n c t i o n of TNF itself has been raised. Evidence against such an internal f u n c t i o n of TNF is b a s e d on the o b s e r v a t i o n that antibodies to a TNF r e c e p t o r (raised against its s o l u b l e form (Engelmann et al., J. Biol. Chem. 1990, in press)) induced T N F - l i k e effects: rapid d o w n - m o d u l a t i o n of T N F - b i n d i n g to cells, k i l l i n g of several cell lines in a way i n d i s t i n g u i s h a b l e from k i l l i n g i n d u c e d by TNF; and formation of p r o s t a g l a n d i n E2. M o d u l a t i o n of the level of TNF receptors, which is one possible way of c o n t r o l l i n g TNF effects, occurs through various mechanisms: r a p i d down m o d u l a t i o n was induced by p r o t e i n kinase C - a c t i v a t i n g phorbol esters and a p p a r e n t l y in a p r o t e i n kinase C - i n d e p e n d e n t w a y by the c y t o k i n e interleukin-l. E x p o s u r e to 8Br-cAMP induced a m o r e g r a d u a l change; w i t h i n several hours TNF-binding was decreased or increased, depending on the type of cell studied. Such d i f f e r e n t i a l m o d u l a t i o n of T N F r e c e p t o r expression, by i n c r e a s i n g the response of c e r t a i n cell types and d e c r e a s i n g that of others, m a y p o s s i b l y impose s e l e c t i v i t y on the a c t i o n of TNF. Dept. Molecular Pharmacology, Medical School Hannover, FRG, and *Dept. M o l e c u l a r G e n e t i c s and Virology, The Weizmann Institute, Rehovot, Israel.

IN VIVO EVIDENCE FOR THE PROTEOLYTIC ACTIVATION OF TUMOR NECROSIS FACTORa M a r c u s NiehGrster A d m i n i s t r a t i o n of l i p o p o l y s a c c h a r i d e (LPS) to Dg a l a c t o s a m i n e ( G a l N ) - s e n s i t i z e d m i c e led to a f u l m i n a n t h e p a t i t i s a n d a d r a m a t i c i n c r e a s e in p l a s m a t r a n s a m i n a s e levels w i t h i n eight h o u r s . T h i s i n f l a m m a t o r y r e s p o n s e to LPS is likely to be m e d i a t e d by s y n t h e s i s of L T D 4 (1) followed by s y s t e m i c r e l e a s e of t u m o r n e c r o s i s f a c t o r a ( T N F a ) (2). In o r d e r to e v a l u a t e t h e role of l e u k o c y t e d e r i v e d p r o t e a s e s and reactive oxygen s p e c i e s ( R O S ) in this m o d e l of h e p a t i c injury, we p r e t r e a t e d mice with a n t i p r o t e a s e s or a g e n t s c o u n t e r a c t i n g R O S . A d m i n i s t r a t i o n of exther a l - a n t i t r y p s i n or eglin C or s u p e r o x i d e d i s m u t a s e or a l l o p u r f n o l or c a t a l a s e p r o t e c t e d a g a i n s t G a l N / L P S - , but not against GalN/TNFa-induced hepatitis. We c o n c l u d e t h a t p r o t e a s e s a n d R O S a r e involved in a mechanism providing bioactive TNFa. We therefore d e t e r m i n e d T N F a - l e v e l s in s e r u m of mice. In n o n e of t h e p r o t e c t e d a n i m a l s , s y s t e m i c T N F a was d e t e c t a b l e in c o n t r a s t to high T N F a - l e v e l s in s e r u m of d i s e a s e c o n t r o l animals. We c o n c l u d e that b i o a c t i v e T N F a is n o t r e l e a s e d u n l e s s a n t i p r o t e a s e s are i n a c t i v a t e d by R O S t h u s e n a b l i n g p r o t e o l y t i c activation of T N F a . (1) G. T i e g s a n d A. W e n d e l . B i o c h e m . P h a r m a c o l . 37: 2569-2573, 1988. (2) V. L e h m a n n , M. A. F r e u d e n b e r g a n d C. G a l a n o s . J. Exp. Med. 1 6 5 : 657-663, 1987. B i o c h e m i c a l P h a r m a c o l o g y , F a c u l t y of Biology, U n i v e r s i t y of K o n s t a n z , D-7750 K o n s t a n z , F R G .

R 78 309 NET-FORMATION OF CHOLIN£ IN INCUBATED ATRIA EVOKED ~ HUSCARINIC RECEPTOR AGONISTS ~ D PHORBOL ESTERS U. Hess, J. LeiBner, R. Lindmar .......................................................... Hydrolysis of choline-(Ch-)containin~ pbospholipids is enhanced by transmitters and hormones as well as by activation of protein kinase C (LSffelholz, Biochem. Pharmacol. 38: 1543-49, 1989}. Both mechanisms lead to the production of phosphatidic acid and diacylglycero! (DAG) and may play an important role in sign~l transduction. In recent years, we proposed on the basis of indirect evidence that mnscarinlc r~cep~or .~timulation activated phospholipase D yielding Ch, phosphatidic acid and BAG. The possibility was not definitively excluded that receptor stimulation enhanced Ch efflux by translocatien of cytosolic Ch rather than net-formation. - In perfused chicken hearts and incubated atria, arecaidine propa~gyl ester (APE) enhanced Ch efflux, the half-maximally effective concentration being 56 nmol/l. This effect which reached a maximum after few min and was maintained for 40 min, was antagonized by pirenzepine (pA~ 8.1). Ill incubated atria, the netformation of Ch (due to hydrolysis of phospholipids) was calculated from tbe Ch efflux during the 40 min-period of dru~ exposure and the changes in,tissue Ch content durin~ this period. It was found that the basal Ch efflux was caused by both translocation of cellular free Ch and net ~ formation of Ch. In contrast, the APE-induced Ch efflux was totally due to net-formation. Almost the same da~a were obtained using ~B-phorbol-12B,13~L-dibutyrate (PDB) as stimulus. This response was blocked in a Ca~+-free~ EGTA-containinE incubation medium, whereas the APE-evoked effect was unchanged. - In conclusion, Ch mobilization by muscarinic receptor stimulation was due to hydrolysis of Ch phospholipids rather than to Ch translocation. The results are compatible with out previous suggestion of the (Ca~*-independent) phospholipase D being the target enzyme. Pharmakologisches Institut, Universit~t Mainz, Obere Zahlbacher Str.67, D-6500 Mainz.

310 IN VIVO ~ ME~L/NE H.N. Deeds

OF ANTIMHSCARINIC DRDGS TO ~ Z E INDUC~ C C ~ S P A ~ IN THE RAT.

~he perasympathetic nervous system has been implicated in the etiology of variant angina. The vascular ~Lu~carinic receptors involved have been characterized by mear~ of in vitro e x p e r ~ (Entzeroth et al., 1989. in i ~ i . Sci. P 34, 1989 in press; Van Charldorp and van Zwieten, Naunyn-Sc~edeberg's Arch P~col. 339:403, 1989) and k~icrggs p r o ~ l y to the M3 subtype. S ~ no in vivo data are available it was the aim of the present study to f r i g a t e the ability of ntlscarinic antagonists to block the r~=t-hacholine i ~ c e d coronary spasm in the anaesthetized rat. Pats w~re anaesthetized and a catheter was inserted via the right carotid artery and advanced closely to the a~c valve in order to inject methacholine near the cstia of the coronary arteries. ECG was monitored and S wave ele%-at/on was t a k ~ as intensity of the c o r o ~ spasm. A n t a g ~ t s were given iv and 5 rain prior to the a d ~ s t r a t i c n of m e - - l i n e . ~e anta~sts produced a dose dependent inhibition of the ~dlhacholine induced S wave elevation. Atropine, 4D A ~ (4-diphe~lacet~-N-~L~thylpiperidine m ~ d e ) i ~ 371 ((+)(6-~(Ir~)-5,1(>-(lihy~5-[ (l-~thyl-4piperidinyl )-acetyl ] - l l H - ~ [ b , e ] [1,4 ]diazepine-11one) were the most potent drugs with -log IDSO values of 7.81, 7.97 ~ 7.06, respecT_ively. Pil~ze4~ine ~ i~/~-4)X 116 (11- [[2- [(diet/~yla;~ )zL~:hyl]- l - p i ~ i d i n y l ]ac~tyl ] -5,1 l - d i h y d ~ i m ] r i d m [2,3-b] [i, 4 ] k ~ a z e ~ i n e - 6 - o ~ e ) p c ~ -log I ~ O ~ralues of 6.05 and 5.44. ~he l ~ t study shows that muscarinic antagonists with high affinity for M3 r ~ z ~ , e.g. 4 - D ~ ~ L ~ A H 371, ar~ potent drugs t o arrtac3~dze methacboline induced c o z z ~

ccr~triction in t~

311 THE DEPENDENCE OF PREPARATION ON VASOMOTION IN THE PORCINE EAR VEIN

ACH

E. Schneider, W. Felix, E. Meyer, J. Paulus and G. Steigerwald Acelycholine causes several different vasomotions on porcine ear veins (Felix et al., Phlebol. Proktol. 17:183 - 91, 1988). The purpose of our study was to characterize the influence of different preparations on ACh induced vasomotion of the porcine ear vein: 1. pedused, isolated veins in an organ bath, 2. simply cannulated, perfused veins in the intact dissected porcine ear and 3, helical stdps. 1. The Dedused. isolated vein in an oraan bath ACh induced three different vasomotions depending on concentrations. ACh 10-s to10-7 M relaxed the precontracted vessels. This relaxation was endothelium dependent (tested by perfusion with distilled water for 5 min), inhibited by atropine (1 t~M), and not influenced by pirenzepine (0.3 ~M) or by gallamine (10 p.M). Up to 10-7 MACh induced a tachyphylactic contraction, which was endothelium dependent and attenuated by indomethacin (10 p,M). Up to 10 ~M, only in 40 % of the veins ACh caused a strong and reproducible contraction which was endotheUum independent, blocked by atropine (1 p,M) and pirenzepine (0.3 MM) and not influenced by gallamine (10 llM). 2. The simolv cannulaled o~dused WiD Precontracted veins developed comparable reactions to ACh as on the isolated pedused veins. Only concentrations differed, A distinct relaxation appeared only up to 0.1 pM ACh. That means the same concentration tachyphylactic contraction staded. Therefore, relaxation could only be realized when the tachyphylactic contraction was exhausted. Damaging the endothelium with distilled water resulted in a conspicuous edema in the surrounding tissue without reducing reactivity to the precontracting agent adrenaline (1 pM). 3. Helical stdos These veins developed reproducible contraction up to 0.5 #M only. The contraction could be blocked by atropine (0.11~M) and pirenzepine (0.3 ~M) but not by gallamine (10 pM), Only 2% of very carefully prepared and precontracted strips relaxed when ACh (0.01 - 0.5 pM) was added. Therefore. we conclude that ACh induces three different muscarinic vasomotions on the porcine ear vein. But the expression of these vasoactive responses is preparation dependent. Walther Straub-lnstitut for Pharmakologie und Toxikologie der Universittit M0nchen, NuSbaumstr. 26, D-8000 MOnchen 2

312 INHIBITION BY THE EPITHELIUM, CYCLOOXYGENASE PRODUCTS AND MUSCARINE RECEPTORS OF THE STIMULATED [SH]ACETYLCHOLINE (ACh) RELEASE FROM THE ISOLATED GUINEA-PIG TRACHEA I. Wessler and G. Hellwig Isolated tracheae were incubated with [SH]choline t 0 label neuronal transmitter stores. After a 70 min washout period tritium efflux was measured in 3 min fractions. To trigger the release of [3H]ACh, tracheae were stimulated electrically (3 Hz, 2 min). In some experiments a $2/SI comparison paradigm was used, to analyse a modulation of [3H]ACh release; substances (oxotremorine, indomethacin) were added 15 min before $2. Separation of radiolahelled compounds was performed by HPLC, to discriminate between the stimulated outflow of [SH]ACh and [sN]phosphorylcholine (Wessler et al. 1989, this journal 340, R39). The tissue contents of [SM]ACh synthesized in epithelium-containing or epithelium-deficient tracheae did not differ (34600 ± 9500 dpm vs 42500 ± 9000 dpm, n = 5). Electrical stimulation of epithelium-containing tracheae, however, released ($1) only small amounts of [SH]ACh (450 + 150 dpm, n = 5), whereas large amounts of [3M]ACh (1800 400 dpm, n = 8) were released from epithelium-deficient tracheae. Likewise the $2/SI ratio increased 3-fold, when the epithelium was removed 12 min before $2. Indomethacin (3 ~mol/l) enhanced evoked [SH]ACh release by 110 ± 15% (n = 4). Under the present experimental conditions the epithelium-derived inhibition could not be transferred between organ baths. Oxotremorine (I pmol/ l) reduced the stimulated tritium outflow from epithelium-deficient tracheae by 64 ± 5 ~ (n = 3). The present experiments demonstrate a marked inhibition by the epithelium of evoked ACh release. Also cyclooxygenase products reduce ACh release. Impairment of the epithelium (bronchitis) or blockade of cyclooxgenase may enhance cholinergic transmission in the airways.

anaesthetized r a t .

A P ~ Research, ~r. Karl Thc~ae G ~ , 5~tr. 65, D-7950 B i b e ~ s s i,

Birker~rfer

INDUCED

Pharmakologisches Institut der Universit~t Mainz Obere Zahlbacher Str. 67, D-6500 Mainz, F.R.G.

R 79 313

315

MUSCARINE PREAND POSTJUNCTIONAL EFFECTS OF RECEPTOR ANTAGONISTS IN THE ISOLATED GUINEA PIG TRACHEA H. Kilbinger, D. Wolf, G. D'Agostino a

PHARMACOLOGICAL CHARACTERIZATION OF MUSCARINIC RECEPTORS MEDIATING CONTRACTIONS OF GUINEA-PIG UTERUS F. DSrje, Th. Friebe, R. Tacke*, E. Mutschler and G. bambrecht

Functional experiments suggest that muscarine autoreceptors i n h i b i t t h e r e l e a s e o f a c e t y l c h o l i n e (ACh) from p u l m o n a r y v a g a l n e r v e s in v i v o (1). We h a v e m e a s u r e d s i m u l t a n e o u s l y t h e o u t f l o w of ACh a n d i s o m e t r i c c o n t r a c t i o n s in g u i n e a pig tracheal muscle strips from which the epithelium was mechanically removed. The preparations were incubated with [3Hlcholine and then superfused (2ml/min) w i t h Tyrode s o l u t i o n c o n t a i n i n g 10 llM h e m i c h o l i n i u m - 3 . The e l e c t r i c a l l y e v o k e d [ 3 H ] o u t f l o w (20 Hz 8 s a t i n t e r v a l s of 30 s; 6 0 0 pulses) was calcium-dependent a n d p r e v e n t e d b y 3 0 0 nM t e t r o d o t o x i n . T h e e v o k e d o u t f l o w of [SH] a s m e a s u r e d In t h e p r e s e n c e of 10 tim n e o s t i g m i n e p l u s 100 nM s c o p o l a m i n e c o n s i s t e d to 104 + 11 ~ (N=4) of [3H]ACh. M u s c a r i n e receptor antagonists enhanced the [3H]outflow and inhibited the muscle contractions caused by electrical stimulation. The - l o g ECho v a l u e s f o r p r e - a n d p o s t j u n c t i o n a l e f f e c t s of s c o p o l a m i n e (8.85; 8.89) a n d p i r e n z e p i n e (7.08; 6.83) w e r e s i m i l a r . T h e M2 s e l e c t i v e antagonists AF-DX 116 a n d m e t h o c t r a m i n e (M) w e r e a b o u t l O 0 t i m e s more p o t e n t on p r e j u n c t i o n a l u u t o r e c e p t o r s ( - l o g ECho A F - D X 116: 7.57; M: 6.88) t h a n on s m o o t h m u s c l e r e c e p t o r s ( A F - D X 118: 5.52; M: < 5). As a c o n s e q u e n c e of t h e s e l e c t i v e b l o c k a d e of prejunctional inhibitory autoreceptors the evoked muscle contractions were significantly enhanced by A F - D X 116 (0.1 pM) a n d M (0.1 a n d 1 ]aM), The r e s u l t s p r o v i d e d i r e c t e v i d e n c e f o r t h e o c c u r r e n c e o f r e l e a s e - i n h i b i t i n g m u s c a r i n e a u t o r e c e p t o r s in t h e g u i n e a pig t r a c h e a , a n d s u p p o r t t h e s u g g e s t i o n (1) t h a t p r e and p o s t j u n c t i o n a l m u s c a r i n e r e c e p t o r s b e l o n g to t h e M2 a n d M3 receptor subtypes, respectively.

Muscarinic (M) receptors mediating contraction of estrogen-dominated guinea-pig uterus have been classified as being o f the M2 type (Eglen et al., BJF 96:497, 1989). In the present study we determined the affinity (pA2 values estimated from Schild plots) of antagonists for M receptors in uterine smooth muscle from immature guinea-pigs which were not pretreated with an estrogen. These data were compared with those obtained in rabbit vas deferens (Ml), guinea-pig atria (M2) and ileum (M3). Uterus Ml M2 M3 Pirenzepine 7.0 8.2 6.8 6.9 4-DAMP + 8.9 9.4 8.4 9.3 Sila-hexocyclium 8.8 9.0 7.6 8.8 Himbacine 7.9 8.2 8.1 7.3 Methoctramine 7.5 6.6 7.7 6.2 +4-Diphenylacetoxy-N-methyl p i p e r i d i n e methobromide.

(1) F r y e r A D, M a c l a g a n J; t h i s j o u r n a l 335: 3 8 7 - 3 7 1 , Pharma~ologisches Institut der I s t i t u t o "di F a r m a c o l o g i a d e l l a

The uterine M receptors displayed high affinity for himbacine and methoctrumine and so did not appear to represent M3 receptors. Despite this high affinity for the two M2 antagonists, pA2 values for 4-DAMP and sila-hexocyclium were not consistent with the presence of functional M2 receptors in the uterus. Finally, the uterine M receptors showed low affinity for pirenzepine suggesting that they were different to the M1 subtype, These results provide evidence that the M receptors in guinea-pig uterus are not either single MI, M2 or M3 subtypes or a heterogeneous mixture of these subtypes. Interestingly, the antagonist affinities for uterine M receptors correspond closely to those found at M4 receptors in rat striatum (M. Waelbroeck et al., this symposium).

1987

U n i v e r s i t l t t , D - 6 S O 0 Mainz; Universita, 1-27100 Pavia

Dept. of Pharmacology, Univ. of D-6000 Frankfurt/M, FRG. *Dept. of Chemistry, Univ. of D-7500 Karlsruhe, FRG.

314

316

MUSCARINICRECEPIDRS IN T H E G U I N E A P I G ~ M. Entzeroth and D. Lef~vre

AFFINITIES OF ACETYLENIC ANALOGUES RELATED TO DIFENIDOL METHIODIDE FOR MUSCARINIC RECEPTOR SUBTYPES N. Retteumayr, N. Wagmer-RTder, C. Strohmann* and L.K. 6%00

In guinea pig lung muscarinic acetylcholine receptors have been characterized in radioligand binding studies (Mak JCN and Barnes PJ, Eur. J. Pharmacol 164: 223-230, 1989; Gies JP et al., J. Fnarmacol. Exp. Tner. 250: 309-315, 1989). Discrepancies, however, exist in the classification of the receptor subtypes. Therefore binding studies to guinea pig peripheral and central lung (enriched in larger airways) were performed using the muscarinic antagonists atropine, AF-DX 116 (11-2((2-((diethylamino) methyl)-l-piperidinyl)acetyl )- 5,ll-dihydr o-6H-pyr ida (2,3 -b) (i,4 )benzodiazepin-6one), 4-DAMP (4-diphenyl-acetoxy-N-methylpiperidine) and pirenzepine. peripheral central NK i nH NK i nH atropine 8.91 0.94 9.03 0.94 AF-DX 116 6.89 0.76* 6.93 0.74* 4-DAMP 8.27 0.83 8.31 0.83 ~irenzepine 6.55 0.96 6.66 0.80* significantly different from unity (p<0.05) [3H]NMS binds to membrane preparation of guinea pig lung with a KD of 0.25 r/4 and a Bmax of 110 fmoles/Mg protein. Displacement curves of AF-DX 116 were shallow in peripheral and central preparations. The PKi-values for the high affinity site were 7.36 and 7.33, for the low affinity site 6.24 and 6.04, respectively. The displacement by pirenzepine in peripheral lung was best described by a 1-site model, while the Hill-coefficient was significantly different from unity in central preparations with ~Ki-values of 8.03 and 6.56 (14:86%). It is concluded that in guinea pig lung MI, M 2 and M 3 receptors are present. The small population of M 1 receptors was found in central lung preparations and may be located in larger airways. In peripheral lung only receptors of the M 2 and M 3 subtype were identified.

Butinol methiodide, the carbon chain of which is constrained to be linear, was more potent (up to 32-fold} than the flexible parent compound difenidol methiodide at the three subtypes. This indicates that the triple bond may contribute to binding and extended rather than folded conformations may be more important for the interaction of difenidol methiodide with the muscarinic receptors. Replacement of one or both phenyl rings in butinol methiodide by cyclohexyl changed the affinity profile of this compound (M1 > M2 = M3). In particular, dihexbutinol methiodide (MI > M3 > M2) showed high affinity for M1 receptors {pA2 = 8.8). Its affinities for M2 and M3 receptors were 25-fold and 6.3-fold lower, respectively.

A Pharma Research, Dr. Karl Thc~nae GmbH, Birkendorfer Str. 65, D-7950 Biberach/Riss i, FRG

Dept. of Pharmacology, Univ. of D-6000 Frankfurt/M, FRG. *Dept. of Chemistry, U n i v . of D-7500 Karlsruhe, FRG.

Affinity profiles of acetylenic conformationally restricted analogues related to difenidol methiodide were investigated at three muscarinic receptor subtypes. Antagonist potencies were determined in rabbit isolated vas deferens (MI), guinea-pig atrium (M2) and ileum (M3). pA2 values obtained from Schild plots are listed below.

C

.0

\/ /C\

H3CxJ~_~

CH2-C.2-CH2-.<~ )--'~

.0

-r-J-

~3~\ /--A

C-- C-Ca2--N~..~

Difenidol methiodide Rs ,R2 =Pbenyl, Cyclohexyt Methiodides of R* Rs M1 M2 Difenidol 7.7 7.6 Butinol phenyl phenyl 9.2 8.3 Dihexbutinol cyclohex¥1 cyclohexyl 8.8 7.4 (R)-Hexbutinol + phenyl cyclohexyl 9.4 8.6 +Data taken from Feifel et al., BJP in press.

M3 7.3 8.3 8.0 8.9

R 80

317

319

IDENTIFICATION OF THE STRIATUM "B" S I T E S A S BELONGING TO THE M4 M U S C A R I N I C RECEPTOR S U B T Y P E . M. Waelbroeck, J. Camus, M. Tastenoy and J. Christophe.

DETERMINATION OF THE EFFECTS OF MUSCARINIC AGONISTS UPON MEMBRANE POTENTIAL AND INTRACELLULAR CALCIUM IN NB-OK1 CELLS. H.W.G.M. Boddeke.

At least three muscarinic receptor subtypes can be discriminated pharmacolog~cally, in rat forebrain. The receptors with a slow [ H]NMS (N-methyl scopolamine) dissociation rate in forebrain, (previously called "B" sites ( I ) ) have an M3-1ike binding profile for pirenzepine (pKi 7.1), AF-DX 116 ([11-(((2[diethylamino)-methyl]-1-piperidinyl}acetyl)-5,11dihydro-6H-pyrido(2 ,3-b) (1,4)benzodiazepin-6-on]) (pKi 6.3), 4 - D A M P (4-diphenylacetoxy-N-methyl piperidine methiedide) (pKi 9.0) and HHSiD (hexahydrosila-difenidol) (pKi 7.8). They however differ from pancreas M3 receptors, by their high affinity for methoctramine (pKi : pancreas 6.0; striatum "B" sites : 7.6) and himbacine (pKi : pancreas 6.5; striatum "B" sites : 8.1). Their binding properties are qery similar to the M4 muscarinic receptor identified in NG 108-15 cells (2) and to the guinea pig uterus smooth muscle receptor (G. Lambrecht et al., this symposium). (I) Waelbroeck, M. et al. Mol. Pharmacol. 32, 91-99

(2)

(1988).

Michel, A.D. et al. Naunyn Schmiedeberg's Pharmacol. 340, 62-67 (1989).

Arch.

Address : Department of Biochemistry and Nutrition, Faculty of Medicine, Universit~ Libre de Bruxelles, 115 Bd. of Waterloo, B-I000 Brussels, Belgium.

Recently, it was demonstrated that NB-OKI cells express ml and m3 muscarinic receptors (Waelbroeck et al., 1988). In the present study membrane depolarization and increase in intraeellular calcium, which are functional responses to muscarinic receptor activation in these cells, were investigated. NB-OK1 cells were grown in RPMI medium supplemented with 10% fetal calf serum and 0.1% penicilline streptomycine solution at 37°C in a humidified atmosphere (5% CO2). Cells were harvested and a suspension of 2xl0cells was prepared. Membrane depolarization was measured using the voltage sensitive dye DIS-C2-(5). For this purpose the cells were incubated for 30 min with the dye. Single concentration response curves of the depolarization -induced changes in absorption of the dye were measured at the wavelength of 650 run. Increases in intracellular calcium were determined using the calcium indicator fura2. The cells were loaded with fura2 (acetylmethoxy form, 5 urn) and washed. Single dose concentration response curves of the calcium-induced changes in fluorescence were made using exicitation wavelength 340 and emission 510 nm. Using these two tests a series of muscarinic agonists was tested. A clear correlation (r=0.97) between depolarization and increase in intracellular calcium induced by musearinic agonists was found. Schild analysis with a series of muscarinic antagonists indicated that membrane depolarization and increase in intraoellular calcium are predominately mediated by ml muscarinic receptors. Waelbroeck et al., FEBS lett. 226; 287-290, 1988 Sandoz Preclinical Research, CH-4002 Basel, Switzerland.

318

320

A G O N I S T - I N D U C E D D E S E N S I T I Z A T I O N OF m - C H O L I N O C E P T O R S ON D I S S O C I A T E D C E L L S OF T H E M O U S E B R A I N L. Stoll

COMPARISON BETWEEN THE ALLOSTERIC AND THE COMPETITIVE ACTION OF A SERIES OF LIGANDS AT roACh-RECEPTORS Kirsten Buschandgrf, K. Mohr. and the late J. Schnekanbur~er* The bispyridinium oxime Uno3 inhibits [SH]NMS-binding to muscarinic acetylcholine receptors probably via a competitive action. Furthermore, it retards [aH]NMS-dissociation via an allosteric action. Derivatives of Uno3 (trimethylene-bis-[4-hydroxylminomethyl-pyridinium]dibromide mono-2,6-dichlorobenzylether) were synthesized with varying structure of the ether-linked subsfituent (indicated in italics). In suspensions of guinea pig cardiac membranes (3mM MgHPO4, 50mM Tris, pH 7.3, 37"C) the Uno--compounds were tested with respect to the action on the equilibrium-binding of [SH]NMS (0.5nM) and on the velocity of [SH]NMS-dissociation visualized by addition of 104M atropine. All Uno-compounds inhibited [SH]NMS-binding concentration-dependently. When applied at 3)1M within the same experiment, they reduced [SH]NMSbinding to 50-30% of the control. Apparently, the Uno-compounds occupied the roACh-receptors with similar atnnities. The dissociation of [ s ~ was retarded by the Uno-compounds at higher concentrations. The factors by which the dissociation was slowed compared with the control condition (tl/2~3min) varied at 30)aM from lfold to 6fold, at 300suM from 2fold to-40fold. Acoordingly, the Uno-compounds differed with respect to the allosteric activity. Uno3 belonged to the potent compounds. A derivative was synthesized which contained an additional 2,6dichlorobenzyl-substituent at the hydroxyiminomethyl-group, which is not substituted in Uno3. Compared with Uno3, this compound inhibited [SH]NMS-binding with the same potency, but had an even higher allosteric activity. In conclusion, the results support the notion that the two actions of the Uno-cempounds on roACh-receptors, i.e. the inhibition of [aH]NMSbinding and the retardation of [SH]NMS-dissociation, are mediated via binding to different sites. Furthermore, the findings demonstrate that the allosteric activity may respond with higher sensitivity to structural variations of ligands than their receptor binding aflqnity.

The concentration of m-cholinoceptors on the cell s u r f a c e is a d y n a m i c f u n c t i o n of the c o n centration of the a g o n i s t in c o n t a c t w i t h the receptors. When dissociated cells of the adult m o u s e brain are i n c u b a t e d w i t h h i g h c o n c e n t r a tions of n a r b a c h o l (I m m o l / l ) and m - c h o l i n o e e p tor d e n s i t y is i n v e s t i g a t e d using the s p e c i f i c b i n d i n g of 3 H - N M S ( N - m e t h y l - s c o p o l a m i n e ) before and after c a r b a c h o l stimulation, the p r e t r e a t ment with the agonist reduces the available number of m - c h o l i n o c e p t o r s by a b o u t 30%. T h i s decrease in the n u m b e r of m u s c a r i n i c receptor binding sites is time-, concentrationand temperature dependent. The ability of several cholinergic agonists to d e s e n i t i z e m - c h o l i n o c e p t o r s correlates fairly w e l l with their a b i l i t y to s t i m u l a t e the h y d r o l ysis of i n o s i t o l p h o s p h o l i p i d s in b r a i n tissue, e.g. e a r b a c h o l , a c e t y l c h o l i n e , and o x y t r e m o r i n e M are most potent, while McN-A-343 and RS 86 are o n l y weakly active. It is s u g g e s t e d that a g o n i s t i n d u c e d d e s e n s i t i z a t i o n of m - c h o l i n o c e p tors on dissociated brain cells represent a simple method to study m - c h o l i n o c e p t o r activation by agonists in brain tissues in vitro. Very i n t e r e s t i n g l y , the rate of d e s e n s i t i z a t i o n induced by the potent agonist carbachol can be diminished by some compounds used in the treatment of a f f e c t i v e disorders. Equilibration of d i s s o c i a t e d m o u s e brain cells w i t h c a r b a m a z epin (i0 mg/l), L i t h i u m (I m m o l / l and I0 m m o l / l ) and L e v o p r o t i l i n (0,i mg/l) s i g n i f i c a n t l y r e d u c e agonist induced desensitization. Department of P s y c h o p h a r m a c o l o g y , Central Ins t i t u t e of M e n t a l Health, D - 6 8 0 0 M a n n h e i m (FRG)

Abt. Pharmakologie, Universittit Kiel, Hospitalstr. 4, 2300 Kiel * Pharmazeutisehes Institut, Universittit Kiel, Gutanbergstr. 76-78 Generously supported by the Frannhofer Gesellschaft, Miinchan

R81 321

323

COMPARISON OF THE EFFECTS OF W84 ON [3H]NMS-BINDING IN PIG AND GUINEA PIG CARDIAC MEMBRANES. Carl-Michael Staschen and Manfred Zieeenhaeen W84 hexamethylene-bis-(dimethyl-[3-phthalimidopropyl]-ammonium bromide) has been shown to be a potent antidote against organophosphate-intoxicalions, when applied in combination with atropine. Binding studies revealed W84 to exert an allosteric effect on mascarlnic receptors in membrane suspensions of rat and guinea pig tissues. The aim of this study was to find out whether this effect also occurs in membranes of a species more closely related to man. The binding studies were, therefore, performed with myoeardiurn of pigs and for sake of comparison also that of guinea pigs. [ate]N-methyl-scopolamine (NMS) was used as muscarlnic ligand and incubated with the membranes for 120 rain at 23°C in 3 mM MgHPO4, 50 mM "Iris, pH 7.3. The results reported below are means of n=2-8 experiments. W84 inhibited the binding of [ZH]NMS concentration-dependently with ICe~-values of 2.5 x 10-GM and 1.3 x 10'~M in pig and guinea pig membranes, respectively. The affinity of [SH]NMS-binding (pig/guinea pig, KD: 1.2 x 10-gM / 1.3 x 10-°M) was reduced in the presence of W84 at its IC~o by a factor of about 2 (KD: 3.0 x 10-aM / 2.2 x 10-PM), whereas the number of binding sites remained unaffected. W84 inhibited strongly the dissociation of receptor-ligand complexes. Under control conditions the half life limes amounted to tv2=15 rain and 14 rain in pig and guinea pig myocardium. W84 at its IC~o prolonged the half life times to t~D=70 min and 55 mln, respectively, i.e. about 4-fold. At 10"4M W84 [aH]NMS-binding appeared to be almost irreversible in both species. In conclusion, the allosteric effect of W84 on muscarinic acetylcholine receptors was almost identical in pig and guinea pig cardiac membranes and is obviously species-independent.

~,-ADRENOCEPTOES OF THE ~,B- AND NOT OF THE 6IA-SUBTYPE COUPLE TO INOSITOL PHOSPHATE (IP) GENERATION IN CORTICAL BRAIN TISSUE Martin C. Michel* and Gerhard Gro~

Dept. of Pharmacology, University Kiel, Hospitalstr. 4, D-2300 Kiel Generously supported by the Fraunhofer Gasellschaft, MOnchen.

322 PMAILMACOKINETICS AI%ID CHOLINESTERASE INHIBITION OF GALANTHAMINE IN MICE IN VITRO AND EX VIVO ~. Bickel, T. Thomsen, J.P. Fischer, and O. Pleul Galanthamine (GAL), an alkaloid isolated from the snowdrop, is currently investigated as a promising agent to relieve the cholinergic deficit in senile dementia of Alzheimer's type. AS many basic enzymatic and pharmacological studies are performed in rodents, we have studied the pharmacokinetics and enzyme inhibition of GAL in mice in vitro and ex vivo. In rive, GAL was administered as a bolus i . v . injection to adult male NMRI-mice (30-45 g b.w.) in doses from 4 to 8 mq/kg. Controls received saline. Animals were decapitated after variable times (i to 180 min). Trunc blood was collected in heparinized tubes and separated into plasma and erythrocytes, The brain was removed and homogenized in PBS• GAL concentrations were determined in extracts from plasma, erythrocytes and brain using a HPLC method with fluorescence detection. Esterase activities were measured radiometrically in cor~esponding~samples. In vitro, GAL concentrations from I0-- M to I0 -~ M were added to native plasma, erythrocytes and brain homogenates to evaluate the relative potency of GAL for inhibition of AChE and butyrylcholinesterase (BuChE) in erythrocytes/brain and plasma, respectively. Terminal half life of GAL in plasma was about 40 min, and first order pharmacodynamics were found. While the concentration in erythrocytes was 1.5-fold compared to plasma, there was 2 to 3-fold accumulation in brain tissue almost immediately after injection• In vitro, pI~0- values of GAL were 5.3, 5.4 and 4•0 for AChE (eryt~rocytes, brain) and BuChE (plasma), respectively. Taking into account the concentrations as reached in vivo, inhibition of AChE in brain and erythrocytes correlated well with the values expected from the in vitro concentration-response Curves at all time points• Maximum GAL concentration (8200 rig/g) after 8 mg GAL i.v. led to an apparent AChE inhibition of 53% in brain homogenate (dilution 1:4, w/v). Institut f0r Klinische Pharmakologie, Freie UniversitGt Berlin, Hindenburqdamm 30, D-1000 Berlin 45

We could recently demonstrate that subtypes of a,-adrenoceptor recognition sites exist in several tissues of the rat as well as in human brain. Determination of the subtype responsible for the generation of IP has so far been hampered by the lack of sufficiently selective antagonists. In the present study we provide evidence that only one of these subtypes (axB) couples to phospholipase C. Binding of [SH]prazosln to al-adrenoceptors on membranes of the rat cerebral cortex was inhibited biphasically by axA-subtype selective drugs like (+)niguldipine, 5-methylurapidil, oxymetazoline and amidephrine. Two-site analysis of the data revealed a 42- to 562-told selectivity of these compounds. About 45 per cent of the sites belonged to the ~*A type irrespective of the drug used for the determination. Pretreatment of cortical brain slices with chloroethylclonidine (CEC) decreased a,B-sites by 89 per cent without affecting a~A-sites. In rat cortical slices prelahelled with [3H]inositol, noradrenaline in the presence of LiCI led to an accumulation of IP. The subtype-selective a,A-antagonists (+)niguldpine and 5-methyl-urapidil failed to block this response in concentrations which occupy s~^- but only a minor proportion of s,s-sites. Complete suppression of IP accumulation was only accomplished by higher concentratidns and by non-selective antagonists like prazosin. In contrast, selectively decreasing a,e recognition sites by CEC pretreatmeut clearly inhibited the generation of IP. These data demonstrate that only a,s- and not ~ A adrenoceptors are linked to phospholipase C and suggest that in contrast to ~- and az-adreuoceptors, ~,-adrenoceptor subtypes may couple to different second messenger systems. Inst. fflr Pharmakologie uud *Biochem. Forsehungslahor der Med. Klinik, Universit~tsklinikum Esseu D-4300 Essen, FRG Supported by the D.F.G.

324

Vasorelaxing. Activity Of Cazvedilol O n Isolated Vessels Precontracteo With Different Vasoconstricting Agents B. Eberwein, M. Kohler, R. G. Hooper and W. Bartsch Introduction; The B-blocki0.g and a-blocking properties of carved ilol (C) have been shown in a variety ot ihvestigations. However, it is not yet clear whether other mechamsms a r e involved in its vasorelaxing property. We therefore studiea m e vasorelaxing activity o f C on isolated vessels precontracted with different vasoconstrictJng agents. Rat aortic spiral strips were precontracted with 10-hM noradrenaline (NA), 4x10-2M K ÷, 10~M Bay-K 8644 or 10-SM PGF2a. The v~orelaxing activity of C was determined ay1 aaaing incremental concentrations to the bath. The resu ts were compared with those obtained with pharmacolozically defined comoounds: o r a z o s i n (P), nicardipine (N), glyceryl t r i n i t r a t e , ( G T l ~ . E quipotent vasorelaxing concentrations ~expresseo as t~t.s.~ ) o t t h e compounds were calculated for all agents and r~lV~ted to those obtained with NA. Results: As expected P and N demonstrated typical activities E~ P N GTN i C

NA

9 x IO-SM > 1 x I04M 3 x i0~ 2 x I(~TM

K +/NA

Bay-K/NA

> IO00 !> iOOO <0.OOOO1 < O.OO18 26 0.5 28

148.0

r~2~/r~ > iOOO < 0.03 20 117

as a~. and Ca+ +:blocking agent~ respectively. G T N was equienective in the 4 O~erent preparations. C is about 20 to 150-times more active on N.~-precontracted strips than on the others, but the difference from P is obvious. It has been shown in additional investigations that C has no vaser e l a x i n g activity against PGF2a in strips preincubated with 10-4M phentolffmine to block the a-receptors, whereas G T N is actwe. It is, therefore ~ that the a-blocking .activit~ .of C i s the m a i n ~ e c ~ s m of its vasoreJamn~ actwlty in large arteries. This may, however, not account entirew tot its actions in m e arterioles, which are responsible for the maintenance of blood pressure. De t.~artment of Pharmacology, Boehringer Mannheim G m b H , • u . ~ox 31 01 20, 6800 Mafinheim 31,-FRG

R 82 325 D~rEI(MINATION OF mRNA CODING FOR B2-ADRENOCEI~TORS IN H U ~ P~IPHERAL MONONUCLFAqR LEUCOCYTES BY IN S I ~ HYBRIDIZATION

B.Liebl,K.Pachmann,W.Liebl,U.RSmer,M.Hallek and B.Emmerich Various clinical studies have demonstrated variations in the expression of 82-adrenoceptor (BAR) sites in human peripheral mononuclear leucocytes (MNL) by means of radioligand receptor binding. A determination of the transcriptional product, the mRNA, may contribute important information concerning the nature of such variations at the cellular level. We therefore investigated the transcriptional activity of the BAR-gene in MNL by in situ hybridization. MNL were obtained from venous blood of healthy volunteers by density gradient centrifugation. Cells were permeabilized by incubation in hypotonic buffer (0.9% trisodium citrate, 5 min), fixated in acetone and attached to adhesive glass slide areas. DNA probes were prepared from hamster BAR-gene inserted in the SV-40 derived expression vector pSV-SAR and coupled to fluorescein-conjugated polyethylene-imide by glutaraldehyde. 10 ~i of DNA-probe in hybridization buffer were incubated with the MNL preparations under sealed cover slips for 48 h at 37°C and washed with IxSSC and hybridization buffer containing 50% formamide. The amount of cloned DNA-mRNA hybrids formed in the cytoplasm of individual cells by in situ hybridization was analyzed by microfluorimetry of specifically bound fluorochrome (difference between the signals of BAR- and control probes). At least 100 cells were analyzed per sample. Ten subjects have been studied so far. In 8 cases MNL were clearly positive for the receptor mRNA. In these cases the proportion of positive cells as well as their signal intensity (i.e. transcriptional activity) revealed a high interindividual variance. Quantification, however, was biased by an unfavourable signal to background relationship in hybridization. Walther-Straub-Institut f~r Pharmakologie und Toxikologie, Nussbaumstr.26, Medizinische Klinik Innenstadt der Universit,t, Ziemsenstr.1, Lehrstuhl f~r Mikrobiologie der Technischen Universit~t, Arcisstr.21, D-8000 M[inchen 2, FRG

326 CYCLIC AMP ANTAGONIZES MITOGEN-INDUCED ACCUMULATIONOF INOSITOL PHOSPHATES IN HUMANPERIPHERAL MONONUCLEARLEUKOCYTES (MNL) IN VITRO L.J.H.van T i ~ . - E . B r o d d e Activation of lymphocytes by mitogens is associated with an early increase in IPs and diacylglycerol (Hadden, Immunol. Today 9: 235, 1988) and the subsequent expression of interleukin 2 (IL-2) receptors and production of IL-2. Several studies have indicated that cAMP can i n h i b i t lymphocyte proliferation (Kammer, Immuno1. Today g: 222, 1988}; moreover, a ~-adrenoceptor (AM) mediated inhibition of IL-2 receptor expression was reported (Feldman et a l . , J. Immunol. 139: 3355, 1987).An antagonistic effect of cAMP on the mitogen-induced generation of second messengers might underlie these phenomena. To test this hypothesis we studied the effects of the ~-AR agonists isoprenaline (IPN), adrenaline (A) and noradrenaline (NA) on [3H]-IPs accumulation in response to various mitogens in v i t r o in human peripheral MNL. The ~-AR agonists inhibited mitogen-induced [3H]-IPs accumulation by about 30 to 50% with a rank order of potency: IPN > A >> NA.This order of potency was identical with that of the ~-AR agonists to generate cAMP; furthermore, inhibi t i o n of IPs accumulation correlated with cAMP generation. The ~-AR agonist-induced inhibition of the mitogen response could be blocked by the selective ~-2 AR antagonist ICI118,551 (erythro-(±)-1-(7-methylindan-4-yloxy)-3-isopropylamino-butan-2-ol; 100 nM) but was not affected by the selective ~-I AR antagonist CGP 20712 A (l-[2-((3-carbamoyl-4hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol;500 nM). On the other hand, the phosphodiesterase inhibitor isobutyl methyl xanthine (IBMX, 100 ~M) enhanced IPN-induced inhibition of the mitogen response resulting in 70 to 90% inhibition of [3H]-IPs accumulation. In conclusion, cAMP antagonizes mitogen-induced IPs accumulation in human peripheral MNL in v i t r o . This could be one possible mechanism of the ant-f?pr--6]-fferative effect of cAMP. Biochem. Forschungslabor, Med. K1inik & P o l i k l i n i k , UniversitBt Essen, D-4300 Essen, Fed. Rep. Germany

327 THE ~-ADRENOCEPTOR SUBTYPE(S) INVOLVED IN THE POSITIVE INOTROPIC ACTION OF DOPAMINEAND ITS N-METHYL-DERIVATIVE, EPININE ON ISOlaTED HUMANRIGHT ATRIUM. H.-R.Zerkowski , A.Daul, and M.Khamssi Dopamine (DA) and DA-analogues are used in the treatment of congestive heart failure. To gain more insight into the mechanism of action of these drugs, we characterized the ~-adrenoceptor (AM) subtype(s) involved in the positive inotropic effect of DA and its N-methyl derivative,epinine (EP,the active metabolite o# ibopamine) on isolated, elect r i c a l l y driven (1.0 Hz, 37uC) right atria obtained from patients undergoing coronary artery bypass grafting (NYHA class I to I I ) . The concentration-effect curve of EP for increases in force of contraction (FC) was shifted to the right by the selective ~-2 AR antagonist ICI 118,551 (erythro-(±)-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol; 3OHM) and the selective ~-I AR antagonist CGP20712 A (1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2propanol; 30OHM) to about the same degree, while only CGP, but not ICI, shifted that of DA to the right. EP caused nearly the same maximumFC-increase as isoprenalJne (IPN) independent of whether neuronal and extra-neuronal uptake were blocked by 5~M phenoxybenzamine(PBZ) or not. On the other hand,only in the absence of PBZ, DA-induced maximum FC-increase was similar to that of IPN, while in the presence of PBZ i t was lower. I t is concluded that EP causes its positive inotropic effect in human heart via direct stimulation of #-I and ~-2 AR to about the same d ~ whereas DA causes its positive inotropic effect predominantly through ~-I AR stimulation; part of the DA-induced positive inotropic effect is indirectly caused through the release of endogenous noradrenaline. ~iochem. Forschungslabor, Med. Klinik & P o l i k l i n i k , and ' Abtlg. Thorax- & Kardiovasku1~re Chirurgie, Universit~t Essen, D-4300 Essen, Fed. Rep. Germany Supported by the Deutsche Forschungsgemeinschaft (DFG Ze 218/1-3).

328 I~lqRT.T.'rl4G

OF

THE ~

B

O

R

~

WITH A

~ I~ERIVI%TIVE J. Michael-Hepp and H.B6nisch* Xylamine is a covalently birding haloalkylamine which as a substrate of the desip ' itive neuronal noradrenaline carrier (uptakel) irreversibly inhibits ~otake 1 (Fischer et al, J Pharmacol Exp ~ner 320:650, 1983). Tritiated Xylamine, a cc~s~mcially not available c ~ , has been shown to label several proteins of PC12 cells (Koide et al, J N e u r ~ e m 47:1277, 1986), a cell line endowed with uptake I; 3H-xylamine or derivatives of this cc~ioound r~%y be useful to label the uptakel-carrier. A series of xylamine derivatives which could be tritiated were synthesized and tested for their "affinity" to the uptakel-carrier of PC12 cells. Desmethyl-xylamine (DMX) and its iodinated derivative (para-I-DMX) ~ e r sibly inhibited the binding of the uptakel-inh/bitor 3Hdesipra~ to PC12 ine~branes and the t r a ~ r t of 3Hnoradrenaline (3H-NA) into PC12 cells. Tritiated DMX was taken up by PC12 cells and the uptake was ~ t u r e - s e n s i t i v e , sodium-dependent and inhibited by the selective uptake 1 inhibitor nisoxetine, i.e. 3H-DMX was transported via uptake I into PC12 cells. Incubation of PC12 cells with ~H-DMX caused a covalent labelling of ~k~ny cytoplasmic proteins and two plasma membrane proteins (32 kDal and 55 kDal); the labelling of the latter was suppressed by nisoxetine or the absence of sodium. PC12 cells cultured for several weeks in the presence of guanethidine lost their ability to transport ~H-NA. Exposure of these cells to ~H-DMX caused no labelling of the 33 kDal and only a very weak labelling of the 55 kDal membrane protein. It is concluded that these two proteins are ccm~ponents of the uptakel-carrier. Supported by the Deutsche Forschungsgemeinsc21aft *Present address: Inst. Pharmakol. University Reuterstr. 2b, D-5300 Bonn, FI~; Inst. Pharmakol. University, D-8700 Wfu--zburg, FRG

Bonn,

R 83 329 THE SPONTANEOUS EFFLUX OF ENDOGENOUS AND EXOGH',/OUS NORADRENALINE (NA)AND ITS MAIN METABOLITES FROM THE RAT VAS DEFERENS C.-L. . . . . . . . . . . . . Sch~nfeld ................................................... Incubation of the densely innervated vas deferens with 3Hn o r a d r e n a l i n e leads to p r e f e r e n t i a l labelling of varicosities c l o s e to the s u r f a c e of the t i s s u e (autoradiography; D. Moura, personal comnmnication). Such an i n h o m o g e n e i t y must influence the composition of e x o g e n o u s and endogenous NA and its metabolites in the s p o n t a n e o u s efflux. After loading of the rat vas deferens with 0.2 gmol/13H-NA for 60 rain and s u b s e q u e n t w a s h - o u t for 100 rain, the eonrposition of e f f l u x was determined as well as the N A - c o n t e n t of the t i s s u e . Tritiated compounds were measured by s c i n t i l l a t i o n counting after separation by column c h r o m a t o g r a p h y ; e n d o g e nous compounds were measured by HPLC and e l e c t r o c h e m i c a l detection. The composition of the s p o n t a n e o u s efflux with regard to NA and the main m e t a b o l i t e s DOPEG (dihydroxy-) and MOPEG (methoxyhydroxyphenylglycol) was (in p m o l / g x m i n ) : NA DOPEG MOPEG exogenous 0.5±0.1 3.4_+0.1 0.9_+0.1 endogenous 0.6+ 0.1 35.0_+IB 25.6*_1.0 During s p o n t a n e o u s efflux the specific activity of NA exceeded that of tissue NA by a factor of 20. 1.5 g m o I / l desipramine, adn~dnistered 40 nrin after the loading (to inhibit the r e - u p t a k e of NA during efflux) increased the efflux of endogenous NA 16-fold, the efflux of exogenous NA 1,8-fold. Efflux of exogenous DOPEG and MOPEG was nearly unchanged. In contrast, the efflux of endogenous DOPEG d e c r e a s e d and that of MOPEG increased. Hence, b e c a u s e of very different diffusion d i s t a n c e s , exogenous N A h a d a good chance to reach the medium, whereas endogenous NA was subject to very efficient u p t a k e l ( f o l l o w e d by v e s i c u l a r s t o r a g e or deamination). The pronounced ?fflux of exogenous NA resulted in a high s p e c i f i c activity of NA efflux. Furthermore, O-methylation of DOPEG to MOPEG increases with increasing diffusion distance. Supported by the DFG (SFB 176} [nstitut fiir Pharmakologie, V e r s b a c h e r Str. 9, 8700 Wiirzburg. PRG

TRANSPORT IN A

E. S c h S m i g a n d J. B a b i n - E b e l l For t h e first t i m e , c o r t i c o s t e r o n e - s e n s i t i v e e x t r a n e u r o n a l t r a n s p o r t of n o r a d r e n a l i n e [ u p t a k e 2 ] h a s b e e n s h o w n to o c c u r in a c l o n a l c e l l l i n e [Caki-1 cellsl. Caki-1 c e i l s w e r e o r i g i n a l l y d e r i v e d from a h u m a n r e n a l c e l l c a r c i n o m a . I n i t i a l r a t e s of 3 H - n o r a d r e n a I i n e t r a n s p o r t i n t o Caki-1 c e l l s w e r e m e a s u r e d a f t e r i n h i b i t i o n of m o n o a m i n e o x i d a s e a n d catechol-O-methyltransferase b y p a r g y l i n e [10 g m o l / 1 ] a n d U-0521 {10 ~ m o l / l ] , r e s p e c t i v e l y . The c o n c l u s i o n t h a t u p t a k e 2 e x i s t s in Caki-1 c e l l s is b a s e d on s e v e r a l f i n d i n g s . [1] The u p t a k e of 3 H - n o r a d r e n a l i n e is s a t u r a b l e , t h e K+IT~ b e i n g 450 {xmol/1. [2] A r e d u c t i o n of e x t r a c e l l u l a r Na a n d C1- d o e s n o t m a r k e d l y i n h i b i t 3 H - n o r a d r e n a l i n e u p t a k e . On t h e o t h e r h a n d , d e p o l a r i z a t i o n of t h e c e l l s b y t h e e l e v a t i o n of e x t r a c e l l u l a r K + m a r k e d l y h i n d e r s 3 H - n o r a d r e n a l i n e u p t a k e . [3] [ n h i b i t o r s of u p t a k e 2 s u c h as corticosterone, O-methyl-isoprenaline, and clonidine inhibit 3 H - n o r a d r e n a l i n e u p t a k e , t h e IC6o'S b e i n g 0.I3, 1.9. a n d 18 ~mol/1, r e s p e c t i v e l y . [4] I n h i b i t o r s of u p t a k e 1, d e s i p r a m i n e [1 Fmol/1] a n d c o c a i n e [J0 pmol/1], f a i l e d to r e d u c e 3 H - n o r a d r e n a l i n e u p t a k e . [5] The IC5o'S of v a r i o u s c o m p o u n d s for t h e i n h i b i t i o n of 3 H - n o r a d r e n a l i n e u p t a k e in Caki-1 c e l l s -are p o s i t i v e l y c o r r e l a t e d w i t h t h e ICso'S for t h e i n h i b i t i o n of u p t a k e z in r a b b i t a o r t a [ r = 0 . 9 9 1 , n = 8 , p<0,001). Supported by the Deutsche Forschungsgemeinschaft [SFB 176 a n d Scho 373]. I n s t i t u t ftir 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 U n i v e r s i t a i t , V e r s b a c h e r Str.9, 8700 W t i r z b u r g .

332

330 TWO TYPES OF INHOMOGENEOUS 3H-NORADRENALINE LABELLING OF THE RAT VAS DEFERENS. U. Trendelenburg

331 EXTRANEURONAL NORADRENALINE HUMAN CELL LINE [CAKI-1 CELLS]

(3H-NA)

Inhomogeneous labelling of an incubated tissue can result from a preferential loading a) of varicesities close to the surface of the tissue (due to a concentration gradient generated by uptakel)(see preceding abstract) and/or b) of vesicles close to the surface of varicosities (due to a concentration gradient generated by vesicular uptake and intraneuronal monoamine oxidase). Because of different diffusion distances, a preferential recapture of endogenous NA may take place during spontaneous efflux: extracellular and axoplasmie NA can be re-incorporated into vesicles; the first (but not the second) type of recapture should be desipramine-sensitive. Any substantial preferential recapture of endogenous NA should result in a specific activity of "total efflux" (= efflux of NA and its metabolites) that exceeds that of tissue NA. If, after inhomogeneous labelling, recapture is prevented, the specific activity of total efflux should equal that of tissue NA. Rat vasa deferentia were loaded (for 60 min) with 0.2 ~mol/l 3H-NA and washed out for 120 min. The specific activity of total spontaneous efflux was then 2.6 times that of tissue NA. When 1.5 ~mol/l desipramine was present from 40 min of wash-out onwards, the same result was obtained. Hence, in addition to the inhomogeneity (a) (see above), there may exist type (b). When (after the usual loading) tissues were exposed to I00 ~mol/l of the reserpine-like compound Re 4-1284 from 130 to 230 min of wash-out, the specific activity of total efflux (collected between 210 and 230 min) was identical with that of tissue NA, although the labelling remained inhomogeneous. Hence, both types of inhomogeneity exist in this tissue. Supported by the DFG (SFB 176) Pharmakologisches Institut, Versbacher Str. 9 D-8700 WHrzburg

TRANSCAPILLARY EXCHANGE OF NORADRENALINE AS INFLUENCED BY

DESIPRAMINE

(DMI) AND

O-METHYLISOPRENALINE

(OMI)

O.Obst, H.Kammermeier Venous(ven) and interstitial(is) concentrations and myocardial uptake rates of noradrenaline(NA) were estimated at equilibrium in saline perfused rat hearts. PerfusJon with 10-810 -7 and 10-6 M NA lead to a transcapillary concentration difference(TCD) of 63 , 4-2, and 22% of the mean vascular concentration and to a removal of 19, 136, and 928 pmol*min-~*g -~. Corresponding to the TCD the is-dose-responserelationships were shifted to the left as compared with the vascular curves.

Blockade of the catecholamine-uptake 1 with 1 p.M DMI

abolished the TCD to 2 , 8, and 9% and decreased the removat to 4-, 4.5, and 321 pmol*min-l*g -1.

Additional blockade of the uptake 2

with 100gM OMI further reduced the TCD to - 3 , 4 , and 3;~ and decreased removal to 1, 8, and 70 pmol*min-t*g -1. By calculation of the transcapillary NA-conductance (expressed as the permeability*surface(P*S)-product), it could be shown that (i) the P-S-product of NA decreases with increasing NA-concentrations, and (ii) that administration of OMI but not of DMI significantly decreased the P - S - product of NA. The findings indicate that the observed TCD (up to 63 %) are the consequences of 1.) Neuronal and extraneuronal catecholamineuptake and 2.) an OMI-sensitive endothelial uptake mechanism, which also increases the apparent NA-P*•-product

below the krn( "~ lgM).

Institute of Physiology, Med. D-5100 Aachen, F.R.G.

Aachen, Pauwelsstr.,

Fac. RWTH

R 84 333 E F F E C T S OF VARIOUS S E C R E T A G O G U E S ON CATECHOLAMINE RELEASE FROM BOVINE CHROMAFFIN CELLS TREATED WITH TETANUS AND BOTULINUM A NEUROTOXINS. P. Marxen, G. Ahnert-Hilger*, Tetanus and botulinum A neurotoxins inhibited the carbachol-induced release of catecholamines from cultured chromaffin cells. The block of exocytosis due to botulinum A neurotoxin could be partially overcome by enhancing the concentration of carbachol. The block due to tetanus toxin remained unaffected. The partial restoration of hormone release was specific for nicotinic stimulation, because muscarinic stimulationdid not initiate e x o c y t o s i s . D e p o l a r i z a t i o n of the m e m b r a n e with increasing concentrations of K + or veratridine failed to restore exocytosis. When cells, blocked by tetanus and botulinum A neurotoxins, were permeabilized by staphylococcal ~-toxin or by the calcium-ionophore A 23187, the Ca++-stimulated exocytosis was inhibited as well: The inhibition was resistent to increasing concentrations of free Ca ++ . Activation of proteinkinase C and of G-proteins by the phorbclester TPA and GMPPNHP, resp., did not restore exocytosis either. It is concluded that carbachol triggers a mechanism beyond the increase in intracellular Ca ++, which bypasses the target of botulinum A neurotoxin. The target of tetanus toxin is probably located even closer to the fusion process, beyond the step affected by botulinum A neurotoxin. Supported by the DFG (Bi 274/4-1 ). Abteilung Toxikologie, Medizinische Hochschule Hannover, 3000 Hannover 61 *Abteilung Anatomie und Zellbiologie, Universit~.t UIm, 7900 UIm

334 DIGITALIS GLYCOSIDES ENHANCE STIMULATION-EVOKED NORADRENALINE OVERFLOW FROM THE HEART BY A DUAL MECHANISM OF ACTION R. KranzhiJfer, C. FiJrster, M. Haass

Digitalis glyccsides facilitate sympathetic neurotransmission. In order to elucidate the mechanism of this enhancing action stimulaticn-evoked overflow of noradrenaline and neuropeptide Y (NPY) from guinea pig in situ perfused hearts with intact sympathetic innervation was determinedoNPY is co-released with noradrenaline from sympathetic nerve endings and has been utilized as marker cf exccytosis (Haass et al.,Naunyn-Schmiedeberg's Arch Pharmacol 339: 71-78). Detection of noradrenaline and NPY was performed by high performance liquid chromatography and radioimmunoassay, respectively Two subsequent electrical stimulations (St, $2~ 5V, 12Hz, 1rain) of the left cervicothoracie ganglion resulted in a comparable overflow of noradrenaline from the heart ($1:186 _* 23,1, $2:178,4 _*

17,0 pmol/g, n=16, n.s.~S2/St-ratio = 1,02) and NPY ($1:191,1± 32,4, $2 224,8 * 32,1 fmol/g, n,s.~ S2/Sl-ratio = 1,19]. Application of ouabain (300 pM) over 5 rain before $2 lead tc enhanced noradrenaline overflow ($2/$1=1,4.4, n=9, p < 0,05) whereas NPY overflow markedly decreased ($2/S1=0,60, p < 0,05). A similar dissociation between ncradrenaline and NPY overflow was observed with desipramine (300 nM), an inhibitor of neuronal oateeholamine uptake (uptake1) (noradrenaline: $2/$1=1,45, n=12, p < 0,06~ NPY: $2/$1= 0,66, p < 0,05). To investigate the effect of ouabain on noradrenaline overflow independent of uptake1, deaipramine was administered both during $1 and $2, Now, ouabain prior to S2ied to a parallel increase in noradrenaline ($2/$1=1,38, n=g, p < 0,05) and NPY ($2/$1=I,4.8, p< 0,05] overflow, Conclusion: Cardiac glycosides enhance accumulation of noradrenaiine in the extraoellular space by inhibiting neuronal catecholamine uptake and by increasing exocytotic transmitter release. Department of Cardiology, University of Heidelberg, Bergheimer Str. 88, D--TgOO Heidelberg, FRG

335 ENERGY DEFICIENCY INDUCES NONEXOCYTOTIC RELEASE OF ENDOGENOUS NORADRENALINE IN HUMAN ATRIAL TISSUE Th. Kurz, W. Said, W. Saggau, G. Richardt, A. Sch~mig Studies in the rat heart demonstrate local metabolic noradrenaline (NA) release in the ischemic myocardium characterized by independence from extracellular calcium and inhibition by uptake 1 blocker such as desipramine (DMI}. The aim of the study was to investigate whether ccmparable nonexccytctio mechanisms cause NA release in the ischemic human heart. Human atrial tissue, obtained during bypass and valve surgery, was incubated in calciumfree Krebs-Henseleit-sclution to avoid interference by exocytotic release. The release of endogenous NA and dihydroxyphenylethylengiycol (DOPEG) was determined by high-performance-liquid-chromatography. The overflow of DOPEG served as indicator of the free axoplasmic amine conoentraticn. Ischemic periods of 60 min resulted in NA release of 84-1 -* 94. pmol/g (n=6), The release was suppressed by the uptake 1 blocker DMI (100 nM) (371 -* 4.0 pmol/g~ n=6), indicating carriermediated outward transport of NA. No major DOPEG release was detected during ischemia. Blockade of energy metabolism by I mM cyanide and glucosefree incubation resulted in increasing NA release starting 25 min after stopping energy supply. NA release was acccmpanied by the overflow of DOPEG formed by monoamineoxidase in presence of oxygen. DOPEG overflow preoeded NA release by about 10 min indicating an early rise cf axoplasmio NA concentration. Cumulative NA release during 60 rain of cyanide intoxication amounted to 596.5 -* 73.0 pmol/g (n=8) without DMI and 288.5 -* 22.7 pmol/g (n=8) in the presence of 100 nM DMI. The results demonstrate nonexocytotic mechanisms of NA release induced by energy deficiency in human heart. The loss of NA from the vesicular stores increases axoplasmio NA concentrations. In a second step axoplasmic NA moves across the cytoplasmic membrane to the extracellular space using the uptake 1 carrier in reverse of its normal transport direction. Department of Cardiology, D-6900 Heidelberg, FRG

University of Heidelberg,

INF 326,

336 PRE- AND POSTJUNCTIONAL EFFECTS OF PHORBOL-12-MYRISTATE13-ACETATE (PMA) IN THE ISOLATED MOUSE VAS DEFERENS M. KASCHUBE Phorbol esters have been repeatedly reported to enhance sympathetic transmission (e.g. Wakade et al., 1985, NSAP 331, 122; Allgaier et al., 1986, EJP 129, 389) by increasing the noradrenaline release in varmous tissues due to activation of protein kinase C (PKC). In contrast, at postjunctional sites of smooth-muscle preparations the effect of PKC-activating phorbols is less uniform: Activation of PKC induces either inhibition (guinea-pig trachea and ileum: Menkes et al., 1986, EJP 122, 19) or potentiation (porcine coronary artery: Mille-e-~-et al., 1986, JPET 239, 38) of receptor-mediated contractions, depending on the type of tissue. We studied the effect of PMA i) on phasic contractions to exogenously applied agon%sts and 2) on stimulationevoked [JH]noradrenaline ([°H]NA) release and 3) on twitches to field stimulation in the mouse vas deferens. PMA (I uM) depressed the maximal contractions to noradrenaline by 24 + 5.5 % (n = 8) and to beth~nechol by 29 ± 5.3 % (n = 8). -- In vasa preloaded with [°~]NA PMA (i0 ~M) increased the fractional release of [JH]NA to field stimulation (I0 volleys of 1 s, 15 HZ, 0.5 ms) by 117 + 18.9 % (n = 5). In contrast to this strong enhancing ~ffect at prejunctional sites, PMA (i0 and 30 pM) did not change significantly the force of contraction to I s volleys of 15 Hz, 0.5 ms and I00 Hz, 0.i ms. However, when applied after an inhibitory agonist, contractions were concentration-dependently enhanced by FMA: In the presence of 1.36 BM FK 33-824* PMA (10 and 30 pM) enhanced the force of contraction to i s volleys of 15 Hz, 0.5 ms by 29 ! 9 % and 53 ~ 15 % respectively (n = 5 each). These results show that in the isolated mouse vas deferens the sustained activation of PKC by PMA enhances the stimulation-evoked release of noradrenaline but leads to suppression of the phasic contractions of the smooth muscle to exogenous agonists. These effects seem to functionally compensate one another when the influence of PMA on contractions to field stimulation is studied. * (D-Ala)2-(MePhe)4-(met(O)-ol)5 - enkephalin. Institut f~r Pharmakologie, Medizinische Universit~t L~beck, Ratzeburger Allee 160, D-2400 LUbeck

zn

R 85

337

339

LOCAL APPLICATION OF DRUGS REVEALS ALPHA2-ADRENERGIC AUTOINHIBITION OF TRANSMITTER RELEASE IN GUINEA-PIG VAS DEFERENS K. S t a r k e , J.A. Brock, T.C. C u n n a n e and C.F. Wardeil

a z - A D R E N O C E P T O R S A N D PGE R E C E P T O R S OF N O R A D R E N E R G I C N E R V E T E R M I N A L S OF R A T B R A I N C O R T E X F U N C T I O NALLY INTERACT, BUT ONLY THE a~-RECEPTORS COUPLE TO N E M - S E N S I T I V E G P R O T E I N S C. A l l g a i e r and T. J & g e r

E x c i t a t o r y j u n c t i o n p o t e n t i a l s (EJPs; i n t r a c e l l u l a r e l e c t r o d e s ) and e x c i t a t o r y j u n c t i o n c u r r e n t s (EJCs; e x t r a c e l l u l a r s u c t i o n e l e c t r o d e s ) e l i c i t e d by h y p o g a s t r i c n e r v e s t i m u l a t i o n (1 Hz) w e r e r e c o r d e d from g u i n e a - p i g i s o l a t e d v a s d e f e r e n s . At a v a r i e t y of s t i m u l a t i o n i n t e n s i t i e s , b a t h - a p p l i e d y n h i m b i n e (0.1 - 1 ~M) did n o t c h a n g e t h e f i r s t EJP in a t r a i n b u t i n c r e a s e d t h e a m p l i t u d e of s u b s e q u e n t EJPs and t h e period of f a c i l i t a t i o n : its e f f e c t was a b o l i s h e d in t i s s u e s from r e s e r p i n e - p r e t r e a t e d g u i n e a pigs. B a t h - a p p l i e d d e s i p r a m i n e (0.1 pM), in c o n t r a s t , s l i g h t l y d i m i n i s h e d t h e EJP a m p l i t u d e s e x c e p t t h e f i r s t in a t r a i n . Yohimbine (0.1 - 1 ~M), when applied locally through the suction electrode, increased the n u m b e r of EJCs p e r g i v e n n u m b e r of stimuli, i.e., e n h a n c e d t h e p r o b a b i l i t y of t h e o c c u r r e n c e of EJCs. It also i n c r e a s e d t h e a v e r a g e EJC a m p l i t u d e s , m a x i m a l l y by 55 %. When d e s i p r a m i n e 0.1 pM was p r e s e n t both in t h e b a t h and in t h e s u c t i o n e l e c t r o d e , t h e a v e r a g e EJC a m p l i t u d e s w e r e d e c r e a s e d . In t h e p r e s e n c e of d e s i p r a m i n e , y o h i m b i n e (0.1 1 pM) i n c r e a s e d t h e n u m b e r of EJC e v e n more m a r k e d l y , and t h e a v e r a g e a m p l i t u d e now was i n c r e a s e d by up to 135 %. N e i t h e r t h e n e r v e t e r m i n a l impulse nor t h e n u m b e r of s p o n t a n e o u s EJCs was c h a n g e d by y o h i m b i n e . T h e s e e x p e r i m e n t s d e m o n s t r a t e a l p h a 2 - a u t o i n h i b i t i o n a t a high d e g r e e of r e s o l u t i o n , i.e., w h e n t h e i n t e r m i t t e n t r e l e a s e of t r a n s m i t t e r from only some t e n v a r i c o s i t i e s a l o n g a s i n g l e axon is m e a s u r e d as t h e EJC (Brock a n d C u n n a n e , J. Physiol. 399, 607, 1988). T a k e n t o g e t h e r with t h e low p r o b a b i l i t y of t r a n s m i t t e r r e l e a s e a t t h e l e v e l of i n d i v i d u a l v a r i c o s i t i e s , t h e r e s u l t s s u p p o r t t h e i d e a of l a t e r a l i n h i b i t i o n by n o r a d r e n a l i n e r e l e a s e d from d i s t a n t v a r i c o s i t l e s r a t h e r t h a n an i n h i b i t i o n due to n o r a d r e n a l i n e r e l e a s e d from t h e s a m e or immediately adjacent varicosities. Pharmakologisches Institut der Universit~t, 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 i.Br.

Stimulation-induced noradrenaline (NA) r e l e a s e in r a t b r a i n c o r t e x is i n h i b i t e d b y a c t i v a t i o n of az-adrenoceptors a n d p r o s t a g l a n d i n E (PGE) r e c e p tors. In the p r e s e n t study, it was i n v e s t i g a t e d w h e t h e r there is a f u n c t i o n a l i n t e r a c t i o n b e t w e e n the a z - and the P G - r e c e p t o r m e c h a n i s m , and whet h e r these r e c e p t o r s are c o u p l e d to N - e t h y l m a l e imide (NEM)-sensitive G proteins. It has b e e n p r e v i o u s l y s h o w n that N E M a l k y l a t e s the P e r t u s s i s toxin (PTX)-sensitive G proteins, thereby inhibiting receptor-mediated signal transduction. [SH]NA release from occipital and parietal cortices was e v o k e d b y e l e c t r i c a l field stimulation. When stimulation occurred under fully developed autoinhibition (36 p u l s e s / 3 Hz), m a x i m u m i n h i b i t i o n w i t h 1.0 ~ M PGEz w a s a b o u t 50%. In the a b s e n c e of a u t o i n h i b i t i o n ( y o b i m b i n e a n d 36 p u l s e s / 3 Hz; 1 pulse; 4 p u l s e s / 1 0 0 Hz), the i n h i b i t o r y e f f e c t of PGEz was m a r k e d l y i n c r e a s e d to about 80%. In addition, PGDg, which did not a f f e c t [SH]NA r e l e a s e e l i c i t e d b y 36 p u l s e s / 3 Hz, became effective in the absence of autoinhibition. As shown previously, presynaptic ~2b r a i n n e u r o n s are a d r e n o c e p t o r s of n o r a d r e n e r g i c c o u p l e d to P T X - s e n s i t i v e G p r o t e i n s . N E M (30 ~M) m a r k e d l y a t t e n u a t e d i n h i b i t i o n of [3H]NA r e l e a s e caused by clonidine, but not the inhibition c a u s e d b y PGEz. T h e s e r e s u l t s indicate a functional interaction b e t w e e n the ~ a n d the PGE r e c e p t o r m e c h a n i s m n o t o c c u r r i n g at the l e v e l of a c o m m o n p o o l of G p r o t e i n s , b u t at s o m e s u b s e q u e n t s t e p of t h e i r p o s t r e c e p t o ~ m e c h a n s i m . Inst.

Pharmacol.,

H.-Herder-Str.5,

D-78

Freiburg

338

34O

AGONIST DISSOCIATION CONSTANTS AT PRESYNAPTIC ALPHA-2 ADRENOCEPTORS IN THE CEREBRAL CORTEX OF THE RAT E. Agneter and E.A. Singer Rats were pretreated with the irreversible alpha-2 adrenoceptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2dihydroquinoline (EEDQ; 0.8 mg/kg i.p.) or saline (I ml/kg i.p.) and killed 18 hours later. Sl~ces of cerebral cortex were prepared, labelled with -H-noradrenaline (0.125~moi/i, 37°C, 30 min) and superfused with physiological salt solution containing I ~mol/l desipramine (0.7 ml/min). Electrical field-stimulation was applied at 8, 36, 64 and 92 min after the start of collection of 4-min fractions ($1-$4; monophasic rectangular pulses, 2ms, 12V/cm, 18rmA). It consisted of 4 pulses(p)/100Hz, 36p/3Hz Or 72p/3Hz. The amount of tritium released by 4p/100Hz was not different in slices of control and EEDQ-treated animals. In contrast, evoked overflow elicited by 36 or 72p/3Hz was markedly enhanced in EEDQ slices as compared to control slices. Alpha-2 adrenoceptor agonists were added at increasing concentrations 20 min before $2, $3 curves were and $4. Cumulative concentration-response generated for 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK 14304) and clonidine in slices of control and EEDQ-treated animals. The agonist dissociation constant (Ka) and the fraction of receptors remaining active (q) were estimated using Furchgott-analysis. Ka's for UK 14304 and clonidine were calculated to be 136 and 50 nmol/l, respectively, when stimulation with 4p/100Hz was used. At 36p/3Hz the values were higher and amounted to 217 and 71 nmol/l. A further shift in the Ka for UK 14304 was observed when 72 p/3Hz were used for electrical stimulation (426 nmol/l). Q was determined to be 0.08 in experiments with 4p/100 Hz, and 0.09 in experiments with 36p/3 HZ. The results demonstrate that the determination of agonist affinftes at presynasptic autoreceptors is complicated by the presence of endogenous transmitter and will lead to an underestimation of agonist affinity.

PROSTAGLANDIN (PG) E 2 INHIBITS N O R A D R E N A L I N E (NA) RELEASE AND POSSIBLY THE RELEASE OF A PURINERGIC CO-TRANSMITTER IN SHR KIDNEYS. L.C. Rump, K. Wilde and P. Schollmeyer

Pharmakologisches Institut der W~hringer Str. 13a, A-I090 Wien.

Universit~t

Wien,

PGE 2 inhibits NA release but enhances p r e s s o r responses in kidneys of Wistar rats (Rump & Schollmeyer 1989, BJP, 97, 8 1 9 - 2 8 ) . This was tested in W K Y a n d S H R ( 5 - 7 ^ w e e k s ) . Kidneys were isolated, i n c u b a t e d w i t h J H - N A and stimulated at 1 Hz. The stimulation induced (S-I) outflow o f radioactivity was taken as a n i n d e x of NA release. S - I o u t f l o w of radioactivity was lower in S H R (21120 (31716 ± 1 8 8 0

± 1028 dpm, n=30) t h a n in W K Y dpm, n=32) b u t S-I p r e s s o r responses were greater in S H R (42 ± 3 mmHg) t h a n in W K Y (28 ± 4 mmHg). Tetrodotoxin (i ~M) abolished S-I outflow of radioactivity and S - I pressor responses in S H R and WKY. PGE 2 (0.6 ~M) i n h a b i t e d S - I outflow of radioactivity in both

SHR

(65 ± 4 % of control,

n=4)

and WKY

(69 ~ 3

however, S-I p r e s s o r responses were decreased in S H R but increased in WKY. P r a z o s i n (0.1 BM) did not not block S - I p r e s s o r responses in S H R but almost abolished %

of

control,

n=4),

them in WKY. Pressor responses to exogenous NA were blocked by prazosin in both strains. The prazosin resistent S-I pressor responses in S H R k i d n e y s were abolished by ~,~-methylene ATP (1 ~M). A c t i v a t i o n of prejunctional PGE 2 receptors i n h i b i t s NA release in W K Y and SHR. ~-I p r e s s o r responses in SHR kidneys are due to neuronal release of a purinergic transmitter. PGE 2 reduces S-I pressor responses in S H R kidneys by possibly inhibiting the co-release of this purinergic t r a n s m i t t e r ; h o w e v e r , a postjunctional effect of PGEg can not be ruled out at present. Innere

Medizin

Hugstetterstr.

IV, 55,

Universit&tsklinik D - 7 8 0 0 Freiburg.

Freiburg,

R 86 341

343

AI)RE~GIC AND PHRIN~GIC COMIK)N~¢~OF NEOROG~klICCC~fRACTIONSOF THE MZ~JSEVAS D ~ d ~ S R. BiLltmannand I. v. K'6gelgen

FURTHER EVIDENCE OF A PRESYNAPTIC EXCITATORY MI NUSCARINE RECEPTOR AT POSTGANGLIONIC CARDIAC ADRENERGIC NERVES U. Altes, Alice Habermeier-Nuth and E. Muscholl

Single pulses or series of low frequency (e.g., 0.5 Hz) pulses elicit biphasic contraction_sof the rodent vas deferens. The transmitterof the second, slow phase is noradresaline; that of the first, rapid phase is not known with certainty. - Isolated vesa deferentiawere electrically stimulatedevery 15 rainby trains of i0 pulses at 0.5 Hz. The putative P2x-antag~st surarainat a concentrationof i0 ~M accentuated the bilgnasicnature of the twitches. The rapid phase was abolished by suramin 100 ~M or a,~-mathylene-ATP1 k~, indicating that its transmitterwas ATP. Prazosin 100 r~ selectivelysupressed the second, adrenergicphase. In order to study rapid purinergic and slow adrenergic phases separately, tissues were superfusedwith either suramin i00 ~ or prazonin i0 hE. The amplitude of the "purinergic twitches" elicited by pulses No. 2-10 amounted to 63 % of the amplitudes of the purinergic response to pulse No. I, while the "adresergic twitches" NO. 2-10 amounted to only ii % of the adrenorgic twitch NO. i. When purinergic phases had been isolatedby prazosin, the response to pulse NO. 1 was slightly decreasedby idazoxan 0.3-3000 rd~ but not changed by yoh/mbine 0.3-3000 DM; in contrast, responses to pulses NO. 2-10 were concentration-dupendestlyincreasedby the ~ antagonists (by up to 210 %). When adrenergi~phases had been isolated by suramin, the response to pulse No. 1 was reduced by both idazoxan and yohimbine (100 % inhibitionat 1 ~); resp~m.sesto pulses No. 2-10 were increasedby lower, but decreased by higher concentrationsof the u~-antagonists (by up to 500 %). Prazosin 0.1-100 nM reduced adrenergic twitches (i00 % inhibitionat 30 r~), - The results indicate that ATP is the transmitterof the first, rapid contractionphase. Idazoxan enhanced purinergic and adrenergic twitches over the same concentration range, and the sar~ was true for yd~.mb~e, indicating a cemmon prespaaptic regulationof noradrenalineand ATP release. The postsynaptic adrenoceptorscomprise both c~- and c~-types, and the sensitivity to low concentrationsof prazonin suggests that the u~receptor may be szB.

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, e.g. 3-10 ms or 200-283 ms, respectively [Habermeier-Muth & Muscholl, J.Physiol. 401: 277-293 (1988)]. VNS applied i00 ms before SNS has no significant effect on NA overflow. In a previous study (Muscholl et al. NSAP 339:Suppl R88, 1989) it was shown that in the presence of the Mr-selective antagonist, pirenzepine (PIR) 80 nM, VNS applied i00 ms before SNS caused a significant inhibition of NA overflow, suggesting that an Mt mediated facilitation of NA release is simultaneously operative that counteracts the Mz mediated inhibition. In the present study NA overflow was determined by HPLC and electrochemical detection. 3 SNS periods ( 3 Hz, 3 min) were carried out at i0 sin intervals. VNS (3 Hz, 3 min) applied simultaneously with SNS 2, but each impulse placed i00 ms before the SNS impulse, had no inhibitory effect on NA overflow (log observed/expected NA overflow, -0.011 ± 0.012, n=8 vs. controls in the absence of VNS, +0.009 ± 0.011, n=10). Increasing concentrations of PIR (0.4 - 21.3 nN present throughout) revealed an inhibition that was maximal at 5.7 nM (-0.084 ± 0.003, n=4, p < 0.02). This concentration corresponds to the 1.4 fold value of the dissociation constant (KB) of PIR at the MI receptor. At 300 nM (1.3 fold Ks at the Mz receptor) PIR also antagonized the inhibition of NA release leading to an overflow value (-0.003 ± 0.016, n=3) not different from the controls. Conversely, the Nz selective receptor antagonist, AF-DX 116 at 800 nM, by blocking the inhibition of NA overflow, revealed a facilitation of NA release (+0.022 ± 0.004, n=6, p < 0.05}. Pharmakologiscbes Institut der Universit~t Mainz, Obere Zahlbacher Strasse 67, D-6500 Mainz

PharmakologischesInstitut, Hermann-Herder-Strasse5, D-78 Freiburg

342

344

I N H I B I T I O N O F E V O K E D S A L I V A T I O N IN C A T S B Y T H E P R E S Y N A P T I C a2 A D R E N O C E P T O R A G O N I S T C L O N I D I N E B U T N O T B Y T H E D2 A G O N I S T B R O M O C R I P T I N E

NICOTINIC FACILITATION OF THE ELECTRICALLY EVOKED RELEASE OF NORADRENALINE F R O M THE GUINEA PIG MYENTERIC PLEXUS E. Plenz

G. Scholtysik, R. Sahmann* and Ch. Pally*

Both clonidine and bromooriptine lower elevated blood pressure and plasma eatecholamine levels in man due to presynaptic c~2 and D2 agodistic actions respectively. Dry mouth is a known side effect of clonidine but not of brnmocriptine. The present study was performed to investigate whether the autonomic nerve supply of feline salivary gland is endowed with (x2 and/or D2 receptors as the heart is. The influence on the salivation by bromocriptine and clonidine was investigated in pithed cats. Parasympatheticallyevoked salivation was induced by regional electrical stimulation in the brain stem using the pithing rod as stimulation electrode (50 V, 1 ms, 0.5 to 32 Hz, each frequency applied during 60 s). For quantification of the evoked salivation three preweighed dental cotton rolls were placed in the mouth, left for 3 min and the dryweight-wetweightdifference was determined. Experiments were performed in Langendorff-perfusedcat hearts where the influence of bromocriptine on the stimulation-induced noradrenaline liberation in the presence of atropine was investigated. In pithed cats the frequency-dependerasalivation was blocked by an-opine(100 pg/kg i.v.) demonstrating the parasympathetic nature of the stimulated salivary flow. Clonidine (10 to 30 ~tg/kg i.v.) inhibited dose-dependently low frequency evoked salivation but bromocriptine (30 pg/kg i.v.) did not. In the heart bromocriptine (1 ~g/min) reduced the stimulation-inducodnoradrenaline overflow in a haloperidol (10 pg/min) :sensitive manner. In conclusion, the parasympathetic salivary gland nerve endings in cats may be endowed with c~2 adrcnoeeptors, stimulation of which by clonidine decreases salivation. Bromocriptine, though inhibiting adrenergic heart nerve transmission, did not decrease salivation, indicating lack of D2 receptors in parasympathetic salivary nerve endings. These results correlate with the clinical observations with the investigated compounds. University of Berne, Vet.-pharm.Inst., CH-3012 Berne * Preclinical Research, Sandoz Ltd., CHM002 Basel, Switzerland

M y e n t e r i c p l e x u s - l o n g i t u d i n a l m u s c l e p r e p a r a t i o n s were i n c u b a t e d in T y r o d e s o l u t i o n c o n t a i n i n g 0.8 BM d e s l p r a m i n e a n d 56 I]M a s c o r b i c acid. The s t r i p s w e r e s t i m u l a t e d e l e c t r i c a l l y (Sl, S2; 35 rain a p a r t ) a t 1 Hz (4 min}. Drugs w e r e a d d e d 25 rain b e f o r e S2. Release of e n d o g e n o u s neradrenaline (NA) was determined using ttPLC with electrochemical detection. Nicotine ( 1 - 3 0 0 BM) did n o t c h a n g e t h e b a s a l outflow of NA b u t f a c i l i t a t e d t h e e l e c t r i c a l l y e v o k e d outflow. T h e m a x i m a l e f f e c t c a u s e d by S00 ~M n i c o t i n e was an i n c r e a s e to 252 ± 28 ~ (N= 4) of t h e c o n t r o l outflow. D e s e n s i t i z a t i o n to t h e e f f e c t of n i c o t i n e w a s n o t o b s e r v e d a n d t h e i n c r e a s e in e v o k e d o u t f l o w of NA by 100 BM n i c o t i n e was n o t d i f f e r e n t a f t e r a p r e i n c u b a t i o n time of 20 s ( i n c r e a s e to 138 ± 5 %; N=4) or 25 rain (131 ± 4 %; N=4), H e x a r a e t h o n i u m (800 pM) a l o n e i n h i b i t e d t h e e v o k e d NA o u t f l o w (by 32 ± 7 ~; N=4), and significantly antagonized the r e l e a s e - e n h a n c i n g effect of 30 llM n i c o t i n e . In t h e p r e s e n c e of 10 pM e s e r i n e p l u s 100 nM s c o p o l a m i n e t h e s t i m u l a t i o n - e v o k e d o u t f l o w of NA was e n h a n c e d to 178 _+ 16 % (N=8), w h i c h s u g g e s t s t h a t e n d o g e n o u s a c e t y l c h o l i n e i n c r e a s e s t h e e v o k e d NA r e l e a s e v i a a c t i v a t i o n of n i c o t i n e r e c e p t o r s . 10 !IM DMPP did n o t a f f e c t t h e b a s a l o u t f l o w of NA b u t d e c r e a s e d t h e e v o k e d o u t f l o w (by 67 % in 2 e x p e r i m e n t s } . H e x a m e t h o n i u m (S00 pM) f a i l e d to a n t a g o n i z e t h e DMPP-induced i n h i b i t i o n which i n d i c a t e s t h a t t h e e f f e c t of DMPP is due to a n a d r e n e r g i c n e u r o n e b l o c k i n g a c t i o n (1). T h e r e s u l t s s u g g e s t t h a t t h e n o r a d r e n e r g i c t e r m i n a l s of g u i n e a pig m y e n t e r i c p l e x u s a r e e n d o w e d w i t h n i c o t i n e r e c e p t o r s w h i c h f a c i l i t a t e t h e e l e c t r i c a l l y e v o k e d r e l e a s e of NA. (1) Wilson A B; J P h a r m Pharraacol 14: 700, 1962 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 U n i v e r s i t ~ t , Obere Z a h l b a c h e r Str. 67, D - 6 5 0 0 Mainz

R 87

347

345 HISTAMINE H3-RECEPTORS ON NORADRENERGIC NERVES OF GUINEA-PIG ATRIA H. Fuder, M. E q e n o l f , M. T o r z e w s k i

BLOOD PRESSURE CHANGES MODIFY THE RELEASE OF CATECHOLAMINES IN THE INTERMEDIATE PART OF THE NTS N.Singewald,A.Klausmair and A.Philippu

Isolated guinea-pig atria_ were loaded with (-)-[°H]-nor~drenaline ([3H]-NA) and the r e l e a s e o f [j H ] - N A a n d of the t o t a l NA evoked b y f i e l d s t i m u l a t i o n (35 p u l s e s at 4 Hz, in t h e presence of cocaine 5, c o r t i c o s t e r o n e 10 a n d p h e n t o l a m i n e I ~ m o l / l ) was d e t e r m i n e d b y l i q u i d scintillation spectrometry or HPLC-ED. H i s t a m i n e (1 o r 3 p m o l / l ) a n d ( R ) - a - m e t h y l h i s t a m i n e (0.03, 1.0 ~ m o l / l ) , a s e l e c t i v e H 3 = a g o n ist, inhibited the s t i m u l a t i o n - e v o k e d [ J H ] - N A a n d the t o t a l NA overflow to a similar extent a n d in a d o s e - d e p e n d e n t m a n n e r . The inhibition induced by h i s t a m i n e 3 ;~$oi/i (38.2 ± 1.8 o r 3 8 . 0 ± 1.0 %, n = 4 for rJ[H]- o r t o t a l NA) w a s u n a f f e c t e d b y the H Ior H2-selective antagonists m e p y r a m i n e o r c i m e t i d i n e (10 ~ m o l / l ) , b u t was abolished in the presence of the H 3s e l e c t i v e t h i o p e r a m i d e (I p m o l / l , 34.5 m i n exposure). The maximum inhibition by (R)-amethylhistamine was similar to that of histamine, and halfmaximal inhibition was observed at 0.03 p m o l / l . In the p r e s e n c e o f t h i o p e r a m i d e (0.1 pmol/1) the i n h i b i t i o n by ( R ) - a - m e t h y l h i s t a m i n e I ~ m o l / l w a s less than h a l f of the m a x i m u m (17.4 ± 0.5 %, n = 4). T h e a g o n i s t w a s inactive at 0.1 pmol/l in the presence of thioperamide, but caused halfmaximal inhibition a f t e r w a s h o u t (54 min) o f t h e a n t a g o n i s t . The results show that inhibitory prejunctional H3-receptors exist on noradrenergio nerves of guinea-pig atria which are blocked by t h i o p e r a m i d e in a r e v e r s i b l e manner. S u p p o r t e d by D F G a n d N M F Z M a i n z .

Previously it has been shown that experimentally induced increases in blood pressure decrease the release rates of noradrenaline (NA) and adrenaline (A) in the rostral part of the nucleus of the solitary tract (NTS). Decreases in blood pressure reduce the release rate of dopamine (DA) in this area (Naunyn-Schmiedeberg's Arch. Pharmaeol.339:R90, 1989). The effects of blood pressure changes in the release of catecholamines in the intermediate NTS (InNTS) have now been studied in anaesthetized cats, in which the InNTS was superfused with CSF through push-pull cannulae. Blood pressure changes were elicited by intravenous injections of drugs, or by a bilateral carotid occlusion. In some animals, vagus, sympathetic trunk and aortic depressor nerve were dissected bilaterally before carotid occlusion. The catecholamines were determined radioenzymatically in the superfusate which was continuously collected in time periods of 3 min. Noradrenaline (3 Bg/kg, i.v.) led to a rise in blood pressure which was associated with a decrease in the release rate of A in the InNTS. Chlorisondamine (3 mg/kg, i.v.) lowered blood pressure and decreased the release rate of DA. A long-lasting (12 min) bilateral carotid occlusion elicited a rise in blood pressure which was accompanied by a decreased release of NA during carotid occlusion and after its termination. Dissection of the nerves abolished the effect of carotid occlusion on the release of NA. The results indicate a similar response pattern of the catecholamine release to blood pressure changes in the two regions of NTS. Moreover, the decreased release of NA on carotid occlusion seems to be due to impulses originating from the aortic arch.

INHIBITORY

PREJUNCTIONAL

Pharmakologisches Institut der Universit~t O b e r e Z a h l b a c h e r Str. 67, D - 6500 M a i n z

Institut f~r Pharmakodynamik und Toxikologie der Universit~t Innsbruck, A-6020 Innsbruck, Austria

346

348

DESIPRAMINE INHIBITS SYMPATHETIC N]~VE ACTIVITY IN ANAESTHETIZED RABBITS A. Schultheiss and B. Szabo The aim of the present study was to determine the sites of action of intravenously administered desipramine (DMI) on the sympathetic nervous system in anaesthetized rabbits (alfadolone + alfaxalone). To determine central nervous and/or ganglionic effects, renal postganglionic sympathetic nerve activity (RSNA) was measured. The clearance of noradrenaline from the plasma (NCL) was determined with an isotope tracer method. From the NCL and the plasma concentration of noradrenaline the noradrenaline spillover rate (NSR) was calculated. These parameters as well as blood pressure (BP) and heart rate (HI{) were measured before (basal values) and at the end of 20 min-infusions of sodium nitroprussside (SNP), which was given in order to modulate efferent sympathetic nerve activity through the baroreceptors. DMI 0.5 mg/kg + 0.05 mg/kg/h and 2 mg/kg + 0.2 mg/kg/h dose-dependently inhibited the basal RSNA and NCL, but had no significant effect on basal BP, NSR or HR. SNP produced hypotension and simultaneously increased RSNA, NSR and HR; NCL was reduced with decreasing BP. The BPRSNA function curve was shifted by DMI in a maturer indicating central sympathoinhibition. DMI did not greatly change the BP-NSR curve, whereas it shifted the BP-HR curve in a manner indicating an enhanc~ent of reflex cardicacceleration. The main new finding of this study is that DMI inhibits the sympathetic outflow centrally. Simultaneously, it enhances the NSR per action potential peripherally (although there was no clear shift of the RSNA-NSR relationship). In our study, the two effects compensated each other, so that DMI had no major effect on the BP-NSE function curve. In the heart, the peripheral effect of DMI outweighed the central sympathoinhibition, hence the enhanced reflex cardicacceleration.

EVIDENCE FOR HYPERNORADRENERGIC [NNERVATION ESSENTIAL HYPERTENSION J. Ludwig, M. Gerlich, T. H a l b r [ i g g e , a n d K . - H . G r a e f e

Pharmakologisches Institut der Universit~t Hermann-Herder-Strasse 5, D-7800 Freiburg i. Br., FRG

IN

We have s t u d i e d t h e r e l a t i o n b e t w e e n p l a s m a d i h y d r o x y p h e n y l g l y c o l (DOPEG), t h e p r i m a r y n e u r o n a l m e t a b o l i t e o f n o r a d r e n a l i n e (NA), a n d p l a s m a NA u n d e r c o n d i t i o n s of g r a d e d o r t h o s t a s i s in 47 n o r m o t e n s i v e s (N) a n d 58 u n t r e a t e d e s s e n t i a l h y p e r t e n s i v e s (H) w i t h no d i f f e r e n c e in a g e a n d sex. The v e n o u s p l a s m a level of DOPEG o b s e r v e d a f t e r 30 rain o f s u p i n e r e s t , q u i e t s i t t i n g a n d q u i e t s t a n d i n g w a s linearly r e l a t e d to t h e p l a s m a level o f NA. In N a n d H t h e s l o p e o f t h i s r e l a t i o n w a s a b o u t unity. H o w ever, w h i l e p l a s m a NA c o n c e n t r a t i o n s w e r e s i m i l a r in b o t h g r o u p s , H e x h i b i t e d h i g h e r p l a s m a DOPEG c o n c e n t r a t i o n s w h e n c o m p a r e d w i t h N [ m e a n s (SEM)]: NA ( p g / m l ) N Supine Sitting Standing

H

203 (14) 222 (17) 307 (24) 345 (16) 475 (25) 524 (25)

DOPEG ( p g / m l ) P

N

H

> 0.1 > 0.1 > 0.1

687 (28) 994 (36) 795 (32) 1127 (38) 980 (40) 1400 (SO)

P < 0.01 < 0.01 < 0.01

Inhibition o f n e u r o n a l u p t a k e by p r e t r e a t m e n t w i t h d e s i p r a m i n e (DMI; 1.5 m g / k g p.o. 3 h p r i o r to t e s t i n g ) r e d u c e d r e s t i n g p l a s m a DOPEG levels by a b o u t 30% in N (n=ll) a n d H (n=12) a n d a b o l ished t h e DOPEG r e s p o n s e to u p r i g h t p o s t u r e in b o t h g r o u p s . M o r e o v e r , the p l a s m a DOPEG o b s e r v e d in the p r e s e n c e o f DMI w a s , in b o t h g r o u p s , v i r t u a l l y identical w i t h the o r d i n a t e i n t e r c e p t of t h e r e g r e s s i o n line r e l a t i n g p l a s m a DOPEG to p l a s m a NA in t h e a b s e n c e o f DMI. Since this D M I - r e s i s t a n t pool o f p l a s m a DOPEG s t e m s mainly f r o m n e u r o n a l NA leaking o u t o f t h e s t o r a g e vesicles r e g a r d l e s s of the level o f s y m p a t h e t i c tone, h i g h e r p l a s m a DOPEG levels in H are in f a v o u r o f t h e p o s s i b i l i t y o f an i n c r e a s e d d e n s i t y o f s y m p a t h e t i c i n n e r v a t i o n in t h e v a s c u l a t u r e o f e s s e n t i a l h y p e r t e n s i v e s . ( S u p p o r t e d by t h e E r n s t und H e d d a W o l l heim-Stiftung) Medizinische Poliklinik, U n i v e r s i t y o f WUrzburg, K l i n i k s t r a B e 8, D-87OO W U r z b u r g , Federal Republic of G e r m a n y

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WHAT IS THE "FUNCTIONAL LINK* BETWEEN SEROTONIN U P T A K E SITES AND PRESYNAPTIC SEROTONIN AUTORECEPTORS? Norbert Limberger and Leni Sp/lth

A 5-HTI-LIKE RECEPTOR MEDIATING CONTRACTION OF GUINEA-PIG ILIAC ARTERIES Philippe Schoeffter and in~i Sahin-Erdemli*

Inhibition of the serotonin (5-HT) re-uptake attenuates the effect of exogenous 5-HT autoreceptor agonists on 5-HT release. This interaotion was attributed to an increased synaptic concentration of released 5-HT after blockade of uptake. Alternatively, a direct connexion between uptake sites and autoreceptors was postulated. W e investigated this issue by studying the release of 3H~5-HT from rabbit brain cortex slices. After preineubation with 3H-5-HT, the slices were superfused and stimulated electrically. Cumulative concentration-response curves of the autoreceptor antagonist metitepin (Meti) and the agonlsts 5-methoxytryptamine (MOT) and 5carboxamidotryptamine (CT) were obtained with the uptake mechanism either intact or blocked [by citalopram I0 /~mol/1 (Cit), fluvoxamine 3 ~mol/l (Flu) or nitroquipazine I /~mol/l (NQ)]. The tritium overflow evoked by 10 oulses/0.033 Hz was increased by Meti and was reduced by M O T with an ECS0 of 30 nmol/l (sigmoid cnrve fitting).In the presence of Fin, the effect of Meti was enhanced, but the EC50 of M O T was increased to I03 nmol/l. The tritium overflow evoked by 4 oulses/100 Hz was hardly changed by Meti, irrespective of whether uptake was intact or blocked. In the absence of uptake inhibitors, M O T (CT) reduced the overflow with an EC50 of 38 (0.64) nmol/1. When uptake was blocked by either Flu, Cit or NQ, EC50 values of M O T (CT) were determined as 46 0.06) nmol/l, 38 (0.95) nmol/l and 35 (0.86) nmol/l, respectively.

It is concluded that there exists no direct connexion between 5-HT uptake sites and presynaptic autoreceptors in rabbit brain cortex slices. It is the increased synaptic concentration of released 5-HT resulting from blockade of 5-HT re-uptake that attenuates the effect of exogenous autoreceptor agonists.

Serotouln (5-HT) induces contraction of blood vessels mainly through stimulation of 5-HT2 receptors. In few cases only, like dog saphenous vein and basilar artery are 5-HT-induced contractions mediated by '5-HTx-like' receptors, We now provide evidence for a '5-HTl-like' receptor subserving contraction in guinea-pig isolated iliac arteries. Four to six rings (2 mm long) from guinea-pig (male albino, 200 g) iliac arteries were suspended in organ baths filled with a Krebs bicarbonate sohtion bubbled with 95% 02-5% CO2 at 37" C. Isometric changes in tension were recorded. A initial tension of 0.5 g was applied, which was repeatedly readjusted during a period of equilibration of 2 h. Rings were then contracted by a maximally effective concentration of prostaglandin F2¢~ (PGF2co 30 ~lmol/1). After washout, tings were allowed a further 60-90 rain period of equilibration. Then a weakly active concentration of PGP2c~ (EClo to EC2o) was given to induce a sustained contraction plateau. Thereafter 5-HT and related agonists were given cumulatively. When studied, antagonists were added 30 rain before PGF2ta. Under these conditions, 5-HT (1 nmolll-1 pmol/l) and the '5-HTl-like' receptor agonists 5carboxamidotryptamin¢ (5-CT, 1 nmol/l-1 I~raol/l) and sumatsiptan (GR43175, 10 nmoUl-10 tamol/1)elicited concentration-dependent conllactiom reaching 40-50 % the amplitude of those induced by PGF2¢~(30 tamol/1).Mean pECso values were 8.2 (5-CT), 7.7 (5-HT) and 6.9 (sumatriptan). The 5-HT2 receptor antagonist, ketanserin (1 ~tmol/l) did not significantly alter 5-HT- and 5-CT-induced contractions, nor did the 5-HT3 receptor antagonist, (ltxH, 3c~, 5c~H-tropan-3-yl)lH-indole-3-carboxylic acid ester (ICS 205-930, 1 ~tmol/l) alter 5-CT-induced contractions. By constrast, methiothepin (0.1 ~tmol/l)produced a rightward shift uf the concentration-response curve for 5-CT, yielding a pKB value of about 8.3, Thus, the 5-HT receptor mediating contraction of guiuea-pig iliac artery fulfil~ the criteria for defining a '5-H%.-like' receptor. Pmclirtical Research, 386/527, SANDOZ Lt& CH-4002 Basel, Switzerland. *Present address: Department of Pharmacology, School of pharmacy, Hacettepe University, Ankara, Turkey.

Pharmakologisches Institut,Hermann-Herder-Strasse 5, D-7800 Freiburg i. Br., F R G

352

350 ANALYSIS OF THE STIMULATORY EFFECT OF SOME BENZAMIDES SMALL INTESTINAL MOTILITY

ON

COMP~ITIlVE ANTAGONISM BY RECOGNISED 5-HT2 RECEPTOR ANTAGONISTS AT 5-HTx¢ RECEPTORS Daniel Hoyer

K.H. Buchhelt and A. Bertholet Benzamides (BA), derived from metoclopramlde (M), llke clsaprlde (C), zacoprlde (Z), renzapride (E) or BEL 20627 [B, (2-~,68,9aa)-(~)-4-amlno-5-chloro-2-metboxy-N-benzamide] stimulate small intestinal motility in man and animals. As these compounds are antagonists at 5-HT 3- and agonlsts at 5-HT&-receptors in the same concentration range, the mechanism for the intestinal proklnetic action has not yet been identifled.'In order to clarify this, the effect of some BA on small intestinal motility was investigated in the isolated guinea pig ileum by means of the Trendelenburg technique which allows the simultaneous measurement of movements of the longitudinal and circular intestinal muscle. Effects on the peristaltic contractions were quantified by integrating the area under the contractlon/tlme curve. All BA enhanced the peristaltic contractions elicited by a constant pressure stimulus. The following order of potency was established: C > R > Z = B > M. All BA affected only the contractions in the longitudinal direction vith the exception of B, which also enhanced circular muscle contractions. ICS 205-930 [(3a-tropanyl)-IH-Indole-3-carboxyllc acid ester] or granlsetron which predominantly block 5-HT 3 receptors did not have an effect on the peristaltic response. 5Methoxy-tryptamlne, a 5-HT 4 agonlst which is devoid of effects at 5-ETa receptors, acted in the same way as C, R , M or Z. 2-Me~hyl-5-HT, which is predominantly a 5-HT 3 agonlst, did not modify the peristaltic reflex at all? Conclusion: These results are consistent with the notion that the prokinetic effect of the BA on the small intestine are mediated by 5-HT 4 receptor agonlsm and not by 5-HT 3 receptor antagonism. Preclinical Switzerland

Research,

SANDOZ

AG,

CH-4002

Basel,

Several antagonists, known to interact with 5-HT 2 receptors (see table) were tested against 5-HT-stimulatod production of inositul phosphate in pig choroid plexus, a 5HTlc receptor model (Hoyer et al, NSAP, 1989, 339:252-258). These antagonists shifted the conosna'ation response curve to 5-HT in a parallel manner without depression of the maximal effecL The calculated pA2 or pKs values were in good agreement with the pKD values determined in radioligand binding studies performed in pig choroid plexus with [3H]mesulergine. Effects of antagonists on 5-HT-induced inositol phosphate accumulation in pig choroid plexus preparations. Antagonists

pA2

Slope

mean pKs

pKD

Ritanserin LY 53857 ICI 169,369 Methysergide Mesulergine Ketanserin

8.24_+0.11 7.99-+0.19 7.40+_0.16 8.29_+0.20 8.87+0.25 5.94_+0.19

1.45+-0.19 1.32+-0.24 1.27+-0.21 1.23_+0.29 0.83±0.14 1.24+-0.23

8.69--.0.10 8.64 8.59+0.11 8.08 7 . 8 5 + - 0 . 0 9 8.03 8.86+0.12 8.61 8.89-+0.06 8.79 6.45+0.10 7.01

The data demonstrate that several thugs described as 5-HT2 receptor selective antagonists, e.g. ritanserin, LY 53857 (4-isopropyl-7-methyl-9-(2-hydroxy-1methylpropoxycarbonyl)-4,6,6A,7,8,9,10,10A-oct ahydroiodelo(4,3 -FG)quinoline maleate) and ICI 169,369 (2-(2-dimethylaminoethylthio)-3-phenylquinoliue hydrochloride) are also potent, competitive and surmountable antagonists at 5HT~c receptors. Thus, the data present further evidence for the pharmacological similarity of 5-HTlc and 5-HT2 receptors. However, in contrast to the situation described with methysergide, ritaoserin and LY 53857 in several 5-HT2 receptor models, none of these antagonists acted in a non competitive or nun surmountable fashion with 5-HTlc receptors. These results rule out the possibility of 5-HTlc receptors undergoing an allosteric modulation, a model proposed for 5-HT2 receptors (Kanmann and F~enkou,NSAP, 1985, 328:295-300). Plecliulcal Research, 3861525, SANDOZ Ltd, CH-4002 Basel, Switzerland.

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CLOSE SIMILARITY BETWEEN CONTRACTION MEDIATING 5-HTI-LIKE RECh3PTORS IN PORCINE BASILARARTERIESAND CANINE SAPHENOUS VEINS K,J. van CharldorD and W. Wouters Contraction-mediating 5-HT1-1ike receptors have been reported to be present in isolated canine saphenous veins (Huz~hrey et al., 1988; Br. J. Pharmacol 94: 1128), canine and primate basilar arteries (Conner et ml., 1989; Br. J. Pharmacol 96: 379) and in human basilar arteries (Parsons et al., 1989; Br. J. Pharmacol 96: 434). It was the aim of the present study to investigate the nature of the 5-HT receptor mediating contraction in porcine basilar arteries. The basilar artery was removed from slaughterhouse-obtained porcine brains. Helieular strips were suspended under i0 mN tension in 10 ml organ baths filled with oxygenated (95% 03 + 5% C02) Tyrode solution (CaCI2.2HeO 1.8 mM, containing indomethacin i0-e M, atropine 10-7 M and propranolol I0-v M, pH=7.4, 37°C). After lh equilibration and two precontractions (i0-~ M 5-HT) a cumulative concentration response curve (CRC) was made for 5-HT. On the same preparation m CRC for an agonist was constructed or another CRC for 5-HT after pretreatment wi~h an antagonist (30 min) was made. The 5-HT induced concentration-dependent contraction was antagonized by the 5-HT-antagonist methiothepin (10-7 M), but not by the a-receptor antagonist phentolamine (10-m M), the 5-HTi/Ic-antagonist ketanserin (i0-s M), the selective 5-HTm-antagonist ic~H,3a, 5aH-tropan-3-yl-3,5-dichlorobenzoate (MDL 72222, 10-6 M), nor by the 5-HTL~zS antagonist (±)-cysnopindolol (I0-e M ) The agonists induced a concentration-dependent contraction with the following order of potency: 5-HT > sumatriptan (GR 43175, 5-HTIB/D) ~ 5-methoxy-3-(l,2,3,8-tetrahydro-4pyrimidinyl IH indole (RU 24969, 5-HTxA/S) > TFMPP (5H T ~ / c ) = 8-OH-DPAT (5-HTIA) > DOI (5-HTIc). We conclude that the contraction mediating 5-HT receptor in porcine basilar arteries does not adhere to the 5-HTs-, 5-HT2- or 5-HT~/csubtype, but closely resembles the 5-HT1-1ike receptors in canine saphenous veins. Duphar B.V,, Dept. of Pharmacology, PO Box 900, 1380 DA Weesp, The Netherlands.

INFLUENCE OF STEREOTAXIC MICROINJECTIONS OF 8-OH-DPAT AND URAPIDIL ON CARDIOVASCULAR PARAMETERS IN THE RAT AFTER CENTRAL SEROTONIN NERVE LESIONS B. Valenta

354 DEPLETION OF BRAIN 5-HT STORES DOES NOT ALTER 0F THE 8-0H-DPAT CUE IN RATS. H.0. Kalkman

The effects of the selective 5-HT I receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino)te~ralin (8-OH-DPAT) and urapidil (U) on mean arterial blood pressure (MAP) and heart rate (HR) of pentobarbital anaesthetized normotensive rats were measured after stereotaxic microinjection into the medial part of the BI/B3 ceil region of the ventral medulla. The mean percentual decreases induced by 8-OH-DPAT (0.2 Bg) and U (3 ~g) amounted to (MAP/HR) -19%/-25% and -13%/-6%, respectively. In a second set of experiments the rats were pretreated either with an intracisternal ( i.c; 0.2 mg in 0.01 ml) or bilateral intraspinal ( i.sp; 5 ug in 0.5 ul between C2/C3) injection of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) 7-10 days before the microinjection of 8-OH-DPAT or U. 60 minutes before the administration of 5,7-DHT or vehicle the animals received the noradrenaline uptake inhibitor desipramine 125 mg/kg i.p.) to prevent nonselective lesion of noradrenergic neurons. The resting values of MAP and HR in animals pretreated with 5,7-DHT were significantly lower than in vehicle-treated controls. The i.c. administration of 5,7DHT resulted in an 80% reduction in serotonin levels in the thoracic spinal cord and a complete abolition of the hypotensive and bradycardic effects of 8-OH-DPAT and U. In animals with i.sp. injection of 5,7-DHT, which caused a 40% reduction in spinal serotonin level, a markedly attenuated effect of 8-OH-DPAT on cardiovascular parameters was observed. The present results support the hypothesis that the central hypotensive and bradycardic effects of U and 8OH-DPAT are mediated via 5-HT I receptors located on bulbospinal projection neurons o8 the ventral medulla. Institute of Pharmacology, University W~ihringerstraJSe 13a, 1090 Vienna, Austria

of

Vienna,

356 RECOGNITION

5-HTIAAGONISTS REDUCE INFARCT SIZE IN RODENT MODELS OF FOCAL CEREBRAL ISCHEMIA G.W. B i e l e n b e r g , M. B u r k h a r d t

The two s e l e c t i v e 5-HT1A r e c e p t o r l i g a n d s , 8 - h y d r o x y - 2 - ( d t n-propylamino) tetraline (8-0H-DPAT) and i p s a p i r o n e , d i f f e r w i t h r e s p e c t to t h e i r e f f e c t s i n r a t s . Compared w i t h 8-OHDPAT, ipsaplrone behaves as a partial agonist in tests believed to reflect stimulation of postsynaptic 5-HTIA receptors, whereas it is a full agonlsts in two models for (presynaptlc) somatodendritic 5-HTIA autoreceptors. Since also in rats trained to discriminate 8-0H-DPAT from saline, ipsa plrone generallsed completely to the 8-0H-DPAT cue (i.e. it was a full agonist), I investigated whether the discriminative stimulus properties of 8-0H-DPAT were resulting from activation of somatodendrltic 5-HTIA autoreceptors. Rats were trained to press levers for food reinforcement according to the procedure of Colpaert et al (Arch int Pharmacodyn Ther 218: 268-276, 1975) on a FR I0 schedule in daily 15 min training sessions. Drug lever appropriate responding to 8-0H-DPAT (0.I mg/kg se) and ipsaplrone (3 mg/kg so) was measured before and after treatment with parachlorophenylalanine (pCPA, 150 mg/kg ip, -3 and -2 days). In biochemical studies, this dose regimen of pCPA was shown to produce a severe (>80%), selective (little effect on noradrenallne and dopamlne) and generallsed reduction of brain 5-HT levels. The recognition of the drug stimulus was not significantly reduced by pCPA, Since similar pCPA pretreatment schedules abolished the hyperphaglc response to 8-0HDPAT, a response considered to result from activation of somatodendrltic 5-HTIA autoreceptors (Dourish et al, Psychopharmacol 89: 467-471, 1986), the result from the present experiments indicates that activation of 5-HTIA autor e c e p t o r s iS of minimal importance to the 8-0H-DPAT (0.I mg/kg) cue. Furthermore, it would characterize the 8-0SDPAT cue as a model for postsynaptic 5-HTIA receptor stimulation with a relatively high receptor reserve, since a partial agonist like Ipsapirone produced a full agonlst response.

5-HT1A agonists have only recently been shown to exert i n h i b i t o r y a c t i o n s on n e u r o n a l t r a n s m i s s i o n . Both p r e - a n d p o s t s y n a p t l c e f f e c t s h a v e b e e n s u g g e s t e d to p l a y a r o l e in this action. Serotonin has been demonstrated to reduce g l u t a m a t e - e v o k e d a c t i v i t y in t h e h i p p o c a m p u s . As t h e e x c i totoxic activity of excessive glutamate in the course of c e r e b r a l i s c h e m i c e v e n t s is w e l l d o c u m e n t e d , i t s e e m e d r e a s o n a b l e to t e s t 5 - H T 1 A a g o n i s t s f o r t h e i r c e r e b r o p r o t e c tive p o t e n c y . C e r e b r a l i n f a r c t i o n w a s e v o k e d b y p e r m a n e n t o c c l u s i o n of t h e l e f t middle c e r e b r a l a r t e r y (MCA-O) in male F i s c h e r - 3 4 4 r a t s or NMRI mice. A f t e r 48 h o u r s , i n f a r c t size w a s d e t e r m i n e d e i t h e r b y i n t e g r a t i o n of t h e i n f a r c t e d a r e a s on s e r i a l s e c t i o n s s t a i n e d w i t h c r e s y l v i o l e t ( r a t ) or b y p l a n i m e t r i c a n a l y s i s of t h e d a m a g e d c o r t i c a l s u r f a c e a f t e r t r a n s c a r d i a c p e r f u s i o n w i t h c a r b o n b l a c k (mouse). D r u g s w e r e a p p l i e d s.c. ( 8 - O H - D P A T ) or i.p. ( o t h e r s ) 30 rain or 15 rain b e f o r e MCAO to r a t s or mice, r e s p e c t i v e l y . In one s e r i e s , i p s a p i r o n e w a s g i v e n to r a t s 1 h o u r a f t e r MCA-O. All 5 - H T 1 A a g o n i s t s i n v e s t i g a t e d (8-OH-DPAT, 1 mg/kg; b u s p i r o n e , 10 mg/kg; i p s a p i r o n e , 10 a n d 30 mg/kg; g e p i r o n e , 10 m g / k g ) c a u s e d a r e d u c t i o n in c o r t i c a l i n f a r c t v o l u m e in t h e r a t MCA-O model. S t r i a t a l d a m a g e w a s n o t i n f l u e n c e d b y e i t h e r c o m p o u n d , l p s a p i r o n e (30 mg/kg) a l s o led to r e d u c e d cortical infarction when applied 1 hour after the vessel o c c l u s i o n . In t h e m o u s e MCA-O model p r e t r e a t m e n t w i t h 8 OH-DPAT (1, 3, a n d 10 m g / k g ) , b u s p i r o n e (80 m g / k g ) , g e p i r o n e (10 m g / k g ) , or i p s a p i r o n e (10 a n d 80 m g / k g ) r e d u c e d t h e cortical damage. O u r r e s u l t s f o r t h e f i r s t time d e m o n s t r a t e c e r e b r o p r o t e c t i v e a c t i v i t y of 5 - H T 1 A a g o n i s t s a n d a d d t h i s m e c h a n i s m t o c u r r e n t a p p r o a c h e s to s t r o k e t h e r a p y .

Precl. Research Sandoz Ltd, PO Box 4002, Basel, Switzerland

" P r e s e n t a d d r e s s : B e e c h a m - W ( l l f l n g GmbH&Co.KG, P o s t f a c h 4, 3 2 1 2 G r o n a u / Leine I n s t i t u t f[lr P h a r m a k o l o g i e , K e t z e r b a c h 68, 3 5 5 0 M a r b u r g

R 90 357 HUMAN CORONARYARTERY SPASM INDUCED BY 5-HYDROXYTRYPTAMINE:ROLE OF RECEPTORSUBTYPES, PLAQUE AND STENOSIS A. d. Kaumann and A. M. Brown Bovine and porcine coronary arteries contract with 5hydroxytryptamine (5-HT) by a c t i v a t i n g 5-HT2 receptors (this journal 328:295,1985;unpublished). 5-HT can also enhance contractions of human coronary a r t e r y , suggesting a role in spasm. However, the involvement of 5-HT and 5-HT2 receptors in spasm has been questioned because the 5-HTz-selective antagonist ketanserin does not prevent Prinzmetal angina (Circulation 69:889,1983). We studied the effects of 5-HT on circumflex a r t e r y dissected from beating hearts obtained from 10 transplant patients (8 ischaemic, 2 myocardiopathic). Up to 15 helicoids from each a r t e r y were set up at 37% (this journal 323:149, 1983) with the endothelium rubbed o f f . Arteries from ischaemic patients showed atheromatous plaques and often contracted spontaneously. The rhythmical contraction of the a r t e r i e s was analysed by i n t e g r a t i n g force over 3 min periods. 5-HT increased the strength and frequency of spontaneous contractions and induced spontaneous or tonic contractions. The blocking effects of ketanserin were e i t h e r marginal (n=3) or incomplete (n=7). A ketanserinresistant component of 5-HT conc e f f e c t curves (usually rhythmical contractions) was surmountably blocked by 100 nM methiothepin. Only tonic 5-HT-induced contractions were observed under methiothepin. The potency of 5-HT appeared unrelated to the existence of plaque. In one ischaemic patient responses to 5-HT in prestenotic segments were predominantly ketanserin-resistant while 5-HT responses of the poststenotic segment were predominantly ketanserin-sensitive. We conclude that 5-HT can induce coronary spasm predominantly through non-SHT2 receptors (blocked by methiothepin). We thank the surgical staff of Papworth Hospital for their help. Smith Kline & French Research Limited, The Frythe, Welwyn, Hertfordshire AL6 9AR and Clinical Pharmacology Unit, Addenbrooke's Hospital, Cambridge CB2 2QQ.

359 NEUROTRANSMITrER CONTENT IN BRAIN AREAS OF RAT OFFSPRING (POSTNATAL DAY 1 - DAY 21) TREATED WITH HALOPERIDOL Rudolf Schwabe, Maike Jiirgens, Xenia Haun, Carmen Munoz The exposure to noxious substances during the developmental period of the brain may result in changes in maturation and various brain functions. The major period of development for central catecholaminergic neurons ts from mid-~estafion to the end of the second postnatal week in the rat. In addition to morphological examinations the determination of various neurotransmitters may be used as an additional sensitive biochemical variable when analysing a possible pre- and perinatal toxic risk of neurotoxicants. Quantification of various transmitters within rat brain areas at different postnatal phases was carried out by HPLC with ELCD (electrochemical detection). Pups were treated on day 1, 4, 7, 14 and 21 with a single dose of haloperidol (1 mg/kg i.p.). The preliminary results of day 21 are shown in the Table (tissue: stfiatum; HP = haloperidol treated; CO = control). DA

Dopac

HVA

3MT

5HT

HIAA

CO

4584 ± 581

551 ± 59

419 ± 41

80 ± 72

438 ± 56

610 ± 123

HP

3966 ±871

1755 ± 496

966 ±279

87 ± 30

382 ± 72

614 ± 130

* = Mean ± S.D.; N = 4; ng]g wet tissue D A = 3-Hydro_xytyramin% D6pac = 3,4-D~y_droxyphenylacetic acid, H V A = Homovanillic acid, 3MT = 3-Metboxyt~ainine, 5-HT = Serotonin, HIAA = Hydroxyindoleacetic acia The data show significant changes in respect to Dopac and HVA. Investigations on earlier days than day 21 (postnatally) will possibly show correlations between developmental stages, content of neurotransmitters in brain areas and the beginning of the influence of haloperidol.

lnstitut far Toxikologie und Embryopharmakologie, Freie Universitiit Berlin, Garystr. 5, D-IO00 Berlin 33

358 CARRAGEENIN-INDUCED TAlL NECROSIS IN RATS IS NOT A SEROTONIN-DEPENDENTTHROMBOSISMODEL W. Witt, B. Harms,P. Woy

360

Kappa-carrageenin-induced tail necrosisin rats (and mice) was introduced as a new thrombosismodel (Bekemeier et al., Agents and Actions 16:446451, 1985). The antinecrotin effect of serotonin antagonistslike ketanserin as well as some enhancing effects of exogenous serotonin on necrosis development were claimed to indicate a serotonin-dependencyof thrombogenesis in this model• We investigated the effects of kappa-carrageenin 10 mg/kg i.v. on blood cell counts, coagulation (aPTT) and fibrinolysis (DBC-LT) in pentobarbitone anesthetized male Wistar rats. We further studied the effects of the 5I-IT2-antagonist ritanserin, the 5HT2/~l-antagonlst ZK 118438 (4-{3-[3-(4-(4-fluorbenzoyl)-l-pipcridinyl)-propoxy]-4methoxyphenyl}-2-pyrrolidon-HC1), the ~l-blocker prazosin, the PGI2-mimetic eicaprost as well as heparin and hydralazin on earrageenininduced tail necrosisin consciousrats. Carrageenin induced a transient thrombocytopenia and leukocytosis, a prolongation of aPTT and a shortening of DBC-LT, and tail necrosis in > 60% of controls. ZK 118.438 and prazosin at equipotent hypotensive doses significantly reduced necrosislength and incidence up to 24 h p.appl, carrageenin.While rltanserin was not effective, elcaprost and hydralazln decreased necrosis incidence at 24 h p.appl, carrageenin. Heparin was not effective at 400 and 1000IU/kg s.c. (+ i.v.) showing reduction of necrosis incidence at 2000 IUA~gi.v. + s.c. accompaniedby major bleeding. Our results suggestthat carrageenin-induced tail necrosis can be inhibited by vasodilatation without any evidence for an effect of 5HT2-antagonism. Acute anticoagulant effects of carrageenin as well as the poor (or absent) efficacy of heparln and pure antiplatelet thugs (Bekemeier and Hirschelmann, Agents and Actions 18"581-585, 1986) cast some doubt on the usefulness of carrageenin-induced tall necrosisas a thrombosismodel.

There is a growing body of evidence that thymus-derived peptides are able to influence the pituitary-adrenalaxis. This is based on in-vivo and i n - v i t r o observations showing that the thymus gland preparation, thymosin fraction 5 (TF-5), exhibits a corticogenic a c t i v i t y at the level of the p i t u i t a r y . In the present study we used a thymus extract (RE) different to TF-5 with regard to an isolation procedure which minimizes proteolytic degradation and did not find any alteration in the release of immunoreactive (ir)-B-endorphin or in proopiomelanocortin gene expression (precursor for ACTH and B-endorphin) in corticotrophs and melanotrophs of primary cultured rat p i t u i t a r y cells, respectively. Thus, the corticogenic a c t i v i t y of TF-5 might be due to peptide composition different to our extract. Since the major peptide components are known, this information might be useful in the identification of this putative corticotropic a c t i v i t y . In contrast, incubating bovine adrenal chromaffin cells with RE resulted in both, an increased release of noradrenaline and ir-met-enkephalin and an activation of proenkephalin gene expression. This effect was mediated by breakdown of phosphoinositides but not by activation of adenylate cyclase. Our findings indicate that thymusderived peptides can activate hormonal systems in the adrenal medulla, thus supporting the concept of interactions between regulatory components of the immune and endocrine systems.

Research Laboratories of Sehering AG, Cardiovascular Pharmacology, MiiUerstr. 170-178, D-1000Berlin 65, West Germany

REGULATION OF OPIOIDPEPTIDE6ENE EXPRESSIONBY THYMUSDERIVED PEPTIDES C.J. Farln , H. Kalbacher+ and W. Voelter÷

~upported by the Deutsche Forschungsgemeinschaft. Department of Neuropharmacology,Max-Planck-lnstitut fQr Psychiatrie, 8033 Martinsried, FRG; +Physiol.-chem. Inst i t u t der Universit~t, 7400 T~bingen, FRG

R91 361

363

INTERLEUKIN-INDUCED RELEASE OF ~-ENDORPHIN AND DYNORPHIN IMMUNOREACTIVE MATERIALS FROM RAT PITUITARY TISSUE IN FITRO

ADRENALECTOMY AND EXPERIMENTAL HYPERCORTISOLISM MODULATE THE BASAL AND CRH- AND AVP-STIMULATED RELEASE OF HYPOTHALAMIC i3-ENDORPHIN

G. Koch, H. Scheffler, V. Brantl, and M. Tesehemacher ........................................................ Recently, in vitro findings have been raised indicating that opioid peptides might have immunomodulatory properties under in rive conditions (Sibiega and Goldstein, Ann. Rev. Immunel. 6: 219, 1988). Since cytokines like interleukin-la or tumor necrosis factor have been shown to induce adrenocorticotropic hormone release from pituitary tissue (Bernton et al., Science 238: 519, 1987; Milenkovic et al., Prec. Natl. Acad. Sci. 86: 4218, 1989) we investigated whether such compounds might also induce the release of opioid peptides from pituitary tissue; this would indicate a functional significance of opioid peptides for the interactions between neuroendocrine and immune systems. Rat pituitary anterior or neurointermediate lobe tissue was incubated together with immunemodulatory compounds and ~-endorphin or dynorphin immueoreactive materials in the incubation medium were determined by radioimmunoassay. Interleukinla induced the release of immunoreactive ~-endorphin from anterior lobe tissue as well as the release of immunoreactive dynorphin from anterior and neurointermediate lobe tissue. Secretion of immunoreactive dynorphin from neurointermediate lobe tissue was also enhanced by serum thymic factor. Further, hemorphin, a fragment of hemoglobin with epioid activity, induced the release of immunoreaetive ~-endorphin from anterior and neurointermediate lobe tissue. These results indicate that opioid peptides might represent messengers within feedback loops existing between neuroendocrine and immune systems. Rudolf Buehheim-Institut f~r Pharmakologie der Liehig-Universit~t, Frankfurter Strasse 107, Giessen.

Juetus D-6300

O.F.X. Almeidat and V. Patchev2 Recent in vitro studies have shown that the release of hypothalamic Bendorphin (B-END), like that of adenohypophsial origin, is enhanced by both corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). However,whereasAVP merely synergiseswith CRH in the pituitary, it seems to be essentialfor the releaseof hypothalamicB-ENDby CRH. The present paper reports on the effects of long-term adrenalectomy (ADX) and subsequent replacementwith high doses of corticosterone (CS) upon the in vitro basal and CRH- and AVP-stimulated release of 6-END from the rat hypothalamus.The basal release of B-END was significantly elevated by ADX, and reduced by CS overdosage,thus showing a resemblanceto data obtained from pituitaries taken from similarly-treated animals. Both ADX and CS replacementsignificantly reduced the stimulatory effect of CRH (10-8 M) upon 8-END release. ADX caused only a slight dimunition (non-significant) in the AVP (10-6 M)-induced releaseof B-END. However,the AVP-stimulatedrelease of B-ENDwas compretelyabolished in ADX rats treated with supraphysiological doses of CS. The hypothalamic content of 8-END was also measuredfollowing ADX and subsequenttreatment with CS. Comparedwith control tissues, those from ADX animals had significantly greater contents of 8-END; the hypothalami from rats in which hypercortisolismwas induced had markedly reduced concentrations of the opioid peptide. These results show that hypothalamic 8-END synthesis and release,and the efficacy of two of the natural secretagoguesof B-END release,are determined by a fine balance in the levels of circulating glucocorticoids. 1Instituteof Pharmacology,Toxicology & Pharmacy,Ludwig Maximilian University, D-8000 M0nchen22, FRG 2Departmentof Neuropharmacology,Max Planck Institutefor Psychiatry, D-8033 Martinsried,FRG Supported by the DFG (SFB 220)

362 PERIPHERAL OPIOID RECEPTORS MEDIATING ANTINOCICEPTION IN INFLAMMATION: ACTIVATION BY ~NDOG~OUS OPIOIDS FROM IMMUNCA~Jnq. C. Stein, A.H.S. Hassan, C. Grausch, R. Przew~ecki Exogenous opioid agonists can produce pronounced antinociceptive effects by activation of local opioid receptors in peripheral inflamed tissue (I). This study demonstrates that these peripheral receptors can be activated by endogenous opioids apparently released from immune cells during cold water swim (6WS). Wistar rats (200-220 g) developed a unilateral inflammation of the right hindpaw following intraplantar (i.pl.) injection of Freund's adjuvant. Four days later, the animals's withdrawal threshold to nocious pressure on both hindpaws was measured using a modified Randall-Sellito test. Subsequently rats received i.pl. (6-18 ~g) or i.v. (18 ~g) naloxone (NAL). 10 rain later they were subjected to CWS for 1 rain. Paw pressure thresholds (PPT) were determined repeatedly following CWS. In an additional experiment, rats were given intraperitoneal (i.p.) injections of cyclosporine A (CsA) (O.75 n~j - 3 mg per injection) or vehicle at 48, 24 and 4 h prior to testing. C%~ produced markedly higher PPT in inflamed than in contralateral noninflamed paws. This effect was dose-dependently attenuated by i.pl. NAL and CsA, but not by i.v. NAL or CsA vehicle. These findings suggest that CWS induces antinociception mediated by local opioid receptors in the inflamed paw. This antineciceptive effect is abolished by the ~ o s u p p r e s s a n t CsA, suggesting that the eD~ogenous opioid(s) involved are released frcm cells of the immune system. (i) Stein C. et al. (1989) J. Pharn~col. Exp. Ther. 248:1269-1275. Dep. of Neuropharmacology, Max-Planck-Institute for Psychiatry, Am Klopferspitz 18a, D-8033 Martinsried, FRG. Supported by Deutsche Forschungsgen~inschaft.

364 FUNCTIONAL ALTERATIONS IN THE RAT BRAIN INDUCED BY 6- and ~-OPIOID RECEPTOR ACTIVATION A.Ableitner The neuronanatomical circuits that may be functionally involved in the pharmacology of 6- a r ~ ~-opioid receptor agonists were examined using the [1- C]-2-deoxyglucose technique. For this purpose, a highly2 specifi~ and potent agonist at 6-receptors (DPDPE: [D-Pen ,D-Pen ]-enkephalin) and a metabolically stable,,dynorphin ~lnalogue (66A-078: [(NmethyI-Tyr ~,N-methyI-Arg r , D-Leu~) dynorphin 1-8 ethylamide]), which displays a high affinity for ~-opioid binding sites, were employed. A comparison of the regional pattern of effects - evaluated as alterations in glucose utilization - produced by DPDPE (5-25#g i.c.v.) and the dynorphinamide (0.5-5#g i.c.v.) revealed conformities but also distinct differences. Thus, glucose utilization was increased by both drugs in limbic forebrain structures (e.g., hippocampal formation, ncl. accumbens anterior thalamic nuclei, mamillary body) and structures involved in central motor regulation (caudate ncl.,globus pallidus, motor cortex). However, in contrast to the effect of DPDPE, glucose utilization in the midbrain raphe nuclei was increased by the dynorphinamide. This latter result parallels that obtained with the selective ~-agonist U-50,488H (trans+3,4dichloro-N- [2-(pyrrolidinyl)-cyclo-hexyl]-benzeneacetamide), as has been shown in recent studies. Based on these findings and the fact that midbrain raphe nuclei are the major source of serotoninergic efferents to forebrain regions, a serotoninergic component mediating the pharmacological properties of ~agonists is inferred. Supported by the Deutsche Forschungsgemeinschaft Institut for Pharmakologie, Toxikologie und Pharmazie, Universit&t M0nchen, KBniginstra8e 16, 8000 M0nchen 2

R 92 365

367

CHRONIC OPIATE TREATMENT ALTERS G-PROTEIN SUBUNIT CONCENTRATIONS IN THE MYENTERIC PLEXUS AND ADRENALS OF THE GUINEA PIG

MOTIVATIONAL EFFECTS OF OPIOIDS II: INVOLVKMI~NT OF THE MESOLIMBIC DOPAMINE SYSTEM. T.S.Shippenberg

H.Ammer,

The motivational (MOT) effects of opioids differ depending on the receptor types with which they interact: ~and 6-agonists function as reinforcers whereas K-agonists induce aversive states. Although the neural substrates mediating such actions are unclear, the ability of opioids to produce reward or aversion after injection into either the ventral tegmental area (VTA) or n. accumbens (NAC) suggest an involvement of the mesolimbic dopamine (DA) system. The present place conditioning study used microinjection and lesion techniques to address this issue. ~-agonists functioned as positive reinforcers in control rats, producing marked preferences for the drug-associated place. In contrast, K-agonists produced aversive effects. 6-hydroxydopamine lesions of the NAC, but not to other brain areas, abolished the MOT effects of systemically applied U- and K-agonists. This treatment did not modify the MOT effects of a non-opioid drug. NAC injections of selective D-I but not D-2 DA receptor antagonists abolished the reinforcing and aversive effects of opioids. It did not, however, alter the MOT effects of other centrally active drugs. Injection of DA antagonists into other brain areas was without effect. These data demonstrate that the mesolimbic system and particularly the NAC D-I receptor are critical for the expression of opioid-induced MOT effects. In view of recent in-vivo microdialysis data, we suggest that opioid reinforcement results from an increase in DA release and increased activation of the NAC D-I receptor whereas a decrease in DA release and the ensuing decrease in D-I receptor tone results in aversive states.

L.Nice and R.Schulz

Guanine nucleotide binding proteins (G-proteins) are known to be linked to the function of opioid receptors, and, thus, may adapt to chronic opioid receptor activation. Guinea pigs were chronically infused with the ~-ligand fentanyl and a highly selective g-opioid receptor ligand, respectively, at doses known to render the myenteric plexus dependent. After 6 days of exposure, G-protein subunits were quantified in the myenteric plexus (separated for nerve somata and terminals) and the adrenals. It was found that G~i/Geo, Gee and GS were significantly increased in the myenteric plexus. These changes were associated with nerve somata rather than nerve terminals. Termination of opiate supply reversed these changes, r e t u r n i n g to control concentrations within 1 to 4 days after commencing withdrawal. Chronic infusion of guinea pigs with a non-opioid, the a 2 - a d r e n e r g i c agonist clonidine, did not result in detectable G-protein concentration changes. Chronic fentanyl treatment resulted in an increase in Gee only, in the adrenal medulla, but not in the cortex. This increase was observed both in cytoplasmic and particulate membrane fractions. These findings suggest that chronic a c t i v a t i o n of both B- and ~-opioid receptors causes an increase of certain G-protein subunits with tissue specific differences. Supported by SFB 220. Institut f~r Pharmakologie, Toxikologie und Pharmazie, Universit~t M~nchen, K6niginstr. 16, D-8000 M~nchen 22, FRG

DepOt of Neuropharmacology, Max-Planck-Institute for Psychiatry, Am Klopferspitz 18a, D-8033 Martinsried, FRG. Supported by the Bundesgesundheitsamt, Berlin.

366

368

MOTIVATIONAL ~'~'hCTS OF OPIOIDS I: NEUROANATOMICAL SUBSTRATES. R. Bals-Kubik, A. Herz

MOTIVATIONAL EFFECTSOF OPIOIDS Ill: THE NEUROCHEMICALSUBSTRATE R.Spanagel

The motivational properties of opioids are well d ~ ted. The opioid receptor types and the neural substrates mediating the motivational effects of opioids remains ill-defined. Accordingly, these issues were addressed by moans of a place preference conditioning procedure; an animal model which permits the detection of both reinforcing and aversive motivational states. Selective ~-(DPDPE) or ~-(DAC~) (I) opioid agonists functioned as positive reinforcers, producing preferences for the drug-associated place. These effects were observed after intraventricular (ICV) or systemic administration. The doses producing these effects were much lower following ICV injections, indicating a central site of action. Pretreatment with the b-antagonist ICI174,864 (I) abolished the reinforcing effect of DPDPE; the selective ~-antagonist CrOP (I) eliminated the effect of DAGO. K-opioid agonist (U50,488H) (I) and opioid antagonists (Naloxone, CTOP) produced clear place aversions. No such motivational effects, however, were observed after injection of 6- or K-antagonists (nor-BNI) (I). Brain mapping studies revealed that the ventral tegmental area (VTA) and its major projection site, the n. acctm~P-ms (NAC) are sites of action of opioids in producing these effects. Thus, microinjection of DAGO into the VTA was reinforcing, whereas injection of U50,488H into either the VTA or NAC produced place aversions. Microinjections into the striatum were without effects. These data indicate that the motivational effects of opioids are centrally mediated and that the VTA and NAC, areas which comprise the mosolimbie system, are critically involved in these opioid effects. (i) For definition of drug symbols see TIPS IO:II,1989.

Behavioural studies have suggested an involvement of the mesollmbic dopaminergic system in mediating the motivational properties of opioid agonists. The influence of epioid peptides upon dopamine (OA) release is however, unclear. This issue was addressed using microdialysis, a newly developed method in the neurechemieal field which allows the direct measurement of neurotransmltter synaptic overflow in-vivo. Microdialysis probes were inserted into the nucleus accul~bens and perfusates were analysed for DA and its metabolites: dihydroxy-phenylacetic acid (OOP~C) and homevanilllc acid (HVA) using for seperation and quantification a reversed phase HPLC-system with electrochemical detection (detection limit for DA: 10 frcol/50 ~l perfusate).Intracerebroventricular administration of the selective ~-agoeist DAGOor the B-agonist DPOPE at doses which were positive reinforcing in the place preference conditioning experiments significantly increased DA overflow. DOPACand HVA levels were also significantly increased. The most pronounced enhancement of DA overflow and metabolites levels was produced by the ~-/&-agonist ~-endorphin. In contrast to ~- and B-agonists, administration of the k-agonist E-2078 (N-CH3-Tyr-GIy-BIy-Phe-Leu-Arg-NH-CH3-ArgD-Leu-NHC2B5), a metabolic resistent dynorphin analog, at doses which produce aversive states significantly decreased DA overflow and metabolites levels. These opiold agonist-lnduced alterations of DA overflow could be blacked by selective antagonists, indicating that these effects are epioid receptor mediated. These data and those reported previously suggest that the differential effects of ~-IBand k~agonists on DA release might be the neurochemica] substrate of their differential effects on reward. Supported by the Bundesgesundheitsamt, Berlin

Max-Planck-Institute for Psychiatry, Dept. of Neuropharmacology, Am Klopferspitz 18a, D-8033 Martinsried, FRG. Supported by the Bundesgesundheitsamt, Berlin.

Department of Neuropharamacology, Max-Planck-lnstitut Martinsried, FRG

flir

Psychiatrie D-8033

R 93

369 SYNTHESIS REGULATION OF ADRENOMEDULLARY SECRETORY PROTEINS AND NEUROPEPTIDES BY STIMULATION OF SPLANCHNIC NERVE AND CELL DEPOLARISATION R.Fischer-Colbrie~ G.H~fle, R.Weiler & H.Winkler The a d r e n a l m e d u l l a s e c r e t e s a complex m i x t u r e of hormones (catecholamines), proteins (chromog r a n i n s A and B, s e c r e t o g r a n i n II) and n e u r o peptides (enkephalin, n e u r o p e p t i d e Y, c a l c i t o n i n gene r e l a t e d p e p t i d e ) i n t o c i r c u l a t i o n . Insulininduced hypoglycemia and r e s e r p i n e treatment, both known t o s t i m u l a t e the s p l a n c h n i c nerve in v i v o , were used to i n v e s t i g a t e the regulation of the biosynthesis of these components in rat adrenal medulla. Hypoglycemia selectively i n c r e a s e d t h e l e v e l s of n e u r o p e p t i d e s in chromaffin cells six-fold, t h o s e of c h r o m o g r a n i n s were unchanged. On t h e o t h e r hand s t i m u l a t i o n of the s p l a n c h n i c n e r v e by r e s e r p i n e t r e a t m e n t e l e v a t e d levels of neuropeptides as w e l l as t h o s e o f chromogranins two-fold. Analogous changes were o b s e r v e d f o r t h e l e v e l s of t h e r e s p e c t i v e mRNA. In p r i m a r y cultures of chromaffin cells depolarisation i n d u c e d by , v e r a t r i d i n e or p o t a s s i u m selectively i n c r e a s e d mRNA l e v e l s of enkephalin and s e c r e t o g r a n i n II but not t h o s e of chromogranins A and B. F u r t h e r experiments revealed t h a t t h i s u p r e g u l a t i o n was m e d i a t e d v i a cAMP and proteinkinase C as second messengers. These results d e m o n s t r a t e t h a t components c o - e x i s t i n g in t r a n s m i t t e r storage organelles are r e g u l a t e d individually via distinct mechanisms and i n d i c a t e furthermore that the r e l a t i v e amounts of p o l y proteins/neuropeptides present in secretory vesicles can vary significantly. Such a modulation should allow a fine tuning of t h e signal transduction across nerves. Department of bruck, Peter Austria.

Pharmacology, Mayr Str.la,

University of I n n s A-6020 Ànnsbruck,

371 ENDOTHELTN RAISES LEVELS OF INOSITOL 1,4,5-TRISPHOSPHATE, INOS[TOL 1,3,4,5-TETRAKISPHOSPHATE AND CYTOSOLIC CA2+ ACTIVITY IN NEURAL CELL LINES G. Reiser, B. Baumann and F. Doni~ The mechanism of action of the vasoconstricting peptide endothelin was investigated in two neural cell lines. In rat glioma cells ( c e l l line C6-4-2), endothelin causes a biphasic rise in cytosolic Ca2+ a c t i v i t y , determined by fura-2 fluorescence, consisting of two separate peaks, a large one with 40 s duration followed by a second smaller one. In the absence of e x t r a c e l l u l a r Ca2+ only the f i r s t peak was detected indicating a contribution of i n t r a c e l l u l a r Ca2+ stores. Measurements of 45Ca2+ fluxes corroborate the conclusion that endothelin induces firstly a release of Ca2+ from internal stores and subsequently a stimulation of Ca2+ entry. In a neuronai cell line (mouse neuroblastoma x rat glioma hybrid c e l l s I08CC15, NG 108-15), endothelin caused a monophasic rise in cytosolic Ca 2+ a c t i v i t y most l i k e l y due to release from internal stores. In the glioma cells the concentrations of both i n o s i t o l 1,4,5-trisphosphate and i n o s i t o l 1,3,4,5-tetrakisphosphate were raised about 2.5 fold by endothelin for ca. 90 s a f t e r addition of the peptide. In the neuronal c e l l s , however, a shorter, smaller rise in inositololigophosphate concentration was induced by endothelin. Thus, endothelin seems to act also as a neuropeptide a c t i v a t i n g phospholipase C and i n t r a c e l l u l a r Ca2+ . Physiologisch-Chemisches Institut der Universit~t T~bingen, Hoppe-Seyler-Str. 4, 74 TObingen F.R.G.

370

372

INCREASED BIOSYNTHESIS OF NEUROPEPTIDE Y IN NIPPOCAMFAL MOSSY FIBERS IN TWO ANIMAL MODELS OF TEMPORAL LOBE EPILEPSY G. Sperk, J.Marksteiner, M.Ortler I and R.Bellmann

OCCURENCE OF ATRIAL NATRIURETIC PEPTIDE ANP) IN LYMPHOID ORGANS OF VARIOUS SPECIES

Hippocampal mossy fibers, which originate in the dentate granule cells and project to the pyramidal cells of the CA3, sprout and establish aberrant connections in animal models of temporal lobe epilepsy, which are thought to contribute to the hyperexcitahility of mossy fibers in epilepsy. Recently we reported pronounced increases in neuropeptide Y (NPY) levels in several brain areas, including the hippocampus, after kainic acid induced limbic seizures and after kindling with pentylenetetrazol in rats. Using indirect immunopsroxidase reaction we now found a pronounced increase of NPY immunoreactivity in the terminal field of mossy fibers in these animals. NPY progressively accumulated in the hilus and the stratum lucidum of CA3, 5 to 60 d after kainic acid and, at the later intervals, extended to the supragranular molecular layer of the dentate gyrus indicating sprouting of these neurons. NPY positive staining of mossy fibers could be reversed by unilateral injection of colchicine into the hilus. In both animal models also markedly enhanced expression of prepro-NPY mRNA was observed by "in situ" hybridization in the granular layer containing the perikarya of the mossy fibers. It is suggested, that sustained expression of the neuromodulatory neuropeptide NPY, in addition to the observed plastic changes, may contribute to altered excitability of hippocampal mossy fibers in epilepsy.

Evidence accumulated over the past that ANP. exerts .biologicalactivities beside the regulati.o.n, of boay fluid balance, uur initial finainq that this peptiae is present in the rat thymus suggested a link of ANP to the immune system. This new concept was more closely studied and, is now further supportedby the s.uosequently prese.meq ooservation: A.NP is present ana moreover synthetizeo in various lympnoia organs of different species incluaing man. This Is oasea on the TOIIowin_q experimental data: TirStly the ANP precursor (1-126) as well.,.as the b!ologically active ANP (99-126). were iaemiTied, by cnromatoqrapnic anaJysis (~epnaaeXoG-50 gelfiltra.don~ RP-HPLC)-and RIA in acia!c extracts or various lymp.noiq organs such as thymus grands or man, mouse, rat anq pig as well as isolated tliymocytes, spleen [mouse anq rat), different lymph nodes of mouse, rat and pig ana moreover in liuman tonsils and adenoids. ~econdly, synthesis of ANP in these organs was ad.dressed 15y searcninq TOr mRNA coding tor ANP by Northern blot hybridiza'~ion. It was found that the thymus of man, rat, mouse and pig, isolated thymocv~es, respectively, human tonsils and adenoids as weir as various lymph nodes of rat mouse and pig express the ANP transcript. .Tissue specific differences are evident. Furthermore, immune orqans of chickens, i.e. bursa of Fabricius, thymus anc] spleen also contain the correspondinCl mRNA for ANP. The demonstration of a widespreac] presence of ANP in the lymphoid or.qans strenghtens the notion of an involvement and mos'{ likely a function of ANP in the immune system. Supported by DFG.

Department of Pharmacology and ~Department Histology and Embryology, University of Innsbruck, A-6020 Innsbruck, Austria

Institut fL~r Pharmakologie Toxikologie und Pharmazie der Universit~.t MSnchen, KSnig nstr. 16, 8000 MSncnen 22, FRG

of

.M, Vollmar

R 94 373

375

THE TACHYKININ RECEPTOR SUBCLASS INVOLVED IN THE SUBSTANCE P (SP)-INDUCED CARDIOVASCULAR DEFENSE REACTION IN CONSCIOUS RATS

T H E E F F E C T S OF C A L C I T O N I N G E N E - R E L A T E D P E P T I D E (CGRP) O N T H E C I R C U L A R M U S C L E (CM) OF T H E GUINEA-PIG COLON H. S c h w 6 r e r , S. K a t s o u l i s , W. S c h m i d t a n d W. Creutzfeldt C G R P is p r e s e n t in m y e n t e r i c a n d s u b m u c o s a l n e u r e n e s . It w a s s h o w n t h a t C G R P r e l a x e d t h e l o n g i t u d i n a l m u s c l e of t h e g u i n e a - p i g g a s t r i c corpus and inhibited gastrointestinal transit in rats. In t h e p r e s e n t w o r k w e s t u d i e d the e f f e c t s of C G R P on t h e C M of t h e g u i n e a - p i g colon. T h e l o n g i t u d i n a l m u s c l e w i t h m y e n t e r i c plexus attached was removed from the colonic segments. The CM was suspended isometrically u n d e r a t e n s i o n of i0 m N in a p h y s i o l o g i c a l s a l t s o l u t i o n . C G R P (1-300 n m o l / l ) c a u s e d c o n c e n t r a t i o n d e p e n d e n t r e l a x a t i o n s of t h e C M (EC50 a b o u t 5 n m o l / l ) . H i g h e r c o n c e n t r a t i o n s of C G R P (i00 a n d 300 n m o l / l ) p r o d u c e d in a d d i t i o n t o t h e i r r e l a x a n t e f f e c t an i n i t i a l c o n t r a c t i o n of t h e CM, w h i c h w a s a b o l i s h e d in t h e p r e s e n c e of t e t r o d o t o x i n (TTx), s c o p o l a m i n e (Scp) or in p r e p a r a t i o n s d e s e n s i t i z e d to CGRP. T h e C G R P i n d u c e d r e l a x a t i o n w a s n o t a f f e c t e d b y Sop, hexamethonium, propranolol, phentolamine, i n d o m e t a c i n o r b y d e s e n s i t i z a t i o n to CGRP. In t h e p r e s e n c e of T T x t h e r e l a x a n t e f f e c t of C G R P (i0-, 30 n m o l / l ) w a s e n h a n c e d b y a b o u t 30 %. In t h e p r e s e n c e of s u b s t a n c e P (SP, i00-, 300 n m o l / l ) w h i c h b y its o w n c a u s e d a d e s e n s i t i z a t i o n - s e n s i t i v e c o n t r a c t i o n of t h e CM, t h e C G R P e f f e c t w a s a l s o e n h a n c e d b y a b o u t 25 %. In c o n c l u s i o n C G R P r e c e p t o r s a r e l o c a t e d on s m o o t h m u s c l e c e l l s of t h e C M and o n c h o l i n e r g i c s u b m u c o s a l n e u r o n e s . It is s u g g e s t e d t h a t C G R P m i g h t r e l e a s e SP f r o m peptidergic nerves. Medizinische Universit~tsklinik G6ttingen Abt. G a s t r o e n t e r o l o g i e und Endok@inologie Robert-Koch-Str. 40, D - 3 4 0 0 G S t t i n g e n , F.R.G.

C. TschSpe,

B. Stau~

In mammalian peripheral tissues three tachykinin receptor subclasses (NK-I, NK-2, NK-3) have been identified. Intracerebroventricular (icv) SP induces a cardiovascular defense reaction (increases in blood pressure (MAP), heart rate (HR) and sympathetic efferent nerve activity, hindlimb vasodilation and mesenteric vasoconstriction) in conscious rats. We sought to identify the tachykinin receptor subclass involved in the central cardiovascular actions of SP in conscious chronically instrumented rats. Icv SP (NK-I agonist) and neurokinin A (NKA) (NK-2 agonist)(0.55-550 pmol) increased MAP and HR dosedependently. Both peptides were equipotent. Icy senktide (NK-3 agonist)(7.4-740 pmol) prominently increased HR but not dose-dependently. Pretreatment with the NK-I selective antagonist, L-668,169 (cyclo(Gln-D-Trp(NMe)Phe(R)GIy[ANC-2]LeuMet)2 , 5 nmol, icy), attenuated the NKA-induced MAP- and HR-responses (34±18 mmHg min, 293±78 bpm min vs 204±34 mmHg min, 1079±440 bpm min in controls, p<0.01, n=7) but not' the SP-induced responses. However, pretreatment with the NK-2 selective antagonist L-659,877 (cyclo(Gln-Trp-Phe-Gly-Leu-Met), 5 nmol, icy) attenuated the SP-responses (65±13 mmHg min, 587±170 bpm min vs 233±26 mmNg min, 1653±383 bpm min in controls, p<0.01, n=6) but not the NKA-responses. These results suggest that the SP-induced cardiovascular defense reaction is mediated by a subclass of receptors specific for the CNS which is different from any subtype of receptors in the periphery.

Dept. of Pharmacology and German Institute for High Blood Pressure Research, Univ. of Heidelberg, Im Neuenheimer Feld 366, 6900 Heidelberg, F.R.G.

374

376 D O S E - D E P E N D E N T A D E N Y L A T E CYCLASE STIMULATION BY A WATER SOLUBLE FORSKOLIN IN POSTMORTEM HIPPOCAMPUS OF CONTROL A N D A D J S D A T P A T I E N T S B.Lemmer, M.Schmitt, J . B o h l a n d T.Ohm

LOCALIZATION OF THE CARDIOVASCULAR DEFENSE REACTION EVOKED BY SUBSTANCE P (SP) IN CONSCIOUS RATS N. Jest, K. Itoi Intracerebroventricular (icy) SP induces a cardiovascular defense reaction (increases in blood pressure (MAP), heart rate (HR) and sympathetic efferent nerve activity, hindlimb vasodilation and mesenteric vasoconstriction) in conscious rats. The aim of this study was to localize the central cardiovascular actions of SP in conscious chronically instrumented rats. First, the effect of an obstruction of the aqueductus cerebri (AC) by cream plugs (eucerine) on icy SP (55 pmol) was examined. The MAP- and HR-increases in the cream-plug-injected (histologically verified after experiments) animals (83.5±15.7 mmHg min, 866.4!232.2 bpm min, n=6) were comparable to the shamoperated animals (146.2±31.0 mmHg min, 1170.5±287.5 bpm min, n=6) indicating that brain areas caudal to the AC are not involved in the SP-response. Second, microinjections of SP into two periventricular areas innervated densely by SP-ergic nerve terminals (anterior hypothalamus (AH, n=8) and ventral tegmental area (VTA, n=8)) were carried out. All injection sites were verified histologically. SPinjection (500 pmol in i00 nl) into the AH elicited an immediate increase in MAP and HR (195.2±41.5 mmHg min, 865.8±206.5 bpm min vs -4.8±13.6 mmHg, 77.5±59.0 bpm min in saline-injected controls, p<0.01), while SP had no effect in the VTA. We conclude from these results that the AH but not the VTA is. involved in the cardiovascular defense reaction evoked by SP.

Dept. of Pharmacology and German Institut for High Blood Pressure Research, Univ. of Heidelberg, Im Neuenheimer Feld 366, 6900 Heidelberg, F.R.G.

In the present i n v e s t i g a t i o n a w a t e r soluble f o r s k o l i n a n a l o g u e [F; f o r s k o l i n - 7 - d e a c e t y l - 7 - b u t y r y l , Calbiochem] was u s e d to a c t i v a t e a d e n y l a t e c y c l a s e [AC] in p o s t m o r tem hippocampi of 8 control a n d 9 AD]SDAT p a t i e n t s . D i a g n o s e s w e r e done b y clinical a n d h i s t o p a t h o l o g i c a l e x a m i n a t i o n s . AC a c t i v i t y was m e a s u r e d b y formation of cAMP from 0.5 mM ATP in t h e p r e s e n c e of 1 mM IBMX a n d an A T P - r e g e n e r a t i n g s y s t e m , cAMP was m e a s u r e d b y r a d i o a s s a y [Learner a n d Witte, E u r J Pharmacol 159:311, 1989]. S u s p e n s i o n s were i n c u b a t e d f o r 6 rain at 37°C w i t h o u t a n d w i t h a d d i t i o n of F [0.1, 1, 10, 30, 100 p_M]. D o s e - r e s p o n s e c u r v e s were a n a l y s e d b y n o n - l i n e a r f i t t i n g u s i n g PHARMFIT p r o g r a m on IBM, p a r a m e t e r s c a l c u l a t e d : Eo=basal AC, Ema~=maximum AC stimulation b y F, ECso and Hill-coefficient. The r e s u l t s a r e summerized in the table (mean + SEM). Control Eo [pmolJmg/min] }E~x[pmol]mg/min] ]ECao[paM] Hill

I

27.3 287.4 4.9 0.72

+ 4.6 + 43.9 + 0.58 + 0.07

AD ] SDAT

t-test

11.5 134.4 4.3 0.62

p<0.01 p<0.01 n.s. n.s.

+ + + ±

2.0 15.5 0.63 0.05

R e s u l t s on r e d u c t i o n in AC in ADJSDAT a r e in acc o r d a n c e w i t h t h o s e p r e v i o u s l y r e p o r t e d u s i n g lipophilic f o r s k o l i n [Ohm et a l . , Alz Dis Rel D i s o r d e r s , Alan Lisa, New Y o r k , 501, 1989]. A n a l y s i s of d o s e - r e s p o n s e c u r v e s do not i n d i c a t e d i f f e r e n c e s in s e n s i t i v i t y to F a c t i v a t i o n b e t w e e n c o n t r o l s and AD/SDAT. Low Hill coefficients in both groups need f u r t h e r investigations. S u p p o r t : DFG a n d D r . P a u l a n d Cilli-Weill-Stiftung. 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, D-6000 F r a n k f u r t ] M 70, FRG.

R 95

377 STRUCTURE-ACTIVITY RELATIONSHIP OF 1,3,7-TRISUBSTITUTED XANTHINES AS SELECTIVE INHIBITORS OF A cAMP-SPECIFIC PHOSPHODIESTERASE FROM BRAIN TISSUE J. G6ring and R. Wilke Methylxanthines (theophylline, caffeine, theobromine) have been known to inhibit cyclic nucleotide phosphodiesterase (PDE) activity for more than 20 years. We have demonstrated that modification of the xanthine structure can give rise to compounds that inhibit relatively selectively one of the three forms of PDE - the cAMP-specific isoenzyme - found in rat and human brain tissue. PDE activities were separated from high-speed supernatant of rat cerebral homogenates by ion-exchange chromatography using a sodium acetate gradient for elutien. Peak three of the elution pattern showed a high affinity for cAMP, and this activity was taken for the estimation of the inhibitory potency of the different trisubstituted xanthines. In a first series, we tested the influence of unbranched alkyl groups - and their lengths - in position I and 3. The strongest PDE-inhibition could be shown by introduction of butyl groups in these positions. Out of a series with butyl substituents in the I and 3 position, maximal potency was reached when an oxopropyl group was in position 7 (= denbufylline). In a comparison between binding to adenosine Ai and A2 receptors and inhibition of cAMP-specific PDE, denbufylline - in contrast to the classical methylxanthines - was a more potent inhibitor of cAMP-specific PDE than an antagonist of adenosine receptors. Of these compounds, denbufylline was chosen for progression to clinical investigation, because it was not only a potent and selective PDE-inhibitor of the cAMP-specific brain PDE isoenzyme, but also showed stronger effects on cyclic AMP accumulation than the classical non-selective methylxanthines. Research Laboratories, Beecham-WOIfing D-3212 Gronau (Leine), FRG

879 EFFECT OF CHOLINERGIC LESION INDUCED BY ETHYLCHOLiNE AZIRIDINIUM ON GLUTAMATERGICNEURONS IN RAT HIPPOCAMPUS H.H~rtnagt and M.L.Berger ........... The withdrawal of cholinergic input to the rat hippocampus induced by ethylcholine aziridinium (AF64A) results in transient changes in noradrenergic and serotonergic function (H~rtnagl et a l . , Neurosci. 22,203,1987; J.Neurochem. 52,853,1989). In the present study we focussed on the question whether the level of the e x c i t a t o r y amino acid glutamate is affected as well. Male Sprague Dawley rats received injections of AF64A (2nmol) or vehicle into each of the lateral ventricles. Rats were k i l l e d 2,4,14 and 65 days after AF64A, the hippocampi were dissected and analysed for glutamate and choline acetyltransferase (CHAT) a c t i v i t y . The i r r e v e r s i b l e loss in ChAT a c t i v i t y was accompanied by a transient decline in the concentration of glutamate as summarized in the following table (results in percent of control-injected rats; *p~O.Ol; **p
Univ.

Wien,

378

380

ISOLATION OF SYNAPTOSOMESFROM PIG BRAIN USING A DISCONTINUOUS FICOLL GRADIENT V. Kliem, G. Erdmann°, H. Weisser*

I~II~-'I~ICS CE • C~IOLINE UPTAKE AND ]~k~,~J%SE BY RAT BRAIN J. Klein, A. K~ppen and E. L~ffelholz

A method f o r the i s o l a t i o n of synaptosomes is described. AI] steps of this procedure were carried out at 4 ° C. Grey matter obtained from pig brain cortex was sampled in a buffer (pH 7.2~ comprising 0.25 M sucrose, 10 mM L - h i s t i dine, 0.5 mM K -EDTA. A 15% (w/v) homogenate in this buffer was made using a Potter homogenizer (4 up-and-down strokes at 1000 rpm). The homogenate was passed through a nylon sieve (pore size 250 ~m) and centrifuged at 650 x g for 10 min. Then the supernatant was centrifuged at 16300 x g f o r 30 min. The p e l l e t was resuspended in the same volume of 7.5% (w/w) F i c o l l in buffer and recentrifuged at 16300 x g for 30 mJn. The p e l l e t was again suspended in the same volume of buffer, and the suspension was layered onto a discontinuous (10%, 15%, 20% (w/w)) F i c o i l / b u f f e r gradient. After centrifugation at 82700 x g for 2 h each of the fractions that had formed at the interfaces was collected, resuspended in buffer (1:3) and centrifuged at 17500 x g for 30 min. The p e l l e t s thus obtained were used for electron microscopic and biochemical examinations.

Choline is an essential precursor for phospholipid and acetylcholine synthesis in the central nervous system. The brain is dependent on the uptake of choline through the blood-brain barrier, and the supply of choline may become rate-limiting for acetylcholine synthesis in diseases which affect the central cholinergic neurons. We have investigated the kinetics of choline uptake and release by the rat brain. Choline levels were determined in blood plasma from a peripheral artery and from the transverse sinus, in the cerebrospinal fluid (CSF) end in total brain homogenate in untreated animals and in animals treated i.p. with 60 mg/kg choline chloride. In untreated rats, the arterio-venous difference of choline across the brain (AVD) was negative (about-2pM) at low arterial blood levels ( (10~M) as reported by several workers in the past. However, in rats with spontaneously high arterial blood levels ( ) 15~M), the AVD was positive indicating a net uptake of choline by the brain. The CSF choline concentration which reflects changes in the extracellular choline concentration also increased with rising plasma levels and closely paralleled the AVD. After acute choline administration, the arterial choline levels rose sharply to 169e20 ~M after ten minutes, and the AVD became markedly positive (+29~15 pM). The CSF levels of choline increased accordingly with a time-lag of several minutes, while the levels of total free choline in the brain were enhanced to a much smaller extent. Therefore, the amount of choline taken up by the brain within 30 rain (510 nmol) was largely stored in a metabolized form and was sufficient to sustain the release of choline as long as the plasmm level remained low. Finally, the mobilization of choline by the brain seems to be influenced by cholinergic activity since muscarinic agonists were able to increase the AVD of choline. Pharmakologisches Institut, Universit~t M~inz, Obere Zahlbacher Str. 67, I)-6500 Mainz.

Eiectronmicroscopy showed that the f r a c t i o n found on top of 10% F i c o l l largely consisted of myelin. Synaptosomes accumulated predominantly on 15% F i c o l l , and a mixed fraction (synaptosomes, mitochondria, g l i a ) on 20% F i c o l l . The bi6chemical markers f o r synaptosomes and organelles - l a c tate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, monoamine+oxldases, cytochrome c oxidase, alcaline phosphatase, Na /K~-ATPase - displayed the patterns t y p i c a l of the individual fffactions. In p a r t i c u l a r , they t e s t i f i e d to the high degree of p u r i t y of the synaptosomal fraction. Abt. Nephrologie, Abt. Toxikologie ° und Zentrum Biochemie*, Medizinische Hochschule Hannover, D - 3000 Hannover 61

R 96 381 ANG II-INDUCED NORADRENALINE RELEASE FROM ANTERIOR HYPOTHALAMUS IN CONSCIOUS RATS. AN IN VIVO BRAIN MICRODIALYSIS STUDY. F. Qadri, E. Badoer, and T. Stadler. We tested the hypothesis that stimulation of periventricular ANGII receptors releases catecholaminergic transmitters in distinct blood pressure controlling brain areas. We utilized the brain microdialysis technique coupled with HPLC-ECD to measure noradrenaline (NA), dopamine (DA) and their respective metabolites DOPEG and DOPAC in the rat anterior hypothalamus (AH) after intracerebre-ventricular (icv) injections of 100ng ANGII. The AH was perfused (2pI/min) with artificial CSF containing the NA reuptake blocker DMI. ANGII induced increases in mean arterial pressure (MAP) (22.80+1.56 mmHg, n=10, P<0.001) together with a significant 62% increase in NA release (n=10, p<0.001) without influencing the DOPEG and DOPAC outflow. DA outflow was not detectable. The ANGII-induced presser response and NA release was abolished by icy pre~reat~ent with the ANGII-receptor-antagonist Sar-,IIe~-ANGII. Our data provide the first in vivo evidence that stimulation of periventricular ANGII receptors stimulates the release of endogenous NA in the hypothalamus. They support the hypothesis that catecholaminergic pathways are instrumental in the central actions of ANGII. Department Heidelberg, Heidelberg,

of Im FRG

Pharmacology, University of Neuenheimer Feld 366, D-6900

383 DIFFERENTIAL EFFECTS OF TETRAETHYLAMMONILIN(TEA) ON AUTOINHIBITION OF DOPAMINE (DA) RELEASE AND OF OPIOID I N H I B I TION-OF OXYTOCIN (OX) RELEASE FRON THE ISOLATED RAT NEUROINTERMEDIATE (NIL) OR NEURAL (NL) LOBE. K. Rackd, A. Fischbach ! U. Haas 1 H. Hof and S. Sperb Impulse-induced release of OA from NILs is inhibited by DA autoreceptors. Impulse-induced release of OX from NLs is strongly inhibited by endogenous opioids. Both i n h i b i t o r y mechanisms can be surmounted by TEA, a potassium channel blocker. Since TEA prolongs the duration of action potent i a l s and thereby f a c i l i t a t e s the impulse-induced release of DA and OX, these d i s i n h i b i t e r y e f f e c t s of TEA may have been caused by a functional i n t e r a c t i o n with the presynapt i c modulation. Therefore, the e f f e c t s of TEA on presynapt i c modulation of DA and OX release was studied uslng high potassium ( i . e . constant depolarizing) stimuli (K+). OX release from isolated superfused NLs was determined by RIA. DA release from isolated, incubated NILs was determined by HPLC with electrochemical detection. Naloxone (10 ~mol/l) which largely increases impulse-induced OX release did net a f f e c t OX release evoked by 30 mmol / I k+ (2 ~U OX) or 45 mmol/l K÷ (13 ~U OX). When 10 mmol/l TEA was present, naloxons increased OX release evoked by 30 or 45 mmol/l K+ 2-3fold, although TEA alone increased already the K+-evoked OX release 2-3fold. The e f f e c t of naloxone observed in the presence of TEA was blocked by barium (500 IJmol/l) or quinidine (300 lJmol/l). ( - ) - S u l p i r i d e (I0 ~mol/l) enhanced the DA release evoked by 30 mmol/l K+ (0.8 pmol DA) and 45 mmol/l K+ (3.1 pmol DA) by 95% and 20%, respectively. TEA, 10 or 30 mmel/l, enhanced DA release evoked by 30 mmol/l K÷ to 1.6 and 2.7 pmol, respectively. In the presence of 10 or 30 mmol/l TEA, ( - ) - s u l p i r i d s increased DA release evoked by 30 mmol/l K+ by 45% and 25%, respectively. In conclusion, opioid receptors i n h i b i t i n g OX release, but not DA autorecsptors, may be linked to TEA-insensitive potassium channels. Pharmakologlsches Institut der UniversitY% Mainz 0here Zahlbacher Sir. 67, D-6500 Mainz, F.R.G.

382 INTRASTRIATAL DOPAMINERGIC GRAFTS: MODULATION OF TRANSMITTER RELEASE 3 AND 12 MONTH AFTER TRANSPLANTATION R. Jackisch*, M. Duschek t and J.P. Herman s Behavioural, electrophysiological and biochemical studies have shown that grafts of fetal dopaminergic cells into the previously lesioned striatum survive in the host tissue, reinnerrate the target area and restore several lesion-induced functional deficits. However, despite their importance for the clinical application of dopaminergic grafts in parkinsonian patients, there are almost no long-term studies on functional parameters in the reinnervated tissue. Recently, we reported that dopaminergic modulation of acetylcholine (ACh) release (evoked by electrical stimulation in brain slices) is partially restored 3 month after grafting of fetal dopaminergic cells in previously lesioned rat striata (Exp. Brain Res. 73:236-248 [1988]). Using the same model, the present investigation shows i) that 12 month after grafting there is a further increase of amphetamine-induced contralateral rotation in these animals. 2) This is accompanied (but most probably not caused) by a slight additional increase of the inhibitory effect of endogenous DA on ACh release. 3) Preliminary studies on the modulation of electrically evoked DA release in grafted striata (3 month after surgery) show no significant differences in autoreceptor sensitivity, whereas facilitatory effects of substance P or neurotensine were lost as compared to control animals. IInst. Pharmacol., Univ. of Freiburg, D-7800 Freiburg, FRG; ZINSERM U259, Univ. of Bordeaux, F-33077 Bordeaux, France

384

2-IODOLISURIDE, AN ERGOT DOPAMINE (DA) ANTAGONIST, SUITABLE FOR IN-VIVO IMAGING OF BRAIN DARICH AREAS H. Wachtel, P.-A. L6schmanno K.-J. Rettiq, G. Sauer, R. Hgrowski, C. Loc'h ~) , M. M a z i ~ r e ~ and B. Mazi~re*; 2-Iodolisuride (2-I-LIS) can easily be prepared by iodination of the ergot DA agonist lisuride. Like bromerguride, another 2-halogenated lisuride derivate, 2-I-LIS exhibited potent DA antagonistic properties as judged from (1) inhibition of locomotor activity in rats, (2) a n t a g o n i s m of DA agonist-induced hyperlocomotion in rats, (3) cataleptogenic activity in mice and rats, (4) antagonism of apomorphine-induced stereotypies in mice and rats, (5) reversal of apomorphine-induced hypothermia, (6) stimulation of prolactin secretion in rats and (7) antagonism of apomorphine-induced emesis in dogs. These findings suggest the blockade of mesolimbic, nigrostriatal, mesohypothalamic, pituitary and medullary DA receptors by 2-I-LIS. The compound had weak serotonin (5-HT2)~antagonistic and no adrenolytic activity in vivo. Iodination of lisuride with the isotope 123I y i e l d e d 2-123I-LIS which was employed for the first time for single photon emission computerised tomography (SPECT)-imaging of basal ganglia in man. Dept. of Neuropsychopharmacology, Sche~ing AG, M~llerstr. 170-178, D-1000 Berlin 65; ";~Service Hospitalier Frederic Joliet, Place du G~n~ral Leclere, F-91406 Orsay, France

R 97 385

387

[~H]HARMAN BINDS SELECTIVELY AND WITH HIGH AFFINITY TO MONOAMINE OXIDASE (EC 1.4.3.4) SUBTYPEA IN RAT AND MARMOSET T. May, M. Pawlik, and U. Immend6rfer

AGENTS WHICH ACT ON GABAA-RECEPTORS MODULATE THE RELEASE OF (3H)GABA FROM SLICES OF RAT NEOSTRIATUM INCUBATED IN VITRO .

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A single high a f f i n i t y binding site was detected in vitro in various tissues including brain of rat and marmoset with [~H]harman (specific activity 26.6 Ci/mmol) in a newly developed binding assay. [3H]Harman bound reversibly and with high a f f i n i t y (Ko~2.2 nM) to crude membranes. Only selective potent inhibitors of MAO A of various chemical groups (e.g. clorgyline, harmaline, harmine, 5-F-~-methyl-tryptamine, brofaromine) inhibited specific [3H]harman binding in the nM range, whereas selective and potent inhibitors of MAOB like l-deprenyl or pargyline exhibited Kcvalues in the yM range. The potency of substances to displace [3H]harman binding correlated very well with that to inhibit MAO A activity ( u t i l i z i n g kynuramine as substrata; r = 0.92, pO.05, n = 15). Similar correlations were found between Bmx- and Vmxvalues from Scatchard vs Lineweaver Burk analysis with homogenates of six different organs of the rat (heart, liver, lung, thymus, spleen, kidney) and six regions of the CNS of rat and marmoset (cortex cerebri, cerebellum, spinal cord, hypothalamus, hippocampus, striatum).

In rat neostriatum, GABA is synthetized by medium-sized spiny projection neurons as well as medium-sized aspiny interneurons. The interneurons are assumed to modulate the a c t i v i t y of the projection neurons and these affect their neighbours via axon collaterals. Thus, there are manifold GABAerglc neuronal interactions via hetero- as well as autoreceptors in the neostriatum Consequently, the modulation of (3H)GABA release from slices of rat neostMatum, which is mainly from GABAerglc projection neurons, can be expected to be quite complex. To reduce the p o s s i b i l i t y for neuronal interactions in the present study, current propagation was blocked by t e t r o d o t o x i n (0.5 t~mol/l). The release of (3H)GABA as synthetized from (3H)glutamine was stimulated by 20 or 25 mmo]/1 K +, Muscimol (0.1 pmol/1) enhanced by approximately 60 % the release of (3H)GABA induced by 20 m m o l / l K +, which was 3 % of tissue content. Bicuculline (1 p m o l / t ) blocked this e f f e c t of muscimol. When bicuculline was used alone, it did not a f f e c t the release of (3H)GABA. In contrast, 0.1 ~mol/1 muscimol did not increase the release caused by 25 m m o l / l K + which was 5.5 % of tissue content. The data show that muscimol affects neostriatal (3H)GABA release. Its mechanism of action is currently under investigation.

Inst. f. Neuropsychopharmakologie,Freie Universit~t, Ulmenallee 30, D-tO00 Berlin ig, F.R.G.

Pharmakologisches Institut der Hermann Herderstr. 5, 7800 Freiburg.

386 CGP 28014, A NEW NON-CATECHOLIC COMT INHIBITOR P.C. Waldmeier, J.-J. Feldtrauer, K. Hauaer, H. Bittiger, S. Bischoff and G. von Sprecher

388 ALTERATIONS IN OENE EXPRESSIONOF CORTICALCHOLECYSTOKININ NEURONS CAUSED IN RATS BY STEREOTAXIC OPERATIONS C. Olenik .

CGP 28014 (N-(2-pyridone-6-yl)-N',N'-di-n-propylformamidine) or its methanesulfonate salt CGP 28014 A was suspected to be a catechol-O-methyl transferase (COMT) inhibitor because it reduced the levels of homovanillic acid (HVA; EDso ~ 10 mg/kg i.p. or p.o.) and increased those of 3,4-dihydroxyphenylacetic acid (DOPAC; at 3 mg/kg i.p. or p.o. and above) in the rat striatum. It was only weakly active as a COMT inhibitor in vitro. However, its effect on striatal HVA and DOPAC was not prevented by pretreatment with proadifen, indicating that, if it acts as a prodrug, its conversion to the active compound is not by oxidative liver metabolism. The in vivo effect of CGP 28014 was substantiated in 2 additional in vivo test systems. Thus, it inhibited the accumulation of 3-methoxytyramine in the rat striatum after MAO inhibition by clorgyline (ED50 ~ 2rng/kg i.p. or p.o.), and the formation of O-methyI-DOPA from exogenously administered DOrA (EDso ~ 3-5 mg/kg i.p. or p.o.). In these tests, it was almost equipotent to tropolone, and also showed a similar duration of action. Similar to tropolone, it increased S-adenosylmethionine levels in the striatum, suggesting that it is not a substrate of COMT. In contrast, pyrogallol, being a substrate, decreased them. No effects were noted on catecholamine and serotonin (5-HT) levels in rat brain; no effects on noradrenaline uptake in rat heart, no effect on 5-HT uptake in rat brain at 30 mg/kg i.p. and above were found. Slight increases of brain tryptophan and 5-hydroxyindoleaeetic acid may indicate a minimal enhancing effect on 5-HT turnover. In receptor binding tests, CGP 28014 showed very weak interactions with ~1- and c~2-noradrenergic receptors and with 5-HT1 receptors (IC50 >> 10 pM); no interaction at all was observed with muscarinic cholinergic, histamine H1,.GABAA, benzodiazepine, opiate, substance P, DA2 and 5-HT2 receptors. Thus, CGP 28014 is a potent and specific inhibitor of COMT in vivo, with a good oral bioavailability. It is presently undergoing tolerability studies in human volunteers. Departement Forschung, Division Pharma, Ciba-Geigy AG, CH-4002 Basel, Schweiz

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In rat neocortex, cho]ecystokinin (CCK) is synthetized by a subpopulation of ~BA-containing interneurons (Jones and Hendry, TINS 9: 71-74, 1986). Since the regulation of their activity by other neurons is unknown, a respective investigation was started. Early experiments showed, however, that opening of a skull area covering the neocortax already transiently enhancedthe expression of the COK-gane in cortical neurons by more than 200%, as measured after extraction of total RNAwith a filter hybMdisetion procedure (Olenik and Meyer, Neuropeptides, in press). In the present study, this phenomenonwas further investigated. All data were obtained from rat brains 2 days after the partial removal of the parietal boneduring eduitensin anaesthesia. 1) In situ hybridisation studies showedthat the unilateral operation enhanced CCK-mRNA levels in neocortical layers It, Ill and V, VI as compared to the contrslateral side which served as control Theseincreases were observed in the frontal, parietal and occipital areas. No changes in CCK-mRNAwere found in subcerticel areas, such as hippcoampus, thalamus, basel ganglia or amygdaloid complex. 2) Neocortical activity of glutamate dacerboxylase,an enzymespecific for e~BA-synthesis, was not changedby bone removal indicating that only the subpopulation of peptida-containing C~BA-interneurons was affected in its activity. 3) The operation-induced increase in CCK-mRNAwas not abolished by daily treatment of the rats with the anti-inflammatory drugs daxamethascne( t mg/kg) or indamethacine ( 1 mg/kg) indicating that local inflammation did not cause the changes in gene expression. ,t) The possible involvement of the "immediateearly onsetgene"c-los, which is known to regulate the transcription of numerous genesand which is also enhancedin its concentration after skull opening, is currently investigated. The obtained data show that opening of the skull alone causes a sequenceof events which leeds to changes in neuronal activity in the cortex. The cortical neurons which inducethesechanga~or are necessaryfor their developmentwill be studied in future experiments. Pharmakologisches Institut dar Universit~t Fraiburg, Hermann Herdarstr, 5, 7800 Freiburg.

R 98 389 ]2~I~BITOI~ ~ll~'uC'1' OF ~T~PHA_q~c AtCE~OLS ~ D E S ~ I T I S A T I C ~ OF THE ~

N-~D-

P~uE~,iOR S Y S T ~

K. Fink and M. G6thert In rat brain cortex slices, low ethanol (E) concentrations selectively inhibit the 5n~DA-evoked NA release leaving that to other depolarizing stimuli unaffected (G6thert and Fink, Naunyn-Sc/3miedeberg's Arch Pharm~ool 340:516-521, 1989). In the present study we ~ whether this effect of E is related to its ability to interact with hydrophobic membrat~ components and whether the desensitisation of the ~ receptor system shown in this model (FJ/3k et al., ibid. 339:514-521, 1989) could be coulqteracted by E.- Rat brain cortex slices, preincubated with 3H-NA were ~ with M~2+-free Krebs-Henseleit solution. The H overflow frcm the slices was stimulated with 300/muol/l ~4DA (S; from the 40th-42nd min of s~oerfusion). When present frcm 20 rain before S until the end of the experiments, i~ethanol, E, propanol, butanol, pentanol and hexanol (at this potency order) inhibited the l@4DA-evoked 3H overflow. The inhibitory potency was correlated with the membrane/buffer (m/b) partition coefficient (r=0.95; p<0.05). Preexposure to i00 ~mol/l NMDA from the 20th-38th min of of superfusion, leading by itself to a transient increase in 3H efflux, abolished the 3H overflow in response to S (in spite of the 2 min interruption of ~ to 5~]A before S); preexposure to 320 ~mol/l E at the same time schedule increased basal efflux, but 2 rain after withdrawal S did not differ from controls; preexposure with E in addition to h~DA abolished the transient increase in 3H efflux found with NMDA alone and restored the response to S. The NMDA receptor antagonist Dl~2-amino-5-phosphonovaleric acid (2-APV) exhibited the same pattern of effects as E. In conclusion, the effects of E on the responses to NMDA may be due to hydrophobic interaction with the NMDA receptor. E may prevent receptor activation or one of the subsequent steps involved in opening of the ion channel and desensitisation. Inst. Rlalnm3kol. Univ. Bonn, Reuterstr. 2b, D-5300 Bonn

390 EFFECTS OF ANTIDEPRESSANTSON THE HIPPOCAMPALSLICE S. B i r n s t i e l and H.L. Haas Recent investigations indicate an i n h i b i t o r y role of t r i c y c l i c antidepressants on the N-methyl-D-aspartate (NMDA) receptor complex (Sernagor E et a l . , Neuron 2:1221, 1989; Reynolds IJ and M i l l e r RJ, Br. J. Pharmacol.95:95,1988). We have studied the impact of antidepressive agents on NMDA-dependent discharges in Mg++-free medium as well as on spontaneous f i r i n g in low Ca++-high Mg++ medium, a measure f o r postsynaptic e x c i t a b i l i t y independent of NMDAchannel mediated Ca++-fluxes. Experiments were performed on the CA I region of transverse hippocampal slices of male and female SpragueDawley rats (150-200 g) as previously described (Rose GM et a l . , Naunyn-Schmiedeberg's Arch. Pharmacol. 332:89, 1986; Psarropoulou C and Haas HL, Naunyn-Schmiedeberg's Arch. Pharmacol. 339:613, 1989). The t r i c y c l i c i m i p r a m i n e and both enantiomers of the t e t r a c y c l i c o x a p r o t i l i n e were used. In Mg++-free medium, the f i r s t and t h i r d population action potential were chosen for evaluation. Imipramine at 10-5 mol/l s l i g h t l y increased both p o t e n t i a l s , an effect that became s i g n i f i c a n t at 5.10 -5 mol/l (20±6% and 12±13% a f t e r 15 min, n=7). After 20 min of perfusion, however, the t h i r d spike declined. The same pattern was found for both oxaprotiline-enantiomers. In low Ca++medium, however, imipramine caused a time and dose-dependent decrease of spontaneous f i r i n g at a l l concentrations tested (10 -5 , 2-10 -5 and 5.10 -5 m o l / l ; 35±1%, 46±12%, 86±11%, n=6-3). Again s i m i l a r effects were produced by (+)- and ( - ) - o x a p r o t i l i n e . At 10-5 mol/l an i n i t i a l decrease in f i r i n g rate (7±4%, n=3; 18±11%, n=6) was followed by an increase a f t e r 25-30 min of perfusion (4±5%, n=3; 20±25%, n=4). An interference of antidepressive agents with NMDA-receptors in rat hippocampus is thus u n l i k e l y . Department of Physiology and Pathophysiology, University of Mainz, Saarstr. 21, D-6500 Mainz, FRG

391 AGE-ASSOCIATED LOSS OF N-METHYL-D-ASPARTATE RECEPTORS IN THE MOUSE FOREBRAIN, PARTIAL RESTORATION BY CHRONIC PIRACETAM TREATMENT

S.A.Cehen and W.E.MUIler One of the many changes detectable in our central nervous system in the course of normal aging is a decrease of the densities of many neurotransmitter receptors. Since many of the physiologicalprocesses in which N-methyI-D-aspartate (NMDA) receptors are usually involved with (e,g. developmental plasticity, memorial acquisation, neurotoxic mechanisms of brain ischemia) are altered during the course of normal aging, we investigated possible age-associated alterations of NMDA receptors in the mouse brain. NMDA-receptor binding was performed using 3H-MK-801 (*-5-methyl10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine) as radioligand. The effect of aging was determined by comparing receptor properties in the brains of 3 months old and 18 months old female NMRI mice. Substantial levels of NMDA receptors were found in homogenates of the mouse forebrain, with a Brnax of about 2.6 pmol/mg protein and a KD value of about 12 nmol/I. Receptor density was redu6ed in the aged mouse brain by more than 30 % with no change of receptor affinity. Moreover, stimulation of specific 3H-MK-801 binding by L-glutamate and by glycine was enhanced in the aged brain when compared with the effects in the brain of young mice. Very interestingly, chronic treatment of aged mice with the nootropic drug piracetam (500mg/ kg/day) elevated NMDA receptor density in the forebrain by about 20 %. These findings support our hypothesis that a restoration of age-associated receptor deficits might contribute to the mechanism of action of nootropic drugs. Department of Psychopharmacology Central Institute of Mental Health; D-6800 Mannheim (FRG)

392 MODULATION OF RESPONSES TO INHIBITORY AND EXCITATORY NEUROTRANSMII"rERS IN CULTURED NEURONS BY ANTICONVULSANT DRUGS

H. Lampe and H. Bigalke

The antispastic and anticonvulsant agents memantine, tizanidine and carbamazepine have been shown to suppress picrotoxinand strychnine- induced hyperactivity of neurons in culture [1]. To elucidate the underlying mechanisms, effects of the drugs on chemosensitive currents were investigated. We used the WholeCell patch clamp technique to record glycine- and glutamateactivated whole-cell currents from mouse spinal cord neurons in culture. Monolayer neuron cultures were continuously superfused with growth medium. Patch pipettes were filled with a solution containing 140 mM KCI, 1 mM CACI2,2 mM MgSO4, 10 mM HEPES and 11 mM EGTA. Either glycine (100/~M) or glutamate (100 #M) were applied by pressure-ejection to cells clamped at - 60 mV membrane potential. The transmitter responses were recorded at different drug-concentrations.The drugs reduced the glutamate-activated currents in a concentration dependent manner. The glycine-induced current was promoted by memantine at concentrations of between100 nM and 5 ~M, whereas it was reduced at higher concentrations of memantine. The effects were fully reversible.Thus the reduction of the hyperactivity in convulsant treated cultures can be explained by a restoration of the inhibitory currents and by a blockade of glutamate-induced currents. [1]: Netzer, Lampe, Binscheck (1989), Naunyn-Schmiedeberg's Arch. Pharm. 339, Suppl.: R 112 Dept. of Pharmacology and Toxicology, Med. School of Hannover 3000 Hannover 61, FRG Supported by the DFG (Bi 274/3-1)

R 99 393

395

SUPPRESSION OF PICROTOXIN-INDUCED HYPERACTMTY IN CULTURED NEURONS REFLECTS MODULATION OF VOLTAGE-GATED SODIUM CHANNELS BY ANTIEPILEPTIC AND ANTISPASTIC DRUGS

E F F E C T OF M E M A N T I N E ON D Y S T O N I C M O V E M E N T S IN M U T A N T S Y R I A N G O L D E N H A M S T E R S C O M P A R E D TO T H E G A B A B - A G O N I S T BACLOFEN G. F ~ e d o w

R. ~ R A N D

T. BINSCHECK

In culturedspinalcordneuronsoffetalmice,10/~M picrotoxininducedhyperactivitydue to a blockof the postsynapticGABA-reccptor.The effectsof the antieonvulsant and antiepileptic drugs phenytoin, carbamazepine, memantine, baclofen and tizanidine on this hyperactivity were compared with each other. Voltage clamp experiments focussed on voltage-gated sodium channels were performed in order to elucidate underlying mechanisms. Patch pipettes (2-4 MI2) containing a low calcium, high potassium salt solution were used for current clamp examinations. Untreated ceils spontaneously generate action potentials (APs) with a frequency of between 0.2 and 2 Hz. After 10 minutes of exposure to picrotoxin bursting activity occured which can be characterized as paroxysmal depolarizing events accompanied by high frequent firing of APs (30-120 Hz). The examined drugs altered this bursting activity in a specific manner. Carbamazepine, memantine, and phenytoin decreased the burst duration. In addition carbamazepine and memantine lowered the frequency of APs. Tizanidine reduced the number of APs, the length and amplitude of the depolarizing shifts remaining unaffected. Baclofen decreased the frequency of bursts, while their duration and the frequency of APs both increased. Voltage clamp examinations in Whole Cell configuration give evidence, that phenytoin, carbamazepine, memantine, and tizanidine block voltage-gated sodium channels. The voltage-dependent inactivation of sodium channels was shifted towards more negative potentials by phenytoin and baclofen. Memantine, tizanidine, and carbamazepine prolonged the refractory period of the sodium channel. It is concluded that distinct reductions of picrotoxin bursts could be explained by modulation of the kinetics of the voltage gated sodium channel.

Dept. of Pharmacol., Toxicol. & Pharmacy, School of V e t e r i n a r y Medicine, 3000 H a n n o v e r 71, F.R.G.

Dept. of Toxicology, Med. School of Hannover, 3000 Hannover 61, FRG

Supported by the DFG ( Bi 274/3-1)

394 EVALUATION OF CGP 37849 ~

Memantine is used in the treatment of s p a s t i c i t y and extrapyramidal m o v e m e n t disorders. The d r u g has s h o w n to be c l i n i c a l l y e f f e c t i v e d u r i n g chronical treatment in some cases of dystonia. In this study we i n v e s t i g a t e d the acute effect of memantine on paroxysmal d y s t o n i c movements in a m u t a t i o n in Syrian g o l d e n hamsters c o m p a r e d to the a n t i d y s t o n i c e f f i c a c y of b a c l o f e n . A g e d e p e n d e n t d y s t o n i c e p i s o d e s , w h i c h last u p to several hours, can be r e p r o d u c i b l y initiated in m u t a n t h a m s t e r s b y p l a c i n g the a n i m a l s i n t o a new, empty cage. M e m a n t i n e (5,10,20 m g / k g i.p.) d o s e - d e p e n d e n t l y i n c r e a s e d the l a t e n c y to the onset of the dystonic attack, while b a c l o f e n had no i n f l u e n c e on the t i m e c o u r s e of d y s t o n i c m o v e m e n t s . In a d d i t i o n , m e m a n t i n e (20 mg/kg) d e l a y e d the d e v e l o p m e n t of the w h o l e d y s t o n i c episode. While b a c l o f e n (i0 m g / k g i.D.) reduced the s e v e r i t y of d y s t o n i c m o v e m e n t s in m u t a n t hamsters, memantine had no significant influence on this parameter. The results show that in the dystonic hamster model memantine exerts acute a n t i d y s t o n i c effects, which, howewer, are less m a r k e d than those of baclofen. D y s t o n i c episodes in m u t a n t h a m s t e r s a r e w o r s e n e d b y d o p a m i n agonists. Therefore it is conceivable that the antidystonic p o t e n c y of m e m a n t i n e is w e a k e n e d by the d o p a m i n e r g i g effects of this drug. Further s t u d i e s w i l l be n e c e s s a r y to i n v e s t i g a t e the c h r o n i c e f f e c t s of m e m a n t i n e in this a n i m a l model.

396 CGP 3 9 5 5 1 ,

TWO COMPETITIVE

NMDA RECEPTOR ANTAGONISTS WITH ORAL ANTICONVULSANT ACTIVITY IN THE KINDLING MODEL OF EPILEPSY D. HSnack

NMDA ( N - m e t h y l - D - a s p a r t a t e ) r e c e p t o r mechanisms h a v e b e e n reported to be i n v o l v e d in p a t h o l o g i c a l phenomena such as e p i l e p t i c d i s c h a r g e s , a n x i e t y , s p a s t i c i t y and ischemic n e u r o n a l d e g e n e r a t i o n . Thus selective NMDA r e c e p t o r a n t a g o n i s t s a r e p o t e n t i a l c a n d i d a t e s for the t r e a t m e n t o f s u c h disorders. CGP 37849 ( D L - ( E ) - 2 - a m i n o 4-methyl-5-phosphono-3-pentenoic acid) and i t s c a r b o x y e t h y l e s t e r , CGP 39551, two n o v e l a n a l o g u e s of 2 - a m i n o 5 - p h o s p h o n o p e n t a n o a t e (APS), are t h e f i r s t 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 i t h oral a n t i c o n v u l s a n t a c t i v i ty. Both compounds were reported to s h o w a n t i c o n v u l s a n t e f f e c t s in e l e c t r o s h o c k - i n d u c e d s e i z u r e s at d o s e s at which no s i d e - e f f e c t s occurred. CGP 39551 w a s s h o w n to d e l a y t h e d e v e l o p m e n t o f amygdala kindled s e i z u r e s in r a t s at d o s e s of 10 mg/kg p.o. In t h e p r e s e n t study, both c o m pounds were t e s t e d in d o s e s from 1 - 1 0 mg/kg in f u l l y kindled r a t s , i.e. a chronic model for focal s e i z u r e s w i t h s e c o n d a r y g e n e r a l i z a t i o n . CGF 37849 o n l y s l i g h t l y reduced s e i z u r e s e v e r i t y at 10 mg/kg, which w a s probably due to t h e marked muscle r e l a x a t i o n occurring at t h i s dose, and d e c r e a s e d t h e a f t e r d i s c h a r g e duration at 2.5 mg/kg. It did n o t a l t e r t h e a f t e r d l s c h a r g e t h r e s h o l d (ADT) s i g n i f i c a n t l y . CGP 39561 e x e r t e d no e f f e c t on s e i z u r e p a r a m e t e r s at d o s e s up to 10 mg/kg and did not a l t e r t h e ADT. T h e s e r e s u l t s i n d i c a t e t h a t CGP 37849 and CGP 39651 did n o t p o s s e s s s u f f i c i e n t e f f i c a c y a g a i n s t f u l l y kindled s e i z u r e s e v e n at d o s e s a t which marked s i d e e f f e c t s occurred. This c o n t r i b u t e s to t h e findings t h a t o t h e r NMDA r e c e p t o r a n t a g o n i s t s , e.g, MK-801, were more p o t e n t a g a i n s t t h e kindling d e v e l o p m e n t t h a n a g a i n s t f u l l y kindled s e i z u r e s . T h i s might s u g g e s t t h a t s u c h compounds are no p o t e n t i a l c a n d i d a t e s for t r e a t m e n t o f p a r t i a l or s e c o n d a r y g e n e r a l l s e d s e i z u r e s in e p i l e p t i c p a t i e n t s . Dept. o f Pharmacol., Toxicol., and Pharmacy, School V e t e r i n a r y Medicine, Bflnteweg 17, D - 3 0 0 0 Hannover 71

of

MINIMUM EFFECTIVE DOSE AND PLASMA CONCENTRATIONS IN MALE MICE FOLLOWING I.P. ADMINISTRATIONOF RALITOLINE (CI-946) A. yon Hodenberg, W. L~scher*, B. Noltlng*, C.P. FaBbender*, I. Fecht-Kempter, C, Taylor** T K.-O. Vollmer Ralitoline (R), a thlazolidlnone, shows antlconvulsant activity In different animal models. The purpose of the study was to determine the minimum effective dose of R In different pharmacological tests, and to determine the corresponding minimum effective plasma concentrations in mlce following IP administration. All tests were carried out in male mice. The following pharmacological tests were performed: Seizure threshold determination with electroshock and IV pentetrazole (PTZ), supramaxlmal electroshock (MES) and SC PTZ seizure test, rotarod and chimney tests for determination of doses causing ataxia. For determination of minimum effective plasma concentrations, blood was collected at time of maximum antlconvulsant a c t i v i t y of R. R prevented electrically induced seizures. Maximal effect was reached 2 min postdose and declined rapidly. Minimum effective dose which significantly increased the seizure threshold In the electroshock seizure test by 2 mA was 0.97 mg/kg. Following IV infusion of PTZ, a R dose of ].9 mg/kg significantly increased the threshold for clonic seizures by 20 %. Tonic seizures were no longer observed following 1.5 to 5 mg/kg of R. EDI, of R in the MES-test was 3.5 mg/kg. In the SC PTZ test, 5 mg/kg of R protected 40 % of the mice. Results of R were compared to those of phenobarbital, primldone, carbamazepine, phenytoin, valproate, ethosuximide, dlazepam and clonazepam. R had the shortest delay between dosing and maximum effect, and the lowest ED,, value in the MES test. Minimum effective R plasma concentration following 0.97 mg/kg, was (mean ± SD) 342 ± 151 ng/ml and 1310 ± 316 ng/ml following 3.5 mg/kg, Minimum effective plasma concentrations of R were lower than of other anticonvulsants except benzodlazepines. Therapeutic R plasma concentration In humans can only be determined from clinical trials. However, based on comparison with marketed anticonvulsants, the effective human plasma concentration of R extrapolated from the mice data is approximately I Rg/ml. G6decke Forschungsinsitut, D-7800 Frelburg, * Dpt. Pbarmacol., Toxicol. School Vet. Med., D-3000 Hannover 71, ** Parke Davis Pharmaceutlcal Research Division, Warner-Lambert Company, Ann Arbor, MI, USA.

R 100

397

399

EFFECT OF CGS 15943A (A TRIAZOLOQUINAZOLINE) UPON THE PROTECTIVE EFFICACY OF COMMON ANTIEPILEPTIC DRUGS AGAINST ELECTROCONVULSIONS IN MICE. S.J. Czuczwar, W. Janusz, B. Szczepanik, and Z. Kleinrok.

PHENYTOIN - A PHARMACOLOGICAL STUDY IN THE AMYGDALA KINDLING MODEL OF FOCAL EPILEPSY C. Rundfeldt

Aminophylline was documented to impair the anticonvulsive activity of common antiepileptic drugs against electroconvulsions (Czuczwar et al., Epilepsia 27:204, 1986). In order to find out whether A1 adenosine receptor blockade may be responsible for this effect, the influence of 5-amino-9-chloro-2-(2-furanyl)-l,2,4-triazolo(l, 5-c)quinazoline monomethanesulfonate (CGS 15943A; a non xanthine adenosine antagonist) was studied on the protection offered by carbamazepine, diazepam, phenobarbital, phenytoin, and valproate against maximal electroshock-induced convulsions. All drugs were injected i.p., phenobarbital and phenytoin 120 min. before the test, carbamazepine and diazepam - 60 min., valproate - 30 min., and CGS 15943A (I mg/kg) - 15 min. prior to electroconvulsions. In no case CGS 15943A influenced the ED50 values of antiepileptic drugs studied. However, valproate (250 mg/kg)-induced sedation was reversed by the adenosine antagonist. It may be concluded that the protection provided by common antiepileptic drugs against electroconvulsions does not seem related to adenosine-mediated inhibition. Dept. of Pharmacol., Med. Sch., PL-20-090 Lublin, Poland

Jaczewskiego

8,

Due to i t s b r o a d a n t i e p i l e p t i c s p e c t r u m a n d i t s v i r t u a l l y a b s e n c e of p e r i p h e r a l side e f f e c t s p h e n y t o i n is one of t h e m o s t f r e q u e n t l y u s e d a n t i e p i l e p t i c d r u g s in h u m a n s . The d r u g is c o m m o n l y u s e d in t h e t h e r a p y of f o c a l a n d c o m p l e x f o c a l s e i z u r e s , b u t i t is a l s o h i g h l y e f f e c t i v e a g a i n s t g e n e r a l i s e d t o n i c clonic s e i z u r e s . However. in c o n t r a s t to its clinical efficacy, conflicting reports exist concerning t h e e f f e c t i v e n e s s of p h e n y t o i n a g a i n s t k i n d l e d a m y g d a l o i d s e i z u r e s , i.e. a model of f o c a l e p i l e p s y . T h e aim of t h i s s t u d y w a s to e v a l u a t e t h e a c u t e a n d c h r o n i c e f f e c t s of p h e n y t o i n on v a r i o u s p a r a m e t e r s of f u l l y k i n d l e d a m y g d a loid s e i z u r e s . After acute administration, phenytoin e l e v a t e d t h e a f t e r d i s c h a r g e t h r e s h o l d (ADT) in a d o s e d e p e n d e n t m a n n e r w i t h a minimum e f f e c t i v e d o s e of 12,5 m g / k g i.p. Up to a d o s e of 75 m g / k g i.p. o t h e r s e i z u r e parameters (seizure severity, seizure duration, afterdischarge duration) were not reproducible altered. During chronic trials, fully kindled rats were treated once daily w i t h p h e n y t o i n , 75 m g / k g on d a y 1, a n d 50 m g / k g on d a y 2 - 15, f o r two weeks. A m y g d a l a s t i m u l a t i o n s w e r e c a r r i e d o u t b e f o r e , d u r i n g , a n d a f t e r t h e p e r i o d of t r e a t m e n t . When a p p l y i n g s u p r a m a x i m a l s t i m u l a t i o n (500 pA, s t a n d a r d s t i m u l u s p a r a m e t e r s ) , no s t a b l e e f f e c t c o u l d be f o u n d . When a d j u s t i n g t h e s t i m u l a t i o n c u r r e n t 20% a b o v e t h e i n d i v i d u a l ADT, t h e s e i z u r e s w e r e i n i t i a l l y t o t a l l y s u p p r e s s e d . D u r i n g t h e two w e e k s of t r e a t m e n t , a n i n c r e a s i n g n u m b e r of a n i m a l s w e r e n o t p r o t e c t e d a n d s h o w e d g e n e r a l i s e d s e i z u r e s , t h u s i n d i c a t i n g d e v e l o p m e n t of t o l e r a n c e . In c o n c l u s i o n , t h e r e s u l t s i n d i c a t e t h a t p h e n y t o i n is h i g h l y e f f e c t i v e in e l e v a t i n g a m y g d a l a k i n d l e d a f t e r d i s c h a r g e t h r e s h o l d a n d b y t h i s w a y of a c t i o n e f f e c t i v e against amygdala kindled seizures. 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, 3 0 0 0 H a n n o v e r 71, F.R.G.

398

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VIGABATRIN INDUCED CHANGES IN AMINO ACID LEVELS IN DIFFERENT RAT BRAIN REGIONS: A COMPARISON TO OTHER GABA-DEGRADATION INHIBITORS AND VALPROIC ACID D.HSrstermann

EFFECTS OF DIAZEPAM D E P E N D E N T NA + C ~ S

V i g a b a t r i n ( g a m m a - v i n y l - G A B A , GVG), a n o v e l c l i n i c a l l y used antiepileptic drug, is l i k e g a m m a - a c e t y l e n - G A B A (GAG) a n e n z y m e - a c t i v a t e d GAHA-transaminase inhibitor. I n s t e a d of t h e s e d r u g s , a m i n o o x y a c e t i c a c i d (AOAA) s e e m s to a c t n o n - s p e c i f i c a / l y v i a p y r i d o x a / p h o s p h a t e - a n t a g o n i s m . The clinically proved antiepileptic drug valproic acid (VPA) r e d u c e s t h e GABA-T a c t i v i t y o n l y in h i g h d o s e s , but additionally increases the GABA-synthesis, In the p r e s e n t s t u d y , t h e e f f e c t s of t h e s e f o u r a g e n t s o n a m i n o a c i d l e v e l s in d i f f e r e n t regions of r a t brain were measured. For that purpose, the drugs were applicated i.p. a t a n t i c o n v u l s a n t d o s e s (GVG 1200 m g / k g , GAG 100 m g / k g , VPA 200 m g / k g a n d AOAA 100 m g / k g ) a n d t h e r a t s w e r e d e c a p i t a t e d a f t e r 4 h (VPA 15 rain). T h e b r a i n w a s d i s s e c t e d i n 12 d i s c r e t e r e g i o n s w i c h w h e r e i m m e d i a t e l y h o m o g e n i z e d in 80 % e t h a n o l . T h e a n a l y s e s w e r e d o n e b y HPLC w i t h f l u o r e s c e n c e d e t e c t i o n . All f o u r s u b s t a n c e s e n h a n c e d t h e G A B A - c o n e e n t r a t i o n , b u t VPA w a s m u c h l e s s a c t i v e t h a n t h e o t h e r d r u g s . GVG, GAG a n d AOAA r e d u c e d t h e g l u t a m a t e , a s p a r t a t e a n d a / a n i n e l e v e l s , w h e r e a s VPA only decreased aspartate and showed no effect on g l u t a m a t e a n d a / c h i n e . S i g n i f i c a n t c h a n g e s in t h e g l y c i n e a n d t a u r i n e c o n c e n t r a t i o n s w e r e o b s e r v e d o n l y in a f e w b r a i n r e g i o n s . I n some b r a i n r e g i o n s , g l u t a m i n e c o n t e n t w a s d e c r e a s e d b y GVG a n d GAG, b u t i n c r e a s e d b y VPA. This investigation demonstrates that vigabatrin acts q u a l i t a t i v e l y q u i t e s i m i l a r to GAG a n d t h e s o - c a / l e d n o n s p e c i f i c AOAA in c h a n g i n g a m i n o a c i d b r a i n l e v e l s . Dept. of P h a r m a e o l . , Toxicol. a n d P h a r m a c y , S c h o o l of V e t e r i n a r y M e d i c i n e , B i i n t e w e g 17, D-3000 H a n n o v e r F.R.G.

AND

FLUMAZENIL

ON

VOLTAGE-

IN N E U R O B L A S T O N A C E L L S G. Trube, K. H. Backus, P. P f l i m l i n

We studied the inhibition of Na* currents in voltage-clamped N2A neuroblastoma cells by the benzodiazepines diazepam (DZ) and flumazenil (FL) and compared it to the effects of lidocaine (LC) and the antiepileptics phenytoin (PT) and carbamazepine (CBZ). DZ, PT, CBZ, and LC reduced the current to 60 - 67 % of the control at a concentration of 100 pM (holding potential = - 80 mV, stimulation rate 0.2 Hz), but FN did not have any effect. With any drug no or little inhibition was seen at 10 DM. In the presence of DZ the Na + channel inactivation curve was shifted in hyperpolarizing direction by 4.8 ± 0.5 mV. PT, CBZ and LC caused stronger shifts of 17.4 ± 2.1, 10.6 ± 0.9 and 17.0 z 2.1 mY, respectively. Inhibition by DZ increased use-dependently during 10 depolarizing pulses repeated at high frequency (200 Hz), whereas use-dependent effects of the other compounds developed less rapidly. At a low stimulation rate (7 Hz) use-dependent block was pronounced with LC, but weak or absent with DZ and CBZ. DZ (100 ~M) was the only compound significantly increasing the speed of Na T current inactivation (control: 0.54 ± 0.11 ms; DZ: 0.41 ± 0.12 ms). It is concluded that DZ causes a faster block and unblock of Na + channels than the other compounds. Effective concentrations, however, are higher than the free plasma levels reached therapeutically,e.g., during the treatment of epileptic seizures. Pharmaceutical Research Department, F. Hoffmann-La Roche AG, Grenzacherstrage 124, CH-4002 Basel, Switzerland

R 101 401

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ENVIRONMENTAL INTERACTIONS TO EXCITATION AND SEDATION IN MICE AFTER APPLICATION OF DIAZEPAM, CAFFEINE, AND COMBIANTIONS Ch. Gerischer

FUNCTIONAL ALPHA-I ADRENOCEPTOR SUPERSENSITIVITY AFTER CHRONIC TREATMENT WITH THE ANTIPSYCHOTIC DRUG HALOPERIDOL Ch. Pifl and O.Hornykiewicz Neuroleptics are considered to exert t h e i r antipsychotic action by blocking the brain D2 dopamine receptor sites. However, in radioligand binding studies most of them show considerable a f f i n i t y for brain alpha-I adrenergic receptors whose number increases in rats treated c h r o n i c a l l y with e . g . h a l o p e r i d o l . To test the functional relevance of this brain alpha-I receptor upregulation we employed the behavioral model (in the rat) of p o t e n t i a t i o n of the dopaminergically e l i c i t e d locomotor a c t i v i t y by an alpha-I adrenergic agonist. St 587 (2-(2-chloro-5-trifluoromethylphenylimino) imidazo l i d i n e ) , a selective alpha-I agonist with good brain permeability (Pichler & Kobinger, Eur.J.Pharmacol. 107, 305, 1985) in the dose of Img/kg i . p . potentiated the locomotor stimulating action of the D2 dopamine agonist l i s u r i d e (O.3mg/kg i . p . ) . This potentiation was completely abolished by prazosin (Img/kg i . p . ) . Under our experimental conditions, subthreshold doses of l i s u r i d e and St 587 (alone or in combination) were 0.15 and O.5mg/kg i . p . , respectively. In rats treated with haloperidol (Img/kg i . p . ) once a day for 4 weeks the subthreshold dose of l i s u r i d e remained i n e f f e c t i v e ; however, combination of this l i s u r i d e dose with the subthreshold dose of St 587 (0.5 mg/kg i . p . ) resulted in strong locomotor hyperactivity. We conclude that chronic administration of haloperidol brings about functional brain alpha-I adrenoceptor supersensitivity, suggesting a functionally significant blockade of brain adrenergic receptors by this c l i n i c a l l y w i d e l y used antipsychotic drug. This behavioral model w i l l be used to establish the rank order of potency of neuroleptics r e l a t i v e to t h e i r c l i n i c a l potency as a n t i psychotic drugs.

The exploratory behaviour of female mice (n = 512) in the open field (OF) was examined after application of diazepam 01); 2, 4, 6 mg/kg), caffeine (C; 10, 20, 30 mg/kg) and combinations (square-shaped experimental design). The first hypothesis to be tested was that both D and C have comparable excitatory actions which superimposed each other in combined treatments. The mice were placed 5 rain p.i. into the OF, for a period of 1 h p.i. square crossings, rearings, and directional changes were measured. Control animals revealed an initial activity of 45 cr/min which gradually decreased to 10 or/rain after an hour. D raised the activity immediately after the placement into the new environment (+25% for 4 mg/kg), but after 5 rain locomotion deelhied abruptly below the control level (-57%). The increase after C was initially poor (+9% for 20 mg/kg) but the level was slowly declining and after 1 h it was 88% higher than that of controls. In concomitant applications initial excitation corresponded to the partial dose of D mad maintained excitation to the C portion, the interaction mode was an effect-additive. A similar relationship was found for rearings. The directional maintenance was enhanced by D but not affected by C. Here combinations of 10 - 20 mg C and 4 - 6 mg/kg D deviated from the effect-additive model (p < 0.01), the mice performed less directional changes than expected. Ia addition the decline of locomotor acti-city was less abrupt. This indicates a differentiated drug interaction. In a second series the hypothesis was tested that after drug application excitation and sedation may coexist simultaneously. To this purpose the mice were placed to the OF 10 rain, 20 rain, and 40 miu p.i. After a delay of 20 rain D-treated mice revealed higher level of activity (p < 0.05) than respective controls (for 2 mg/kg: +52%, for 4 mg/kg: + 20%) although animals treated in the same way but put into the OF five rain pi. were by this time already sedated (-46% and -57%, resp. cp. controis). These results prove that excitation and sedation can be triggered at the same time. Corresponding actions of C were not found. In D applications (4 mg/kg and 6 mg/kg) the effect is superimposed by the time course of the drugs's sedative action. The initial activity level is relatively high with mice placed to the OF 5 mln pi. (53 er/mia, 4 mg/kg), it declines with mice put to the OF 10 rain p.i. (40 er/mln) and is finally (40 min p.i.) raised to 60 cr/min). Addion of C prevents the effect.

Supported by a grant of the Bundesgesundheitsamt, lnstitutfar Arzneimittel Iast. f. Neuropsychopliarmakologie FU, Ulmenallee 30, D-1000 Berlin 19, F.R.G.

402 EVALUATION OF LOCOMOTOR ACTIVITY: INTRA. AND INTERSPECIES DIFFERENCES IN MICE I b r a h i m Chahoud, Frank Iversen, Reinhard Meister

The measurement and analysis of the locomotor activity is important for experimental studies on effects of agents on central nervous systems, especially if no morphological alterations are observed. A device (using four infra-red light photocells p e r cage) which simultaneously monitors the motility of 120 individual rodents was developed at our institute (Thiel et aL, 1989, Neuro Tox 10: 1625-1632). The locomotor activity of 55 adult female mice ( H a n : N M R I ) was measured. The counts of the light b e a m interruptions (LBI) during 5 minute intervals were summarized and recorded for at least 24 hours. In order to analyze the circadian rhythms the day was divided into 4 equal time periods and the counts of LBI were calculated for each period absolutely and relatively to the overall counts of the day. Based on the observation a n d analysis of the LBI values the mice could be classified into three groups: hypoactive ( < 2000 counts), normoactive (20005000 counts) and hyperactive ( > 5000 counts). Considering the relationsip to the overall value during 24 hours, no significant differences could be registered for all time periods between the three groups. W h e n comparing two mouse strains, H a n : N M R I mice were found to be more active (32% activity) over a 12-h light period than e.g. D B A / 2 J mice (14% over the same time period. While the D B A / Z I mice showed only about 14% of their locom o t o r activity during the light period (12 hrs), the activity of the H a n : N M R I mice was m o r e than one third in the same period. These results demonstrate that the nocturnal behaviour in the D B A / 2 J strain is m o r e pronounced than that in the H a n : N M R I straim After treatment with the neurolepfic haloperidol ( 1 . 5 / 3 / 6 m g / k g s.c.) the relationship between certain time periods changed in both strains and at each dose.

Institut far Toxikologie und Embryopharmakologie, Freie Universitlit Berlin, Gatystr. 5, D-IO00 Berlin 33

I n s t i t u t for Biochemische Pharmakologie der Univ. Wien, Borschkegasse 8A, A-I090 Wien (Osterreich)

4O4 PHARMACOLOGICAL MODEL FOR ANTIPSYCHOTICS USING LSD ON RATS SIMULATING NEUROCHEMICAL CHANGES IN SCHIZOPHRENIA L.Hetey The acute effects of LSD on functional parameters (uptake, release) of the dopaminergic system in rats were investigated as a pharmacological model i. for characterizing antipsychotic drugs and 2. for modelling pathoneurochemical changes in schizophrenia. In e _ ~ v i v o experiments, LSD induced a decrease in K -stimulated DA release and increased DA uptake in n. accumbens synaptosomes of rats. In in vitro experiments, LSD was able to induce similar alterations: decreased DA release (IC-50 = 0.07 ~M) and increased DA uptake (at i ~M LSD: Km = 21%, Vmax = 62% of controls) mediated by DA-autoreceptors and 5HT-heteroreceptors. The in vitro LSD effects on n. accumbens synaptosomes represent a suitable model for screening and characterizing classical and atypical antipsychotic drugs. In post-mortem n. acc u~+ens of schizophrenics (Type I) synaptosomal K -stimulated DA release is similarly decreased and DA uptake increased. These alterations are due, however, to other kinetic changes of uptake (Km = 245%, Vmax = 282% of matched controls) and to supersensitivity of DA-autoreceptors. Thus, besides differences in triggering mechanisms, the changes in the LSD-model and in schizophrenia are similar underlining the usefulness of this model. Dept. Neuropsychopharmacology, Schering AG, i000 Berlin 65, Postfach 65 03 ii

R 102 405 CONDITIONINS OF APOMORPHINEEFFECTS SN CORTICAL EEG ACTIVITY IN RATS W. Kropf and K. Kuechinsky .

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407 PLASMA CATECHOLAMINE STRESS ALCOHOL CONSUMPTION IN R A T S . T a y l o r a n d N. H a r r i s

RESPONSES AND W.H. Vogel, J.

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The possible conditioning of pharmacological effects of apomorphine on cortical electroencephalogram (EEG) was studied using telemetric recordings in rats. Previous studies have shown that apomorphine-induced stereotyped behaviour can be conditioned: after repeated pairings of defined environmental stimuli (conditioned stimuli,

CS) with the drug effect, the presentation of the s t i muli alone elicited stereotyped s n i f f i n g , licking and gnawing. Since apomorphine, an agonist at dopamine receptors, also produces characteristic alterations in the EES pattern with increase of power in the range of 7.00-9.50 Hz (alpha-1 band), the possibility that this effect can be conditioned as well was studied. In fact, conditioning seven times with mpomorphine (0.5 mg/kg, s.c.) led to a significant increase in the number of short-lasting periods with enhancement of the power in the alpha-I band in the presence of the CB alone when the results obtained by the conditioned group were compared with those of con'trole ("pseudo-conditioned"). In addition, extinction experiments were performed, in which the conditioned stimuli were repeatedly uncoupled from apomorphine administration. Extinction occurred rapidly, and during the forth extinction session, the level of conditioned alpha-1 activation had decreased to the level of pseudoconditioned controls. The results demonstrated that a characteristic alteration in the EEG pattern produced by apomorphine can be conditioned. Institute of Pharmacology and Toxicology, Faculty of Pharmacy, University of Marburg, Ketzerbach 63, D-3550 Marburg, FRG.

Stress has been implicated in t h e c o n s u m p t i o n of alcohol and the etiology of alcoholism. To determine the possible role of stress in alcohol consumption, we offered alcohol in a free choice two bottle situation ( b o t t l e I: water; bottle 2: 5% a l c o h o l ) to 2 groups of rats. These rats were bred selectively for "low" or "high" plasma catecholamine stress responses. Marked variations in alcohol intake were found among animals. However, "high" responders consumed more alcohol than did "low" responders ( 3 . 0 ± 0 . 4 vs 1 . 7 + 0 . 4 g / k g ; p <0.05). After an alcohol-free p~riod of 4 weeks, these rats were offered the same alcohol solution. Again, the "high" responders consumed more alcohol than did the "low" responders (2.9~0.4 vs 1.8~0.4; p <0.05). High correlations were found between the drinking habit of the animals during both alcohol periods (r=0.6487; p <0.05) and between the extent of the plasma eatecholamine stress response and the amount of alcohol consumed (E: r = 0 . 6 0 5 1 ; p < 0 . 0 5 ; NE: r = 0 . 4 4 1 2 ; p <0.0S). After alcohol, cocaine (0.02%) w a s offered; no difference was seen between "low" and "high" responders and no correlation was apparent between alcohol or cocaine intake. T h u s , a l c o h o l i n t a k e is h i g h l y c h a r a c t e r i s t i c for a given animal, correlates positively with the plasma catecholamine response and shows no correlation with cocaine intake. (Supported by Grant AA06107).

Supported by a grant (Ku 39514-I) of the DFS.

Department of Pharmacology Jefferson Medical College Philadelphia, PA 19107, U S A

406

408 PHYSICAL AND PSYCHIC DEPENDENCE OF RATS ON ETHANOL AFTER FORCED OR VOLUNTARY LONG-TERM CONSUMPTION A.Heyne

8-CARBOLINE LEVELS ARE INCREASED IN BLOODOF ALCOHOLICS H.Rommelspacher, S.Lutter, and H.Damm Based on the hypothesis that condensation products of neurotransmitters are involved in the pathogenesis of alcoholism aromatic B-carbolines (harman and norharman) were measured in the blood plasma and erythrocytes of alcoholics and nonalcoholics. The concentration of harman in plasma was about twice as high in alcoholics as in nonalcoholics not reaching significant difference. Loading of nonalcoholics with ethanol (ig/kg within 10 min) did not induce a change of the levels during the observation period (8 h). In contrast to the findings with harman, the concentration of norharman in the plasma of alcoholics was significanty higher than in nonalcoholics ( 110 pg/ml plasma n=12 vs. 40 pg/ml n=lO). Loading with ethanol did not a l t e r the levels in nonalcoholics. Harman was detected in erythrocytes of human subjects as well. Loading with ethanol caused a slight increase of the level after four hours (Rommelspacher et a l . , Alcohol, in press). The findings suggest a t r a i t marker role for B-carbolines in alcoholism. Since the biosynthesis and the metabolism of norharman is unknown, i t remains to be elucidated whether the synthesis or the breakdown of norharman is disturbed in alcoholics. Only a few studies h a v e been published investigating the biological function of norharman. I t produced proc o n f l i c t behaviour in a Geller Seifter paradigm while clearly higher doses e l i c i t e d tonic-clonic convulsiv episodes. Autoradiographic experiments revealed the preferential concentration.of h i g h - a f f i n i t y binding sites in limbic-hypothalamic and limbic-cortical structures (Pawlik et al. J. Chem. Neuroanat. 3, 1990, in press). Inst. f . Neuropsychopharmakologie,Freie Universit~t, Ulmenallee 30,D-1000 Berlin lg,FRG

To study possible intcrrclationshlps between physical and psychic dependence on ethanol (ETOH) long term experiments with male Wistar rats were performed. For one day every week all rats (N=80) had the choice between tap water and three ETOH solutions (5,10,20 Vol%) as drluidug fluid. During tho other 6 days they were either forced to drink ETOH solutions (5% and 20%) or received water.The "forced drhLkers" were housed in groups (FG),contact cages (FC),or individual c~es (FI). "Voluntary drinkers" (V) were kept in groups. During the 8 month period of ETOH consumption the forced intake amounted to 3.4 g/kg/d. The voluntary intake differed significantly among the groups (FI: 1.8 g/kg/d,FC: 1.2 g/kg/d, FG: 1.0 g/kg/d). V rats dcloped the highest preference for ETOH : 3.2 g/kg/d.All the testgroups revealedan incroasingtrendof consumption ( +0.03 g/kg/dper week).After8 months E T O H supplywas interruptedfor 3 months.To studysymptoms of withdrawalalgcsicthresholdand behaviorin dyadicencouters were registereddailyfrom 4 days beforeto 4 days afterwithdrawal.In allgroups the algcsicthresholdrevealeda significantdeclineduringthe firstthreedrug-free days (-13%) which recoveredat the fourthday. This resultindicateda physical dcpandence on ETOH. In thebehavioralteststhe ratsperformedlessoxploration, socialinteractionsand rapidlocomotionduringthe firstthreedays of withdrawal. In contrastto hyperalgesiathe behavioraleffectswcrc dlffercntiallyexpressed among the groups:FI > FC > F G > V. Afterthe drug-fromperiodallthegroups were submlttcdto a frce-cholccparadigma (5,i0,20Voi% E T O H and tap water) for 4 weeks.During the subsequent4 weeks E T O H solutionsbut not waterwere spoiltwkh quiuine (0.1 g/l).Rats having developed a psychicdependance on E T O H were expectedto takehighdosesof E T O H even afteradditionof quinine. V ratsmatched thiscrltcrinn.Whon E T O H was re-offeredthey consumed 3.Z g/kg/d(1.8g/kg/dduringthe quininephase) FI ratsalsorevealedsignsof psychic dependence whereas FC and F G did not meet the demands (2.3and 2.4 g/kg/d, 1.1 and 0.9g/kg/dwithquinine).The differencesbetween the experlmcntalgroups indicatethatphysicaland psychicdependence on E T O H arc not coupledto each other.In particularforced consumptionof high quantitiesof E T O H for a long periodcausedphysicaldependancobut did not alwaysleadto psychicdepcndeuco whereas rats which were enabled to take E T O H intermittentlyby theirown choicerevealedsignsofpsychicdependence.Thus the mode of drug takingrather than the dosesalonedeterminesthe dovelopmentof psychicdependance. Inst.f.NeuropsychopharmakologioFU, Ulmonallce30,D-1000 Berlin19,F.R.G.

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411

VOLUNTARY INTAKE OF ETHANOL AND DIAZEPAM BY RATS: RELATIONSHIPS TO DRUG EXPERIENCES AND INDIVIDUAL BEHAVIORAL FEATURES J. Wolffgramm

FLURARIZIHE DELAYS THE Z/C~ACELLULAR OVERFLOW OF ASPARTATE AND gLUTAMATE DURING GLOBAL CEREBRAL ISGHEHIA D, Scheller, U. Heister and T. Peters The amino acids aspartate (Asp) and glutamate (Glu) are considered to act cytotoxlc under conditions of cerebral ischemla and reperfuslon. For both amino acids a considerable release has been observed under the respective conditions. Antagonists of Asp and Glu at the NMDAreceptor appear to inhibit ischemlc cell death. Since the release of Asp and GIu from presynsptle nerve terminals is induced by ealclum-lnflux, calcium antagonists could inhibit this release thus ellcltlng protection. In order to test this hypothesis, a mlcrodlalysls probe was implanted into the cortex of anaesthetized rats and the extracellular concentrations of Asp and Glu were determined. A DC-electrode was placed in the immediate vicinity of the mlerodlalysls tip. Cerebral Ischemla was induced by cardiac arrest. Asp and Glu were determined in the mlcrodlalysls fractions obtained in 2 mln intervals by HPLC. As a calcium antagonist flunarlzlne was administered i.v. (I0 mg/kg) 30 min prior to induction of cardiac arrest. Under control conditions the latency from onset of cardiac arrest to membrane depolarization was ii0.i ± 21.02 see (n = 9). Flunarlzlne significantly prolonged this latency to 197.1 ± 24.46 sec (n = 7). Concomitantly with depolarization a transient increase in extracellular Asp and Glu levels could be observed In the majority of the control experiments which was followed by a second continuous and pronounced rise. After pretreatment with flunarlzlne the transient peak was either abolished or delayed whereas the subsequent massive and continuous increases of Asp and Glu were considerably postponed. The described actions of flunarlzlne may contribute to the beneficial effects of this compound on delayed neuronal death.

Male Wistar rats had the choice between water and ethanol (El solutions (5, 10, 20 Vol %) for a period of nine months. Controls received water during the same time. After a drug-free period of 3 months diazepam (D) containing solutions (50 and 100 rag/l) were offered to both groups in addition to water (3 months). After a second drug free period (3 months) both groups received water and E solutions. Two hypotheses were tested: (a) the experience with E influences the attitude towards D; (b) animals preferring E also preferably take D solutions. During the first period of E experience (9 months) the rats developped an increasing tendency to consume E. When D was offered no differences were detected between E-experienced and naive (control) rats. In contrast to the first hypothesis the intake of D was not affected by previous experiences with E. This result was not due to an extinction of E preference since experienced rats revealed an E choice after the second drug-free period which was twice as high as in E-naive rats. The second hypothesis, however, coald be verified. In E-experienced rats the correlation between E a~d D intake doses was significantly (!0 < 0.01) positive indicating a common individual disposition for both drugs. Such individual dispositions might be related to characteristical behavioral features. To identify such features "tetradic" encounters (4 rats) in the open field (1 m x 1 m) were performed before drugs were offered. Each encounter session (15 rain) was analyzed aceordiug to six behavioral types: rest, ambulation, exploration, social investigation, play, and aggrcssiou. The individual intake doses of both E and D measured several weeks later correlated significantly (p < 0.01) to aggressive performance and social reelpience. These two behaviottral features represented the dominance rank of an individual. Dominant rats took about 50 % less E and D than non-domlnant ones. Thus social dominance can serve as a predictor for drug taking behavior of rats. Inst. f. NeuropsychopharmakologieF U , Ulmenallee 30, D-1000 Berlin 19, F.R.G.

JANSSEN RESEARCH FOUNDATION,

D-4040 Neuss 21, FRG

410

412

I N C R E A S E IN L O C A L C E R E B R A L G L U C O S E U T I L I Z A T I O N A N D D E C R E A S E IN L O C A L C E R E B R A L B L O O D F L O W IN R A T C A 1 - S U B F I E L D 7 D A Y S A F T E R I S C H E M I A R. R i s c h k e a n d J. K r i e g l s t e i n S e v e n d a y s a f t e r a 10 r a i n f o r e b r a i n i s c h e m i a in t h e r a t a m a r k e d n e u r o n a l n e c r o s i s c a n b e o b s e r v e d in t h e h i p p o campal CAl-subfield. This phenomenon is c h a r a c t e r i z e d by a delayed onset of neuronal cell death, and by the appearence of astroglial cells. The question arose how these histological changes could influence the local cerebral glucose utilization {LCGH/ and the local cerebral blood flow (LCBF). T h e r e f o r e , t h e L C G H w a s d e t e r m i n e d 2, S, 7 a n d 14 d a y s post-ischeraia in m a l e W i s t a r r a t s ( o c c l u s i o n o f c a r o t i d arteries combined with hypotension of 40 ramHg) with the [14Cl-deoxyglueose method. The LCBF was determined 7 days after ischeraia with the [14C]-iodoantipyrine technique. In t h e h i p p o c a r a p a l CAl-subfield we further counted the glial ceils after irarauuocytochemical staining procedures, T h e L C G H w a s d e c r e a s e d in v a r i o u s c o r t i c a l a n d t h a l a raic brain structures 2 a n d S d a y s a f t e r i s c h e r a i a b e f o r e it reached control values 7 days post-ischemia. In c o n trast, the LCGtl of CAl-subfield decreased 2 days postischemia, normalized 5 days post-ischemia and increased 7 a n d 14 d a y s p o s t - i s c h e m i a . L C B F w a s d e c r e a s e d in t h e CAl-subfield 7 days after ischemia, lramunocytochemical s t a i n i n g r e v e a l e d p r o l i f e r a t i o n o f g l i a l c e l l s 7 a n d 14 d a y s after ischemia. The neuroprotective vinpocetine was cap a b l e o f p r e v e n t i n g t h e c h a n g e s in L C G H a n d L C B F . T h e r e s u l t s s u g g e s t t h a t t h e i n c r e a s e in L C G H 7 d a y s a f t e r i s c h e m i a is c a u s e d b y g l i a l m e t a b o l i s m , and that the d e c r e a s e in L C B F is c a u s e d b y c a p i l l a r y r e d u c t i o n in t h e CAl-subfield. Institut fiir Pharraakologie und Toxikologie, Ketzerbach 63, D - 3 5 5 0 M a r b u r g , F R G

FAILURE OF A LIPID PEROXIDATION INHIBITOR TO IMPROVE BRAIN MORPHOLOGY IN CEREBRAL ISCHEMIA IN THE RAT Th. B e c k , G. W a g e n e r O n l y r e c e n t l y , t h e r o l e of f r e e r a d i c a l g e n e r a t i o n a n d t h e e n s u i n g p e r o x i d a t i o n of m e m b r a n e l i p i d s h a s b e e n a d v o c a t e d a s a m a j o r d e t r i m e n t a l f a c t o r in c e r e b r a l i s c h e m i a (Hall ED e t el. S t r o k e 19: 9 9 7 - 1 0 0 2 ) . The p r e s e n t s t u d y w a s s e t u p to a s s e s s t h e e f f i c a c y o f t h e l i p i d p e r o x i d a t i o n i n h i b i t o r U 7 4 . 0 0 6 F in g l o b a l a n d f o c a l c e r e b r a l i s c h a r a i a in t h e r a t . Male W i s t a r r a t s w e r e s u b j e c t e d t o 10 rain o f f o r e b r a i n lschemia by clamping both common carotid arteries and s i m u l t a n e o u s l y l o w e r i n g b l o o d p r e s s u r e t o 40 mm Hg ( 2 v e s s e l o c c l u s i o n ; 2 - V O ) . A f t e r 7 d a y s , p y r a m i d a l cell l o s s in t h e h i p p o c a m p a l CA1 s e c t o r w a s d e t e r m i n e d h i s t o l o g i c a l l y . F o c a l i s c h e m i a w a s i n d u c e d in F i s c h e r - S 4 4 r a t s b y p e r m a n e n t o c c l u s i o n o f t h e middle c e r e b r a l a r t e r y (MCA-O). Q u a n t i f i c a t i o n o f t h e r e s u l t i n g i n f a r c t size w a s p e r f o r m e d a f t e r 48 h. U 7 4 . 0 0 6 F ( 2 1 - [ 4 - ( 2 , 6 - d i - l - p y r r o l i d l n y l - 4 - p y r l m i d i n y l ) - 1 - p i p e r a z i n y l ] - 16 - m e t h y l p r e g n a - 1,4,9( 1 1 ) - t r i e n e - 8 , 2 0 dione; 10 m g / k g i.p.) w a s g i v e n 10 min p r i o r to a n d a g a i n 8 h o u r s a f t e r t h e i n s u l t in t h e 2 - V O model a n d 80 rain b e f o r e a n d 2, 6 a n d 24 h o u r s a f t e r v e s s e l o c c l u s i o n in t h e MCA-O model. In t h e 2 - V O model, U 7 4 . 0 0 6 F f a i l e d to i n f l u e n c e n e u r o n a l s u r v i v a l in t h e CA1 s e c t o r (18.7_+5.5 {mean_+SEM} s u r v i v i n g n e u r o n s / r a m in t r e a t e d vs. 32_+11.4 in c o n t r o l s ) . In t h e r a t MCA-O model a p p l i c a t i o n of U 7 4 . 0 0 6 F did n o t s i g n i f i c a n t l y r e d u c e c o r t i c a l i n f a r c t size t h o u g h d r u g t r e a t m e n t c a u s e d a t e n d e n c y t o w a r d s l o w e r i n f a r c t v o l u m e s (60.8_+11.9 mm 8 in t r e a t e d vs. 8 8 . 7 ± 1 0 . 0 mm 3 in c o n t r o l s {p<0.1; U - T e s t } ) . O u r r e s u l t s i n d i c a t e a f a i l u r e of U 7 4 . 0 0 6 F to i m p r o v e h i s t o l o g o g i c a l o u t c o m e in 2 i s c h e m i a models in t h e r a t . T h e d a t a don't support the view that lipid peroxidation plays a major r o l e f o r f i n a l n e u r o n a l n e c r o s i s in t h e c o u r s e o f c e r e b r a l ischemic events. ' P r e s e n t a d d r e s s : D e p a r t m e n t of P a t h o l o g y , C o l u m b i a U n i v e r s i t y , New York I n s t l t u t ffir P h a r m a k o l o g i e , K e t z e r b a c h 68, D - S S 5 0 M a r b u r g

R 104 413 INTRATHECAL SALICYLIC ACID DEPRESSES C FIBRE-EVOKED ACTIVITY IN RAT THALAMUS AND SPINAL CORD I. J u r n a A c e t y l s a l i c y l i c a c i d (ASA) d e p r e s s e d t h e t a l l - f l i c k r e s p o n s e in r a t s (TL Y a k s h , in HJM B a r n e t t e t al (eds), A c e t y l s a l i c y l i c Acid: New A s p e c t s f o r a n Old Drug, R a v e n P r e s s , New York, pp 137, 1982) a n d l y s i n e a c e t y l s a l i c y l a t e (LAS) c a u s e d p a i n r e l i e f in t u m o r p a t i e n t s (JC D e v o g h e l , J I n t Med Res 11:, 90, 1988) w h e n t h e two d r u g s w e r e a d m i n i s t e r e d b y i n t r a t h e c a l (i.t.) i n j e c t i o n . It w a s t h e r e f o r e s t u d i e d if e i t h e r t h e a c e t y l g r o u p or t h e s a l i c y l i c a c i d (SA) m o i e t y is e s s e n t i a l to p r o d u c e t h e a n t i n o c l c e p t i v e or a n a l g e s i c e f f e c t a t t h e s p i n a l l e v e l . The e x p e r i m e n t s w e r e c a r r i e d o u t 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 . A c t i v i t y w a s e l i c i t e d in s i n g l e n e u r o n e s in t h e d e 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 (VDM) of t h e t h a l a m u s a n d in a s c e n d i n g a x o n s of t h e s p i n a l c o r d b y s u p r a m a x l 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 s u r a l n e r v e . D r u g s w e r e i n j e c t e d l.t. b y a c a t h e t e r i n s e r t e d i n t o t h e l u m b o s a c r a l s u b a r a c h n o i d s p a c e . LAS, ASA, a n d SA s i g n i f i c a n t l y r e d u c e d t h e a c t i v i t y e v o k e d in t h a l a m i c n e u r e n e s b y a f f e r e n t C f i b r e s t i m u l a t i o n . The m a x i m u m d e p r e s s i o n c a u s e d b y t h e t h r e e d r u g s a m o u n t e d to a b o u t 50% of t h e a c t i v i t y e v o k e d in t h e c o n t r o l s . T h e d e p r e s s i o n l a s t e d 4 0 60 rain. The e f f e c t s of ASA a n d SA w e r e d o s e - d e p e n d e n t ; EDso of ASA: 37 p g (0.28 p m o l ) / r a t ; ED~o of SA: 87 tlg (0,27 p m o l ) / r a t . SA (37.5 p g / r a t ) d e p r e s s e d C f i b r e - e v o k e d a c t i v i t y in a s c e n d i n g a x o n s , w h i l e AI3 f i b r e - e v o k e d a c t i v i t y w a s n o t m a r k e d l y c h a n g e d . T h e o b s e r v a t i o n t h a t i.t. i n j e c t i o n of SA d e p r e s s e d n o c i c e p t i v e a c t i v i t y in t h e t h a l a m u s a n d s e l e c t i v e l y r e d u c e d C f i b r e - e v o k e d a c t i v i t y in a s c e n d i n g a x o n s s u g g e s t s t h a t (1) t h e e f f e c t s of LAS a n d ASA r e s u l t from a n a c t i o n o f SA, (2) n o c i c e p t i v e t r a n s m i s s i o n f r o m p r i m a r y a f f e r e n t s to t h e t h a l a m u s c a n be i n h i b i t e d b y t h i s a c t i o n a t t h e s p i n a l l e v e l , e n d (8) p r o s t a g l a n d i n s a r e n o t i n v o l v e d in t h i s a c t i o n . I n s t i t u t ffir 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 des Saarlandes, D-6850 Homburg/Saar

der

Universitgt

414 DIFFERENCESIN ANALGESICEFFECTSOF IBUPROFEN(ACID)AND IBUPROFENLYSINATEUSINGAN EXPERIMENTALEVOKEDPOTENTIALPAIN MODEL Th. Hummel, H. Huber, E. Pauli,and G. Kobal

415 EVIDENCE FOR A NON-OPIOID COMPONENT IN

THE ANAhGESIC ACTION OF TRAMADOL B. Driessen~ H. Schl6tz and W. Reimann Tramadol enhanced extracellular noradrenaline concentration following stimulation of rat brain cortex slices probably by inhibition of noradrenaline uptake (Naunyn Schmiedeherg's Arch. Pharmacol. 339, R89, 1989). We therefore eharacterised the uptake inhibition in a purified synaptosomal preparation. The in vivo relevance of noradrenergic activation was tested in the rat tail flick model after intrathecal drug injection. Purified synaptosomes were p~epared from rat brain hypothalamus and incubated with H-noradrenaline for 2 min at 37°C. Polyethylene catheters were inserted intratheeally in rats (Yaksh and Rudy, 1976), two days prior to tail flick test using radiant heat. Desipramine inhibited noradrenaline uptake in a 5iphasic manner. The high potency component of uptake inhibition by desipramine (ICgn 0.8 nM) constituted 72 % of total uptake inhibition, ~ d is probably related to uptake in norsdrenergie nerve terminals. Cocaine inhibited the total uptake with an IC50 of 0.16 pM and tramadol with 2.8 pM. Maximum inhibition of total uptake at I00 pM was 93 % by cocaine and 87 % by tramadol. In vivo, intrathecal tramadol as well as morphine prolonged tail flick latencies; significant effects were observed with 12 pg and 3 pg, respectively. Potentiation of opioid analgesia by uptake inhihitors was shown by combining subthreshold doses of morphine and desipramine. This effect, like tramadol amtinociception, was antagonised by 1 mg/kg i.p. yohimhine; the effect of 12 pg morphine alone was not inhibited. The fact that yohimbine antagonises the antinociceptive effect of tramadol, and of morphine combined with an uptake inhibitor, hut not that of morphine alone, provides evidence for a noradrenergic component to the antinociceptive action of tramadol; this may he the result of noradreaaline uptake inhibition. Gr6nenthal OmhH, Abtlg. Pharmakologie, Zieglerstr. 6, D-5100 Aachen, FRG

416

Maximal plasma concentrations are reached sooner in case where ibuprofen is administered(p.o.) as the lysinate than when administered in acid form (GeisslingerG. et al., Int. J. Clin. Pharmacol.Ther. Tox 27(7):324328, 1989). The aim of the present study was to test, whether the analgesic effects of these two preparations are also different. 16 young and healthy volunteers (8 female, 8 male) participated in the study, which followed a controlled, randomized, double-blind, 3-fold cross-over design (ibuprofen acid 600 mg, ibuprofen-lysinate 1000 mg, and placebo). Measurements were performed before, 30, 60, and 90 min after administration of the test medication. 30 phasic CO2-stimuli of two painful concentrations were applied to the left nostril and 20 tone burstswere presentedto the left ear. A continous cool and dry air stream was used in order to study the effects on responses to tonic pain stimuli. The EEG was recorded from 8 sites of the 10/20 classification. Following are the main results of the experiments:No changes in acoustic evoked potentials and cardiovascular parameters (heart rate and blood pressure)were observed.A short lasting decrease in the alpha-bandof the EEG 30-60 min after administrationof both ibuprofen preparationswas found when comparedwith placebo (p < 0.05), as well as a decrease in intensity estimates to tonic pain stimulation which was more pronounced for ibuprofen lysinate than for ibuprofen acid (p<0.05). Estimates of the tonic pain stimuli did not differ after administration of both test medications.Latencies of pain-relatedresponsesobtained 30 min after ibuprofen lysinate administration increased more than after ibuprofen acid and placebo administration (p<0.05). Plasma concentrations mainly correlated with amplitudes of the pain-related potentials from the frontal leads. Based on the data of this ext~eriment it can be assumed that ibuprofen lysinate occasioned a better/sooner analgesic effect than ibuprofen in acid form. The induction of tonic nociceptive activity which is generated in a mildly inflamed tissue seems to lead us to a well suited human pain modelfor investigatingNSAIDs.

AGE-RELATED CORTISOL RESPONSE TO H-CRH IN DEXAMETHASONE PRETREATED PATIENTS WITH DEPRESSION U. v, Bardeleben Human corticotropin-releasing hormone (h-CRH),administered as a bolus of 100 I~g at 3 PM, fails to induce a substantial release of cortisol in young controls pretreated the day before at 11 PM with 1.5 mg dexamethasone. In the present study we performed this combined challenge in 17 healthy men (age 41.5 + 15.9 (SD) years) and in 20 newly admitted male inpatients with a majordepression (age: 45.2:h8.9), Blood samples for cortisol determination by radioimmunoassay were drawn at 2:00 PM, 2:30 PM, 3:00 PM before infusion of h-CRH and thereafter every 15 minutes until 5 PM. Cortisol secretion after h-CRH assessed as area under the curve was significantly increased in patients with depression when compared to controls (14.8 + 11.9 ng x 1000 x min/ml vs. 4.1 + 3.5 ng x 1000 x min/ml). Whereas regression analysis in the normal controls did not point to a major influence of age on cortisol secretion after hCRH, multiple regression analysis in the patients revealed a significant impact of age as well as of severity of depression. The pathophysiology of this illness- and age-related cortisol escape from dexamethasone induced suppression is yet unknown. At the pituitary site, the elevation of endogenous CRH and of peripherally circulating glucocorticoids in hypercortisolemio patients would induce a down-regulation of CRH-receptore, leading to a blunted ACTH response, However, at the suprapituitary sites of the brain-pituitary-adrenocortical (BPA) axis, hypercortisolemia might induce a down-regulation of glucocorticosteroid receptors in the hippocampal neurons, thus increasing their vulnerability and reducing their capacity to shut off BPA activity.

Departmentof Pharmacologyand Toxicology, Universityof Erlangen-Niirnberg, Universit~.tsstr.22, D-8520 Erlangen,FRG

Department of Psychiatry, University of Freiburg, Hauptstrasse 5, D 7800 Freiburg, FRG

R 105 417 PRECISION AND ACCURACY OF HIGH PERFORMANCE UQUID CHROMATOGRAPHY (HPLC) AND FLUORESCENCE POLARIZATION IMMUNOASGAY (FPIA) TECHNIQUES FOR THE MEASUREMENT OF FLECAINIDE IN PLASMA - A COMPARISON BASED ON INTERNALQUALITY CONTROL Kr0dewaoen. B.. HonDO.U.. Hertramof. R. Precision and accuracy of HPLC and FPIAfor the measurement of fleceinide plasma levels were compared below, In and above the therapeutic range (200 - 1000 ng/ml). Following a calibration crossover study for the determination of method correlation, 5 Internal standard solutions (ISS) of varying flecainide concentrations and 99 flecainide containing p~asmasamples of 24 In-patients (PSP) were analyzed wIth both methods. The results show that within the range of approx. 500 - 1500 ng/ml the two methods did not differ in either precision (expressed as coefficient of variation, CV) or accuracy (expressed as relative error, RE). Close to the lower limit and below the therapeutic range

however, HPLC provided higher precision and accuracy than FPIA. 18~2Or¢. Ina/ml) mean (r~/roll

100 250 500 1000 1500

93 252 513 999 1506

PSP-Con¢. Ir~/m~ m e ~ (t~/mB

1-15O 151-350 351-75O 751-1250

125 238 464 847

HPT.£ cv~ 1,0 4,2 4,0 3,0 2,6 so (,,a/m~ 24 52 94 72

FPIA RE i'W4

mean lrralm~

CV t ~

I ~ (*/*)

-7,0

73

7,0

-27,0

+1,0 +2,6 -0,1 +0,4

210 481 1006 1486

5,2 4,5 3,1 2,6

-16,3 -3,8 +0,6 -0,9

.

mean (nll/rflB

~D [na/mll

13 59 25 2

105 218 454 880

53 53 104 60

n

13 59 25 2

As the table shows, especially flecainide concentrations < 350 ng/ml tended to give lower results with FPIA than with HPLC. This characteristic however should not be considered of clinical relevance because generally it will not cause different therapeutic consequences. Combining sufficient reliability and low effort, FPIA is a good alternative to HPLC In clinical routine. HPLC however should be preferred for scientific analyses because of Its higher precision/accuracy and wider range of measurement.

419 ABSORPTION OF COLLOIDAL BISMUTH SUBCITRATE (CBS) IN PATIENTS WITH NORMAL AND IMPAIRED R E N A L FUNCTION DURING TREATMENT G.Treiber, U.Gladziwa, S.Walker and F.Schweinsberg Since the clinical significance of Campylobacter pylori (CP) in the development of ulcer and gastritis becomes increasingly important, recent interest has focused on bismuth (Bi) preparations because of their antibacterial and cytoprotective effects. As there exist only few pharmacokinetie studies, we investigated the disposition of CBS (216 mg Bi bid) in patients with normal and impaired renal function during 4 weeks treatment. 12 patients (23-66 years, mean 47.7 + 12) with gastric/duodenal ulcer or gastritis type B were divided into two groups: I creatinine-clearance (CLot) • 75 ml/min, mean 115 _ 37 ml/min and II CLef 10-65 ml/min, mean 38 -+ 18 ml/min. 11 of them were followed up for four weeks after treatment. In the beginning and 4 weeks after treatment an upper gastrointestinal endoscopy with a CP urease test was performed and only CP+ patients were included. Bi was determined in plasma and urine by atomic absorption spectrophotometry with hydride generation. The absorption profile of CBS (6 blood samples for the first 2 h, 24 h urine collection) was evaluated following the first dose, 2 and 4 weeks treatment, as well as 2 and 4 weeks after cessation of therapy (1 blood and urine sample only). The mean results are listed below: week

predose plasma levels (ug/l) after therapy 0 2 4 6 8

Ae (ug/24h) 2 4

group I n=7

5.6

11.8

15.4

5.6

5.3

1114

1175

II n=5

4.6

22.7

25.9

11.1

7.9

1273

1502

Absorbed amounts of Bi as estimated by the plasma AUC (0-2 h) demonstrated a wide intra- and interindividual variability indicating no differences between both groups and no alterations during the treatment course of 4 weeks. However, an accumulation during this period especially in patients with impaired renal function could be noticed, showing a relationship between CLer and plasma Bi levels. Plasma level monitoring appears advisable to avoid unnecessary toxic risks in long-term treatment and/or renal insufficiency.

Abteilung for Expedmentelleund K]inische Pharmakologie, InstItut for Arznelmittel, Bundesgesundheitsamt, $eestraBe 10, D-1000 Bedin 65

Dr. Margarete Fischer-Bosch Institut for Klinische Pharmakologie, Auerbachstr. 112, D 7000 Stuttgart - 50 The study was supported by the Robert Bosch Foundation.

418

420

BLOOD LOSS IN PHARMACOKINETICSTUDIES. C. de Hey, D. Enterling, G. Hanft and H. Wesche

THE A B S O R P T I O N OF SULFAMERAZINE FROM DIFFERENT REGIONS OF THE HUMAN SMALL INTESTINE T. Grammatt~, B. Terhaag, and K. Feller

The precision of estimated pharmacokinetic variables depends on the extent of blood sampling. Hany studies involve several profiling days. We assessed the course of serum hemoglobin values (Hb,g%) retrospectively for 22 such studies with estimated blood sampling ranging from 150 to 1050 ml over 3 to 6 profiling days over a time frame of 16 to 58 calendar days, in 166 young males and 57 females. Assessments were based of the data on recruitment (RC : i.e. 2 weeks prior to study start), on the morning of the 1st profiling day before dosing (DI), 24 hrs later (D2) and at the end of the study (DL). The mean (+range) of Hb at these times is detailed below: time males females RC 15.45 (13.3,17.8) 13.96 (12.1,16.4) D1

15.00 (12.3,17.3)

13.46 (10.9,14.9)

D2 15.11 (12.6,17.9) 12.97 (10.9,15.2) DL 14.86 (12.4,17.8) 13.10 (11.4,15.3) The point and interval estimates (95% CI) for the relative changes are detailed here below : DI-RC -0.45 (-0.56,-0.33) -0.51 (-0.73,-0.29) D2-RC -0.34 (-0.48,-0.21) -1.00 (-1.27,-0.74) DL-RC -0.59 (-0.76,-0.42) -0.86 (-1.14,-0.59) 02-01 +0.10 (-3.06,+0.23) - 0 . 4 9 (-0.75,-0.23) DL-D2 -0.24 (-0.38,-0.10) +0.13 (-0.05,+0.32) I t thus appeared that potentially c l i n i c a l l y relevant reductions in Hb indeed occurred over the course of such studies (i.e. from RC to DL), but, that the main drop already took place between RC and D1 ( i . e . without sampling or study exposure). L i t t l e further loss took place during actual blood sampling (D2-DI and DL-RC). This suggests that recruitment screens (which are performed under different environmental conditions) might be misleading. SK&F-Institute for Applied Clinical Pharmacology, Hi]debrandt-str. 30, D-3400 G~ttingen, FRG.

Small intestinal perfusion with three lumen tubes is one of the established methods to investigate local a b s o r p t i o n along human intestine. This method was used to evaluate absorption of sulfamerazine from different regions of small intestine: d u o d e n o - j e j u n a l junction (A), middle jejunum (B), and middle ileum (C). Sulfamerazine was chosen as a weak acid with low lipid solubility as well as low water solubility. Preliminary results

(3 h e a l t h y volunteers)

reported in this abstract show no d i s t i n c t differences in the mean amount absorbed (mg/cm x hr): A = 2.9, B = 2.3, and C = 1.9, resp.. In the same way, blood c o n c e n t r a t i o n - t i m e profiles m e a s u r e d simultaneously during the perfusion period display no local differences. No clear relationships were detectable between drug disappearance and w a t e r movement in the intestinal segment under study as well as intraluminal bile acid concentrations.

Institut fHr Klinische Pharmakologie, Medizinische Akademie, DDR 8019 Dresden

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INFLUENCE OF LIPOSOME ENCAPSULATION ON BETAMETHASONE DIPROPIONATE EFFICACY M. S c h ~ f e r - K o r t i n g , H.C. K o r t i n g , H. Z i e n i c k e , a n d O. B r a u n - F a l c o

RADIORECEPTOR ASSAY AS MEANS OF ASSESSING BIOEQUIVALENCE AND PREDICTING EFFECT-KINETICS OF TWo ATENOLOL FORMULATIONS IN MAN G. Sitzler*, B. Helbel+ and P. L~cker+

L i p o s o m e s a r e f r e q u e n t l y c o n s i d e r e d as d r u g d e l i v e r y s y s t e m s for s y s t e m i c a l l y a p p l i e d drugs. Recently topical administration to the skin has r e c e i v e d a t t e n t i o n , too. To evaluate whether liposome preparations imp r o v e t h e a c t i v i t y of t o p i c a l glucocorticoids, the effects of a liposomal preparation of betam e t h a s o n e d i p r o p i o n a t e (0.03%, BDP) w a s compar e d t o t h o s e of a c o m m e r c i a l propylene glycol c o n t a i n i n g gel w i t h 0.05% BDP. In a d o u b l e b l i n d randomized paired trial the preparations were a p p l i e d t o i0 p a t i e n t s w i t h a t o p i c eczema and i0 p a t i e n t s with psoriasis vulgaris for 14 days. S~rmptoms w e r e r a t e d b e f o r e entering the s t u d y as w e l l as on d a y s 4, 7, and 14 during treatment. In eczema, t h e l i p o s o m e p r e p a r a t i o n tended to reduce erythema and scaling more than the conv e n t i o n a l gel t h e d i f f e r e n c e b e i n g significant with the latter parameter on day 7 (p
Single o~al doses of two atenolol formulations (200 mg Tenormin , T and 200 mg of duraatenolol , D) were given to eight healthy male volunteers (double blind randomized cross-over design) in order to evaluate t h e i r bioequivalence. Plasma samples were obtained up to 48 hours after drug administration. Their antagonistic a c t i v i t i e s on 3H-CGP 12177 binding to 61- and ~2-adrenoceptors (6AR) were determined by means of a radioreceptor assay (RRA; J. Pharm. Exp. Ther. 246, 328, 1988). After both regimens plasma samples drawn within the f i r s t 12 hours after drug intake exerted an antagonistic effect at 61AR. Thereafter antagonistic a c t i v i t i e s were beyond detection l i m i t . Apart from peak values two hours after atenolol administration antagonistic effects at g2-AR were not detectable reflecting an at least lO-fold &I-AR selectivity of both, T and D. At ~I-AR there was no difference between both regimens in terms of pharmacokinetic (AUC, t l / 2 , ±max, Cmax) and pharmacodynamic (receptor occupancy) parameters. From these parameters the time course of ~I-AR mediated effects in vivo was predicted. The prediction made was in good correlation with the time course of heart rate reduction observed after ergometric exercise up to 12 hours after intake of both formulations. Thus, the relative biosquivalence of T and D was established by RRA in terms of pharmacokinetic and pharmacodynamic parameters. In contrast to other methods RRA allows for a direct prediction of effect-kinetics in man. As determined by RRA ~2-AR do not contribute to these effects.

P h a r m a k o l o g . I n s t i t u t f. N a t u r w i s s . d e r U n i v e r sit~t Frankfurt, Theodor-Stern-Kai 7, D-6000 Frankfurt/M., Dermatol. Klinik u. Poliklinik der Universit~t M~nchen

Kiln. d. Univers. Frankfurt, Z. Pharm., Theodor-SternKai 7, D-6000 Frankfurt + IKP, Richard-Wagner-Str. 20, D-6718 GrOnstadt

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BIOAVAILABILITY OF TWO INTRAMUSCULAR AMPICILLIN PRODUCTS IN RABBITS. M. Ollin~, A.G. Rauws. The bioavailability (BA) of products for intramuscular (i.m.) administration should not be taken for granted. To investigate the suitability of the rabbit as a model for studies of i.m. availability, we compared the BA in rabbits (6 m, 6 f) of two i.m. ampicillin products found to show different BA in calves. Before administration of the products the intravenous pharmacokinetics of ampicillin (I0 mg/kg) in rabbits was studied. Results: tl/2 (h): 0.74 ± 0.ii; AUC (mg.h/l, males): 9.51 ± 1.29; (fem.): 17.24 ± 3.64; MRT (h): 0.61 ± 0.14; Vd (I/kg, males): 0.26 ± 0.04; (fem.): 0.13 ± 0.02; C1 (I/kg.h, males): 1.07 ± 0.13; (fem.): 0.60 ± 0.Ii. The BA (20 mg/kg) was studied using a two-way cross-over design with a wash-out period of 14 days. Plasma was sampled for 24 h. In the ist session the products were injected in the left hind leg, in the 2nd in the right one. At the end of the 2rid session the animals were killed and ampicillin was determined in the plasma samples and in liver, kidney, muscle and right hind leg injection site. Results: Product A Product B Prob. AUC (mg.h/l, males) 20.28 ± 4.31 9.09 ± 3.10 p <0.01 AUC (mg.h/l, fem.) 31.56 i 5.56 11.65 ± 4.94 p <0.01 Fabs (%) ca. 100% ca. 40% liver (mg/kg) 1.22 ± 0.38 0.03 ± 0.01 kidney (mg/kg) 5.13 ± 2.80 0.21 ± 0.08 muscle (mg/kg) 0.29 ± 0.33 < 0.01 inject, site (mg) 0.08 ± 0.13 1.66 ± 1.27 The results demonstrate the necessity of exact knowledge of the pharmacokinetics of the relevant substance in both sexes of the animal used. In the second place they confirm the difference in the bioavailability between both products found earlier in calves. It may be wordth while to extend the validation of this model with other products.

DOES NON-ENZYMIC GLYCOSYLATION OF HUMAN SERUM ALBUMIN INFLUENCE DRUG PROTEIN BINDING ? W. W6rner, J.-W. Bae, S. Pfleiderer and N. Rietbrock

National Institute of Public Health and Environmental Protection, Unit Biotransformation, Pharmaco- and Toxicokinetics, P.O. Box I, 3720 BA, Bilthoven, The Netherlands.

Department of Clinical Pharmacology, University Hospital, Theodor-Stern-Kai 7, D-6000 Frankfurt/M. 70

Previous studies using dansylsarcosine as a marker for the benzodiazepine binding site on albumin indicate that glycosylation of albumin leads to changes in the tertiary structure and that this phenomenon can alter drug binding kinetics (I). A reduction in sulfonamide binding in diabetic patients has been reported. We have investigated the influence of glycosylation on the binding of warfarin, furosemide, tolbutamide, diazepam and dig±toxin. The degree of glycosylati0n was varied by mixing I00 % glycosylated albumin (obtained e.g. by incubating fatty acid-free albumin, 8.0 g/dl, with 20 g/dl glucose for two weeks) with non-glycosylated albumin. The solutions were incubated with the drugs at 37 °C and albumin binding determined by equilibrium dialysis and ultrafiltration. Free drug was measured photometrically, fluorimetrically or by radioimmunoassay. With equimolar concentrations of albumin and drug (3.32"10 -4 mol/l) the percentages of free drug for 7.5 % and I00 % glycosylated albumin, measured by equilibrium-dialysis, were for warfarin 4.2 % and 4.1%, for furosemide 8.3 % and 7.7 %, for tolbutamide 9.3 % and 10.3 %, for diazepam 8.0 % and 7.6 %, and for dig±toxin 1.5 % and 2.0 % respectively. Therefore these studies involving the three primary binding sites on albumin do not indicate a major alteration of binding kinetics following non-enzymic glycosylation of albumin even up to 100 % glycosyfat±on is not to be expected. These results do not support the view that albumin glycosylation in diabetics has a major effect on drug binding. (i) W6rner, W., Pfleiderer, S. (1989) Naunyn-Schmiedeberg's Arch Pharmacol 340, Suppl.

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CEPHALOSPDRIN INDUCED HYPOPROTHROMBINEMIA: DOES A CORRELATION EXIST BETWEEN BLOOD LEVELS OF N-METHYL-TETRAZDLE-5THIOL (NMTT) AND DISTURBANCES OF HAEMOSTASIS? THE ANSWER IS: NO W. Christ, W. Hecker, Marianne Jacobsen, Heidemarie Junge

CLEARANCE OF FLECAINIDE IN CONGESTIVE HEART FAILURE (CHF) H. Stein, U. Gundert-Remy

Different NMTT-containing cephalosporins are used therapeutically, e.g. cefemandol, latamoxef, cefoperazone, cefmenoxime. These compounds con produce hypoprothrominemia especially in vitamin K-deficient patients. The hypothrombinemia is due to an inhibition of the vitamin K1-2,3epoxide reductase. The mechanism of hypoprothrombinemia has been investigated in a randomized clinical pilot study with 14 hospitalized patients (inclusion criteria: age 50 years, urinary tract infection, normal prothrombin time). Therapy groups: latamoxef 2 g bid (n = 5), cefoperazone 2 g bid and cefotaxime 2 g tid (n = 4). Duration of treatment: 7 days. Methods: NMTT was determined besides of the parent cephalosporins in blood samples, which were drawn immediately before the first infusion and during the 7th day of treatment. For determination, a specific end sensitive HPLC method was developed in our laboratory. Results: NMTT levels were higher in,serum of patients receiving latamoxef (43.1 ± 6,8 ~mol/l) than in patients treated with cefoperazone (29 ± 8.8 ~mol/1). In contrast to these findings, the effects on clotting factors were more pronounced under cefoperazone therapy. Two patients under cefoperazone exhibited a significant increase of prothrombintime, accompanied by the appearance of PIVKA II (prothombininduced in vitamin K absence). Both cefoparazone (in 4 patients) and latamoxef (in 3 patients) caused the appearence of endogenous vitamin K1- 2,3-epoxide, whereas cefotaxime did not. This confirms the hypothesis that NMTT-cephalosporins are inhibitors of hepatic vitamin K epoxide reductase. (For details see: Schiller, Naber, Adam, Arzneim.-Forsch. 1989; 39 (II): 1156 - 1162.) Institut for Arzneimittel des Bundesgesundheitsamtes, Seestra6e 10, D-IO00 Berlin 65

PATIENTS

WITH

One p r o b l e m of the class Ic antiarrhythmic drug flecainide is the narrow therapeutic range combined with the high interindividual v a r i a b i l i t y of clearance. The purpose of our observational study in patients was to analyze the influence of some factors on the pharmacokinetics of this drug. Patients with CHF were classified according to NYHA. We measured flecainide trough levels in 30 patients on the first six days of therapy using HPLC. Clearances (el) were calculated with a computer program assuming the open-onecompartment model with first-order-absorption. Patients with values lower than i0 ml/min were excluded from the analysis. Patients classified as NYHA I class (4) had a mean Cl of 492 ml/min, such of NYHA class II (4) 450 ml/min and the NYHA class III group (3) had a mean Cl of 376 ml/min. Comparing the combined data of patients with CHF to those of 18 patients without CHF (mean Cl of 414 ml/min) we did not find a statistic difference, which is in contrast to reports from the literature (Cavalli et al.clin Pharmacokin.14:187,1988). However, compared to healthy volunteers (CI mean 1041 ml/min) (Mikus et al.Clin. Pharmacol. Ther 45:562,1989), the clearances of all patients were considerably lower. We conclude from our results that a lower degree CHF does not reduce the clearance of flecainide. Abt. f. Experimentelle und Klinische Pharmakologie, Institut f. Arzneimittel, Bundesgesundheitsamt, Seestr.10, D-1000 Berlin 65

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STEREOSELECTIVE IN VITRO METABOLISM OF R, S AND R / S NITRENDIPINE BY MICROSOMAL FRACTION OF H U M A N LIVER C. Fischer and P.Janicki

R E L A T I V E B I O A V A I L A B I L I T Y OF S U B L I N G U A L G L Y C E R Y L TRINITRATE AND EFFECT ON PULMONARY ARTERIAL PRESSURE AND DIGITAL PULSE WAVE

The in vivo metabolism of nitrendipine exhibits stereoselective first pass metabolism in humans (1).The bioavailability (F) of the active Senantiomer is on average two to three times higher than the F of the R-nitrendipine. In order to see which metabolic pathways are responsible for the stereoselective first pass metabolism, the metabolism of R, S and R/S-nitrendipine was studied in microsomal fraction of human liver. Separation and quantification of the numerous metabolites was achieved using two newly developed HPLC systems. The metabolites recovered were unequivocally identified by comparison with reference compounds using highly sensitive and specific GC/MS. In addition to the nitrendipine metabolites which have been extensively studied (MI,M2a, M2b,M3a,M3b) four previously unreported metabolites were found. Chiral carboxylic acids are formed by oxidative cleavage of the ester linkage with retention of the 1,4dihydropyridine structure after 10 minutes incubation. This pathway exhibits remarkable stereoselectivity with preferential deethylation of the R-enantiomer of nitrendipine resulting in an R / S ratio of about 3. In addition, in vitro reduction of the Y-nitrophenyl moiety to the corresponding amino analogues of nitrendipine and of MI could be detected. Formation of M1, the pyridine analogue of nitrendipine, exhibited no stereoselectivity. Using the racemic mixture of aitrendipine as substrate an even more pronounced stereoselectivity was found for the formation of chiral metabolites indicating an interaction of the enantiomers during metabolism. The 0attern of metabolism which resulted from these in vitro experiments is in contrast to the findings of Guengerich et al. (2) who postulated that oxidation.of the 1,4-dihydropyridine to the corresponding pyridine is the primary metabolic step. Our findings support the complex metabolic pathways suggested by K a n n et al.(3). 1. Mikus et.al.: Br. J. Clin. Pharmacol.24 (1987): 561 2. Guengerich et al.: J. Biol. Chem. 263 (1988): 8168. 3. K a n n et al.: J. Cardiovasc. Pharm. 6 (1984) $968 Dr. Margarete Fischer-Bosch Institut fiir Klinische Pharmakologie, Auerbachstr. 112, 7000 Stuttgart 5 0 / F R G This study was supported by the Robert Bosch Foundation.

A.Wie@and,

K.Schnellbacher , A.Rehe,

E.J~hnchen

This study was designed (I) to determine the relative bioavailability of a new spray formulation of glyceryltrinitrate (GTN) containing 0.4 mg/stroke vs. a reference spray with 0.8 mg/ stroke and (2) to measure simultaneously time course and magnitude of effect of GTN on diastolic pulmonary arterial pressure (PADP) and on the a/b ratio of the digital pulse wave. A randomized double-blind cross-over study was performed in 12 patients suffering from congestive heart failure (NYHA II-III) and elevated PADP at rest (> 16 mmHg) during a diagnostic right heart catheterization. Following administration of one stroke of either spray, plasma concentrations of GTN and its metabolites (GC-MS), PADP (Swan-Ganz catheter) and the a/b ratio of the digital pulse wave (photo electric device at the middle finger) were measured up to 30 min. The 2nd experiment started about 15 min. after the ist experiment. The mean time courses of PADP and of the a/b-ratio following sublingual application of the two spray formulations were parallel. By comparing the areas under the effect curves the relative bioavailability of the test spray was 147 ± i17 % (PADP) and 161 ± 54 % (a/b ratio). The bioavailability obtained by comparing the areas under the plasma concentration curves was 157 ± 59 %. There were considerable variations between the patients in the ratios of the integrated effects obtained by both pharmacodynamic methods. Thus, in patients with CHF the a/b ratio of the digital pulse wave predicts the time course but not the magnitude of effect of sublingually administered GTN on PADP. Abteilung f~r Klinische Pharmakologie, Rehabilitationszentrum, D-7812 Bad Krozingen

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B E T A - A D R E N O C E P T O R B L O C K I N G A C T I V I T Y OF THE E N A N T I O M E R S OF D I P R A F E N O N E IN MAN

PHARMACODYNAMICS AND PHARMACOKINETICS PRANOLOL FOLLOWING PHYSICAL EXERCISE

D. Trenk

M. Buschmann,

The class I antiarrhythmic drug diprafenone exhibits distinct 6-adrenolytic properties following single oral doses of the racemio mixture. We therefore investigated the 6-antagonistic activity of the R-(+)- and the S-(-)-enantiomers of diprafenone in comparison with the racemate in ten healthy male volunteers in a placebocontrolled double-blind randomised study. The 6adrenolytic activity was determined by using the standardized isoproterenol test. Two hours following ingestion of single oral doses of 150 mg racemic diprafenone-HCL, 150 mg R-(+)-diprafenone-HCL or 150 mg S-(-)-diprafenone-HCL increasing i.v.-bolus doses of isoproterenol were repeatedly administered until the heart rate at rest increased by at least 25 beats per minute. The chronotropic dose of isoproterenol which increased heart rate by 25 b.p.m. (CD25) was calculated from dose-response curves using the Hill-equation. The CD2S-values following R-(+)diprafenone (1.35 ± 0.82 pg) did not differ from those obtained during the placebo period (1.67 ± 1.23 Hg), In contrast to that finding, significantly higher doses of isoproterenol were necessary to increase the heart rate to the same extent following pretreatment with the same doses of S-(-)-diprafenone ( C D 2 5 : 1 5 . 7 7 ± 16.95 #g, p=0.002) or racemic diprafenone (CD25: 18.81 ± 19.86 #g, p=0.002).

The effect of modest bicycle ergometer exercise on the pharmacokinetics and the hemodynamic effects of propranolol was investigated in eight healthy volunteers after oral administration of a single dose of 80 mg propranolol. In a randomized cross-over study the subjects either sat or exercised on a bicycle ergometer at about 75 watts for 2 hours after drug ingestion. After 2 hours usual indoor activities were allowed. The cross-over experiment was performed at least one week apart. Plasma concentration of propranolol as well as blood pressure and heart rate were determined repeatedly up to 12 hours. Hemodynamics were compared statistically starting three hours after drug intake. On the exercise day propranolol did not affect heart rate or blood pressure, while on the rest day heart rate was reduced up to i0 hours following drug intake (p<0.05). The area under the plasma concentration - time curve (AUC) and the maximal plasma concentration (Cmax) of propranolol were considerably reduced on the exercise day (AUC: 233 ± 148 vs 124 ± 86 ng'hr/ml; Cmax: 52.4 ± 32.4 vs. 36.0 ± 17.5 ng/ml; p<0.05). Binding of propranolol to plasma proteins was not affected by exercise. It is suggested that the reduced splanchnie blood, flow during exercise might have increased the first-pass extraction of the high clearance drug propranolol, which leads to a reduced bioavailability and diminished hemodynamic effect.

These results demonstrate that the 6-sympathicolytic properties of diprafenone following oral doses can be ascribed only to the S-(-)~enantiomer. Abteilung f%r Klinische Pharmakologie, tationszentrum, D-7812 Bad Krozingen

OF

PRO-

H. Roskamm

Abteilung f~r Klinische Pharmakologie, tationszentrum, D-7812 Bad Krozingen

Rehabili-

Rehabili-

430 HEMODYNAMIC EFFECTS OF CELIPKOLOL VERSUS METOPROLOL IN MAN ON THE BASES OF COMPARABLE D-RECEPTOR OCCUPANCY K. Breithaupt, J. Schloos, J. Baehr, G.G. Belz, D. Palm Celiprolol is a 8x-selective adrenoceptcr-antagonist supposed to have ancillary properties. Its hemodynamic effects were compared with the full ~-selective adrenoceptor-antagonist metoprolol. 5 healthy male volunteers in a cross-over, double blind study received either 1200 mg celiprolol, 600 mg metoprolol, or placebo as single oral doses. Resting hemodynamics (blood pressure, heart rate, total peripheral resistance, electro-mechanical systole (qS=c) and pre-eJection period (PEP)) as well as exercise blood pressure and heart rate were determined up to 48h after drug intake. Plasma samples were obtained at the respective time points and D-receptor occupancies were measured usinE ~x- and ~=-radioreceptor assays. Both applied substances yielded comparable B-receptor occupancies (in maxi-

mum about 95~). The respective pharmacokinetic halflifes were 3.2h for metoprolol and 5.2h for celiprolol. Due to differences in pharmacokinetic profiles of the two drugs, the effects had to be compared on the basis of ~-adrenoceptcr occupancy. Resting heart rate was increased by celiprolol (+ 10-15 bpm) whereas metoproiol showed a slight drop (- 2-3 bpm). Exercise heart rate was less inhibited by celiprolol compared to metoprolol. Cardiac performance, as visible from PEP and QS=c, was enhanced after celiprolol but not after metoprolol, total peripheral resistance showed a decrease on celiprolol. In this study in addition t o its D-antagonistic effects and in contrast to metoprolol celiprolol showed vasodilating and cardiac performance enhancinE properties. These differences cannot be explained by the ~xadrenoceptor antagonism of both drubs but must be due to ancillary, e.g. ~-adrenoceptor agonistic and/or direct vasodilatin8 properties of celiprolol. Zentrum fur KardiovaskulMre Pharmakologie, Mathildenstr. 8, Mainz and Zentrum der Pharmakologie, Klinikum der J.W. @oethe-Universit~t, Theodor-Stern-Kai 7, Frankfurt.

432 NON-INVASIVELY MEASUREDHEMODYNAMICPARAMETERS ON VARIOUS H2-RECEPTOR ANTAGONISTS W. Kirch, H. Hinrichsen, A. Halabi, G. HUbner In a previous study famotidine appeared to exert negative effects on cardiac performance (Kirch et al. Gastroenterology 96: 1388-92, 1989). Therefore the question arose how the older H2-antagonists cimetidine and ranitidine would alter non-invasively measured hemodynamic parameters compared with famotidine. In a randomized placebo controlled double blind study lO healthy volunteers (age 26,5 ± 2.3 yrs.; ~ ±ST) were treated o r a l l y for one week each with cimetidine 800 mg once daily, ranitidine 300 mg once daily and famotidine 40 mg once daily. After l week's treatment heart rate, blood pressure, systolic time intervals (STI) and parameters of impedance cardiography (IC) were measured before and 2 and 6 hours after the morning dose. Heart rate and blood pressure values were not altered by any of the H2-antagonists investigated. There were also no significant differences in the data of STI and IC comparing cimetidine, ranitidine and placebo treatment. Famotidine however significantly decreased stroke volume, cardiac output and the heather index in IC and significantly increased PEP/LVET ratio in STI 2 hours after the morning dose compared with placebo (p<0.05). For example 2 hours after administration stroke volume was reduced by -8.8 3.5 cm3 (~ + _ S~) on famotidine (placebo +5.5 + _ 3.1 cm3) compared with the baseline values. 6 hours after administration most of these effects could not be seen anymore. The observed changes of hemodynamic parameters under famotidine administration confirm that this H2-antagonist exerts negative effects on cardiac performance whereas for cimetidine and ranitidine such influences could not be shown. I. Medizinische 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 SchittenhelmstraBe 12, 2300 Kiel l , FRG

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RANDOMIZED PLACEBO-CONTROLLED STUDY OF FOUR WEEKS TREATMENT WITH CRATAEGUS ON CARDIOCIRCULATORY RESPONSE AFTER EXERCISE STRESS TEST AND CHALLENGE BY NORADRENALINE (NOR) OR ISOPROTERENOL (IPR) IN HEALTHY SUBJECTS. R. Saller, C. R~ckbeil, M. B[ihring, D. Hellenbrecht*

Sparteine/Debrisoquine Polymorphism of Drug O~ddation: The 11~ kb Allele Is the Result of a Deletion of the 111)6 Gens on Chromosome 22

Extract of crataegus oxyacantha ~CRAT) is claimed to have regulatory effeczs on caraiocirculatory dysfunctions. Nine volunteers (c.f.table) received CRAT (Kneipp Pflanzen - Dragees Weissdorn) 3 t.i.d, and 9 others (c.f.table) too~ placebo over 4 weeks. Heart rate (HR, b.p.m.), bleed pressure !BP¢ mm Hg) a~d rate - pressure proauct (RPP, sys~oilc BP x ~ . units x 10- ) was tested by incremental upright cycle ergometry (2 min @teps of 50,75, 100, 125, 150 W]. (-)NOR.HCI (5 min seeps of 20, 40, 100, 200, 400 ng/kg/min) for response on BP and (-)IPR for_response on HR (5 min steps of 5, 10, 25, 50 ng/kg/min) were given by constant infusion. Table: arithmetic mean CRATAEGUS PLACEBO Female/male 4 / 5 2 / 7 Age/body weight 35 yrs. / 72 kg 35 yrs. / 70 before after before after kg Resting HR 68 3 65.3 63.1 64.8 Restinq BP 116/75 121/70 118/72 118/78 HR at 150 W 155 144 133 131 BP at 150 W 161/78 151/73 144/72 155/68 RPP at 150 W 24.8 21.6 19.3 20.0 a) Sumk~f RPP 94.6 80.8 76.0 75.0 ED ~{ of NOR 110 89 170 110 NS ED ~ of IPR 18.2 20.8 25.1 25.7 NS a) = mean differences CRAT vs. Dlacebo p = 0.04 (Wilcoxon two-tailed rank %est); N~=D>0.05 b) geometric mean effective dose (ng/kgTmin) to increase mean BP to 110 mm Hg c) = geometric mean effective dose (ng/kg/min) to increase HR by 20 b.p.m. The shift of exercise RPP to lower values after CRAT may indicate a salutary effect on circulatory stress tolerance. The mechanism of this effect m a y be located at the heart or blood vessels, or both. acting directly or by reflexes. Peripheral aarenergic receptors are probably not involvee, since responsiveness to exogenous catecholamines was not altered. Centre of Internal Medicine and Pharmacology * . U n i v . Clinics, Theodor-Stern-Kai 7, D-6000 Frankfurt/Main

434 NEW HPLC-METHODS FOR EVALUATION OF PROPOSED GENETIC POLYMORPHISMS IN METABOLISM OF CARBOCYSTEINE IN MAN B. Staffeldt, *E, Kraus, I. Roots The assumption of a genetic polymorphism (i) in sulfoxidation of carbocysteine (CMC) has stimulated further investigations and divergent results have been obtained in a study on healthy volunteers (2) using analysis of the CMC metabolites by HPLC. Detection was, however, limited to metabolites containing a primary amino group. Now a procedure was developed, which allows quantitation of acetylated and deaminated metabolites of CMC and of the CMC-metabolite thiodiglycolic acid as well. Methods: Procedures using precolumn derivatization of the metabolites via their carboxylic groups were developed with the fluorescent reagents 9-anthryldiazomethane, 4-diazomethyl-7methoxycoumarin or l-pyrenyldiazomethane. Furthermore, a method with l-ethyi-3-(3-dimethylaminopropyl)-carbodiimide-activated coupling of the carboxylic acids to 7-amino-4-methylcoumarin was established. Samples were not extracted prior to or after derivatization, interfering compounds were separated by the following HPLC on columns filled with reversed phase material (Shandon Hypersil ODS, 3 ~unparticles) using a gradient from eluent A (15 mM triethylamin-phosphate, pH 2.5; 20% methanol) and eluent B (acetonitrile). Results: The methods have been applied for urine analysis of samples from 30 healthy volunteers, who got a single oral dose of I.I g Carbocysteine and collected their urine for 8 hours. Results are given as percentual recovery of dose in urine (median and range). CMC: 20 (I0-30), (R)-CMC-sulfoxide: 0.i (0.1-0.3), (S)-CMC-sulfoxide: 0.4 (0.1-0.6), methylcysteine: not detectable, N-acetyl-CMC: 0.2 (0-0.5), (R)-N-acetyl-CMCsulfoxide: 0.i (0-O.l), (S)-N-aeetyl-CMC-sulfoxide: not detectable, Thiodiglycolic acid: ii (4-40}. Conclusion: These methods may be of interest also in other areas of drug and drug-metabolite analysis, since fluorescent derivatization allows high detection sensitivity. So far, a himodal distribution of excretion values for CMC-sulfoxides that might point for genetic deficiencies has not been evaluated. I. S.C. Mitchell et al., Br J Clin Pharmacol 18, 507-521 2. J. Brockm611er et al., Fur J Olin Pharmacol 228, 70 Institut ffr Klinisohe Pharmakologie, Klinikum Steglitz, Hindenburqdamm 30, 1000 Berlin 45, *E. Merck AG, Darmstadt.

A. Gaedigk, M. Blum*, U.A. Meyer* and M. Eichelbaum The clinically important genetic deficiency of o:ddative drug metabolism known as sparteine/debrisoquine polymorphism affects 5-10% of Caucasian populations and is due to the absence of e~..ochrome P450 IID6 and consequent decreased metabolism of over 25 rudely used drugs. The gene locus on chromosome 22 consists of two pseudogenes (IID7, IID8) and one functional gene (IID6). Analysis of genomic DNA from poor (I'M) and extensive (EM) metabollzers by RFLP-analysis (restriction fragment len~rth polymorphism) indicates that the gene locus is highly l?olymorphid. XSaI restriction enzyme was used to determine the freqaenc,es of polymorphic fragments assoeiated with the PM and EM phenotype: polymorphic fragments

PMs n 62

frequencies in PMs

11.5/115 44/44 44/11.5 44/16 + 9 29/29 29/11.5 29/44 29/16 + 9 29/9

2 4 8 2 18 5 21 1 1

0.032 0.065 0.129 0.032 0.290 0.081 0.339 0.016 0.016

l~olymorphic tragments

EMs n frequencies 88 in EMs

not observed 29/29 29/11.5 29/44 29/16 + 9 not observed

62 10 14 2

0.705 0.114 0.159 0.023

RFLPs predict the phenotype in 25.8% of PMs, in 71% at least one identifiedmutant allele could be demonstrated. In the present study ganomic DNA of a PM homozygons for the 11.5 kb allele was used to elucidate the molecular mechanism leading to this phenotype. To that end a genomic X EMBL3 library was constructed and overlapping clones isolated which span the entire gene locus. Sequence analysis andalignment with the sequences of the wild-type allalC revealed a deletion of the entire functional I ~ 6 gene, explaining the absence of IID6 protein. One of the deletion braakpoints could be localized. Further characterization of the other mutant alleles should allow the development of specific probes for routine detection of all alleles. ~Skoda et al., 1988, PNAS, 85.5240-5243 2Kimttra et al., 1989, Am. J. Hum. Genet., in press Dr. Margarete Fischer Bosch-Institut fiir Kliulsche Pharmakologie, 7000 Stuttgart 50, FRG *Dept. of Pharmakology, Biocentar, University of Basel, 4056 Basel, Switzerland The study was supported by the Robert Bosch Foundation.

436 PHARMACOKINETICS OF THE DEHYDROMETABOLITES OF SPARTEINE W. D. P a a r , G. M i k u s a n d W. H i n t z e n ............................................... Sparteine is oxidized polymorphically in man. Extensive metabolizers (EM) of s p a r t e i n e (SP) e l i m i n a t e m o s t of the d r u g b y o x i d a t i v e m e t a b o lism, l e a d i n g to 2 - d e h y d r o s p a r t e i n e (2DHS) a n d 5-dehydrosparteine (5DHS). Poor metabolizers (PM) e x c r e t e m o s t of the drug unchanged into urine. Whereas pharmacokinetics of S P h a s b e e n s t u d i e d extensively, data on the pharmacokineties of the d e h y d r o m e t a b o l i t e s a r e rare. In the p r e s e n t s t u d y p l a s m a a n d u r i n e l e v e l s of SP, 2DHS a n d 5DHS w e r e d e t e r m i n e d b y g a s e h r o m a t o g r a p h y in 10 h e a l t h y v o l u n t e e r s . SP d o s e w a s 1 0 0 mg ( s p a r t e i n e s u l f a t e , D e p a s a n S ) . E i g h t v o l u n t e e r s w e r e i d e n t i f i e d as E M of SP, 2 v o l u n t e e r s w e r e PM. In E M m a x i m u m p l a s m a c o n c e n t r a t i o n s of 2 D H S a n d 5DHS w e r e r e a c h e d a l r e a d y 1.4 h a n d 1.1 b a f t e r i n t a k e of SP. T h i s i n d i c a t e s fast m e t a b o l i s m . T h e A U C of SP a n d 2DHS w e r e in the s a m e r a n g e (768.7 v e r s u s 724.6 n g / m l - h ) , the c o r r e s p o n d i n g v a l u e for 5 D H S w a s 132.1 ng/ml-h. Mean residenee time of SP w a s 3.2 h ( S E M : 0 . 5 h). T h e mean residence times of the metabolites were c o m p a r a b l e : 2 D H S : 3 . 7 h ( S E M : 0 . 3 h), 5DHS: 3.0 h (SEM: 0.4 h). In E M a p p a r e n t m e t a b o l i c c l e a r a n ce of S P w a s 1 4 5 8 . 6 m l / m i n . Apparent metabolic c l e a r a n c e of SP in the 2 P M a m o u n t e d to 18.3 a n d 10.0 m l / min. M e a n r e s i d e n c e t i m e of s p a r t e i n e w a s 9.0 h in b o t h PM. Medizinisehe Universit~tsklinik Bonn, S i g m u n d F r e u d - S t r . 25, D - f i 3 0 0 B o n n 1 . Dr. M a r g a r e t e Fiseher-Bosch-Institut fur K l i nische Pharmakologie, Auerbaehstr. 112, D - 7 0 0 0 S t u t t g a r t 50.

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COMPARISON OF METHODS FOR DEBRISOqUINE POLYMORP~ISM P H E N O - AND OENOTYPINO IN HEALTHY VOLUNTEERS M. Hildebrand, K. Oro~, D. Oro~, W. Seifert, D. Heger-Mahn

IS CYCLOSPORIN A AN INHIBITOR OF D R U G METABOLISM? G. Li, J. Meinshausen, J. Wolf, and U. Klotz

The determination of debrisoquine metabelizer status is of growing importance in drug development. A group a healthy volunteers (n ~ 450, 299 m, 151 f; age: 1878 yr), which regularly participated in clinical phase I trials, was classified by the dextromethorphan (DMP) test. In 8 h-urine the metabolic ratio (MR) of dextrorphan (DOP) / DMP was determined. Poor metabolizers (PM) exhibited a MR < i, intermediate metabolizers (IM) a MR of 1-10 and extensive metabolizers ( ~ ) a MR > i0. Each 5 I of the population were IMs and PMs and these groups were retested by debrisoquine (D). For phenotyping with D a MR of D]4-Hydroxy-D in 5h-urine was determined and a MR < 12 classified the EM- and a MR > 12 the PM-type. In addition restriction fragment length polymorphism (RFLP) patterns were determined in IMs and PMB of DMP using the cytochrome P~450 dbl cDNA provided by U.A. Meyer (Basle). All PMs of D M P w e r e c o n f i r m e d as PMs of debrisoquine. Interestingly the IMs of DMP could be attributed to either EM or PM type of debrisoquine. MR of D[4-Hydroxy-D varied from 0.2 to 140. Different RFLP patterns were seen in the PMs of D. Restriction fragment combinations in the PMs were: 4 and 29 khases (kb), 4/29]44 kb and 4[44 kb as well as several rarely observed patterns. Most EMs exhibited a 4/29 kb-pattern. The present data demonstrated that PMs of DMP (MR < i) w e r e unambiguously classified as PMs of dehrisoquine. EFLP analyses revealed genetic heterogeneity especially in the PM group. Research Laboratories, Schering AO, ROB 650311, D-1000 Berlin 65 Inst. fur Klin. Pharmakologie, Klinikum Steglitz, Hindenburgdamm 30, D-I000 Berlin 45

In a combined in vitro/in vivo approach the potential of cyclosporin A to inhibit drug metabolism has been investigated. Since the same cytochrome P-450 isozyme seems to be involved in the metabolism of cyclosporin A and midazolam, this new benzodiazepine was used as a probe. In vitro, the binding of cyclosporin A to male rat microsomal cytochrome P-450 was evaluated. The observed K,-value of 0.4 uM indicated that the cyclic peptide antibiotic might interfere with hepatic drug metabolism. Direct in vitro experiments with rat and human liver microsomes using midazolam as substrate demonstrated that its hydroxylation to o d - O H - , 4 - O H - and d i - O H midazolam was inhibited by cyclosporin A with ICso-values of 6.1, 7.8 and 70 uM respectively (rat); with human microsomes the corresponding ICsn-values were 324 and 65 uM for the formation of o C - O H - afl~l 4-OH-midazolam respectively. In vivo, the pharmacokinetics of midazolam following a single intravenous dose of 0.075 m g / k g was studied in nine patients who received routinely cyclosporin A to prevent rejection of their transplanted kidney. Averaged cyclosporin A steady state blood levels in those patients, as assessed by RIA with specific monoclonal antibody, varied during a dosing interval between 175 and 600 ng/ml. Hepatic elimination of midazolam as characterized by a mean t½ (_+ SD) of 2,3 + 1.2 h or a total plasma clearance (CL) of 414 + 95 ml/min was not different in the patients studied from that in age - matched controls (t½ = 1.5 to 4 h, CL = 350 to 700 ml/min), indicating that no interaction between cyclosporin A and midazolam occurs under clinically relevant conditions. From the various experiments carried out it is concluded that cyclosporin A possesses a weak potential for the inhibition of drug metabolism. Therapeutic blood levels in patients', however, do not appear to be sufficient to result in an effective impairment of the hepatic elimination of drugs given concomitantly. Dr. Margarete Fischer-Bosch Institut for Klinische Pharmakologie, Auerbachstr. 112, D-7000 Stuttgart 50, F R G The studies were supported by the Robert Bosch Foundation

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INTERINDMDUAL VARIATION OF N-PROPYLAJMALINE DOSE REQUIREMENT IN PATIENTS WITH VENTRICULAR ARRHYTHMIA IN RELATION TO METABOLIC CAPACITY OF SPARTEINE Klaus M6rike, Elke Hardtmann*, Peter Heimburg*

PHARMACOKINETICS OF CYCLOSPORIN AFTER INTRAVENEOUS AND ORAL APPLICATION IN LIVER TRANSPLANT RECIPIENTS J. Brockm611er, C. O6tze, N. Reisinger, W-O. Bechstein*, R. Raakow*, P. Neuhaus*

N-Propylajmaline (NP) is a widely-used class I antiarrhythmic drug. Following the administration of the recommended dose (20 mg t.i.d.) high Interlndividual variability in plasma concentrations was observed and arrhythmia suppression achieved in only 35 % of the patients. NP disposition has beon shown to cosegregate with the sparteine/debrleoquine polymorphism. The metabolic capacity of an individual can be expressed as the metabolic ratio (MR). We investigated whether steady state plasma concentrations of NP are related to MR. Twenty patients with symptomatic premature ventricular complexes (PVCs) were included In this trial. After obtaining the individual sparteine MR NP was admln stared. The initial dose of 20 mg t.i.d, was raised to 60 mg t.i.d. until a >70% suppression of PVCs was achieved. Inter-dose steady state pharmacokinetics, PQ and QRS intervals, and arrhythmia suppression were quantified during a dosing interval. Results: A linear correlation between dose-corrected steady state plasma concentrations and log MR was observed (r=0.66; p
Dr. Margarete Flscher-Bosch-lnstitut for Klinische Pharmekologie und *Robert-Bosch-Krankenhaus (Zentrum for Innere Medizin, Abt Kardiologie), D.7000 Stuttgart 50/FRG

Cyclosporin (CyA) is mostly applied orally, however absorption of CyA requires bile production. Therefore the drug has to be given intraveneously in the first week following liver transplantation. Between different centers, the doses ~ e administered in varying velocities from 2 hour- to contineous 12 hour-infusions. Typical side effects (eg. neurotoxicity) observed during CyA infusions may be attributable to high blood concentrations following rapid infusions. Patients and methods: Kinetics of two infusions (4 and 8 hours) of equal doses (i or 2 mg/kg body weight) was determined in eight patients. In addition, kinetics after 4 days of oral treatment was measured. Concentrations of CyA and its metabolites i, 17, 18 and 21 were analysed by HPLC. Results: In 4 Patients, CyA concentrations measured during the 4 hour infusion were significantly above levels predicted from the concentration time course determined for their 8 hour infusion (assuming linear kinetics). Peak blood levels of CyA (median and range) were 650 pg/l (540-1400) at end of the 4 h infusions and 360 Dg/l (280-700) at the end of the 8 h infusions. Ratios of the AUC of the metabolites 1 and 17 to CyA increased under oral therapy by the factors of 3.6 and 1.6 in comparison to intraveneous therapy. Conclusions: The overproportional high drug concentrations observed in some patients are suggesting low infusion rates as the most save therapy. Generally, in CyA therapy trough levels are measured. As shown, peak levels of the drug at the end of infusions should also be determined. In each patient, there was a considerable shift in the ratio of CyA to its metabolites when changing therapy from intraveneous to oral dosage. Thus, immunological methods not differentiating between CyA and its metabo!ites are questionable as the sole analytical method in therapeutic drug monitoring. Institut for Klinische Pharmakologie, Klinikum Steglitz, Hindenburgdamm 30, 1000 Berlin 45, *Chirurgische K l i n i k , Klinikum Rudolf Virchow. Freie Universit~t Berlin

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PHARMAOOKINETICS OF IFOSFAMIDE (IF) AND 4-OH-IFOSFAMIDE (4-OH-IF) IN PATIENTS ON ORAL AND I N T R A ~ U S IF-THEIRAPY Kurowskir V. and Wagner t T.*

Q u a n t i t a t i o n of a n e w a n t i n e o p l a s t i c hexadecylphosphocholine I. R u s t e n b e c k a n d S. L e n z e n

Enzymatic hydroxylation of the oxazaphosphorine IF forms its cytostatically active metabolite 4-OH-IF. The pharmacokinetics of IF and 4-OH-IF were investigated in 9 patients with various malignant diseases. All patients received oral and i.v. IF i D a randc~nizwd sequence on day I and 3 at a dose of I g/mz or 1.5 g/m z. Blood samples were drawn 20 rain to 32 h after IF application. The determination of IF plasma levels was performed by means of gaschromatography (NPFID) following derivatization of IF with heptafluorobutyric acid. Whole blood levels of 4-OH-IF were measured using a HPLC-assay with fluorometric dectection of 7-OH-quinoline, which is formed by condensation of 4-OH-IF-derived acrolein with maminophenol. On day I (independent of the dose and the mode of application) IF plasma concentrations declined with a terminal half-life of 6.5 h (range 5.2 - 9.1 h) and reached the detection limit 24 h after dosing. On day 3 (when the second dose was given by the alternate route) in 5 out of 9 patients the terminal half-life decreased by 1 .I - 4.4 h, thus indicating self induction of hepatic IF metabolism. Therefore the oral bioavailability of IF could not be calculated by intraindividual ccmloarison of oral and i.v. pharmacokinetics. After oral application IF absorption proceeded rapidly. Peak concentrations that were in the range of concentrations found after i.v. application were reached within 45 rain. 4-OH-IF was already present in the 20 rain samples and 4-OH-IF blood concentrations paralelled those of the parent ccmlpound IF at constant levels ranging interindividually between 0.12 - 5.9 %. In the individual patient the amount of metabolite formed was always higher after oral than after i.v. application (Cmax p.o./Cmax i.v. = 2.43; AUC p.o./AUC i.v. = 1.85). These results indicate that - probably due to first - pass metabolism orally administered IF may be more cytotoxic than IF given intravenously.

agent,

Some s y n t h e t i c p h o s p h o l i p i d s h a v e b e e n s h o w n to possess antineoplastic activity. A newly synt h e s i z e d c o m p o u n d of this c l a s s is h e x a d e c y l phosphocholine (HePC, p r o p o s e d INN: M i l t e f o s i n e ) . H e P C has b e e n u s e d s u c c e s s f u l l y in the t r e a t m e n t of c u t a n e o u s m e t a s t a s e s of m a m m a r y c a r c i n o m a a n d f u r t h e r i n d i c a t i o n s for its u s e are u n d e r i n v e s t i g a t i o n . We e s t a b l i s h e d a n o n r a d i o m e t r i c m e t h o d for the d e t e r m i n a t i o n of H e P C in plasma, w h i c h c a n a l s o be u s e d to s t u d y the a b s o l u t e a m o u n t s of H e P C in t i s s u e a n d s u b cellular fractions. Lipids were extracted by vortex-mixing a sonicated sample (homogenates f r o m l i v e r cells, p l a s m a , w h o l e b l o o d or w a s h e d e r y t h r o c y t e s ) w i t h the t w e n t y - f o l d v o l u m e of hexane-isopropanol (3:2, v/v). A f t e r d e s a l t i n g the c o n c e n t r a t e d e x t r a c t on a S e p h a d e x G 1 5 column, the s a m p l e s w e r e s t r e a k e d o n a s i l i c a gel 60 H P T L C p l a t e a n d d e v e l o p e d w i t h chloroform-methanol-triethylamine-water (30:35:34:8, v/v). A f t e r c h a r r i n g the l i p i d s w i t h a c u p r i c s u l f a t e r e a g e n t , the v i s i b l e l i p i d b a n d s w e r e s c a n n e d at 530 n m in a d e n s i t o m e t e r . H e P C was b a s e l i n e - s e p a r a t e d from lysophosphatidylcholine and s p h i n g o m y e l i n e . T h e l o w e r l i m i t of d e t e r m i n a t i o n was 25 pmol, the r e c o v e r y 89%. N e a r l y all H e P C in b l o o d was f o u n d in the plasma, w h i l e o n l y t r a c e s c o u l d be d e t e c t e d in w a s h e d e r y t h r o c y t e s . 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 , Universit~t GBttingen, D-3400 G6ttingen

Institut f[[r Pharmakologie und *Klinik f[ir Innere Medizin, Medizinische Universit~t zu L'dbeck, Ratzeburger Allee 160, D-2400 Lilbeck

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PLASMA P H A R M A C O K I N E T I C S OF H I G H - D O S E BUSULFAN I N THE COURSE OF AUTOLOGOUS BONE MARROW T R A N S P L A N TATION IN ADULTS. H.E. Scheulen, G. B a l s , D.W. B e e l e n , 3. K § d i n g , K. Ouabeck, U.W. S c h a e f e r

CONCENTRATION DEPENDENCY OF N-METHYLNICOTINAMIDE RENAL CLEARANCE: USE OF SYSTEM APPROACH W. Weber, S, Toussaint, M. Nitz,and H, Kewitz

Plasma pharmacokinetics of high-dose busulfsn was s t u d i e d i n 5 a d u l t p a t i e n t s w i t h h e m a t o l o g i c malignancies (3 AML, 1 ALL, 1MDS) a f t e r oral doses of 4 x 1mE/ks/day for 4 days before autologous bone marrow transplantation. After reaction of the m e t h a n o l - d e p r o t e i n i z e d p l a s m a w i t h

sodium diethyldithiocarbamate the b u s u l f a n derivative was extracted w i t h e t h y l a c e t a t e and quantitatively d e t e r m i n e d by U V - s p e c t r o m e t r y at 278nm after s e p a r a t i o n by HPLC on M e r c k L i C h r o spher C18 in a c e t o n i t r i l e / w a t e r (75Z/25Z). Half life was 2 . 1 7 ~ 0 . 5 1 h (range 1 . 5 0 t 2 . 7 3 h) and AUC afte~ last dose was 7 . 2 7 - 2 . & 8 pg h/ml {range &.08-I0.01 pg h/ml). When busulfan was given exactly in B-h~urs intervals (four p a t i e n t s } C(min) was 0 . 3 3 - 0 . 2 5 p g / m l and C(max) 1 , 7 7 - 0 . 3 & p g / m l (C(mean) O . g O - 0 . 3 0 pg/ml). In contrast, in one p a t i e n t w i t h v a r i a b l e i n t e r v a l s {&-13 hours) C(min) was 0.11 p g / m l and C(max) w a s 3.24 pg/ml (C(mean) = 1.18 pg/ml). Thus. w i t h r e s p e c t to the short h a l f life of b u s u l f a n a e u r a t e 6-hours intervals b e t w e e n a p p l i c a t i o n s m a y be m a n d a t o r y for low toxic side e f f e c t s . The i m p a c t of the d o s e s c h e d u l e on m y e l o t o x i c i t y has to be d e f i n e d by the d e t e r m i n a t i o n of the i n t r a c e l l u l a r busulfan M i n e t i c s in b o n e m a r r o w cells. (Supported by Deutsche Forschungsgemeinschaft: SFB i 0 2 . ) Innere Klinik (Tumorforschung) und K l i n i k for Knochenmarktransplantation, Universit~tsklinikum Essen, H u f e l a n d s t r . 55. D - & 3 0 0 Essen I

Endogenous N-Methylnicotinamide (NMN), an organic cation, is mainly excreted in urine both by glomerular filtration and proximal tubular secretion. In the rat only a negligible amount was tubular reabsorbed, It was suggested that the endogeneous NMN would be a useful probe to estimate renal plasma-flow (RPF) without constant infusion of exogenous substances. This hypothesis was tested in 8 humans. We examined the renal clearance of NMN at two steady state concentrations: endogenous and after a continuous infusion following an iv-b01us loading dose. The plasma concentration time course was described as response to a unit step input. This response was used as input to the kidney, w i t h the renal excretion-rate as output. The effect of the kidney, treated as a black box, is described by a mathematical operation, i.e. multiplication of input by a renal clearance (CIR) term. Because input and output were experimentally determined, a suitable mathematical operator had to be found to characterize the black box. In the case of NMN, a nonlinear operator was determined. Renal clearance at physiological levels of 0.2 (0.1-0.3) ~mol/l (median, 95% confidence interval) was 273 (165-478)ml/min, and increased to 641 (505-776) ml/min at steady state concentrations of 3.8 (3.3-4.8) ~mol/l during infusion. Introducing a lag-time for the delayed response, the time courses of plasma concentration (C) were linked to the renal excretion-rates by an empirical concentration dependent operator: ClR = CiRmax (I - exp(-C/Km)) with CiPanax = 624 (412-730)ml/min and Km = 0.4 (0.2-0.9)Dmol/l. With increasing plasma levels, renal clearance increased to a constant value in the order of RPF. This effect can be interpreted as saturation of an effective NMN tubular reabsorption. It's concluded, that RPF in humans can only be estimated with an infusion of exogenous NMN. Institut f~r Klinische Pharmakologie, Hindenburgdamm 30, D-1000 Berlin 45

Klinikum Steglitz,

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M U L T I P L E D O S E P H A R M A C O K I N E T I C S OF G A N G L I O S I D E GMI (GMI) A F T E R I.V. A N D I.M. A P P L I C A T I O N I N H E A L T H Y VOLUNTEERS K.L. Rost, S. B r a u n e *

B-HT 920 - A NOVEL DOPAMINE A U T O R E C E P T O R AGONIST IN THE TREATMENT OF PATIENTS WITH SCHIZOPHRENIA

Clinical studies indicate that GMI a c c e l e r a t e s the r e c o v e r y of n e u r o l o g i c a l d e f i c i t s in stroke. Besides early administration after the e v e n t an optimal dosage scheme based on p h a r m a c o k i n e t i c d a t a m a y be i m p o r t a n t to reach consistent treatment efficacy. G M l - p h a r m a c e k i n e t i c s w e r e e v a l u a t e d a f t e r 21 days of a p p l i c a t i o n and 21 days w a s h - o u t in 5 h e a l t h y m a l e v o l u n t e e r s a g e d 27 - 33 years. I00 m g h i g h l y p u r i f i e d GMI (Fidia, A b a n o Terme, Italy) w a s giv e n d a i l y to t h r e e v o l u n t e e r s b y 0.5 h - i . v . - i n f u sion and to two v o l u n t e e r s b y i.m. route. S a m p l e s were t a k e n f r o m plasma, urine, and faeces. GMI was d e t e r m i n e d b y an s p e c i f i c e n z y m e l i n k e d comp e t i t i v e b i n d i n g a s s a y u s i n g the s p e c i f i c b i n d i n g of GMI to c h o l e r a toxin. Pretreatment l e v e l s of GMI w e r e 0.i to 0.4 D g / m l in p l a s m a a n d 0.5 to I0 D g / g d r y w e i g h t of faeces. GMI was not d e t e c t e d in u r i n e d u r i n g the w h o l e study. G M l - l e v e l s in faeces d i d n o t c h a n g e a b o v e p r e t r e a t m e n t l e v e l s w i t h t~e dosage. P l a s m a trough l e v e l s r a n g e d f r o m 25 to 80 u g / m l plasma. Plasma data were described by concentration-time functions achieved by repetitive input functions of r e c t a n g u l a r pulses (i.v.) or f i r s t o r d e r abs o r p t i o n (i.m.) and the r e s p o n s e f u n c t i o n of an open two-compartment model, f i t t e d b y the i t e r a t i v e l y r e w e i g h t e d l e a s t s q u a r e m e t h o d . B - h a l f lifes r a n g e d f r o m 60 to 120 h, i r r e s p e c t i v e of the w a y of a p p l i c a t i o n . The v o l u m e of d i s t r i b u t i o n of a b o u t 0.06 1 / k g r e s e m b l e d the p l a s m a volume. Total c l e a r a n c e was c a l c u l a t e d to b e I - 4 m l / m i n . Inst. of Clin. P h a r m a c o l o g y , H i n d e n b u r g d a m m 30, I000 B e r l i n 45; * F i d i a P h a r m a f o r s c h u n g , M O n c h e n

K.Wiedemann, O.Benkert and F.Holsboer In an open clinical trial the azepine derivative B-HT 920 (6-allyl2-amino-5,6,7,8-tetrahydro-4H-thiazolo(4,5-d)-azepine) was administered to patients suffering from schizophrenia, paranoid type (according to ICD-9 criteria), to examine whether dopamine autoreceptor stimulation exerts antipsychotic effects, as can be assumed by clinical studies with low dosed apomorphine. Twelve patients participated in the study and received the test drug orally for up to 28 days in a dose range from 0.3 to 1.2 rag/day. The following results emerged: in four patients a significant reduction of psychotic symptomatology was observed; eight patients remained without improvement of psychopathology. Psychomotor activation was observed in seven patients, and prompted termination of the trial in two cases. No other marked adverse effects of B-HT 920 were noted, including EEG, ECG and clinical chemistry parameters. Treatment with B-HT 920 did not lead to any extrapyramidal symptoms which are characteristic for traditional neuroleptics. As was to be expected from the pharmacology, plasma prolactin concentrations were significantly reduced two hours after oral application of a single dose of the drug. It remains to be clarified whether chronic treatment with B-HT 920 induces antipsychotic effects within as yet unidentified subsamples of schizophrenic patients. The observed activating effects of BHT 920 may focus future investigative efforts toward study s~mp~es where negative symptoms predominate. Dept. of Psychiatry, University of Freiburg, HauptstraSe 5, D-7800 Freiburg, FRG

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T H E R A P E U T I C C O N C E N T R A T I O N S OF L I T H I U M AND C A R B A M A Z E P I N E I N H I B I T cGMP A C C U M U L A T I O N IN H U M A N LYMPHOCYTES T. S c h u b e r t

EXCRETION OF URINARY NOREPINEPHRINE (NE) METABOLITES IN YOUNG PATIENTS WITH ANOREXIA NERVOSA (AN) H.-U. MUller and A. Rothenberger

Lithium and carbamazepine are commonly used drugs in the p r o p h y l a c t i c t r e a t m e n t of a f f e c t i v e disorders. D e s p i t e of a wide v a r i e t y of b i o c h e mical e f f e c t s w h i c h are r e l a t e d to these compounds, the molecular mechanism underlying their t h e r a p e u t i c e f f i c a c y is not yet known. Our data i n d i c a t e that both drugs in t h e r a p e u t i c c o n c e n t r a t i o n are able to i n h i b i t s o d i u m n i t r o prussid induced cGMP accumulation in human lymphocytes. This i n h i b i t i o n is very s p e c i f i c for lithium and carbamazepine, since other t r i c y c l i c a n t i d e p r e s s a n t s do not a f f e c t a c t i v a ted l y m p h o c y t e guanylate cyclase. The e f f e c t s of l i t h i u m and carbamazepine show p r o n o u n c e d interindividual variations beween healtly volunteers. The r e s u l t s are d i s c u s s e d in the view of a common mechanism of action of l i t h i u m and c a r b a m a z e p i n e in the t h e r a p y of e f f e c t i v e disorders. Furthermore it is s p e c u l a t e d that the individual sensitivity of the cGMP generating system of human lymphocytes to l i t h i u m and c a r b a m a z e p i n e could be a h e l p f u l model for p r e d i c t i n g therapeutic r e s p o n s e in p a t i e n t s with e f f e c t i v e d i s o r d e r s . D e p a r t m e n t of P s y c h o p h a r m a c o l o g y , C e n t r a l Instirue of M e n t a l Health, D - 6 8 0 0 M a n a h e i m (FRG)

It has been reported that urinary excretion of the NE metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) is low in untreated patients with AN. However, it was unclear whether low MHPG excretion is due to changes in central or peripheral NE metabolism. Differential determination (i) of urinary MHPG-sulfate and MHPGglucuronide provides possible evidence of central or peripheral NE metabolism respectively (2). Thus, we measured MHPG conjugates and peripheral NE metabolite vanilmandelfc acid (VMA) in the urine of ]0 AN inpatients (DSM III criteria) on two different steps of the treatment (tl, with very low weight in the first week; t2, with weight gain). Mean percentage of ideal body weight increased high significantly from (tl)~ 72,4% to (t2) 90,9% (pC O.001) Mean MHPG-sulfate excretion was not significantly different between (tl) 1,3 ~g/mg creatinine and (t2) 1,4 wg/mg creatinine, in contrast mean MHPG-glucnronide excretion increased significantly from ( t l ) 0,8 ~g/mg creatinine to (t2) 1,5 ~g/mg creatinine (Wilcoxon Test, p
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S A F E T Y OF R E V E R S I B L E M O N O A M I N E O X I D A S E I N H I B I T O R S (MAOI): I N T E R A C T I O N O F B R O F A R O M I N E W I T H S Y M P A T H O M I M E T I C D R U G S IN H E A L T H Y V O L U N T E E R S . B. M ~ h l b a u e r r G. G r a d i n - F r i m m e r , P. B l a c k

AMPHETAMINE-LIKE E F F E C T S IN H U M A N S OF T H E K H A T A L K A L O I D C A T H I N O N E

Hazardous hypertensive episodes by interaction of t h 9 o l d e r M A O I w i t h s y m p a t h o m i m e t i c d r u g s h a v e D e a n r e p o r t e d . It s e e m e d i m p o r t a n t t o inv e s t i g a t e if b r o f a r o m i n e (BROF), a n e w s e l e c t i v e a n d r e v e r s i b l e i n h i b i t o r of t y p e A of M A O w o u l d a l s o s h o w s u c h i n t e r a c t i o n . In a c o n t r o l l e d trial, blood p{essure effects of phenylephrine (PE) a n d p n e n y ± D r o p a n o ± a m i n e (PPA) w e r e i n v e s t i g a t e d in ~ h e a l t h y s u b j e c t s b e f o r e , d u r i n g a n d a f t e r t r e a t m e n t w ~ t h B R O F (75 m g b.i.d) f o r i0 d. P E (nose drops) a n d P P A (capsules) w e r e g i v e n in i n c r e a s i n g doses. F o r p r o l o n g e d d e l i v e r y o f P P A (6 - 8 m g / h * i0 h) one o r a l o s m o t i c d r u g d e l i v e r y s y s t e m I P P A - O R O S , A c u t r i m a) w a s a d m l n i s t e r e d . A c u t r i m ~ is u s e d c l i n i c a l l y f o r c o n t r o l of a p p e t i t e . D o s e s of t h e a m i n e s w h i c h increase systolic blood pressure (SBP) b y 30 m m H g (PD~0) a n d a m b u l a t o r y BP a f t e r P P A - O R O S w a s e v a l u a t e d f o r 10 h ( O x f o r d M e d i l o g R A S P ) . Results: PD3Q v a l u e s ( n p ~ A 6 1 m g ] Treatment P E [mgj ............................................. Control 16.7 ± 7.5 62.5 ± 1 2 . 5 B R O F (d9) 9.0 ± 6.5 2 5 . 0 ± 10.2 ............................................. PD30 ratio 2 . 5 ± 0.6 3.0 ± 1.4 C l i n i c a l l y u s e d d o s e s of P E (2.5 mg) c a u s e d n o i n c r e a s e mn SBP b e z o r e a n d a u r i n g BROF. S e v e n f o l d h i g h e r d o s e s o f P E in c o n t r o l s a n d 3 . 5 - f o l d h i g h e r a o s e s d u r i n g B R O F r a i s e d SBP b E 30 ram Hg. T h e p r e s s o r e f f e c t of an a c u t e d o s e of P P A w a s p o t e n t i a t e d 3-fold. T h i s e f f e c t w a s r e v e r s i b l e w i t h i n 2 to 3 days. P P A - O R O S e ± e v a t e d m e a n SBP b y i0 m m H g d u r i n g B R O F ~ M a x i m a ± i n c r e a s e of SBP w a s s e e n f r o m 115 t o ~ u ram Hg. Conclusion: BROF causes no relevant potentiation of Erie p r e s s o r e f f e c t o f P E a n d P P A - D R O S in clinically used dosages. Combined administration a p p e a r s t o b e safe. Human Pharmacology Waldh6rnlestr. 22,

Institute, CIBA-GEIGY 7 4 0 0 T ~ b i n g e n , FRG.

GmbH,

P. K a l i x I , H.-U. F i s c h 2 , U. K o e l b i n g 2 , S. G e i s s h ~ s l e r 3 and R. B r e n n e i s e n 3

In certain countries of East Africa and of the Arab Peninsula, fresh leaves of the khat shrub are widely used as a stimulant. The effects of this drug are supposed to be due to cathinone, a phenylpropylamine alkaloid that has been shown to be, in experiments with animals and with isolated tissues, a potent amphetamine-like compound. In view of these findings, we decided to evaluate the effects of cathinone in humans, and we administered the alkaloid per os at a dose of 0.5 mg/kg to six healthy volunteers in individual tests using a placebo-controlled balanced experimental design; this dose corresponds to the content of a portion of khat of average quality. The administration of eathinone produced clearcut increases in blood pressure and heart rate, and the observed changes were concomitant with the presence of cathinone in blood plasma; cathinone was rapidly inactivated by transformation into norephedrine. For determining the psychological effects of cathinone, the subjects were required to self-report physical and mental changes that they felt to occur by answering sets of questions from the Addiction Research Center Inventory; the responses indicated that cathinone has in humans marked psychostimulant and euphorigenic effects. 1 Department University 2 Department University 3 Department University

of of of of of of

Pharmacology, Faculty of Medicine, Geneva, Switzerland Psychiatry, Faculty of Medicine, Berne, Switzerland Phytochemistry, Institute of Pharmacy, Berne, Switzerland

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PHARMACOKINETICS OF THE POTENTIAL NOOTROPIC CGS 5649 B IN GERIATRIC PATIENTS AND IN YOUNG HEALTHY VOLUNTEERS B. EN~LIN-HAASISr K.H. ANTONIN, *R. HEINRICH, and T. SCHNEIDER

TIME COURSE OF THE REACTION TIME MEASURED BY COMBINED COMPUTERIZED ATTENTION TASKS AFTER A I.V. BOLUS OF MIDAZOLAbI

The potential nootropic CGS 5649 B (6-(2-Isopropylaminopropyl)-3-pyridinol fumarate was given in an open study to 6 female geriatric patients (mean: 79, range 62 to 88 years, mean weight: 50, range 38 to 69 kg) and to 6 healthy young male subjects (mean: 28, range 22 to 33 years, weight 71, range 61 to 77 kg). Patients received a single dose of 600 mg and healthy subjects doses from 900 to 2400 rag p.o. Plasma concentrations of unchanged CGS 5649 B and the two conjugates were measured by specific HPLC. Results: Cmax/dose (m#an ± SD) [nmol/(L*mg)] Patients

AUC/dosa [nmol*h/(L*mg)]

tl/z [hi

(n = 6)

unchanged sulfate glucuronide

22 ± 19 57 ± 20 28 ± i0

Healthy volunteers unchanged sulfate glucuronide

3 5 ± 12 459 ± 286 322 ± i18

2.23 ± 0.30 4.36 ± 2.27 7.44 ± 3.92

21 ± 6 117 ± 16 180 ± 44

1.49 ± 0.46 4.14 ± 0.99 5.18 ± 1.41

(n = 6)

8 ± 2 20 ± 5 19 ± 6

Patients had a higher Cmax/dose and increased AUC/dose (p ~ 0.05) values. Half-lives did not differ. Possible explanations such as differences in sex, age, body weight, elimination and distribution will be discussed. Human Pharmacology Institute, CIBA-GEIGY GmbH, Waldh6rnlestr. 22, 7400 Tfibingen, FRG. *Medizin.-Geriatrische Klinik, Marienhospital, 4690 Herne, FRG.

G o t t s c h a l k , D.*, T o l k s d o r f , W.*. Dehus, O. *$ FrShlich, J.** and Theisohn,M.*** 12 h e a l t h y , male s t u d e n t s ( a g e : 2 1 - 3 2 y , b o d y w e i g h t : 6 5 90 kg) t a k e p a r t i n t h i s p l a c e b o c o n t r o l l e d c r o s s o v e r s t u d y ( w a s h - o u t t i m e 1 wk), r e v i e w e d b y t h e r e s p o n s i b l e ethical committee, after giving informed consent. After training with the Computerized Attention T a s k s (CAT) b a t t e r y (aim: < 5 % e r r . i n r e a p . , < 15 % s t d . d e v , o f t h e Reaction Time (RT) f o r e a c h t a s k ) t h e i n v e s t i g a t i o n started in the morning with 0.5 h training phase followed by 0.5 h base line estimation. Thereafter, midazolam ( 0 . 0 5 mg/kg, 3 . 2 5 - 4 . 5 mg) o r p l a c e b o was g i v e n i . v . f o r s e d a t i o n , f o l l o w e d i m m e d i a t e l y by t h e r e c o r d i n g o f RT. 30 min. l a t e r a 2nd i n j e c t i o n was g i v e n ( p l a c e b o o r f l u m a z e n i l ) . The p r o c e d u r e s o f m e a s u r i n g RT w i t h 9 combined CAT of different difficulties in information processing ( V i s u a l S e a r c h (VS) a n d F o c u s s e d A t t e n t i o n (FA); f o r e p e riode duration 1 sac, stimulus presentation 1 sac) takes 12 m i n . a n d was r e p e a t e d e v e r y 30 m i n . up t o 8 h . In r e l a t i o n t o t h e AT t h e RT a f t e r p l a c e b o was 4 0 0 - 4 8 0 msec. c o m p a r a b l e t o b a s e l i n e . A f t e r midazolam RT s i g n i f i c a n t l y ( p < 0 . 0 1 , ANOVA) i n c r e a s e d b y 120-135 msec. i n t h e VS and b y 1 4 0 - 1 6 0 msec. i n FA t a s k s . The p r o l o n g a t i o n o f t h e RT decreased like a e-function (lst order process, r between 0 . 9 1 a n d 0 . 9 8 ) w i t h a t / 2 o f 3 5 - 4 6 min. f o r VS and 4 3 - 5 0 m i n . f o r FA d u r i n g 2 h o u r s . T h e s e t / 2 ' s of the effect kinetic are similar to the reported t/2 of distribution ( a l p h a ) o f midazolam b a s e d on a two c o m p a r t m e n t model. I t c o u l d be shown t h a t m e a s u r i n g RT w i t h t h i s new CAT battery describes the effect kinetic similar to the theoretical expected plasma levels of midazolam after a single dose. S u p p o r t e d b y H o f f m a n n - L a R o c h e , G r e n z a c h - W h y l e n , FRG * K l i n i k ffir A n a e s t h e s i o l o g i e , ** I n s t . f f i r P s y c h o l o g i e I , RWTH A a c h e n , and *** P h a r m a k o l . I n s t . , U n i v . K61n.

R 114 CI TEACHING PHARMACOLOGY WITH DRUG INFORMATION SYSTEMS COMBINED WITH SIMULATION MODELS ON PERSONAL COMPUTERS G. Kobal, Th. Hummel, and G. Geisslinger In order to teach pharmacology via multi-sensory inputs and, at the same time, dispensing with experimental animals, it has become necessary to employ the advantages of novel computer systems. Based on a computerized pharmacological data bank we developed a new system to gain direct access to drug information, which, in addition, enables the medical student to study in an interactive way. The underlying reason to employ such a system is not the possibility to obtain written information or to make use of test questionnaires by way of a computer monitor - a book would always be the superior medium to draw upon in these cases - but to enable the student to determine the mode as well as the direction of his studies himself. In other words, students would not have to follow a prescribed way of acquiring knowledge, but could proceed with their studies, if desired, in a transversal manner. Should it become necessary, students could revert to illustrations, or else they could study functional principles by employing simulation models. At the same time, the entire pharmacological data bank would be at their disposal. By way of example a program on cardiac glycosides shall illustrate the advantages of such a system. In addition to their basic mode of action, pharmacokinetic changes due to organic disorders will be simulated. Subsequently, the results of the simulation process can be utilized in order to establish the optimal dosage regimen. Department of Pharmacology and versity of Erlangen-NOrnberg, 22, D-8520 Erlangen, FRG

Toxicology, UniUniversit~tsstr.