R1

CADMIUM-AND ZINC-DISTRIBUTION AND ENZYME ACTIVITIES IN RATS SUBACUTELY TREATED W~TH CADMIUM (Cadmium- und Zink-Verteilung und Enzymaktivlt~ten bei Ratten nach subakuter Behandlung mTt Cadmium) C. Alsen and J. Schneider Male rats were treated with 0.3 and 1.0 mg Cd~/kg (CdCI2) resp. i.p. For 3-27 days. After 4,7, 14,21 and 28 days the animals were sacrificed. Cd- and Zn-concentrations in liver, kidneys, testes, erythrocytes and serum were measured and the activities of SGOT, SGPT and alkaline phosphatase in serum as well as the activity of carbonic anhydrase in erythrocytes and kidneys were determined. In llver, kTdneys and erythrocytes the Cd-concentration was found to be lineary related to the administered doses of Cd, while the Cd-concentraHon in the testes and serum displayed a more complex pattern. On the 4 th, 14th and 28th day of treatment, 48, 69 and 61% resp. of the totally applied amount of Cd could be recovered in the investigated organs mentioned above. The concentration of Zn, also, increased in liver and kldneys reaching a maximum on the 14th day of treatment with Cd, whereas it declined in the testes. During the period of treatment the activities of SGOT, SGPT and carbonic anhydrase remained unchanged. The activity of alkalln~.phosphatase, however~ had decreased to 69% (0.3 mg Cd++/kg) and 52% (1.0 mg Cd /kg) already after 4 days. Later on changes in ~he actTvTty of this enzyme could not be observed. Dr. C. Alsen, Abteilung FUr Toxikologle, Instltut fur Pharmakologle , Christian-AlbrechtsUnlversitst und Untersuchungsstelle fur Umwelttoxikolog|e (Sozialmin|sterium des Landes Schleswig-Holstein) 2300 Kiel~ Hospitalstr. 4-6 West Germany

EFFECT OF GLUCOSE AND M E T ~ BLUE (MB) ON NADP,NADPH AND INSULIN SECRETION OF ISOLATED PANCREATIC RAT ISLETS (Der EinfluB von Glucose und Methylenblau (MB) auf NADP,NADPH und Insulinsekretion isolierter Pankreasinseln der Ratte) H.P.T.Ammon Glucose metabolism via the pentosephosphate shunt in the B-cell has been suggested to be important for the secretory mechanism. The possible role of its product N A D P H in insulin secretion was studied using collagenase isolated islets in vitro.Methods: 5 or q0 islets/flask were incubated a) in the presence of 3mg/ml glucose and 0-0.5-2.0 or 5.0 ~ g / m l MB~ b) in the presence of 0-~-3mg /ml glucose. Insular NADP,NADPH and i m m u n o r e a c t i v e i n s u l i n (IRI) released into the medium were measured after 60 or 90 min incubation.Results: a) NADP: ~.07+0.05 vs ~.~I+0.09 vs ~.56+0.07 vs q . 7 2 + O - - ~ - N / ~ O islets (n=q~);NADPH: 0.7~+0.04 vs 0.6~+0.06 vs 0.38+--0.07 vs 0.36+-0.06 pM/~0 islets;NADPHT~ADP: 0.gg v ~ 0.62 vs 0.24 vs 0.20; IRI: 566+5o vs 409+35 vs 282+4~ vs 236+27 p U / 5 islets/9o min (n=20). b)--In the presence of ~mg/ml g l u ~ o s @ t h e NADPH/NADP ratio increased when compared to Omg/ml glucose by q24%.But no furtlher increase could be observed employing 3mg/ml glucose. Insulin release was detected only in the presence of 3mg/ ml glucose.Conclusion:Inhibition of glucose induced insulin release b y FIB coincides with a decrease of the NADPH/NADP ratio. However,this ratio does not coincide with the stimulatory effect of glucose on insulin secretion, it is suggested that the NADPH/ NADP ratio plays the role of a necessary cofactor in the mechanism of insulin secretion,but does not play the role of a signal. Priv. Doz. Dr.H.P.T.Ammon,Pharmakologisches Institut der Universit~t, D-852 Erlangen,Universit~tsstra~e 22

R2 INHIBITION AND ACTIVATION OF HISTAMINE METHYLTRANSFERASE BY HI - AND H2RECEPTOR ANTAGONISTSAND METHYLATEDHISTAMINES (Hemmung und Aktivierung der Histaminmethyltransferase durch H1- und H2-Rezeptorenblocker und methylierte Histamine) H. Barth~ I . N,iemey,er Hl-receptor antagonists like mepyramine, chlorpyramine, dimethpyrindene and promethazine were found to inhibit (I.D.50=O.I - lmM) the 15-fold purified histamine methyltransferase (HNT) from pig antrum mucosa(method: Snyder, BBA 111, 416 (1965)) Burimamide, the first H2-receptor antagonists (Black, Nature 236, 385 (1972)) in 0.I mM concentration also inhibited this enzyme to 50~. Thus, there was no striking difference in inhibition of HNT by H I- and H2-receptor antagonists. In 5 0 ~ M concentration, all antihistamines acting on the H]-receptor activated the HMT from pig antrum mucosa by 30-60~. 8urimamide was %he only antihistamine, which was unable to enhance HMT activity, l-methyl-, 2-methyl- and 3methylhistamine (I-MH,2-MH,3-MH) were strong inhibitors of HMT too. With a 50~ inhibition a% 0.1 mM concentra• they behaved like the H 1- and H2-receptor antagonists. Like burimamide, they were not able to activate HMT. 5-methyl-, N'-methyl- and N'-,N'-dimethylhistamine (5-MH, N'-MH, N',N'-DNH) were weak inhibitors of HMT (I.D.50=3mM), but, like the H lreceptor antagonists, they could activate the enzyme by 50~ in a 50 ~M concentration. Thus I-MH, 2-MH and 3-MH acted on HMT in the same manner as the HI-receptor antagonists, whereas 5-MH, N-MH and N',N'-DflH in their action resembled the H2-receptor antagonist burimamide. Dipl.-Chem. H. Barth, Ab•163 u. Pathol.Biochemie der Universit~t, D-3550 Marburg/Lahn, Robert-Koch-mitre 8

THE EFFECTS OF THE TRICYCLIC ANTIDEPRESSANTS DESMETHYLIMIPRAMINE, DOXEPINE AND IPRINDOLE ON THE ISOLATED PERFUSED RABBIT HEART (Die Wirkung der trizyklischen Antidepressiva Desmethylimipramin, Doxepin und Iprindol am isolierten, perfundierten Kaninchenherzen) N. Barth Ventricular rate, atrial and ventricular tension were recorded transversely (Fozard & Muscholl, Arch.Pharmak. 27o, 319-325, 1971) on rabbit hearts with the sympathetic nerves attached. Desmethylimipramine (DMI), doxepine (DOX) and iprindole (IP) (each 1.5 pg,/ml and n=6) reduced ventricular tension by about 60 ~ . Atrial tension was decreased by 4 8 + 6 . 7 ~ (mean +_ S.E.) after DMI but remained unaltered after DOX and IP. DMI, DOX-and IP reduced ventricular rate by 15-32 ~ . The postganglionic sympathetic nerves were stimulated (STIM.) electrically (1 msec, maximal strength, 15 sac) with different frequencies at intervals of 180 sec before (control period) and 20 rain after infusion of DMI, DOX and IP. STIM. after DMI and DOX caused supraventricular arrhythmias (arrh.) of the following mean durations: STIM. Incidence of arrh. Duration of arrh. (sec) Hz DMI DOX DMI DOX 0.25 1/6 0/6 30 + 30.0 w 0 w recording of 0.5 2/6 I/6 31 ~ 29.8 w 2 + 2.0 arrh. terminated 1 4/6 4/6 38 ~ 28.7 w 22 ~ 8.0 180 sec after onset 3 6/6 5/6 87 ; 31.8 w 101 u 28.7 w of STIM. 10 6/'6 6/6 117 ~ 29.4 w 152 ~ 21.6 w No arrh. were caused by STIM. in the control period, otter IP (1.0-2.5/Jg/'ml) or after 1.0 p~/'ml DMI. The method described may be used to test tricyclic antidepressants for their arrh. producing potency. Dr. N. Barth, Pharrnakol.lnst.d.Univ. D-65 Mainz, Obere Zahlbacher Str. 67

R3 PLATELET AGGREGATION INDUCED BY A HIGHLY SPECIFIC SUBSTANCE (DAS) IN VITRO: SOME INVESTIGATIONS ON ITS MECHANISM OF ACTION (Untersuchungen zum Wirkungsmechanismus einer hochspezifisch aggregationsinduzierenden 8ubstanz (DAS) auf Thrombocyten in vitro) K.U. Benner~ K.A. Schumacher I L. Hedler I I. L u s t e r - H a ~ e n e ~ In earlier investigations the ioV. injection of the depressor active substance (DAS) (Hedler and Marquardt 1961) into cats was followed hy a rapid increase in pulmonary vascular resistance combined with a marked decrease in platelet count (Schumacher and Classen 1972). Therefore, the effect of DAS on platelets of men, cats, pigs~ dogs, rats, and rabbits has been studied using the turbidimetric method of Born. Only in human and feline platelet rich plasma DAS was found to induce platelet aggregation (PA), the degree of which was comparable to that induced by ADP or adrenaline. DAS induced PA resembles ADP induced aggregation in many ways: (I) the shapes of the aggregation curves induced by both agents are similar; (2) both forms of aggregation require Ca++; and (3) they are inhibited by adenosine, tosylargininemethylester, and sulfhydryl inhibitors such as parachloromercuribenzoate. However, its species specifity and a marked tachyphylactic action on feline and human platelets also pharmacologically makes DAS clearly discernible from all the other PA inducing substances which have been studied so far. Evidence suggests that DAS in the presence of Ca ++ causes a release reaction of cellular substances known to enhance PA in a second phase. Dr~ K.U. Benner, Institut f~r normale und pathologische Universit~t, D-5000 K~ln 41, Robert-Koch-Str. 39

Physiologle

der

DRUG AND STEROID BINDING AND ELECTRON TRANSPORT IN HUMAN PLACENTAL MICROSOMES (SubstratbTndung und Elektronentransport in menschlTchen Plazentamikrosomen) P. Bergheim and K.J. Netter Cytochrome P 450 was first demonstrated in placental tissue fractions by Meigs and Ryan(BBA 165,476,1968). Using 11 term placentas we found a P450 content of 0.1 nmoles+0.014(SEM) mZ~-gprotein(range0.05-0.2 ) as compared to0.27 nmoles/mg protein in human liver (P.Rai e t a l., Chem. biol. Interact.3, 303, 1971). The concentration of cytochrome b5 in two cases was determined to be 0.067 and 0.061 nmoles/mg protein(0.2+0.02 in human liver). NADPHcytochrome c-reductase activity was found to be 87 and 90 nmoles cyt.c reduced/mg prot/ rain which ~s in the same range as in human and rat liver. It is slightly inhibited by metyrapone (Mp). 1.5+0.19 (SEM) mg microsomal protein was isolated per gram of perfused placental tissue. Th~s represents 5% of the recovery from human liver. Anillne, Mp, p-aminophenol show type 11 spectra,while hexoberbital,aminopyrine,ethylmorphine and naphthalene show no type I spectrum. Mp did not cause a Soret absorption in reduced microsomes. No P 450 dependent metabolism could be demonstrated for aniline, acetanilide, p-nitroanisole and amphetamine. 17-~- OH-progesterone and progesterone yield type I spectra, 17-o(-OH-pregnenolone and pregnenolone type II spectra, while androstenediole testosterone and tr-dehydro-androsterone give a mixed spectrum with maxima at 387, 427 and a minimum at 413 nm. A concentration dependent inhibition of NADH oxidation by androstenediole and testosterone is observed suggesting a possible interference with the transfer of the second electron. For this a critical conc. (1 pM) was found below which cyt. b5 could not be kept in the reduced state. (Supported by grants from Deutsche Forschungsgemeinscha~t and Stiftung Volkswagenwerk) Depto of Pharmacology, Section on Toxicology, Un~v.oF Mainz, D 65 Mainz, Germany

R4 GAS CHROMATOGRAPHIC ANALYSES OF BARBITURATES IN SMALL AMO UNTS OF BLOOD (Gaschromatographische Barbituratanalysen in kleinen Blutvo{umina) E. Bergheim-lrps, W. Dunges and H. Heinemann Up to now a minimum of 1 ml of blood is required for the g c analysis of barbiturates. This is partly due to the nonldeal g c behaviour of the free barbituric acids in the submicrogram range. The following method allows the analysis of ppM levels of barbiturates in small blood volumes. 2o ,ul blood is extracted with an organic solvent. Ether/acetone is used (N.C. Jain and P.L. Kirk, Microchem. J. 1_22, 249, 1967). Using special mlcrotechniques the extract is brought to 5 pl. With the aid of a newly developed "microrefluxer" (VV. DUnges, Anal. Chem., in press) the barbituric acids are alkylated, using a new derivatisation method ON. DLinges, Chromatographia, in press) which allows the direct injection of the reaction mixture. Qualitative and quantitative analyses appear to be possible since good recoveries and blank values have been achieved. Dr. W. DUnges, Pharmakologisches Institut der Universit~t Mainz, D 65 Mainz, Obere Zahlbacher Str. 67

EFFECTS OF LITHIUM ON CYCLIC 3~5'-AMP METABOLISM IN RAT BRAIN (Wirkungen von Lithium auf den Stoffwechsel von cyclischem3~'5CAMP im Rattenhirn) S. F. Berndt Since lithium has been well established as an effective therapeutic agent in the treatment of mania, it was of interest to examine its influence on cyclic 3',5'-AMP (cAMP) metabolism in brain. Using Gilman's protein binding method I1970), the cAMP levels were measured after acut and chronic administration of lithiumchloride (LICI) in rat frontal brain and its urinary excretlon. In addition we examined the influence of LICI on the activity of purified cAMP-phosphodiesterase (PDE) and adenyl cyclase in rat cerebral cortex homogenates. The intravenous as well as the intraperitoneal injection of LiCI (1 and 5 mmoles/kg) failed to change cAMP levels in rat frontal brain. Only after feeding the rats with 2 mmoles/kg of LICI for 8 days was a slight decrease in the cAMP content noticeable. In spite of this, the urinary excretion of cAMP did not differ from the controls. LICI showed neither an influence on the activity of the high km-phosphodiesterase nor on the low km-enzyme~ but inhibited the adenyl cyclase activity in a dose dependent manner. Our results suggest that the psychotropic effects of lithium are related to the decrease of cAMP content in brain by inhibition of brain adenyl cyclase activity. Dr. S. F. Berndt, Neurologische Klinik der Universit~t, D-8700 WUrzburg, Josef-Schneider-StraSe 2

R5 INHIBITION AND INDUCTION BY Hoe 879 OF MICROSOMAL DRUG-HYDROXYLATING ENZYME SYSTEMS IN MOUSE LIVER. F. E. Beyhi t E. Lindner Male white mice were iniected 150 mg Hoe 879 (2-ethyl-2-hydroxybutyric acid diethylamide) pro kg i. p. 40 minutes and 24 hours after the administration, Hexobarbital-sleeping time (I) and the activity of liver microsomal enzymes (N-Demethylase (II), 0-Demethylase (III), and NADPH:-Cytochrome C-0xidoreductase (IV)) and the cytochrom P-450 (V) content were determined. 40 minutes after in}ection I was increased, the activities of II and III were depressed, whereas IV and V remained constant. 24 hours later I was diminished and II, III, IV and V were increased. The sleeping times after other anaesthetics were influenced in the same manner (except of inhalation anaesthetics). Similar results were obtained from rat experiments. From this results, we conclude that Hoe 879, a fatty acid derivative, is capable to inhibit microsomal drug-hydroxylating enzyme systems (short time effect) and to induce their biosynthesis (long time effect). Dr. F. E. Beyhl, Farbwerke Hoechst A.G., Biochemisches Laboratorium,

6230 Frankfurt (M)

80.

DEVELOPMENT OF PHYSICAL DEPENDENCE ON MORPHINE IN RESPECT TO TIME AND DOSAGE AND QUANTIFICATION OF THE PRECIPITATED WITHDRAWAL SYNDROME IN RATS (tTber die Bedeutung van Zeit und Dosis bei der Entwicklung der Morphinabh~ngigkeit und Quantifizierung des provozierten Entzugssyndroms bei der Ratte) J.Blisiq,S.Zieqlq~nsberger. Different amounts of morphine pellets (containing 10-75 mg base) were implanted s.c. into rats at intervals of 1 (short-term experiments), 3 or 5 days (long-term experiments). The abstinence syndrome was precipitated by levallorphan or naloxone (0.01-i0 mg/kg i.p.)between 1 and 30 days after the ist implantation. The frequency of different symptoms was counted (e.g. jumping, teeth chattering, wet dog shaking, w r i t h i n ~ and the presence of others (e.g. diarrhoea, ptosis, chewing, scream on touch) was checked. The amount of morphine absorbed from the depot was determined. The short-term experiments show that not all the individual withdrawal symptoms increase monotoneously with the degree of physical dependence. While writhing and wet dog shaking are most intense in slightly dependent animals~ these symptoms decline or even vanish as dependence gets stronger and jumping increases. A similar shift of symptoms was obtained when higher doses of the antagonist were applied. In the long-term experiments some symptoms (e.g. jumping, teeth chattering) tended to plateau while others decreased (wet dog shaking) or increased (writhing). Because of the interference between the various symptoms a quantification of the withdrawal by simple scoring seems impossible. Dr. J. Bl~sig, Max-Planck-Institut 40, Kraepelinstra~e 2

fur Psychiatrie,

8000 M~nchen

R6 CHANGES ! N S U B S T R A T E S P E C I F I C ! T Y O F LIVER MICROSOMALUDP-GLUCURONYLTRANSFERASE(S) AFTER TREATMENT OF RATS WITH PHENOBARBITALOR 3 - M E T ~ 0 L A N T ~ (~uderung der Substratspezifit~t derUDP-glucuronyltransferase(n) in Lebermikrosomen dutch Vorbehandlung yon Ratten mit Phenobarbital oder 3-Methylcholanthren) K. W. Bock, I. Abt, B. Rexer Male Sprague-Dawley rats were treated either with phenobarbital (I00 mg/kg, daily) or with 3-methylcholanthrene (80 mg/kg) and liver microsomes were prepared on the 4th day. After phenobarbital treatment glucuronyltransferase activity increased differently depending on the substrate: ca. 50 % with pnitrophenol and 1-naphthol, ca. i00 % with bilirubin and ca. 350 % with chloramphenieol. Methylcholanthrene treatment did not alter glucuronyltransferase activity with chloramphenicol and bilirubin whereas enzyme activity with pnitrophenol and 1-naphthol was increased ca. 300 %. These changes in enzyme activity were found in freshly prepared or in 0.05 % (w/v) Triton X-IO0 activated microsomes as well as in desoxycholate solubilized microsomal preparationa The increase in enzyme activity was prevented when protein synthesis was inhibited by cycloheximide. In addition, kinetic analysis of the enzyme indicates that the altered enzyme activity is not correlated with a marked change in the apparent K M for UDP glueuronic acid and p-nltrophenol. The results point to distinct differences between the enzyme(s) glucuronylating p-nitrophenol or 1-naphthol on one side and chloramphenicol or bilirubin on the other side. Dr. K. W. Bock, Institut fur Toxikologie der Universit~t, D-7400 TUbingen, Wilhelmstra~e 56

UPTAKE AND METABOLISM OF ISOPRENALINE IN THE ISOLATED PERFUSED HEART O F T H E RAT AND GUINEA PIG (Aufnahme und Metabolisierung von Isoproterenol im isoliert Perfundierten Ratten- und Meerschweinchenherzen) H. BSnisch, W. Uhli$ 3 Isolated hearts were perfused with (Z) H-isoprenaline (3H-ISO) and the removal of the a~ine from the mediumas well as the formation ~f the O-methylated metabolite (~H-OMI) was measured. On perfusion with I0 -M -H-ISO the rate of removal of amine declined exponentially and reached a constant rate after 10-20 min corresponding to the rate of production of H-OMI. In the rat heart this rate was lO.times higher than in the guinea-pig hear~. In the presence of U-0521 (IO-4M) to block COMT the rate of removal of H-ISO declined exponentially to zero in both spcies. For kinetic analysis of removal and O-methylation of 3H-ISO the rat heart was used. Initial ra~es of removal, determined by extrapolation of the removal rate obeyed Michaelis-Menten~kinetics (app. K = 20xlO VM. V = 38 nmoles/min.g). 9 J . m . x . . . . The total formatlon of H-OMI was llnear over a perzod om~ 30 mln zndzcatzng a constant rate of O-methylation during this time. This rate of O-methylation was dependent on the amine concentration in the medium and showed saturation kinetics (app. K = 5xlO- M, V = 1.8 nmoles/min.g). 9 m . . m g x . .. Cortlcos~erone was a competltlve inhlbltor of both the removal and O-methylation of H-ISO. Rat hearts were perfus~d with IO-6M 3H-ISO for 30 min and then washed with amine-free medium ( I O - M U-O521 present throughout). After clearance of the extracellular space efflux was biphasie (t/2 = 9 and 21 min, respectively). When corticosterone was present during the wash out, efflux was monophasic (t/2 = 29 rain). H. BSnisch,/Institut fHr Pharmakologie und Toxikologie der UniversitNt, 87 WGrzburg, Koellikerstr. 2

RZ THE ACCUMULATION P A ~ 0 F E T H ~ T R A D I O L A N D M E S T R A N O L M E T A B O L I T E S IN RELATION TO COVALENT PROTEIN BINDING (Kovalente Proteinbindung von Mestranolund AthinylSstradiolmetabollten als Ursache ihres Kumulationsverhaltens) H. M. Bolt Accumulation of the radioactivity of 4-14C-estradiol, 6,7-3H-ethynylestradiol and 6,7-3H-mestranolunder chronic treatment (2.5 Mg daily for 19 days) was studied in brain, ~ m g and liver of mice. Compared with estradiol, ethynylestradiol and mestranol caused 2-3 fold higher levels of radioactivity in these organs. After ending administration, the radioactivity of ethynylestradiol and mestranol disappeared within 4 days. Comparison with previous results obtained after a single dose presents evidence that an accumulation of metabolic products occurs during treatment with ethynylestradiol or mestranol. In contrast to 4-14C-estradiol, radioactivity in the liver during chronic treatment with 6,7-3H-ethynylestradiol or 6,7-3H-mestranol was tightly bound to tissue and could not be removed by solvent extraction or hot acid hydrolysis. Details are given elsewhere ("Xenobiotica" 2, 489~ 1972). In addition, liver microsomes were prepared from rats dosed wit/~6,7-2H-estradiol and 6,7-3H-ethynylestr~liol 48 h before sacrifice. After removal of the labile metabolites from the microsomes with dextran-coated charcoal the amount of e~vnylestradiol r~lioactivity firmly bound to the microsomes was twice as much as that found after dosing with3H-estradiol. Further evidence for the kind of the retained ethynylestradiol radioactivity was obtained by trypsin treatment of the microsomes and chromatography of the tryptic peptides on DOWEX resin, suggesting a covalent binding of ethynylestradiol metabolites to the microsomal proteins. Dr. H. M. Bolt, Institut ~dr Toxikologie der Universit~t, D-7400Tdbingen, Wilhelmstra~e 56

D O S I S A B H ~ N G I G E R E I N F L U S S VON A D R E N A L I N UND N O R A D R E N A L I N AUF DIE ~ I C H T I N D U Z I E R T E N E T Z H A U T A K T I V I T A T ( The d o s e - d e p e n d e n t i n f l u e n c e of e p i n e p h r i n e and n o r e p i n e p h r i n e on the retinal a c t i v i t y after light s t i m u l a t i o n ) U. B o r c h a r d U n t e r s u c h u n g e n von H ~ g g e n d a l und M a l m f o r s (1963) haben gezeigt, dab C a t e c h o l a m i n e in g e r i n g e n M e n g e n im z e n t r a l n e r v ~ s e n N e t z h a u t gewebe vorkon~en. Uber den Einflu8 von A d r e n a l i n und N o r a d r e n a l i n auf aas ~ e l i c h t u n g s p o t e n t i a l (ERG) b e s t e h e n w i d e r s p r O c h l i c h e Aussagen. Die n a c h R e s e r p i n a p p l i k a t i o n b e o b a c h t e t e a n f ~ n g l i c h e Potentialsteigerung, gefolgt yon einer langsamen stetigen P o t e n t i a l a b na~ne, legte den SchluB nahe, dab die b i o g e n e n Amine eine direkte ~ i r k u n g auf die n e u r o n a l e n S t r u k t u r e n der N e t z h a u t ausdben. Wir unt e r s u c h t e n daher die d o s i s a b h ~ n g i g e W i r k u n g von A d r e n a l i n und Nora d r e n a l i n auf das ERG der i s o l i e r t e n F r o s c h r e t i n a . Unsere Ergebnisse m a c h e n deutlich, dab A d r e n a l i n in einem K o n z e n t r a t i o n s b e reich von o,oi bis io ~Mol/l eine zunehmende S t e i g e r u n g der Potent i a l g r O B e bewirkt, die yon der L i c h t i n t e n s i t ~ t a b h ~ n g i g ist. Bei loo pMol/l erfolgt eine schnelle, sich als r e v e r s i b e l e r w e i s e n d e P o t e n t i a l a b n a h m e . Fdr N o r a d r e n a l i n gelten analoge Aussagen, w e n n auch die b e o b a c h t e t e Zunahme des B e l i c h t u n g s p o t e n t i a l s g e r i n g e r ist als fHr Adrenalin. Die V e r s u c h s e r g e b n i s s e liefern eine E r k l ~ rung fdr die b i s h e r b e s c h r i e b e n e n w i d e r s p r H c h l i c h e n Befunde. Die F o r m v e r ~ n d e r u n g des ERG l~St sich auf Grund einer K o m p o n e n t e n a n a lyse des P o t e n t i a l v e r l a u f s mit dem u n t e r s c h i e d l i c h e n E i n f l u B der C a t e c h o l a m i n e auf die T e i l k o m p o n e n t e n des B e l i c h t u n g s p o t e n t i a l s deuten. Dr. U. Borchard, P h a r m a k o l o g i s c h e s K~in. 5 K61n 41, G l e u e l e r Str. 24

Institut der U n i v e r s i t ~ t

zu

R8

THE BINDING OF BENZYLPENICILLIN TO THE SURFACE OF SENSITIVE A N D RESISTANT STAPHYLOCOCCI (Die Bindung von Benzylpenicillin an die 0berfl~chenstrukturen von sensiblen und resistenten Staphylokokke~ W.Bruns~ H.Keppeler Auf Grund frSherer Befunde kSnnen wir fSr die penicillinase-unabh~ngige Form der Penicillin-Resistenz eine Permeationsbarriere d i ~ kutieren. Wir haben deshalb die Bindung yon 14C-Benzylpenicillin (14C-BP) bei 2 sensiblen (SG 511 und S 5939) und 2 resistenten Staphylokokkenst~mmen (P 2o9 u. Oe 45o3) vergleichend untersucht und insbesondere das Verhalten von Zellwand (ZW) und Zytoplasmamembran (PM) studiert. Die Bakterien (io mg/ml) wurden in Puffer, pH 7,3, mit 0,6 pg/ml 14C-BP bei 37oc 3o und 6o Min. inkubiert. Die Gewinnung der ZW erfolgte nach Bleiweis et al. (J.Bacteriol. 88, 1198 (1964)), die der PM nach Hitachi et al. (Biken J. 14, ii (1971)) unter Verwendung von Lysostaphin. Bei den sensiblen St~Lmmen betrug die Bindung an die ZW durchschnittlich 0,045 pg/g Bakt.-Trockengew. (BTG). Die PM nahm 0,o6 ~ g / g BTG auf. Das Maximum der Bindung war nach 30 Min. erreicht. Bei den resistenten St~mmen lagen die 3o Min.-Werte n i e d r b g e r u n d zeigten eine Abh~ngigkeit vom Resistenzgrad. Und zwar wurden an die ZW nur 5 % bzw. 10% und an die PM 20% und 65% gebunden. Die Aufnahme war hier nach 30 Min. noch nicht abgeschlossen. Doch auch die 60 Min.-Werte lagen noch erheblich unter den Maximalwerten der sensiblen St~tmme. Ein weiterer Unterschied bestand darin, dab bei den resistenten Staphylokokken die Gesamtaufnahme an 14C-BP um den Faktor 3 bzw. Io vermindert war. rlv.Doz.Dr.W.Bruns, ?harmako!ggisches Institut der Universit~t, -~ooo K61n 41, Gleuelers~r. z~

~

EVIDENCE FOR THE PRESENCE OF ~-RECEPTORS IN HUMAN SAPHENOUS AND DOG FEMORAL VEINS (In vitro-Nachweis yon ~-Rezeptoren in der Vena saphena des Menschen und der Vena femoralis des Hundes) Th.J.Bucher, E.MHller-Schweinitzer, E.Stdrmer Freshly obtained human saphenous veins (normal and varicous) and canine femoral veins were helically cut and immediately suspended in organ baths (Krebs-Henseleit solution, 37~ 95 % 0 2 5 % CO2, tension 0.5 g). Changes in the tone of the preparatio-ns were monitored isometrically by a Statham force transducer and a Linseis multichannel writer. The submaximal contractions of equilibrated vein strips elicited by 10 -7 g/ml noradrenaline were relaxed by the subsequent addition of isoprotere~ol. Pretreatment with either 3xlO -6 g/ml pronethalol or 3xlO- g/ml pindolol inhibited the relaxing effect of isoproterenol. The results provide further evidence for the existence of ~2receptors in veins. Dr.E.StUrmer, Medizinisch-Biologische CH-4OO2 Basel

Forschung,

Sandoz AG,

Rg EFFECTS OF RESERPIN~ AND PRENYLAMINE ON THE NORADRENALINE FLUXES OF ISOLATED SPLENIC NERVE GRANULES (Wirkung von Reserpin und Prenylamin auf die Aminfluxe isolierter Noradrenalingranula aus Milznerven) A. Burger Noradrenaline (NA) granules prepared from postganglionic sympathetic splenic nerve trunks of cattle, contalnung > 5 nmoles NA/mg protein, exchanged 85% of their endogenous NA during incubation with ATP-Mg ++ and NA. The granules were preloaded with labelled NA and the unidirectional NA-fluxes were calculated by measuring the NA content and the exponential decline of the radioactivity of the granules during incubation with ATP-Mg ++ and unlabelled NA. Identical flux measurements were obtained when the initial linear increase of the radioactivity was measured in NA-equilibrated granules exposed to tracer amounts of labelled NA. Preincubation (15 min, O~ and subsequent incubation (20 min, 28~ with reserpine or prenylami~e (IO-SM - Io-bM either) did not influence the NA content of the granules, while inhibiting, to the same extent, both influx and efflux. In the absence of drugs, granules incubated with 1.5.10-5M NA and ATPMg ++ exchange 16.5 + 2.6 ng NA/mg protein/min (~ + s~; n = i0). Under these conditions Io-bM reserpine or prenylamine completely blocked the NA-exchange. The IC50 for both drugs was about IO-7M. In the presence of 1.5.10-5M NA, Io-SM of either drug did not influence the fluxes, whereas at I.IO-6M NA (exchange in the absence of drugs: 11,4 ~ 3,1 ng NA/mg protein/min; n = 8) this concentration of both drugs inhibited the fluxes by 30-40%. Dr. A. Burger, Institut fHr Pharmakologie und Toxikologie der Universit~t, D-8700 WHrzburg, Koellikerstr. 2

DIFFERENCES IN DOSE-RESPONSE CURVES OBTAINED BY CUMULATIVE AND N O N CUMULATIVE TECHNIQUE (Abh~ngigkelt der Dosis-Wirkungs-Kurven vom methodischen Vorgehen) G.O. Carrler, Y. Lezard and A. Ziegler According to the occupation drug-receptor theory identical dose-response-curves (D-R-C) should be obtained when applying the drug in a cumulative or a non-cumulatlve manner. Consequently most investigators (see i.e. van Rossen, Arch. Int. Pharrnacodyn. 143,299, 1963) have favoured the cumulative technique for methodical reasons. Recently evidence was presented which provided doubts on the validity of the occupation theory (see i.e0 Ziegler, Acta pharmacol, toxicol. 2.99r 65, 1971). It seems more reasonable to assume that the effect is correlated to a concentration of a transient rather than a stable drug receptor complexr since otherwise experimentally observed desensitization remains ununderstandable. The desensitization phenomenon becomes more pronounced with increasing drug concentrations and time of exposure. Using isolated longitudinal ileal muscle strips (isotonic and isometric recordings) and atria of gulnea-plgs~ D-R-C's for chollnerglc drugs (carbachol, pilocarplne~ arecaidlne-butyl-ester, N-methyl-pyrrolldinol-acetate-CH 3 J) were established by the two different procedures mentioned. Pronounced differences (ED50 and maximum response) became evident when the D-R-C's obtained by the different techniques were compared. In addition, the shape of the cumulative D-R-C's depended not only on the drug in questlon but also on the dose schedule (concentration and time interval). It is concluded that the data to evaluate "parameters" like affinity and intrinsic activity depend upon the method of determination. The validity of these "parameters" to characterize a drug, therefore become:; highly questionable. Dr. A. Ziegler, Instltut fL~r Pharmakologie, Christian-Albrechts-Universit~t 2300 Kiel, Hospitalstr. 4-6 West Germany

R 10

A SIMPLE AND RAPID SPECTROPHOTOMETRIC DETERMINATION OF MONOAMINE OXIDASE ACTIVITY (Eine einfache und schnelle spekfrophofometrische Bestimmungsmethode der Monoamin-Oxidase)W. Christ~ D. Rakow This method is based on the high extinction of 4-hydroxy-3-methoxy-benzaldehyde (vanillin) in alkali solution at 345 nm. This product is formed in the course of action of MAO on 4-hydroxy-3-methoxy-benzylamine when used as substrate. Tissues (brain, heart and liver) are homogenized in isotonic KCI. Incubation mixtures consisted of enzyme (20 - 50 mg wet weight) and from 5 . 10-7 moles to 5 9 10-6 moles of 4-hydroxy-3-methoxy-benzylamine in a total volume of 5.0 rnl (phosphate buffer 0.1 MS pH 7.0). Samples were incubated for 30 minutes at 37~ and the reaction was stopped by the addition of HCIO 4 (60 %). 4-Hydroxy-3-methoxy-benzaldehyde formed was extracted into toluene and from toluene re-extracted into i m K2CO 3 solution. The K2CO 3 solution was assayed for the product at 345 nm. The reaction was linear with time for at least 50 rain and with enzyme concentration over a range of I0 - i00 mg of tissue (brain, heart, liver). 4-hydroxy-3-methoxy-benzylamine is readily degraded by MAO of brain, heart and liver - but not of blood serum - and its

primary product~ the vanillin, does not s e e m to undergo further enzymic oxidation. The enzyme specificity of this assay was investigated by determining the effect on rat brain m o n o a m i n e oxidase activity of pretreatment in vivo with iproniazid, pargyline~ tranylcypromine and amphetamine. Dr. W. Christ, Institut ffir Neuropsychopharmakologie der Freien Universit~ Berlin, D-looo Berlin 19, Ulmenallee 30

RESORPTION AND STORAGE OF MAGNESIUM AFTER THE ADMINISTRATION OF MgC12, MgSO4 AND OF Mg-ASPARTATE AS BASE OR AS ITS HALOGEN HYDROGEN ACID COMPOUNDS (Resorption and Speicherung von Magnesium nach Zufuhr yon MgCI~, MgSO4 sowie yon Mg-Aspartat als Base oder in Form yon Halogenwasserstoffve-rbindungen) H.-G. Classen, M. Sp~ith The enteral resorption of MgC12 (I), MgSO4 (II), Mg-aspartate (IH), Mg-aspartate-HF (IV}, Mg-aspartate-HC1 (V), Mg-aspartate-HBr (VI) and Mg-aspartate-HJ (VII) was studied in cats and rats. - In cats the continously determined Mg--content of the whole blood increased by 0.2 to 0.4 mg% after the intraduodenal administration of 10 mg/kg Mg given as (I), (IH), (IV), (V) and (VII); (II) proved tess and (VI) most active (+0, 6 mg%). In rats measurable increases in plasma-Mg were found after 250 mg/kg Mg per os or 125 mg/kg Mg applied into the duodenum: again, (II) was absorbed less, followed by (IH), (IV) and (VII); (I), (V) and (VI) proved most effective. In rats the administration of daily 2x250 mg/kg Mg during 2 days showed that (I), (V) and (VI) increased the Mg-content of plasma and bones significantly more than (III). Comparable Mg-eoncentrations in plasma and bones were found after (I) and (V); however animals treated with (I) scarcely gained body weight in contrast to (V) indicating that, for therapeutical purposes, (V) might be superior to (1), (II), (III), (IV) and (VH). - T h e highest Mg-concentrations were observed after (VI), however, these animals were remarkably_ sedated, an effect which could be reduced by the additional administration of C1 . Univ. -Doz. Dr. H. -G. Classen, Iastitut fur experimentelle Therapie der Universitiit D-78 F r e i b u r g / B r . , Hugstetter Stra6e 55

R 11 CHEMOTACTIC AND PERMEABILITY-INCREASING E F F E C T S OF H I G H L Y P U R I F I E D H O G A N A P H Y L A T O X I N IN V I V O ( C h e m o t a k t i s c h e und p e r m e a b i l i t i t s s t e i g e r u d e W i r k u n g yon h o c h g e r e i n i g t e m S c h w e i n e - A n a p h y l a t o x i n in vivo) B. D a m e r a u , W. V o g t H i g h l y p u r i f i e d h o g s e r u m a n a p h y l a t o x i n (AT) a b i o l o g i c a l l y active c l e a v a g e p r o d u c t of the fifth c o m p o n e n t of c o m p l e m e n t - was s t e r i l i s e d b y m e m b r a n e f i l t r a t i o n and infused s u b c u t a n e o u s l y into the b a c k of g u i n e a pigs,. At a rate of as little as 1.8 #g AT/h (0.2 ml/h) infused for 10 hours, a dense a c c u m u l a t i o n of PMN leuk o c y t e s was p r o d u c e d at the site of injection. In h i s t o l o g i c a l s e c t i o n s from c o n t r o l i n f u s i o n s with sterile i s o t o n i c saline ~nly a few P M N ' s were found at most. Single i n j e c t i o n s of A T (10 and 20 ~g) into the p l e u r a l c a v i t y of g u i n e a pigs p r o d u c e d an e x u d a t e rich in l e u k o c y t e s a f t e r 4 hours. Both the n u m b e r of cells and the v o l u m e of e x u d a t e i n c r e a s e d with the dose o f AT given. I n j e c t i o n s of b r a d y k i n i n (18 Bg) led to ~ u ~ a c c u m u l a t i o n but no l e u k o c y t e e m i g r a t i o n The a m o u n t of E v a n s blue which a f t e r i.v. i n j e c t i o n d i f f u s e d into the p l e u r a l c a v i t y was i n c r e a s e d by i n t r a p l e u r a l A T i n d i c a t i n g an i n c r e a s e in local v a s c u l a r p e r m e a b i l i t y . T h i s effect lasted for a bout 3 hours. ~y a s s a y on g u i n e a pig ileum AT a c t i v i t y was r e g u larly d e t e c t e d in the p l e u r a l fluid 2 h o u r s a f t e r i n j e c t i o n , b u t only in I out of 6 e x p e r i m e n t s a f t e r 4 hours. The r e s u l t s i n d i c a t e that hog s e r u m AT is c h e m o t a c t i c a l l y a c t i v e and i n c r e a s e s p e r m e a b i l i t y , in vivo, at c o n c e n t r a t i o n s which m a y well be a t t a i n e d by i n f l a m m a t o r y s t i m u l i u n d e r n a t u r a l conditions. Dr. B. D a m e r a u , A b t e i l u n g B i o c h e m i s c h e P h a r m a k o l o g i e , M a x - P l a n c k Institut fur e x p e r i m e n t e l l e M e d i z i n , D - 3 4 0 0 G S t t i n g e n , H e r m a n n Rein-StraBe 3

T H E E F F E C T OF P R O B E N E C I D ON THE I N T E S T I N A L A B S O R P T I O N OF C A R D I A C G L Y C O S I D E S . ( E i n f l u S y o n P r o b e n e c i d auf die e n t e r a l e R e s o r p t i o n yon H e r z g l y k o s i d e n ) K.H.Damm, W.Braun

The i n t e s t i n a l a b s o r p t i o n of 3 H - D i g i t o x i n , D i g o x i n and O u a b a i n (30 ~ g / m l ) has b e e n s t u d i e d d u r i n g 90 m i n p e r i o d s by m e a n s of e v e r t e d sack p r e p a r a t i o n of the m o u s e gut u n d e r the i n f l u e n c e of P r o b e n e c i d . As k n o w n f r o m o t h e r s p e c i e s a b s o r p t i o n was h i g h e s t w i t h D i g i t o x i n . D i g o x i n and O u a b a i n were only p o o r l y a b s o r b e d . The D i g i t o x i n t r a n s p o r t rate was r e d u c e d in the cold (0 ~ C ) , w i t h nitrogen,Dinitrophenol and in the a b s e n c e of g l u c o s e . P r o b e n e c i d was fo u n d to have s t r o n g - r e v e r s i b l e - i n h i b i t o r y e f f e c t s only in the t r a n s p o r t of D i g i t o x i n but not on D i g o x i n and O u a b a i n abs o r p t i o n . I00 m g % (3,5 mM) P r o b e n e c i d r e d u c e d the D i g i t o x i n t r a n s p o r t rate to a b o u t 30% of c o n t r o l v a l u e s w h e r e a s w i t h 5 0 m g % P r o b e n e c i d the i n h i b i t i o n was still h i g h e r than 50%~ B e s i d e s on D i g i t o x i n transport, P r o b e n e c i d was f o u n d to have i n h i b i t o r y effects also on g l u c o s e and w a t e r a b s o r p t i o n ~ W i t h all P r o b e n e c i d e f f e c t s a c e r t a i n d e p e n d e n c e on p o t a s s i u m m e d i u m c o n c e n t r a t i o n was o b s e r v e d : D o u b l i n g the p o t a s s i u m conc e n t r a t i o n r e s u l t e d in h i g h e r i n h i b i t o r y r a t e s of D i g i t o x i n absorption. T h e s e r e s u l t s s u g g e s t that like in b i l i a r y e x c r e t i o n d i f f e r e n t t r a n s p o r t m e c h a n i s m s s e e m to m e d i a t e D i g i t o x i n a g a i n s t D i g o x i n and O u a b a i n a b s o r p t i o n . S i m i l a r r e s u l t s were o b s e r v e d u n d e r in v i v o c o n d i t i o n s in rat e x p e r i m e n t s . Dr.KoH.Damm, Pharmakologisches Institut D - 2 0 0 0 H a m b u r g 20, M a r t i n i s t r a S e 52

der U n i v e r s i t ~ t

Hamburg

R 12 THE ACTION OF D I M E T H Y L S U L F O X I D E (DMSO) ON THE RESTING P O T E N T I A L (RP) OF I S O L A T E D FROG NERVE ( Uber die W i r k u n g yon D i m e t h y l s u l f oxid auf das R u h e p o t e n t i a l des i s o l i e r t e n F r o s c h n e r v e n ) E.Ch. Dittmann, W. H e n n i @ e s To e l u c i d a t e the action of DMSO on the rp of the i s o l a t e d frog nerve we used the sucrose gap m e t h o d (St~mpfli 1954). Since DMSO proved to be a d e p o l a r i z i n g agent we c o m p a r e d its e f f i c a c y w i t h that of p o t a s s i u m chloride (KC1). Our e x p e r i m e n t s w e r e p e r f o r m e d on d e s h e a t h e d N . i s c h i a d i c u s of rana temporaria. Using nerves from w i n t e r - and summer-frogs, 3 o % - D M S O - R i n g e r s o l u t i o n caused no different d e p o l a r i s a t i o n s w h e r e a s the rp by 2o mMol/l K C i - R i n g e r solution was s i g n i f i c a n t l y more d e p o l a r i z e d in w i n t e r - than in summernerves. The v e l o c i t y and course of DMSO- and K C l - d e p o l a r i s a t i o n s were f u n d a m e n t a l l y different. E x a m i n i n g the e f f i c a c y of DMSO as a function of time the nerves were p e r f u s e d for about 5 or 20 min with 30% D M S O - R i n g e r solution; following K C l - d e p o l a r i s a t i o n s in w i n t e r - and s u m m e r - n e r v e s w e r e rev e r s i b l y m o d i f i e d only by 5 min e x p o s u r e to DMSO. In s u m m e r - n e r v e s w i t h 20 min e x p o s u r e to DMSO the f o l l o w i n g p e r f u s i o n w i t h Ringer solution frequently caused a d e p o l a r i s a t i o n . F r o m the data obtained we p o s t u l a t e d i f f e r e n t s t r u c t u r e s and k i n e t i c s in the m e m brane by w h i c h the DMSO- and K C l - d e p o l a r i s a t i o n is m e d i a t e d and supported. A p o s s i b l e m o d e of action of DMSO may be a s p e c i a l i z e d d i f f u s i o n of d i m e t h y l s u l f a t i z e d K--ions through a p o s s i b l e leakage in the membrane. The season d e p e n d e n t K C l - e f f e c t s may be contributed to seasonal changes in m e m b r a n e ATPase a c t i v i t y (Kennedy and Nayler 1965). Dr. D r . E . C h . D i t t m a n n , P h a r m a k o l o g i s c h e s Institut der U n i v e r s i t ~ t zu K61n, 5 KOln 41, G l e u e l e r Str. 24

MUSCARINIC INHIBITION OF NORADRENALINE RELEASE EVOKED BY POTASSIUM IONS. M.P. Dubey, E. Muscholl In previous investigations it was shown that the noradrenaJine (NA) release from the perfused rabbit heart evoked by sympathetic nerve stimulation or nicotinic drugs is dose-dependently decreased by muscarinic compounds. In the present experiments rabbit hearts were perfused at 35~ with a constant volume of 30 ml/min. NA release into the perfusate was elicited by raising the potassium concentration of the Tyrode solution to 135 mEq,/l for 10 min (equivalent substitution of NaCI by KCI). From 0 - 10 min the perfusates contained 1956 + 132 ng NA (mean + S.E., n = 5). In the presence of atropine (1.4 x 10-6 M) the NA releasing effect of high potassium was unaltered (1810 + 188 ng; n = 3). Various concentrations of methacholine (MCh) were added 2 minbefore and during perfusion with 135 mEq K+/'I. While 1.25 and 5 x 10- 6 M MCh did not significantly alter NA release after potassium (2063 + 167, n =5 and 2484 + 289 ng, n = 6, respectively), 4 x 10-5 and 3.2 x 10-4 M-MCh decreased NA release dose-dependently (1009 + 65, n = 3 and 545 + 85 ng, n = 3, respectively). The inhibition of NA release b y MCh (3.2 x 10-4 /V~ v~s fully reversed by perfusion with atropine, 1.4 x 10-6 M (2381 + 91 ng, n = 4). - Since potassium releases NA independent of propagation of impulses along the axons the muscarintc inhibition must have occurred at the terminal adrenergic fibre. Similarly, the muscarinic inhibition of NA release after nerve stimulation or nicotinic drugs may occur at the site of the physiological stimulus-secretion process. Dr. M.P. Dubey, Pharmakologlsches lnstitut der Universit~it D-65oo Mainz, Obere Zahlbacher Str. 67

R 13

THE BIOLOGICAL DETERMINATION OF CATECHOLAMINES IN URINE FOR THE DETECTION OF PHEOCHROMOCYTOMA (DiebiologischeBestimmung yon Catecholaminen im Urin zum Nachweis eines Ph~iochromocytoms) H. Ebel, P._:_.Marquardt, K.-H. l>ieper Analogicalrecordings of changes in pulse-rate, blood-pressure and blood-pressuree f f e c t s ~ f A p (t) ] i n cats allow a s e m i - q u a n t i t a t i v e d s t e r m i n a t i o n of c a t e c h o l a m i n e s (CA) in u r i n e down to c o n c e n t r a t i o n s of about O. 5 # g / m l . P r e t r e a t m e n t of the a n i m a l s with a d r e n e r g i c b l o c k i n g agents and the continuous m e a s u r e m e n t of g l u c o s e - and p o t a s s i u m - c o n c e n t r a t i o n s i n whole blood (Auto-Analyzer) a r e a d a e q u a t e m e t h o d s of d e t e r m i n i n g and d i f f e r e n t i a t i n g e p i - and n o r e p i n e p h r i n e . - I m m e d i a t e a c i d i f i c a t i o n of the u r i n e (2n HC1) is n e c e s s a r y to s t a b i l i z e CA, a pH b e t w e e n 3 . 0 and 2 . 0 b e i n g o p t i m a l . C i r c u l a t o r y effects due to h i s t a m i n e c a n be b l o c k e d by m e p y r a m i n e m a l e a t e . T o exclude o t h e r i n t e r f e r i n g effects no d r u g s should be t a k e n d u r i n g the w e e k p r e c e d i n g the c o l l e c t i o n of the u r i n e . - In the l a s t 13 y e a r s the u r i n e s of 1260 p a t i e n t s w e r e e x a m i n e d : 13 c a s e s of p h e o e h r o m o c y t o m a w e r e detected; 10 t u r n o u t s w e r e v e r i f i e d l a t e r by o p e r a t i o n o r autopsy. T h e e x c r e t i o n of CA was : > 1000 /~g i n 6 p a t i e n t s ; 750 to l o o o pg: 1 p a t i e n t ; 500 to 750 #g: 1 patient; 250 to 500 /~g: 1 p a t i e n t . In 1 c a s e the e x c r e t i o n was found to be l e s s t h a n 260 /~g/24 h. One r e c u r r e n c e ( p h e o c h r o m o b l a s t o m a ) is included in t h e s e 13 c a s e s ; 2 p a t i e n t s w e r e r e l a t i v e s . - T h e C A - c o n t e n t of 8 t u m o u r s was e x a m i n e d biologically: C o n c e n t r a t i o n s v a r i e d b e t w e e n 0 . 0 5 and 7 . 4 m g C A / g t u m o u r d r y - w e i g h t ; 1 t u r n o u t c o n t a i n i n g s o l e l y e p i n e p h r i n e and two o t h e r s s o l e l y n o r e p i n e p h r i n e ( c o r r e s p o n d i n g to the e x c r e t i o n found in the u r i n e s ) . It s h o u l d b e noted t h a t the e x c r e t i o n of v a n i l l y l m a n d e l i c acid was < 10 r a g / 2 4 h i n 5 of the iO p a t i e n t s . H. E b e l . I n s t i t u t f . e x p . T h e r a p i e d. Universit~it, D-78 F r e i b u r g / B r . , H u g s t e t t e r S t r .

ON THE SIGNIFICANCE OF ADENOSINE IN THE REGULATION OF CYCLIC AMP LEVELS AND LIPOLYSIS IN ISOLATED FAT CELLS (Uber die Bedeutung des Adenosins FUr die Kontrolle des cyclischen AMP-Spiegels in isolierten Fettzellen) R. Ebert Stimulation of cyclic AMP accumulation and glycerol production by hormones was dependent on the concentration os fat cells in the incubation medium. After addition os noradrenaline cyclic AMP levels in diluted fat cell suspensions (20 000 cells/ml) reached I0-s hi/her levels and declined much more slowly than in concentrated cell suspensions (100 000 cells/ml). An inhibitor was released from the fat cells into the medium, which caused a dose-dependent inhibition. Analysis of the medium by acid extraction, gel filtration, thin layer chromatography, and gas chromatography showed that the inhibitor released was adenosine. This nucleoside r e , b e d maximal levels (0,2 nmol/ml/105 cells) in the medium after 20 min of incubation of the Fat cells. Addition os 0,01 to 0,1 bM os adenosine decreased cyclic AMP accumulation and lipolysis induced by noradrenaline. Although dipyridamole completely blocked the uptake os adenosine-3H, it was unable to counteract the effect os this nucleoside on lipolysis and cyclic AMP accumulation; hence, the site os action os adenosine might be on the cell membrane. It is tentatively suggested that adenosine released from Fat cells plays an important role in the regulation of cyclic AMP accumulation and lipolysis. Dr. R. Ebert, Institut fur Pharmakologie, Medizinische Hochschule Hannover, 3000 Hannover-KleeFeld, Karl-Wiechert-Allee 9

R 14

EFFECT OF CORTICOSTERONE ON EXTRANEURONALUPTAEE IN, AND EFFLUX •H(+)-NORADRENALINE H (NA) FROM RABBIT E AORTIC STRIPS (Der3Effekt yon

OF Corticosteron-auf die extraneuronala Aufnahme und den Efflux yon H(~)-Noradrenalin am isollerten Aortenstreifen des Kaninchens) E. Eckert, M. Henselin$

Aortic strips were either obtained from reserpine-pretreated rabbits or made "nerve-fr~e" according to Maxwell and Bevan (1968). They were exposed to 200 ng/ml of ~H-NA for 30 min and then washed with amine-free solution in order to study efflux. Cocaine (I0 ~g/ml) was present throughout the whole experiment in order to block residual neuronal uptake. When COMT was blocked by U-0521 (18 ~g/ml), accumulation of total radioactivity at the end of the incubation was nearly equal after reserpine pretreatment and in "nerve-free" preparations (T/M ratio of about 1.6). Under these conditions corticosterone (C)(30 wg/ml) reduced accumulation of radioactivity by 40-50%. The effect of C on the production of deaminated metabolites was much more pronounced than on accumulation of unchanged amine. Apparently, there is extraneuronal MAO, and access to at least part of the enzyme activity is sensitive to C. After clearance of the extracellular space, efflux originated from at least 2 compartments. When C was added during efflnx only, the rate of efflux of total radioactivity was slowed during the first 30 min and accelerated subsequently, The results are compatible with the view that C increases the t/2 of the efflux from the compartment characterized by a short t/2; C seems to have little or no effect on the efflux from the second compartment (long t/2). Moreover, during the late phase of efflux C changed the efflux ratio "noradrenaline/deaminated catechols" in favor of noradrenaline. Dr. E. Eckert, Institut fGr Pharmakologie und Toxikologie der Universit~t, 87 Erdrzburg, Koellikerstr. 2

ANTIPYRINEHALF-LI~E TIME, A N T I P Y R I N E M E T A B O L I S M AND THYROID F U N C T I O N IN N A N ( A n t i p y r i n h a l b w e r t s z e i t , Antipyrinmetabolismus und S c h i ! d d r G s e n f u n k t i o n beim Menschen) N. E i c h e l b a u m ~ G. Bodem und Ch. S c h n e i d e r - D e t e r s A n t i p y r i n e plasma hs~--T~-l-ife time and cumulative u r i n a r y excretion of 4 - h y d r o x y s n t i p y r i n e (4-OH-A) has b e e n studied in patients s u f f e r i n g from h y p e r t h y r e o s i s or h y p o t h y r o i d i s m f o l l o w i n g a single oral dose of Sooomg of a n t i p y r i n e (A). In each patient tO.5 and cumulative urinary e x c r e t i o n of 4-OH-A was studied during h y p e r t h y r e o s i s or h y p o t h y r o i d i s m and after n o r m a l l z s t i o n of t ~ e t h y r o i d f u n c t i o n by s t r u m e c t o m y end s u b s t l t u t l o n w i t h thykrOid hormones, respectively. In h y p e r t h y r o i d patients the mean plssms tO.5 of A of 9.8• 1.6h was a p p r e c i a b l e lower than tO.5 of 12,9 h messured in normal h e a l t h y volunteers. The decrease in tO.5 correlated well w i t h the severity of the h y p e r t h y r e o s i s . A ~ t e r n o r m s l i z a t i o n of thyroid function tO.5 i n c r e a s e d t0+13.3 - 1.4 h. H y p o t h y r o i d patients h s d s mesh tO.5 of 17.3 - 1.1 h. A f t e r s u b s t i ~ u t l o n w i t h thyroid h o r m o n e s p l a s m a tO.5 of A decreased to 12.0 - 0.5 h. No difference was observed in the cumulative urinary e x c r e t i o n of 4-OH-A in h y p e r t h y r o i d patients during h y p e r t h y r e o s l s (43.9% of the dose) and after s t r u m e c t o m y (44.3% of the dose). In contrast h y p o t h y r o i d i s m m a r k e d l y influenced the e x c r e t i o n of 4-OH-A. In these patlents n o r m a l i z a t i o n of thyroid f u n c t i o n caused an increase of the urinary e x c r e t i o n of 4-OH-A from 30.9% to 41.5% of the dose. M. Eichelbaum, M e d i z l n l s c h e P o l l k l l n l k der U n l v e r s l t ~ t D 63oo Giessen, ~ r i e d r i c h s t r a s s e 2V

R 15

COMPETITIVE INHIBITION BY SPIROLACTONE~ OF CORTICOSTEROID BIOSYNTHESIS IN VITRO (Kompetitive Hemmung der Corticosteroidbiosynthese durch Spirolactone in vitro) H. Erbler Quartered adrenals of rats were incubated with and without the addition of stimulators of corticosteroid synthesis: I) Experiments with radiolabelled precursors revealed a significant inhibition by the spirolactone aldadiene of the incorporation of 140 _ progesterone into corticosterone, 18-hydroxycorticosterone and aldesterone. The effect of spironolactone was less pronounced than that of aldadiene. The inhibitory effect of spironolaetone and aldadiene on the incorporation of 14C-progesterone into eorticosteroids was reduced by a simultaneous stimulation of cortlcostercid biosynthesis. 2) The production of aldosterone, 18-hydroxycorticosterone, 18-hydroxydesoxycorticesterone and corticosterone was inhibited by aldadiene between 5x10-b and I0-4M in a dose-dependent manner. 3) After incubation of adrenal quarters with aldadiene polar metabolites of aldadiene were detected in the tissue as well as in the incubation medium. The production of the aldadiene metabclites increased with the aldadiene concentration in the incubation medium and was reduced by a simultaneous stimulation of the oortlcosteroid synthesis. The mass spectrometric analysis of two aldadiene metabolites revealed a hydroxylation of aldadiene in two different positions most likely at the carbon in position 11 or 18 respectively. It is concluded that the inhibition of eortioosteroid synthesis is due to a competition between corticostsroid precursors and aldadiene for mitochondrial hydroxylases. Dr.H.Erbler, Pharmakologisches Institut der Medizinischen Hochschule,3000 Hannever-Kleefeld, Karl-Wiechert-Allee 9

INVESTIGATIONS C~ THE NECHAN!SM OF ALCOHOL-INDUCED FATTY INFILTRATION OF THE LIVER (Untersuchungen Gber den Nechanismus der alkoholischen Leberverfettung) C.-J. E s t l e r In mice a single dose of 4 mg/g ethanol i.v. causes an increase of the hepatic triglyceride content by 11 mg/g within 2 h~ which is accompanied by an increase of the non-esterified fatty acids in the serum (N~:FA) by 15% and a decrease of the esterified fatty acids in the serum by 14%. Theoretically three mechanism~ may be responsible for the fatty infiltration of the liver: I. A diminished release of triglycerides from the liver into the blood, 2. an increased uptake of fatty acids from the blood into the liver~ and 5- an enhanced synthesis of fatty acids and subsequently of triglycerides within the liver itself~ due to a rise of the hepatic NADH/NAD ratio in ethanol-treated animals. Experiments in mice pretreated with Triton WR ~339 show that indeed alcohol reduces the release of esterified fatty acids from the liver by 25%~ but it appears that this effect cannot fully account for the rise of the hepatic triglyeeride content.Pretreatment of the mice with 540 /ug/g pyrazole15 min prior to ethanol inhibits completely the alc6hol oxidation and the rise of the NADH/NAD ratio, On the other hand, adipose tissue lipolysis is not affected by pyrazole~ the NEFA level still rises by 15% and the hepatic triglyceride level increases by 13 mg/g. These results suggest that not the shift of the hepatic NADH/NAD ratio but an increased supply of fatty acids from extrahepatic sources is the major reason for the elevation of the fat content of the liver. Prof. Dr.C.-J.Estler, Pharmakologisches Institut der Universit~t Erlangen-N~rnberg~ D-8520 Erlangen~ Universit~tsstr. 22

R 16

THE REACTION OF 4-DIMETHYLAMINOPHENOL WITH HEMOGLOBIN (Die Reaktlon yon 4-Dimethylaminophenol mlt H~moglobln) P. Eyer, M. Kiesep N. Weser The reaction was studied with 4-dlmethylaminophenol (DMAP) 14Clabelled in the methyl groups or in the ring. In the presence of oxygen DMAP quickly transformed many equivalents of ferrohemoglobin into ferrlhemoglobln and disappeared within a few mln. Finally about half the methyl radioactivity was found to be bound to hemoglobin. About 70% of the radioactivity not bound to hemoglobin was recovered as dimethylamine and about 20% as formaldehyde. Virtually all of the ring of DMAP was tightly bound to globln and was found mainly in the B-chalns. Reaction of hemoglobin with Nethylmaleimide or pCMB prior to DMAP diminished the binding of DMAP to hemoglobin. In 0.2 m phosphate pH 7.4 under air at 37 ~ DMAP disappeared only slowly. In addition to dlmethylamlne and formaldehyde the following reaction products were isolated and identified: hydroqulnone, 4-aminophenol, 4-methylamlnophenol, 2-methylamlno-l,4-benzoquinone, [2-(p-hydroxyphenyl) methylamino]-1,4-benzoquinone and a yellow dye which may also be produced by oxidation of the latter compound and whose structure is not yet fully elucidated. Dr. Peter Eyer, Pharmakologisches Institut der Ludwig-MaximiliansUniversit~t, 8 M~nchen 2, Nussbaumstrasse 26

THE E F F E C T O F CHRONIC CANNABIS AND CANNABINOID ON T H E H E P A T I C DRUG M E T A B O L I Z I N G SYSTEM OF THE flu2 c h r o n i s c h e r A p p l i k a t i o n y o n C a n n a b i s und C a n n a b i n o i d e n m i f f e l - a b b a u e n d e S y s t e m d e r R a f f e n l e b e r ) M . F e r n a n d e s ~ R.

APPLICATION R A T (Der E i n auf d a s A r z n e i H i l l a S. Kluwe

C a n n a b i s and s y n t h e t i c / ] 9 _ T H C w e r e a p p l i e a t e d f o r 9 m o n t h s by m e a n s of d r i n k i n g f l u i d s to 40 m a l e W i s t a r r a t s w h i c h w e r e r a n d o m l y a s s i g n e d into f o u r g r o u p s r e c e i v i n g the f o l l o w i n g t r e a t m e n t s : t a p w a t e r s v e h i c l e (0. 5 % c r e m o p h o r s o l u t i o n ) , synthetic/19-THC(10 m g / k g / d a y ) a n d c a n n a b i s e x t r a c t ( s t a n d a r d i z e d on l 0 m g / k g / 1 9 - T H C / d a y ) ~ r e s p e c t i v e l y . As c o m p a r e d to t a p - w a t e r - a n i m a l s no c h a n g e s w e r e found in the v e h i c l e and the T H C - t r e a t e d a n i m a l s with r e s p e c t to the m i c r o s o m a l c o n t e n t of p r o t e i n , Cyt-P-450j Cyt b 5 and the activities of morphine demethylase~ aminopyrine demethylase and aniline hydroxylasej respectively. In the cannabis group Cyt b 5 and Cyt-P-450 were slightly elevated. W h e n the animals were killed immediately after stopping the treatment marked decreases in the activities of m o r phine- and aminopyrine-demethylases were to be found, probably due to cannabinoids present in the microsomal preparation. These decreases could not be detected 8 days after stopping the treatmentj whereas eytoehrome P-450 increase still persisted. B y daily i.p. injection of purified cannabinoids (10 rag/ kg/day) for 20 days the effects of the extract were established to be produced by cannabidiol (CBD). This is consistent with the acute experiments~ which had shown C B D to be m o r e active on the hepatic drug metabolizing system than cannabinol and the psychotornimetic A g-THC. Dr. M. Fernandes, Institut f~ir Neuropsyehopharmakologie der Freien Universit,it Berlin~ D-looo Berlin 19~ Ulmenallee 30

R 17

EFFECT OF CYCLOPHOSPHAMIDE ON EXOCRINE PANCREAS FUNCTION OF THE RAT (Die Wirkung von Cyclophosphamid auf die exkretorisehe Funktion des Rattenpankreas) K. F ! e i s c h e r ~ R. F i e d l e r Exoerine pancreatic s e c r e t i o n was s t u d i e d i n r a t s t r e a t e d w i t h cyclophosphamide for three days in different dosages schedules (40, 80, 120 mg/kg/d i.p.). Weight, total protein, amylase (U/h), trypsin (mU/h) and lipase activities were determined in pancreatic secretion. Excretion was followed for two hours after eannulation of the pancreatic duct with two sampling periods of one hour duration. Maximal stimulation of exocrine pancreatic secretion was achieved by infusion of secretin (25 U/kg/h) and panereoeymin (25 U/kg/h). Volume of pancreatic secretion was reduced by 120 mg/kg cyelophosphamide, it was not changed by the lower dosages. Total protein content, amylase, trypsin and lipase activities were significantly decreased by treatment with 80 and i20 mg/kg cyclophosphamide. The effect of cyclophosphamide on pancreatic exoerine secretion of the rat was dose-dependent. This indicates an impairment of protein synthesis and enzyme secretion under the experimental conditions. Dr. K. Fleischer, Medizinische Universit~tsklinik WUrzburg, D-8700 WGrzburg, Josef-Schneider-Str. 2

ABSORPTION OF PHENYLBUTAZON AND GUINEA PIG (Resorption offenen Analogon bet Ratte

AND A NON-CYCLIC yon Phenylbutazon und Meerschweinchen).

Forth~

Specht,

W.~

Seebald~

H.

and

ANALOGUE IN RAT und seinem ring-

W.

Absorption of phenylbutazon and a non-cylic analogue ~umadizon: Butylmalonic acid-mono-(l,2-diphenylhydrazid-calcium-semihydrate~ was investigated in rats and guinea pigs in vitro and in vivo. In vivo, the highest absorptive activity for both phenylbutazon and the non-cyclic analogue in rats shows the jejunum. In guinea pigs the amount absorbed of both compounds is 0nly 1/3 of that which was measured in rats. In stomach, jejunum, ileum and colon of rats the amount absorbed of either compound is determined by the amount of the lipid-soluble form available at the different pH values and the permeability propert~es of the different sections os the gastrointestinal tract. In vitro, phenylbutazon inhibits in high concentrations(175260 nmoles/ml) the absorption of water, electrolytes and glucose in jejunal segments of the rat, but not in those of guinea pigs. In the same concentrations the non-cyclic analogue does not influence the absorptive activity of jejunal segments in rat. Simultaneously, a disintegration of the epithelium could be observed in rat jejunal segments after perfusion with pheny~ butazon containing fluid, whereas segments of guinea pig remained unaffected. W. Forth: Abteilung ffir spezielle des Saarlandes, 665 H o m b u r g / S a a r

Pharmakologie

der

Universit~t

R 18

I N F L U E N C E OF C A R B O N D I S U L F I D E AND T H I U R A M S O N , E T H A N O L E L I M I N A T I O N AND A C E T A L D E H Y D E P R O D U C T I O N (EinfluB von S c h w e f e l k o h l e n s t o f f und T h i u r a m e n auf die ~ t h a n o l e l i m i n a t i o n und A c e t a l d e h y d b i l d u n g ) K.J.Freundt, H.Netz A f t e r i . p . e t h a n o l a d m i n i s t r a t i o n (2 g/kg) 8-hr e x p o s u r e of adult female W i s t a r rats to 20 and 400 ppm CS~, resp. does not alter the alcohol e l i m i n a t i o n rate in the blood; ~ linear fall of the ethanol level is a s s o c i a t e d w i t h a rise of the a c e t a l d e h y d e (AC) conc e n t r a t i o n (determined s i m u l t a n e o u s l y by GC) w h i c h v i r t u a l l y plateaus for 4 hr w i t h a m a x i m u m at 1.5 times the control level. Rep e a t e d 8-hr e x p o s u r e s at 400 ppm (3,6 and 12 e x p o s u r e s at 2-day intervals) do not change the ethanol e l i m i n a t i o n and yield the same b l o o d AC c o n c e n t r a t i o n s as after single exposure. 20 min after i.p. doses of d i m e t h y l d i t h i o c a r b a m a t e and d i e t h y l d i t h i o c a r b amate (50 m g / k g each) AC levels in the blood are i n c r e a s e d (ca.3 times those found in the controls) in the course of u n a f f e c t e d ethanol e l i m i n a t i o n and p e r s i s t for 4 hr. Oral doses of tetram e t h y l t h i u r a m d i s u l f i d e (0.O6-1.O g/kg) p r o d u c e a iO-fold increase, w h i l e ethanol e l i m i n a t i o n is slightly, t h o u g h s i g n i f i c a n t l y retarded only after 0.25 and 1 g/kg. D i s u l f i r a m (i g/kg o r a l l y at 16 hr b e f o r e ethanol loading) raises the b l o o d AC level up to 5-fold at u n a f f e c t e d ethanol elimination. The half life (i min, 45 sec) of i.v. AC (i mM/kg) is p r o l o n g e d by n e a r l y 1 m i n by identical doses (w) of CS^ and thiurams. The o b s e r v e d effects are thought to be due to i n h i b i t i o n of a l d e h y d e dehydrogenase. (Supported by a grant from the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t . ) P r i v . - D o z . D r . K l a u s J.Freundt, Institut fGr 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 der U n i v e r s i t ~ t , D - 8 7 O O WGrzburg, K o e l l i k e r s t r a B e 2

THE INFLUENCE OF Ca ++ ON THE Na+-ACTIVATED, ATP-DEPENDENT UPTAKE OF CARDIAC GLYCOSIDES BY A MICROSOMAL FRACTION OF GUINEA PIG HEARTS. (Uber den Einflu~ von Ca++-Ionen auf die Na+-aktivierte, ATP-abh~ngige Bindung von Herzglykosiden an eine myokardiale Mikrosomenfraktion. U. Fricke, R. Alken und W. Klaus The uptake of 3H-digitoxin (DTO) and 3H-digoxin (DO) by a microsomal fraction isolated from guinea pig hearts by a two-step-procedure (differential centrifugation (10 O00g, 15 min) followed by gel chromatography (sepharose 4B) of the supernatant) was studied under different experimental conditions. The microsomal fraction was shown by electron-microscopy to be free of mitochondrial particles, the contamination by succinic dehydrogenase was in the range of 3% based on the total myocardial amount. Na+-K+-ATPase activity was 18.3• uM Pi/mg prot.x hr (i.e. 41.2• % of total ATPase activity). The following results were obtained: I. the uptake of DTO and DO was ATP-dependent (p
R 19 UDP-GLUCURONYLTRANHFERASE ACTIVITY IN ISOIATED PERFUSED RAT LIVER AND IN MICROSOMES (UDP-Glucuror~vltransferaseaktivit~t in der isoliert perfundierten Rattenleber und in Mikrosomen) W. Frb%Llin$

l-naphthol glucuror~,ltransferase activity in isolated perfUsed rat liver was estimated from the linear appearance of l-naphthol gl~curonide in perfusate and bile. A radioactive assay was used to determine l~C-l-naphthol and l-naphthol glucuronide which involved counting in a dioxane and toluene based scintillation fluid. In the toluene system free naphthol can be determined, whereas naphthol conjugates are retained in the aqueous phase and their radioactivity is completely quenched. In the dioxane system the sum of unmetabolized l-naphthol and l-naphthol conjugates is measured, l-naphthol glucuronide is discriminated from other conjugates after digestion with sulfatase-free ~-glucuronidas e. Under saturating conditions of 0.5 mM l-naphthol an activity of 0.09 ~unoles 1-naphthol glucuronide formed/min x g liver was found. With the same methc~1 l-naphthol real system at 0.5 mM 1 naphthol lar level). An activity of 0.7 and without 0.05 % (w/v) Triton

glucuronidation was determined in the microsoand 0.3 ~M UDP-glucuronic acid (the intracelluand 0.07 p~moles/min x g liver was found with X-100, respectively.

~'ne activity in the isolated perfused liver corresponds well with the activity in microsomes which are not activated by detergents. Dr. W. Fr~hling, Institut f'dr Toxikologie der Universit~t, D-7400 TUbingen, WilhelmstraSe 56

I N T E R A C T I O N OF F U R O S E M I D E WITH A M I L O R I D E AND OUABAIN IN FROG SKIN ( K o m b i n a t i o n s v e r s u c h e von F u r o s e m i d mit A m i l o r i d e und Ouabain an der Froschhaut) G. F~l~raff, W.-D. Gulden It was the purpose of our studies to examine the h y p o t h e s i s i n more detail that f u r o s e m i d e (fu) p r i m a r i l y acts by i n c r e a s i n g Na p e r m e a b i l i t y (W.D. Rudroff, G. FOigraff; Arch. Pharmak., Suppl. 270~ R 117, 1971). P o t e n t i a l d i f f e r e n c e (pd) and short circuit current (scc) were m e a s u r e d in ventral skin of rana e s c u l e n t a m o u n t e d in a lucid double chamber. It is w e l l e s t a b l i s h e d that amiloride (am) added to the b a t h i n g m e d i u m on the e p i t h e l i a l side decreases pd and scc by b l o c k i n g the entry of Na into the t r a n s p o r t i n g cells. Fu, hQwever, increased pd and see in the p r e s e n c e of 10 -6 and 10 -b M am to the same extent as in the control experiments r e l a t e d to the initial values. The r e l a t i v e decrease of pd and scc by i n c r e a s i n g concentrations of am, on the other hand, was not a f f e c t e d by 3.10-" and 3"10 -3 M fu. Ouabain (ou), w h i c h inhibits d i r e c t l y the transport m e c h a n i s m when given to the corial bathing medium, d i m i n i s h e d pd and scc to the same values in controls and in the p r e s e n c e of fu. Fu given to the outer surface has no s t i m u l a t i n g effects on pd and scc in the p r e s e n c e of ou in the corial b a t h i n g solution. The results are compatible with the p r e v i o u s l y p r o p o s e d h y p o t h e sis that f u r o s e m i d e p r i m a r i l y increases the s o d i u m permeability. Prof. Dr. G. FOlgraff, A b t e i l u n g P h a r m a k o l o g i e der M e d i z i n i s c h e n F a k u l t a t der T e c h n i s c h e n Hochschule, 51 Aachen, M e d i z i n i s c h - T h e o r e t i s c h e Institute, M e l a t e n e r s t r . 213

R 20 THE INFLUENCE OF METHYLXANTHINES ON EXPERIMENTAL CEREBRAL EDEMA IN CATS (Die Beeinflussung des experimentellen Hirn8dems der Katze dutch Methylxanthine) V. Ganser, I. Boksa 7 Cerebral edema was induced in anesthetized cats by a standardized freezing lesion made on the exposed dura of the right hemisphere. The right hemisphere is designed as experimental hemisphere, the left as control hemisphere. Dry weight and sodium and potassium content of white matter and cerebral cortex were determined from samples of both traumatized and control hemispheres. Theophylline-ethylene-diamine (T) and ~,7-Dimethyl-l-(5-oxohexyl)-xanthine (BL 191) were Given intravenously, starting the treatment 2~ hours before the lesion was made. The treatment with BL 191 and T 72 and 96 hours after the lesion was made caused a significant reduction in the increase in sodium content and that of the decrease in potassium content in the injured hemisphere. In the experimental cerebral edema in the cat BL 191 proved more favourable when compared with T. The mechanism involved in the action of Methylxanthines on cerebral edema has not yet been clarified. The increase of cerebral blood flow and the inhibition of cyclic AMP-PDE could play a rSle in the beneficial effect of the Methylxanthines on the Brain edema~ other mechanisms can, however, not be excluded. Dr. V. Ganser, Chemische Werke Albert AG, Pharmazeutische Abteilung, 62o2 Wiesbaden-Biebrich, Postfach 91ol

COMPARATIVE STUDIES ON LACRIMAL AND SALIVARY SEORETION IN DOGS (Vergleichende Untersuchungen zur Tr~nen- und Speichelsekretion beim Hund) H. Ganz r I. Weisse~ R. Bauer~ H. StGtzer Bei 222 klinisch gesunden Beagle-Hunden wurde mit dem modifizierten Schirmer-Test die Tr~nensekretion gepr~ft. Ein Verglelch zwischen dem in der Veterin~r-Ophthalmologie gebr~uchlichen EinMinuten-Test und dem in der Human-Ophthalmologie angewendeten 5Minuten-Test ergab eine gute ~bereinstimmung. Uuter Einhaltung bestimmter Kautelen kann mit diesem Test elne quantitative Aussage ~ber die Tr~nensekretion beim Hund gemacht werden. Nach i.v.-Gabe yon Antlcholinergica bei 30 wachen H u n d e n w u r d e mit dem Schirmer-Test die Hemmung der Tr~uensekretion und bei 15 narkotisierten Hunden die Hemmung der Speichelsekretion getestet. Die mit beiden Methoden ermittelten ED~^-Werte erlauben einen Vergleich ~qui-effektiver Desert. So e r ~ b slch z.B. fur Atropln eln Verh~ltnis von ca. 1:10 (ED=o-Speichelsekretionshemmung : ED~o_Tr~nensekretionshemmung). 9 Di@ Ergebnisse zeigen, dab der Schirmer-Test eine einfache Methode ist, am wacheu Hund die sekretionshemmende Wirkung von Substanzen im pharmakologisch-toxlkologischen Experiment zu bestimmen. C.H. Boehringer Sohn, 6507 Ingelheim/Rh., Pharmaforschung Biologie

R 21 SYNTHESIS, DEGRADATION, AND ACTION OF CYCLIC 3'~5'-AMP IN RED BLOOD CELLS FROM RATS (Synthese, A b h a u n n d W i r k u n g ~ o n cyclischem 3',5'-AMP in Erythrocyten yon Ratten) D. Gauger, D. Palm

Previous investigations (Gauger et ai., J. de Pharmacol. (3), 25, 1972) have shown that adenyl cyclase activity in rat erythrocytes is mainly localized in premature red blood cells~ i.e. in the reticulocytes. Mature erythrocytes seem to contain only negligible enzyme activity. The enzyme is specifically stimulated by sympathomimetics in the order D(-)-isoproterenol) D(-)-adrenaline ~ D(-)-noradrenaline. Definite enzyme activities were also found in cell fractions obtained by centrifugation on an isopycnic discontinuous dextran gradient; the respective blood samples from control and 1-acetyl-2-phenyl hydrazine-pretreated rats had been freed from leukocytes and thrombocytes by filtration through cotton wool. The activity of the cyclia AMP inactivating enzyme phosphodiesterase (kM - I 0 - 5 M) was found to be present both in ghost membranes and in the supernate from red cell lysates. Enzyme activity was several-fold higher in preparations from reticulocyte-rich blood. Similar results were obtained when the activity of a cyclic AMP-stimulated protein kinase was measured. From these results, it may be concluded that cyclic AMP-mediated 5-sympathomimetic effects play an essential yet unknown role in premature red blood cells. Zentrum der Pharmakologie, Universit~t Frankfurt, D - 6000 Frankfurt 70, Theodor-Stern-Kai 7

DIE W I R K U N G VON P H E N O B A R B I T A L AUF DIE F E T T S ~ U R E N D E R R A T T E N L E B E R G L Y K O L I P O I D E ( Effect of p h e n o b a r b i t a l on the fatty acid compo-

sition in rat liver glycolipids ) W. Gielen, H. S t e i n b a c h Die i.p. A p p l i k a t i o n yon Phenobarbital in einer Dosierung yon 75 mg/kg in o.9 % NaCl Ober einen l~ngeren Zeitraum fHhrt zu signifikanten V e r ~ n d e r u n g e n im F e t t s ~ u r e m u s t e r der neutralen Glykolipoide der Rattenleber. Eine Zunahme von C14-, C16- und C l 8 - F e t t s ~ u r e n ist begleitet yon e n t s p r e c h e n d e r V e r m i n d e r u n g der C2o-, C22- und C24Fetts~uren. Von allen untersuchten G l y k o l i p o i d e n weisen die Cerebroside, die die H a u p t k o m p o n e n t e im G l y k o l i p o i d g e m i s c h darstellen, die gr~Bten V e r ~ n d e r u n g e n auf. Die Fetts~uren im G l u k o s y l c e r a m i d der P h e n o b a r b i t a l - b e h a n d e l t e n Tiere zeigen die folgende prozentuale Zusammensetzung: C14:o 8.2 (5.3); C16:o 28.o (16.4); C18:o 36.1 (15.5); C18:1 7.2 (4.o); C2o:o 3.1 (4.1); C22:o 7.8 (20.7); C23:o 0.3 (1.2); C24:o 9.5 (32.6). Die Zahlen in Klammern geben die F e t t s ~ u r e v e r t e i l u n g in den L e b e r g l u k o s y l c e r a m i d e n unbehandelter Ratten wieder. Yon gleicher Art, zwar w e n i g e r ausgepr~gt, aber d e u t l i c h nachweisbar, sind die V e r s c h i e b u n g e n im F e t t s ~ u r e m u s t e r der Ceramiddihexoside, -trihexoside und -tetrahexoside. Die Glykolipoide, die bevorzugt in der Plasmamembran lokalisiert sind, dHrften als S t r u k t u r k o m p o n e n t e n nicht ohne funktionelle B e d e u t u n g fHr die P e r m e a b i l i t ~ t der Membran sein. Die Ganglioside der Rattenlebet sind nach P h e n o b a r b i t a l a p p l i k a t i o n q u a l i t a t i v und q u a n t i t a t i v unver~ndert. Ein vermehrter Einbau von N - G l y k o l y l - N e u r a m i n s ~ u r e in L e b e r g a n g l i o s i d e wird nicht beobachtet. Offenbar ist die Acetyln e u r a m i n a t - N - A c e t y l - H y d r o x y l a s e in der Rattenleber durch Phenobarbital nicht zu induzieren. Prof. Dr. ~. Gielen, Pharmakologisches Institut der U n i v e r s i t ~ t zu K~in, 5 K~In 41, Gleueler Str. 24

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PLASMA OORTICOIDS IN RABBITS IMMUNIZED AGAINST VARIOUS ADRENAL STEROIDS (Plasmacorticoide naeh Immunisierung gegen verschiedene Nebennlerenrindenhormone) K.H. Gless, P. Vecsei, M. Hanka-Posztook~, E. Knott Rabbits were immunized by injection of a steroid-hemisuooinate-albumin complex every three weeks. Repeated determinations of plasma cortisol (F) and plasma corticosterone (B) were done by means of radioimmunoassay. In control animals, plasma ~ and B concentrations were below 3.5~g/i00 ml. In animals having an antibody titre for F and B above 1:3000, an increase in the plasma levels of the corresponding hormones was ohserved. In four rabbits immunized against F, plasma P concentration was 85+ii~g/i00 ml. In two rabbits immunized against B, plasma B concentrations were 3 7 ~ g / 1 0 0 m l and 3 0 ~ g / 1 0 0 ml, respectively. In three rabbits immunize~simultaneously against P, B, DOC, and aldesterone, the plasma concentrations of F and B were above 40 ~g/100 ml. The ratios of plasma F to plasma B concentrations were 5.0 in rabbits immunized against F, below 0.4 in rabbits immunized against B, and 1.3 in rabbits immunized with four corticoids. Plasma P and B concentrations were positively r the antibody titre of the respective steroids. Three hours after administration of AOTH, plasma F concentrations increased to 130-214~g/i00 ml in rabbits immunized against F. D~. K.H. Gless, Pharmakologisches Institut der Universit~t, D-6900 Heidelberg, Hauptstr. $7-51

THE DISTRIBUTION OF (• (NA) OR (• (MA) IN THE ADRENERGIC NERVE ENDINGS OF THE RAT VAS DEFERENS (Die Verteilung yon (~)-Noradrenalin und (~)-Metaraminol in den adrenergen Nervenenden des Vas deferens der Retie) K.H.Graefe~ F.J.E.Stefano~ S.Z.Lan~er (Institute de Investigaclones Farmacolo~icas, Buenos Aires) Pairs of isolated rat vasa deferentia were incubated with tritiated NA or MA and then washed by transferring them every I0 and later every 2 min into new tubes each containing 2 ml of amine-free medium. From the efflux of radioactivity the fractional rate of loss of amine (FRL) was calculated. It was found that the FRL decreased rapidly witktime and remained constant from the IOOth min onwards indicating a single exponential decline of the amount of amine remaining in the tissue with a half-life (t/2) of 300-400 min. The long t/2 strongly suggests a neuronal origin of this efflux. When, 128 min after the beginning of the washout procedure, the neuronal re-. uptake was blocked by the addition of Cocaine to the medium (3xlO-- to lxl0-4M) the FRL of MA increased markedly (up to 20-fOld), while that of NA remained unaltered. However,under conditions in which monoamine oxldase (MA0) and cateehol-O-methyl transferase (COMT) were inhibited cocaine caused a moderate increase (3-foldIAin the FRL of NA. The time course of the FRL observed in the presence of I0 -M cocaine was biphaslc (sharplncrease followed by a phasic decline) both for MA (normal conditions) and NA (MAO and COMT inhibited) indicating an inhomogeneous distribution of the amines in the nerve endings. The striking difference between the effects of cocaine on the neuronal efflux of NA and MA demonstrates a different intraneuronal distribution of these amines. Dr. K.H.Graefe, Institut f~r Pharmakologie und Toxikologie der Universit~t 87 W~irzburg, Koellikerstr. 2

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THE EFFECT OF HYPOTHERMIA ON RENAL FUNCTION AND THE D~URETIC EFFECT OF FUROSEMIDE IN ANAESTHETIZED RATS (Der EinfluS yon Hypothermie auf Nierenfunktion und diuretische Wirkung yon Furosemid in Ausscheidungsversuchen an narkotisierten Ratten) J. @reven Cooling of anaesthetized rats in an ice bath to a colon temperature of 30 ~ C diminishes glomerular filtration rate, total renal plasma flow~< urine flow and renal excretion of potassium and sodium. When the rats are cooled to 26 ~ C~ these parameter are furthe~ reduced. At this temperature blood pressure and plasma potassium concentration are also decreased, whereas plasma sodium concentration increases. Fractional tubular sodium reabsorption remains unchanged at all temperatures. The impaired renal function in hypothermia is thought to be caused by a reduced cardiac output and an increased tonus of the renal blood vessels. Renin probably does not play a major role in the renal vasoconstriction. When the rats are depleted of renin by a sodium rich diet, hypothermia causes the same change of glomerular filtration rate as in controls. At 30 ~ C, the diuretic effect of furosemide is diminished and at 26 ~ C completely abolished. This is thought to be the consequence of the reduced glomerular filtration rates in hypothermie, which ape further decreased by injections of furosemide. Dr. J. Greven, Abteilung for Pharmakologie der Medizinischen Fakultat der Technischen Hochschule Aachen, 51 Aachen, Melatenersir. 213.

STUDIES ON THE EFFECT 0 F A M A N T A D I N E 0 N N E U R O N A L MEMBRANES (Untersuchungen Uber die Membranwirksamkeit yon Amantadin) W. Grossmann Experiments performed on nigro-striatal system of rats suggested that amantadine acts indirectly on dopaminergic systems (Jurna, I. et al, Europ. J. Pharmacol., in press). Moreover, experiments performed on spinal motor activity indicated that amantadine exerts a direct effect on neuronal membranes (Sontag~ K.-H., personal communication). The action of amantadine on the membranes of sensory nerve fibres was studied on dorsal root L6 or SI isolated from rats. Action and resting potentials were determined by means of the sucrose-gap technique. Amantadine hydrochloride I0-5M reduced the amplitude of the action potential by 20.8 • 11.2 ~ of the control values (n=5) without changing the resting potential. The membrane resistance was increased by 5.2 • 2.3 ~ in comparison with the control. This effect is not significant on account of the small number of experiments (n=4). The repetitive activity was studied with long-casting depolarizing pulses. The number and the amplitudes of subsequent action potentials were reduced after amantadine. The interval between two action potentials was shortened. In calcium-free Loche solution no repetitive spike discharge was observed, which may be due r o a n asynchronous activity of each nerve fibre in the dorsal root. Amantadine added to the calcium-free Loche solution elicited a repetitive discharge of spike potentials. Dr. V. Grossmann, Institut fdr Pharmakologie der Ruhr-Universit~t Bochum (present address: Institut f~r Pharmakologie,D-665 Homburg)

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ISOLATION O F S M O O T H MUSCLE C O N T R A C T I N G SUBSTANCES FROM LUNG WASHING IN RATS (Isolierung von glattmuskul~r k o n t r a h i e r e n d e n Stoffen in der L u n g e n s p G i f l G s s i g k e i t bei der Ratte) M . G R U N S P A N In a previous paper (Grfinspan and Pallade, 1971) we have described smooth muscle contracting substances in the lung washing (pulmonary surfactant) of rats. In order to study further the p h a r m a c o l o g i c a l properties of the pulmonary surfactant, we have investigated the role of phospholipids in this reaction. The lung w a s h i n g from 2o rats (3 ml Tyrode from each animal) were pooled and lyophilysed. The lyophilysate was e x t r a c t e d with chlor o f o r m - m e t h a n o l 2:1 and the p h o s p h o l i p i d s e s t i m a t e d by thin layer chromatography. We have isolated 4 fractions: sphingomyeline, phosphatidylcholin, phosphatidylserine, p h o s p h a t i d y l e t h a n o l a m i n e . Each p h o s p h i l i p i d fraction was scraped from the c h r o m a t o g r a p h y p l ~ re. The same p h o s p h o l i p i d fractions from several plates were pooled, eluted, dried, r e d i s s o l v e d in saline and tested s u b s e q u e n ~ ly on the guinea pig ileum. The p h o s p h a t i d y l c h o l i n fraction non elicited a contraction, but p h o s p h a t i d y l s e r i n e exhibits a sensitizing action of the ileum to histamine. We think these are similarities to the findings published by Brown, Poyser and Telford (1972) who have found that a crude extract consisting of p h o s p h a t i d y l s e r i n e and p h o s p h a t i d y l e t h a n o l amine induced the same phenomenon on the guinea pig ileum. The rate of p h o s p h o l i p i d s in pulmonary surfactant is discussed. Dr.M.GrGnspan, Med. Institut f. L u f t h y g . u . S i l i k o s e f o r s c h g , DGsseld.

CONSUMPTION AND HEAT PRODUCTION OF K+-ARRESTED HEARTS UNDER THE INFLUENCE OF CA, 0UABAIN AND ADRENALINE (Sauerstoffverbrauch und W ~ r m e 9 + . . . . ~+ produktlon K -stlllgestellter Herzen bel Var!atlon der Ca -Konzentratlo~ sowie unter dem Einflu~ von Strophantin und Adrenalin) K. G~ttler, M. Theisohn Die fr~her vorgestellte Methode (I) zur Messung der W~rmeproduktion isolierter Kaninchenherzen wurde dutch die gleichzeitige, polarographische Messung der av-O~-Diff, erg~nzt. Das Verhalten der beiden Parameter 9 L. e wurde bestlmmt bel 8 [K)^ (5.4 - 8o mM) unter Reduktlon der [Na]-^ auf 75 mM (osmot. Ausgleich ~urch Saccharose) bei 4 Versuchsb~di~gung~n: Kontrolle (o.9 mM Ca); e r h ~ t e ~a]e (3.6 mM)i Adrenalin (2*1o-~ o.9 mM Ca); Strophantin (1o-bg/ml, 0.9 mM Ca); Adrenalin und Strophantin bei max. pos.-inotrop wirkender Dosierung. 02-Verbrauch und W~rmeproduktion nehmen bei steigender [K+] e unter allen Bedingungen gleichsinnig zu. Der W~rmeproduktion yon I cal entspricht ein 02-Verbrauch yon o.1-o.15 ml 02 . Bel einem Energie~quivalent yon 4.85 kcal/ll 02 wUrden ca. 2/3 dieser durch den 02-Verbrauch gewonnenen Energie als Warme abgegebeno Die Minimalwerte belder Parameter fanden sich bei Io - 2o mM [K~] e. Sie lJegen f~r die Kontrollen signifikant niedriger als fur die 3 anderen Bedingungen. Die minimale W~rmeproduktion betr~gt I/4 bis 2/3 der fr~her (2) aus Regressionsgeraden am kontrahierenden Herzen gesch~tzten Ruhew~rme. Diese Differenz erkl~rt sich durch den hier 9 fehlenden Anteil an kontraktionsunabh~ngiger Aktivierungsw~rme. Die ~nderung der beiden Parameter wird im Zusammenhang mit der zu erwartenden ~nderung der freien [Ca]• und der daraus folgenden metabolischen Auswirkungen diskutiert. (I) Theisohn et al. Naunyn Schmiedeberg's Arch.Pharm. Suppl.274 R116(72) (2) Klaus et al. Naunyn Schmiedeberg's Arch. Pharm. Suppl. 274 R 65 (72) K. G~ttler, Pharmakol. Instit., Med. Hochschule Hannover OXYGEN

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PINDOLOL (VISKENR), PHARMACOKINETICS AND RELATION 0F P~ASMA LEVEL TO B - B ~ O C ~ D E IN MAN (Pindolol: Phermskokinetik und Korrelation zwischen Blutspiegel und B-Blockade beim Menschen) R. Gu~ler~ W. Herold P~armacoklnetlc data on pindolol were obtained using s fluorometric determination method as described by PACHA (Exper~entia 25, 8o2, 1969). ~he elimination rate constant is o.2o h- after ~ a l and 0.24 h- after intravenous administration, corresponding to plasma half llfe times of 3.65 h and 3.13 h, resp. Comparing the cumulative urinary excretion after i.v. and oral administration an absorption rate of 92 % can be estimated. The mean volume of distribution is 139 l~ The invasion rate constant after oral administration is 2.36 h-', the corresponding invasion half life ~ims is 0,47 h. Cltot. is 425 ml x min --,'Clren. 163 ml x min- . In addition to these studies an attempt was made to correlate the B-receptor blocking activity of the agent to its actuel plasma level. In 8 volunteers a close correlation was found between log plasma concentration and decrease in exercise tachycsrdia for each subject. Although the single regression lines differed from each other considerably in the elevation, the curves of all subjects tested showed a parallel course as calculated by an analysis of variance. The results indicate that for the same degree of ~-blockade each subject needs a different plasma concentration, but responds to a distinct change in the plasma level by an accurately predictable change in the B-blocklng activity. R. Gugler, Medizinische Pollklinik, D 63oo GieBen, ~rledrichstrasse 27

STUDIES ON CHANGES OF SERUM-ENZYME ACTIVITIES AFTER INTRAMUSCULAR IRON APPLICATION (Untersuchungen ~ber Aktivit~ts~nderungen yon Serumenzymen naeh intramuskul~rer Eisenverabreichung) K. Gutsehow ~ A. Plendl Nach intramuskul~rer Verabreichung yon 133 mg Fe(III) in einem Volumen yon 1,3 ml pro kg K~rpergewich~ in Form von 5 handelsblichen Eisenpr~paraten mit einer LD50/24h yon 7787 (I), 4150 II), 860 (III), 410 (IV) iAud 8 (V) mg Eisen pro kg Maus bei intraperitonealer Applikation (K. Gutschow, A. Schmid: Arch. Pharmakol., Suppl. to Vol. 270, R 50, 1971) wurde folgende statistiseh signifikante (p~ 0,05-0,001) Steigerung der Enzymaktivit~t im Serum (Bioehemiea Mikrotest Boehringer Mannheim) von 224+ 30 g IX+Sx) schweren Sprague Dawley Ratten eigener Zucht ermit--telt (G~schlecht und Kontrollaktivit~t nach Applikation yon 0,9 %iger NaC1-LSsung bei jedem Enzym in Kl~mmern angegeben): GOT (m 44 mU~ml, w 45 mUZml): 1 14%, II 17%, IV 17%, V 1366%; GPT. (m 20 mU/ml, w I'7 mU/ml): V 1222%; LDH (m 317 mU/ml, w 456 mU/ ml): II 240%, III 240%, IV 208%, V 266%; LDH-l-Isoenzym (m 93 mU/ml, w 90 mU/ml): III 73%, IV 64%, V 309%; OPK-aktiviert (m 71 mU/ml, w 62 mU/ml): I 35%, III 52%, IV 68%, V 189%, alkalische Phosphatase (m 66 mU~ml,.w 34 mU/ml). V 60%, saure Phosphatase (m 30 mU/ml, w 24 mU/ml). III 42%, V 130%. - Die Ergebnisse zeigen, da@ toxische Eisenpr~parate die Serumaktivit~t der LDH-1 und der sauren Phosphatase erhShen und dab ein Aktivit~tsanstieg der GPT sowie der alkalischen Phosphatase auf eine ungew~hnliche Giftigkeit solcher PrQparate hinweist. Dr~ K. Gutschow, Institut f~r Pharmakologie, Toxikologie und Pharmazie der Universit~t, 8000 MGnchen 22, Veterin~rstr. 13

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R 26

BESONDERHEITEN DER CHRONOTROPEN, DROMOTROPEN UND INOTROPEN WIRKUNG VON CA-ANTAGONISTEN (VERAPAMIL~ D 600, NIFEDIPINE) AM HERZEN IN SITU UND AN ISOLIERTEN VORH~FEN SOWIE DEREN BEEINFLUSSUNG DURCH EXTRA-CALCIUM UND ISOPROTERENOL. (Characteristic chronotropic~dromotropic and inotropio properties of Ca-antagonistlc drugs (verapamil, D 500, nifedipine) on hearts in situ or isolated atria and their susceptibility to extra-calcium or isoproterenol). H.-P. Haastert Hohe Dosen yon Ca++-Antagonistensenken am isolierten Vorhof und am Herzen in situ die Spontanfrequenz des Sinusknotens und die Kontraktionskraft des Myocards. Im EKG yon Meerschweinchen werden bei Applikation steigender i.v. Dosen von Ca++-Antagonisten bis zum Herzstillstand folgende Ver~nderungen beobachtet: (I) Verlangsamung des Sinusknotens bei konstanter QRS-Zeit (einfache Lokalan~sthetica wie Procain, Procainamid und Xylocain reduzieren dagegen bei vergleichbarer Frequenzsenkung die ventrikul~re Erregungsausbreitung erheblich). (2) Auftreten von AV-Dissoziationen und AV-Rhythmus bei zunehmender Hemmung des Sinusknotens, in wenigen F~llen Wenkebach'sche Perioden oder AV-Block. (3) Knotung der RZacke und QRS-Verbreiterung im Terminalstadium. Extra-Calcium restituiert am Vorhofs-PrMparat die Kontraktionskraft ohne die abgesunkene Frequenz anzuheben. Dagegen kann Isoproterenol sowohl die Kontraktionskraft als auch die Frequenz normalisieren. Am Ganztier fGhren Extra-Calcium oder Isoproterenol zur Erholung der Sinusknotenfunktion und der Kontraktilit~t. Au~erdem k6nnen beide Stoffe in geeigneter Dosis am Ganztier die Toxizit~t der geprGften Ca ++Antagonisten auf etwa I/4 reduzieren. Die Wirkungsmechanismen wetden er~rtert. Dr.H.P.Haastert,Physiol.Inst.,Univ. Freiburg i.B~

PHARMACOLOGY OF PENBUTOLOL (HOE 893 d): A NEWSTRONGAND LONG-LASTING EFFECTIVE B-SYMPATHOLYTIC (Zur Pharmakologle von Penbutolol (Hoe 893 d): ein neues, stark und lang anhal%end wirksames B-Sympatholy%ikum) G. H d r t f e l d e r t J. Kaiser The new B-sympa%holytic penbu%olol was found in a series of a l k y l a m i n o s u b s t i t u t e d cyclopen%ylphenoxypropanols by means of usual pharmacological methods (isoproterenol antagonism in the anesthetized dog, the isolated a u r i c l e , and the tracheal s p i r a l of the guinea pig; d i r e c t effects on heart rate and dp/d%, e t c . ) . In contrast to most of the known B-sympat h o l y t i c s , the compound has a t e r t i a r y butylamine rest in the side chain. I t i s a levoro%atory isomer, The B-sympatholytic effects are approximately 10=times stronger than those of the Optic antipode. As compared with some known B-sympatholy%ics, penbu%olol reveals an absolutely and r e l a t i v e l y stronger B-sympatholytic a c t i v i t y when administered i n t r a - . venously or en• The most impressive property of penbu%olol, which can c e r t a i n l y be used in therapy, is the long duration of effect. G. H~rtfelder, J. Kaiser, Farbwerke Hoechs% A.G., Abteilung fUr Pharmakologle H 821, D-6000 Frankfurt/Haln 80

R 27 EFFECTS OF .~-ADRENOLYTICS ON CENTRAL CARDIOVASCULAR CONTROL (Beeinflussung der zentralen Steuerung des Kreislaufes dutch Z-Adrenolytica). G. Haeusler The effect on blood pressure of uni- or bilateral, supramaximal electrical stimulation of the carotid sinus nerves was measured in rats anaesthetized with urethane. By doubling stepwise the rate of stimulation from 2 to 32 shocks/sec a frequency-dependent decrease in blood pressure was obtained. The maximum fall of blood pressure occurred at a rate of 16 shocks/sec and was on an average 25 and 40 mm Hg for uni- and bilateral stimulation, respectively. Injection into a lateral brain ventricle of the ~-adrenolytics phentolamine, phenoxybenzamine or piperexan caused a dose-dependent inhibition of the stimulation-induced depressor baroreceptor reflex, phentolamine being most and piperoxan least potent with respect to this central effect. Similar observations were made in cats. These results indicate that the baroreceptor reflex pathway may contain at least one adrenergic neurone. Further support for this was provided in cat experiments in which the posterior hypothalamus, the locus coeruleus or the nucleus fastigii were stimulated with bipolar electrodes. The stimulations increased the sympathetic discharges recorded from the splanchnic and renal sympathetic nerves. Due to the activation of the depressor baroreceptor reflex by the accompanying blood pressure rises the discharges in the sympathetic nerves showed a characteristic pattern. This discharge pattern was changed in an identical manner by intraventricular injection of~-adrenolytics and b ~ section of the buffer nerves. The observations support the hypothesis put forward previously by Haeusler and Finch (1972) that clonidine exerts its hypotensive effect by an activation of the depressor baroreceptor reflex. G. Haeusler, Department of Experimental Medicine of F. Hoffmann-La Roche & Co. Ltd., CH-4002 Basel~ Switzerland.

INFLUENCE ON THE ACTIVITY OF 6-AMINOLEVULINIC ACID DEHYDRATASE BY LEAD AND GLUTATHION (Beeinflussung der Aktivit~it der 6-Aminol~vulins~ureDehydratase duroh Blel und Glutathlon) H,-J. Hapke, E. Prigge Die Feststellung einer Blelwirkung erfolgt dutch Messung der Ausscheidung der 6 -AminoIdvulin~ure (ALA) im Harn und der Aktlvit~it der 6-Aminol~ivulin~ure-Dehydratase (ALA) im Blur. In Tien, ersuchen (Ratten, Schafe, Rinder) liel3 sich feststellen, dab bel nur geringer Blelbelastung beide Parameter die Tendenz zur Normalisierung nach 4 - 7 Wochen der Bleiexposition zeigten. Dieser als Adaptation interpretierte Vorgang wurde durch Messung der ALA-D-Aktivlt~it in vitro und in vivo gep~ft. Blei hemmt in vitro die ALA-D dosisabh~ngig, aber nicht kompetit;v. Der durch Blei inaktivierte Anteil der ALA-D konnte in vitro durch Glutathlon zumindest teilweise wieder aktiviert werden. Sowohl hinsichtlich der Aktivit~it der ALA-D wie der Reaktivierbarkeit bei nicht vermehrt bleikontaminierten Ind;viduen bestanden erhebllche tierartliche Untersehiede. Der dutch Blei blockierte Anteil der ALA-D war bei vermehrt bleikontaminierten Versuchstieren h~her als bei Kontrolltieren. Dagegen bestanden im freien Anteil keine signifikanten Unterschiede trotz der Bleiaufnahme, die zu einer betrdchtlichen Ansammlung von Blei im K~rper flJhrte. Somit erscheint die Messung des duroh Blei inaktivierten, durch Glutathion reaktivierten Anteils dieses Ferments eher geeignet, eine chronische Bleieinwlrkung festzustellen, als die Messung clef ALA-Ausscheidung im Ham oder als die der ALA-D-Aktivitdt im Blur. Prof. Dr. Hapke, Inst. fur Pharmakologis, Toxikologie u. Pharmazie der Tier~irztlichen

Hochschule D-3 Hannover, Bischofsholer Damm 15

R 28 THE USE OF G L U C O S E F O R G L Y C O P R O T E I N SYNTHESIS O F G L O M E R U L A R B A S E M E N T M E M B R A N E ( Z u r V e r w e n d u n g yon G l u c o s e beirn Aufbau d e r G l y k o p r o t e i n e d e r g l o r n e r u l ~ r e n B a s a l m e m b r a n ) A . H e i L h e c k e r , F . v. B r u c h h a u s e n In d e r g l o m e r u l f i r e n B a s a l m e r a b r a n d e s R i n d e s l i n d e n s i c h a l s s t a r k z u c k e r h a l t i g e K o m p o n e n t e n K o l l a g e n und G l y k o p r o t e i n e . B e i A n g e b o t yon U14C G l u c o s e an R i n d e r g l o m e r u l i v e r t e i l t s i c h d ie R a d i o a k t i v i t ~ t in den d a r a u s i s o l i e r t e n B a s a l m e m b r a n e n s t a r k e r auf L i p o i d e und G l y k o p r o t e i n e a l s auf d a s z u c k e r h a l t i g e K o l l a g e n . Auf~er G l u c o s e t r e t e n auch m a r k i e r t e M a n n o s e , G a l a k t o s e , F u c o s e u. S i a l i n s ~ u r e auf. I m G e g e n s a t z z u r b i s h e r i i b l i c h e n E r f a s sung d e r G l y k o p r o t e i n e an Hand i h r e r p r o t e a t i s c h e n P e p t i d e w u r d e n nun n a c h E x t r a k t i o n d e r K o l l a g e n k o m p o n e n t e 2 G l y k o p r o t e i n e I und II f r a k t i o n i e r t . I i s t zu e t w a 15%, II zu e t w a 6o% a m Aufbau d e r B a s a l m e m b r a n b e t e i l i g t . B e i d e F r a k t i o n e n sind b e i A n g e b o t r a d i o a k t i v e r G l u c o s e s o w o h l in i h r e m Z u c k e r a n t e l l w ie in i h r e m A m i n o s ~ u r e n a n t e i l r n a r k i e r t . D i e t r y p t i s c h e A u f s p a l t u n g d e s k l e i n e r m o l e k u l a r e n G l y k o p r o t e i n s II fiihrt b e i F r a k t i o n i e r u n g fiber D E A E C e l l u l o s e zu 4 gr61~eren und w e i t e r e n k l e i n e r e n F r a k t i o n e n , yon d e n e n n u r e i ne einen beachtlichen Zuckergehalt besitzL Die V e r s u c h e l a s s e n erkennen, da b G l u c o s e auf vielf~iltigen S t o f f w e c h s e l w e g e n z u m Aufbau v e r s c h i e d e n e r B a salrnembrankomponenten v e r w e n d e t wird. Die s t ~ r k e r e Markierung d e r Glyk o p r o t e i n e d e u t e t d a r a u f hin, dab i h r e B i o s y n t h e s e und e x t r a z e l l u l ~ i r e V e r wendung in d e r B a s a l m e m b r a n b e i e i n s t i i n d i g e r Inkubation e r f a ~ b a r i s t . P r o f . D r . r n e d . F . yon B r u c h h a u s e n , 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 F r e i e n U n i v e r s i t ~ t B e r l i n , 1 B e r l i n 33, T h i e l a l l e e 6 9 - 7 3

EFFECT OF SYMPATHOMIMETIC8 AND A b-RECEPTOR BLOCKING AGENT ON FLUID TRANSPORT IN ISOLATED GUINEA PIG GALL-BLADDER (Wirkung von Sympathomimetica und eines ~-Rezeptorenblockers auf den FINssigkeitstransport der isolierten Gallenblase des Meerschweinehens) K. Heintze, K. U. Petersen, O. Heidenreich To evaluate the influence of sympathomimetics and -lytics on fluid and solute transport in reabsorbing tissues the isolated guinea pig gall-bladder was used. Orciprenaline, in the range from IO-8M to IO-4M, applied from the serosal side inhibited fluid transport concentration dependently. Surprisingly it was ineffective from the mucosal side. Propranolol 10-4M did not a~ter fluid transport but inhibited completely the effect of 10-~M orciprenaline. Noradrenaline applied from the serosal side had a slightly inhibiting effect in the highest used concentration of IO-~M. Because many effects of orciprenaline are mediated by cAMP, we tried to mimic the serosal effect of orciprenaline with c-AMP in the concentration 10-4M Or with dibutyryl-c-AMP 10 -5 and 10-4M. These concentrations failed to influence transport. To exclude the possibility that c-AMP was degraded by a highly active phosphodiesterase, theophylline 10-7M to 10-2M was administered to the serosal side. It had no effect in concentrations up to 10-4M but inhibited fluid transport concentration dependently in the range from 3.3 10 -4 up to 10-2M. The possible relations between ~-sympathomimetic stimulation, c-AMP and inhibition of fluid transport are discussed. Dr. K. Heintze, Abteilung Pharmakologie 51 Aachen, Melatenerstr. 213

der RWTH Aachen,

R 29

GAS CHROMATOGRAPHIC DETERMINATION AND PHARMACOKINETICS OF PH]ENPROCOUMON ( Gaschromatographische Bestimmung und Pharmokokinetik von Phenprocoumon - Marcumar ) N.Heni~ P.Glosner Es wird 5bet eine quantitative gaschromatographische Methode zur Bestimmung von Phenprocoumon in Serum und Urin berichtet. Als innermr Standard bei der Extraktion umd Gaschromatographie wird ein i n p a r a - S t e l l u n g am Benzolring chloriertes Phenprocoum o n verwendet, das bei den verschiedenen Aufarbeitungsschritten ( XAD o - S~ule, D~nnschicht- und Gaschromatographie ) stets ein gleiches Verhalten wie Phenprocoumon zeigt. Vor der Gaschromatoraphie erfolgt eine Silylierung mit einem BSA-TMCS Gemisch 99:1 ), verwendet wird ein Varian G~schromatograph Typ 1704 ( S~ule 3% OV I, S~ulentemperatur 230 ). Die pharmakokinetischen Untersuchungen wurden nach intravenSser Gabe von 20 mg Marcumar an gesunden Probanten durchgef~hrt. Die Anfangskonzentrstionen lagen um 5 ug /ml Serum, entsprechend einem Verteilungsvolumen von ca. 5 Litern. Danach f~llt der Serumspiegel mit einer schnellen (I) und einer langsamen (II) Komponenten ab. Die Halbwertszeiten lagen bei 5 (I) und 150 (II) Stunden. Phenprocoumon wird zu 90 Prozent als Glucuronkonjugat im Urin ausgeschieden.

~

Dr. N. Keni, Medizinische Hugstetterstrasse 55

Universit~tsklinik,

D-7800 Freiburg,

INFLUENCE OF EThaNOL ON TRICHLOROETHYLENE METABOLISM IN MAN (EinfluB von ~thanol auf den Trichlor~thylen-Umsatz im Menschen) D.Henschler, Th.Urban Six male volunteers inhaled 50 ppm trichloroethylene (Tri) for 6 h/day on 5 consecutive days with and without simultaneous consumption of ethanol to maintain a blood level of 0.6%0 throughout the 6-h periods. Measured by the plasma (or blood) levels, the formation of trichloroethanol (TCE) was inhibited by ethanol to abt.50%, that of trichloroacetic acid (TCA) to abt.60% of normal. This is confirmed by the concomitant reduction of the urinary metabolites TCE (mostly as the glucuronide) and TCA. Detailed analysis of the blood (plasma) levels reveals that after the rapid elimination of ethanol an additional transformation of Tri to TCE and of TCE to TCA takes place resulting in irregular elimination characteristics of both metabolites: the plasma concentrations continue to rise during the first 4 hrs after the exposure but decline in the control series. This deviation from the normal pattern is more pronounced with the formation of TCA as compared to that of TCE, indicating that the enzymatic conversion of Tri to chloral is inhibited to a greater extent than that of TCE to TCA. This accords well with the finding of SELLERS et al. (Clin.Pharmacol. 13, 37; 1971) that ethanol enhances the formation of TCE from chloral hydrate. D.Henschler, Universit~t,

Institut fur Pharmakologie und Toxikologie D-8700 W~rzburg, KoellikerstraBe 2

der

R30 HYPERTENSIVE ACTION 0P 9~-PLUOROOORTISOL IN THE RAT (Hypertensive Wirkung yon ~-Fluorocortisol bei der Ratte) R. Hepp, F. Gross Unilaterally nephrsct0mized male Wistar rats received daily injections of 9~-fluorocortisol ( ~ - ~ ) in doses of I mg (group z) and 0.5 mg (group II) during three weeks. Controls had daily injections of peanut oil. Pellets containing 100 mEq/kg NaC1 (Ssniff) were given as diet and 0.9% saline as drinking fluid. In both groups, blood pressure rose already three days after the beginning of treatment. After three weeks, mean blood pressure was 178+5.07 SE (group I) aad 179.4+3.77 SE (group II) (control group, 132.5+2.67 SE). In contrast to the lack of dose-dependence for blood pressure, effect on body weight was related to the dose. In group I, body weight decreased, in group II a markedly reduced weight gain was found compared with control rats. All rats on 9~-FF had a diminished food intake. At the end of the experiment, plasma renin concentration was very low in both groups (group I: 25.65+1.88 SE ng Al/ml; group II, 25.55+1.27 8E ng AI/ ml; control group: 181.4+19.1 SE ng AI/ml). Similarly, kidney renin content was reduced (group I: 27.4+5.46 SE ~g/kidney; group II: 27.4+ 4.44 SE ~g/kidney! control group: [64.6+17.34 SE ~g/kidney). Haematocrit was elevated in some of the rats treated with 9u-FF and reduced to ~.ubnormal values in some others. High doses of 9~-FF induce both mineralo- and glucocorticoid-like symptoms, and in the rat the glucocorticoid effect is prevailing. Dr. R. Hepp, Pharmakologlsches Institut der UniversltKt, D-6900 Heidelberg, Hauptstr. 47-51.

M O D I F I C A T I O N OF PRECIPITATED W I T H D R A W A L IN THE RAT BY INFLUENCING C A T E C H O L A M I N E R G I C MECHANISMS ACUTELY A N D C H R O N I C A L L Y (Ver~nderung des p r o v o z i e r t e n M o r p h i n e n t z u g s an der Ratte durch akute und chronische Beeinflussung k a t e c h o l a m i n e r g e r Mechanismen) A. Herz, K. Reinhold The effect of various drugs on the morphine withdrawal syndrome was investigated by the use of a m e t h o d for q u a n t i t a t i v e assessment of withdrawal in rats (BLASIG, ZIEGLGANSBERGER, this meeting). Within i0 days 6 morphine pellets (each 75 m g base) were implanted s.c. Withdrawal was p r e c i p i t a t e d by 1 m g / k g levallorphan i.p..Brain amines were estimated fluorimetrically. "Acute" inhibition of catecholamine synthesis within 24h before withdrawal by ~ - m e t h y l - p - t y r o s i n e (AMT) markedly reduced a series of symptoms ~.g. jumping, wet dog shaking, chewing, ptosis)."Acute" inhibition of noradrenaline synthesis by FLA-63 has a similar effect. After "chronic" AMT-treatment (up to 48h b e f o r e withdrawal) the syndrome was completely d e v e l o p e d . " C h r o n i c " d e g e n e r a t i o n of catecholaminergic nerve terminals by 6-hydroxydopamine intensified some symptoms. Acute administration of cocaine and amphet a m i n e enhanced some symptoms (e.g. jumping) others were inhibited (e.g. wet dog shaking, chewing~ ptosis). A similar shift of individual symptoms occurred when the rats had developed a higher degree of dependence (see BLASIG). These results point to an important role of catecholaminergic mechanisms in the manifestation of morphine withdrawal syndrome while in respect to the development of drug dependence the involvement of such m e c h a n i s m s seems to be questionable. Prof. Dr.A.Herz,

Max-Planck-Inst.

f. Psychiatrie, 8 MHnchen 40 KraepelinstraBe 2

8~ INVESTIGATIONS

ON METABOLISM

OF ETHANOL

( U n t e r s u c h u n g e n Gber den S t o f f w e c h s e l (A.G. H i l d e b r a n d t und M. Speck)

IN

RAT

LIVER

MICROSOMES.

yon ~thanol. )

C o n f l i c t i n g reports a p p e a r c o n c e r n i n g the role of a m i e r o s o m a l ethanol o x i d i z i n g system (MEOS). (Lieber and DeCarli, JBC 245 (1970) 2505, T h u r m a n et al., Eur. J.Bioch. 25 (1972) 420). On the a s s u m p t i o n that MEOS r e f l e c t s a c o m b i n a t i o n of c a t a l a s e and N A D P H oxidase activity, we compared rate of N A D P H d e p e n d e n t H202 f o r m a t i o n in m i c r o s o m e s w i t h ethanol or m e t h a n o l m e t a b o l i s m , u s i n g a special device to allow the s i m u l t a n e o u s d e t e r m i n a t i o n of H20~, HCHO and CH$CHO. The f o l l o w i n g results have b e e n obtained: I) the rate of H202 f o r m a t i o n as d e t e r m i n e d in p r e s e n c e of 2 x 1 0 - 4 M azide is n e i t h e r i n f l u e n c e d by C 2 H s O H , C H 3 O H , nor m a n y s u b s t r a t e s of m i c r o s o m a l m i x e d f u n c t i o n oxldase activity. 2) Cons e q u e n t l y i n h i b i t i o n of catalase by 2xio -4 azide a b o l i s h e s methanol o x i d a t i o n completely, but d e c r e a s e s m i c r o s o m a l ethanol oxid a t i o n by 30-50 p e r c e n t only; s u g g e s t i n g an additional, H202 ind e p e n d e n t ethanol d e g r a d a t i o n . E t h a n o l o x i d a t i o n becomes either s t i m u l a t e d or s l i g h t l y i n h i b i t e d by m e t a b o l i s m of other drugs d e p e n d e n t on the s u b s t r a t e chosen and on the rate l i m i t i n g step e s t a b l i s h e d . This d e p e n d e n c e seems to be of p r i m a r y i m p o r t a n c e for the i n t e r p r e t a t i o n of c o n f l i c t i n g results. (A.G. H i l d e b r a n d t , H i n d e n b u r g d a m m 30)

Inst.f.

Klin. P h a r m a k o l o g i e ,

1 Berlin

45,

E f f e c t of ~ - G l y c y r r h e t i n i c Acid on C o r t i c o s t e r o n e P r o d u c t i o n Rats (Einflu8 v o n ~ - G l y c y r r h e t i n s ~ u r e auf die C o r t i c o s t e r o n B i l d u n g bei Ratten) M. H i l f e n h a u s

in

C a r b e n o x o l o n e a s u c c i n y l d e r i v a t i v e of ~ - g l y c y r r h e t i n i c acid has b e e n shown to exert a n t i - i n f l a m m a t o r y a c t i v i t y in rats (for r e v i e w see K h a n and Sullivan, 1968). The effect of 2 - g l y c y r r h e t i n i c acid (GA) on the basal and s t i m u l a t e d p r o d u c t i o n of c o r t i c o s t e r o n e was i n v e s t i g a t e d in rat a d r e n a l s in vitro. At c o n c e n t r a t i o n s of 5xi0-4 to 1x10-3M GA i n c r e a s e d the b a s a l c o r t i c o s t e r o n e p r o d u c t i o n by 3-fold, w h i l e the c o r t i c o s t e r o n e p r o d u c t i o n s t i m u l a t e d by A C T H (SynacthenR: 10 -~ and 10-7 r e s p . ) was d e c r e a s e d s i g n i f i c a n t l y by GA. An inhibitory effect was also obtained, w h e n the c o r t i c o s t e r o n e production was s t i m u l a t e d by d i b u t y r y l - c A M P . In e x p e r i m e n t s w i t h C - 1 4 - p r o g e s t e r o n e as a p r e c u r s o r of c o r t i c o steroid synthesis GA under basal c o n d i t i o n s i n h i b i t e d the conv e r s i o n to 18-0H.-corticosterone, a l d o s t e r o n e and 1 8 - 0 H - d e s o x y c o r t i c o s t e r o n e , while there was no s i g n i f i c a n t i n h i b i t i o n of the c o n v e r s i o ~ to c o r t i c o s t e r o n e . W h e n s t i m u l a t e d by A C T H (I0-7M S y n a c t h e n n ) , however, the c o n v e r s i o n of C - 1 4 - p r o g e s t e r o n e to c o r t i c o s t e r o n e was s i g n i f i c a n t l y d e c r e a s e d by GA. Dro M. H i l f e n h a u s , Hochschule, D-3000

I n s t i t u t fGr P h a r m a k o l o g i e , M e d i z i n i s c h e Hannover, K a r l - W i e c h e r t Allee 9

R 32

ON THE NATURE OF SUBSTRATE BINDING TO MIXED-FUNCTION OXIDASES FROM RABBIT'S LIVER (0ber die Natur der Bindung yon Substraten an mischfunktionelle Oxygenasen aus Kaninchenleber) P.Hlavica The pK values of groups involved in the binding of aniline (Type II) and N,N-dimethylaniline (Type I) to hepatic microsomal mixed-function oxidase have been found to be 7.07 for aniline and 6.72 for N,N-dimethylaniline at 36 o. The heats of ionization determined for both groups were 6,310 cal/mole and 7,575 cal per mole, respectively. Photooxidation of the enzyme in the presence of rose bengal obeyed first order kinetics. Aniline as well as N,N-dimethylaniline were protective against photoinactivation. Plots of first order rate constants for inactivation against pH yielded dissociation curves of groups with a pK around 6.0. These findings univocally hint at the involvement of a histidyl residue in the binding of both compounds to the oxidas~ probably constituting an axial ligand in cytochrome P-450. The results furthermore suggest at least one common binding site for Type I and Type II substrates. An additional lipid binding site of faciliative character for Type I compounds is discussed. Thus binding of aniline to the enzyme is an endothermic process (AG 3-5 kcal/mole; A H = + 11.8 kcal/mole; A S = + 49.9 cal/mole per degr.), whereas binding of N,N-dimethylaniline is an exothermic reaction (&G = - 5-5 kcal/mole; A H = - 13.0 kcal/mole; A S = -25 cal/mole per degr.). P. Hlavica, Institut fGr Pharmakologie und Toxikologie der Universit~t, D-8000 M~nchen 2, Nussbaumstrasse 26 -

NEGATIVE REINFORCING PROPERTIES OF MORPHINE-ANTAGONISTS IN NAIVE RHESUS MONKEYS (Aversive Eigenschaften yon Morphinantagonisten bei nicht abh~ngigen Rhesusaffen) F.Hoffmeister, W.Wuttke 7 rhesus monkeys were trained to press a lever to turn off a white light which was associated with a drug infusion of 10 sec scheduled to occur at 30 sec after the onset of the light. Each response during the light period terminated the light for a one-minute time-out period (avoidance), a response during the injection terminated the injection (escape). Under this conditions the monkeys tolerated a high number of saline injections. Then saline was replaced by different unit doses of nalorphine, cyclazocine, codeine and cocaine each for six successive daily two hour sessions~ Doses from 500 to 10 mcg/kg/inj, nalorphine and doses from 10 to 2.5 mcg/kg/inj, cyclazocine generate and maintain avoidanceescape behavior while codeine and cocaine in unit doses of 50 mcg/kg/inj, are tolerated by the subjects, that is, lever pressing behavior extinguished. The results show, that in naive rhesus monkeys, that is, subjects without any drug experience prior to the experiment the morphine-antagonists nalorphine and cyclazocine have negative reinforcTng properties. Prof.Dr~

BAYER AG, 56 Wuppertal I, Postfach 130105

R 33 H E M O D Y N A M I C E F F E C T S OF C H E M I C A L S Y M P A T H E C T O M Y W I T H D 0 P A M I N E IN C O N S C I O U S D O G S ( W i r k u n g yon c h e m i s c h e r mie auf d e n K r e i s l a u f des w a c h e n H u n d e s ) J. H o l t z , E. B a s s e n g e , W. v. R e s t o r f f , K. 0 v e r s o h l

6-HYDROXYSympathekto-

In t r a i n e d dogs h e a r t rate (HR), c a r d i a c o u t p u t (CO), m e a n a r t e rial p r e s s u r e ( M A P ) , p e r i p h e r a l r e s i s t a n c e (PR), total b o d y 0 2 - c o n s u m p t i o n (V02) , and c o r o n a r y f l o w (CF) w e r e r e c o r d e d u s i n g chronically implanted flow transducers. Chemical sympathectomy w i t h 6 - h y d r o x y - d o p a m i n e (6-OH-DA) was p e r f o r m e d in the f o l l o w i n g m a n n e r : 6 - 0 H - D A in i n c r e a s i n g d o s a g e s icy. (i~ 2; 4; 8; 15; 30 mg/kg) w i t h i n 4 hours. F o l l o w i n g the f i r s t i n j e c t i o n M A P imm e d i a t e l y rose up to 230 m ~ g , there was an i n c r e a s e of 400 in PR, H R d r o p p e d d o w n to a p p r o x i m a t e l y 30 b e a t s / m i n d u e to vagal a c t i v a t i o n . P e a k r e a c t i o n s of all p a r a m e t e r s a f t e r the next i n j e c t i o n s w e r e similar, but d u r a t i o n of e f f e c t s was s h o r t e n e d i n s p i r e of i n c r e a s i n g d o s a g e s . 3 to 7 days a f t e r 6 - 0 H - D A sympat h e c t o m y was v e r i f i e d by t y r a m i n e i n j e c t i o n s up to 1 m g / k g a n d by s t e l l a t e g a n g l i o n s t i m u l a t i o n ( a n e s t h e s i a ) . At rest and d u r i n g s t e a d y state e x e r c i s e PR and CF per unit of CO w e r e red u c e d s i g n i f i c a n t l y c o m p a r e d to c o n t r o l s (at i d e n t i c a l V02) p r i o r to s y m p a t h e c t o m y . C a r d i o v a s c u l a r a d j u s t m e n t to e x e r c i s e was e x t r e m e l y d e l a y e d . H o w e v e r , w h e n e x e r c i s e was g r a d u a l l y increased, a 400 % rise of V02 d u r i n g s t e a d y state e x e r c i s e was tolerated. Dr. J. Holtz, Priv. Doz. Dr. E. B a s s e n g e , P h y s i o l o g i s c h e s I n s t i rut der L u d w i g - M a x i m i l i a n s - U n i v e r s i t ~ t , 8000 M ~ n c h e n 2, P e t t e n k o f e r s t r a B e 12

INVESTIGATION BY VARIATION STATISTICS OF SERUM-ELECTROPHORESIS DATA AT WISTARRATS ( V a r i a t i o n s s t a t i s t i s c h e Untersuchungen mit Serum-Elektrophoresewerten bei Wistar-Ratten) H. Jacobi, P. Breier Im Rahmen der tierexperimentellen PrUfung von Antiphlogistika i s t die Beeinflussung der SerumeiweiBkonzentration in Abh~ngigkeit vonder Dosis der zu untersuchenden Pharmaka von zus~tzlicher Bedeutung fur die Beurteilung der entzUndungshemmenden E f f e k t i v i t ~ t der Wirkstoffe. Um ein Bezugssystem schaffen zu k~nnen, wurde bei u~behandelten Wistar-Ratten unter konventioneller Haltung bzw. unter SPF-Bedimgungen durch Elektrophorese die Albumin- bzw. Globulinkonzentration im Serum bestimmt. Es wurde gefunden, dab m~nnliche konventionell gehaltene Wistar-Ratten einen h~heren Globulinanteil im Serum besitzen als die weiblichen Tiere der gleichen Species. Bei SPF-Tieren war - unabh~ngig vom Geschlecht - die Globulinkonzent r a t i o n im Serum niedriger als bei m~nnlichen Ratten unter konventionellen Bedingungen. Die Unterschiede der Werte der Tiere der letztgenannten Gruppe gegenUber anderen zum Vergleich anstehenden Versuchstiergruppen war s t a t i s t i s c h im X-Test als auch im t-Test mit einer I.W. < o , I % gesichert. Bei der Sicherung mit v a r i a t i o n s s t a t i s t i s c h e n Testmethoden konnte gezeigt werden, dab die Albumin- und Globulinkonzentration der Ratten a l l e r Gruppen nicht einer Normalverteilung f o l g t , sondern einer Mischverteilung. Dabei war es gleichgUltig, ob der SerumeiweiBgehalt in Prozent oder in g/loo ml aufgetragen wurde. Der Befund, dab m~nnliche konventionell gehaltene Wistar-Ratten mehr Globuline als Albumine im Serum besitzen, wurde unabh~ngig davon erhalten, ob das Blut zur Untersuchung aus dem Unterzungenvenenplexus oder durch Herzpunktion gewonnen wurde. Dr. H. Jacobi, Abteilung f u r experimentelle Pharmakologie der Troponwerke, D-5ooo K~In 8o, Berliner Str. 22o

R84 EFFECT OF SUBSTANCES ONPARAVASCULARPAINRECEPTOR~OF THE PERFUSEDRABBIT EAR (Wirkung yon Substanzen aufparavascul~re Schmerzrezeptoren des durchstr6mten Kaninchenohres) H. Juan~ F. Lembeck Bei Injektion yon verschiedenen Substanzen (s. Tab.) in das isoliert durchstrOmte, nur mehr durch den N. auricularis m. mit dem K6rper verbundene Kaninchenohr kommt es zu reflektorischem Blutdruckabfall und zu Atmungssteigerung. Um die Erregung der paravaskul~ren Schmerzrezeptoren genauer erfassen zu k6nnen, wurden die Aktionspotentiale des N. auric, registriert. Die Schwellendosen f/Jr die Aktivierung yon Aktionspotentialen und for den reflektorischen Blutdruckabfall sind nahezu identisch (I). Ein reflektorischer Blutdruckabfall um mehr als 20 n~n Hg erfolgte auf die 4 his 10fache Schwellendosis (II).

(I) Histamin Serotonin ACh ATP SP (Darmextrakt) SP (Hirnextrakt "Fa") SP (synthetisch) Bradykinin

5OO~g 200 ~g 5 ~g 200 ~g 5 E 5 E 100 ng 50 ng

(II) 3 2 20 2 20 20 5OO 200

mg rag ~g mg E E ng ng

Die Ergebnisse zeigen a) die Schmerzausl~sung durch eine Vielzahl yon Substanzen und somit die geringe Spezifit~t dieser Rezeptoren und b) die hohe Wirksamkeit yon Bradykinin und Substanz P. Dr. H. Juan, Univ.-Institut ffir Pharmakologie A-8010 Graz, Universit~tsplatz 4, ~sterreich

Pi{ARMACOLOGICAL CHARACTERIZATION IN SYMPATHETIC GANGLIA OF cAMP INCREASE DEPENDENT UPON SYNAPTIC TRANSMISSION (Pharmakologisehe Charakterisierung einer dureh synaptische t~bertragung ausgelSsten Zunahme des zyklischen AMP in sympathisehen Ganglien) P. Kalix, D. McAfee and P. Greensard It has been shown previously that electrical stimulation of the preganglionic fibres to the rabbit superior cervical ganglion results in an increase in the content of cAMP in this tissue, and that this increase is associated with synaptic transmission (McAfee, Schorderet and Greengard: Science 171, 1156(1971)). It has been possible to characterize in vitro the pathways involved in generating this increase in cAMP by use of various transmitter agonists and antagonists. Thus the cAMP concentration was increased in ganglia incubated in the presence of carbachol (202+9% of controls) or bethanechol (175~15%). Atropine but not hexamethonium antagonized the increase in cAMP caused by either cholinomimetic agents or preganglionic stimulation. Furthermore, the m-adrenergic antagonist, thymoxamine, and the dopaminolytic agent, spiroperidol, antagonized the carbachol induced increase in tissue cAMP concentration. The phosphodiesterase inhibitors theophylline and Ro 20-1724 potentiated the increase in cAMP generated by either carbaehol or preganglionic stimulation. These observations are consistent with the idea that the observed increases in cAMP are dependent upon muscarinie activation of a dopaminergic interneuron. According to this hypothesis, the dopamine released from this interneuron is responsible for the activation of a Postganglionic adenylate cyclase system. Dr. P, Kalix, Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA

R 35 2-HYDROXYLATIONOF ETHYNYLESTRADIOL AND SUBSEQUEN~fREACTIONS DUE TO THE MICROSOMALMIXEDFUNCTION 0XIDASE (Bedeutung der mikrosomalen mischfunktionellen Oxidase Cdr die 2-Hydroxylierung yon Athinyl~stradiol und anschlieHende Folgereaktionen) H. Kappus, H. Remmer

The metabolism of ethynylestradiol by rat liver mierosomes in the presence of a NADPH regenerating system has been studied. The major pathway, the 2-hydroxylation of the aromatic ring, was measured by the labillsation of tritium from 2,4,6,7-3H-labelled ethynylestradiol. Hydroxylations at C-6 and C-7 were found to be of minor importance. The amount of tritium, removed from C-2 and C-~ was increased two-fold by pretreatment of the animals with phenobarbital. The addition of glutathione to the incubation resulted in a 50 % increase of the mobilization of the 2,~-tritlum due to the formation of glutathione conjugates at position #. The 2-hydroxylatlon of ethynylestradiol required NADPH and could be inhibited by CO and SKF 525 A. Thus, it was concluded that the microsomal drug metabolizing mixed function oxidase is responsible for the 2-hydroxylation of ethymylestradiol. Microsomal incubations of 6,7-3H-ethynylestradiol with glutathlone resulted in the formation of polar metabolites, which was also dependent on NADPH and inhibited by CO and SKF 525 A. Furthermore, part of the metabolized ethynylestradlol was bound firmly to the mlcrosomes and could not be removed by solvent extraction, dialysis and charcoal adsorption. A similar binding to albumin has been detected if albumin was added to the incubation. The protein binding which could be abolished by glutathlone was also increased two-fold by phenobarbital pretreatment. This reaction also required NADPH and was inhibited by CO and SKF 525 A. Dlpl.-Biochem. H. Kappus, Institut ~dr Toxlkologle der UnlversltKt, D-?~00 Tgblngen, WllhelmstraSe 56

EFFECT OF SOME GASTRIC STI~ILANTS ON TNE P R O D U C T I O N OF CYCLIC A M P IN M I N C E D GASTRIC T I S S U E ( U b e r die W i r k u n g einiger S t i m u l a t o r e n der M a g e n s a f t s e k r e t i o n aus die B i l d u n g yon c y c l i s c h e m AMP in isoliertem M a g e n g e w e b e ) H.O.Karppanen, P.T.Neuvonen, E . W e s t e r m a n n M i n c e d gastric tissue os rats or guinea pigs was i n c u b a t e d in Tyrode solution. The cAMP content in the tissue and m e d i u m was d e t e r m i n e d by the p r o t e i n binding assay os G i l m a n (Proc.Nat.Acad. S c i . 6 7 : 3 0 5 : 1 9 7 0 ) . T h e o p h y l l i n e (10 -4 - 5x10-2M) caused a dosedepen--~ent i n c r e a s e in the level os cAMP in the tissue. H i s t a m i n e or p e n t a g a s t r i n alone had a h a r d l y d e t e c t a b l e es163 on the tissue cAMP. In the p r e s e n c e os t h e o p h y l l i n e (IO-JM), h i s t a m i n e (I0-3M) e l e v a t e d tissue cAMP w i t h i n 5 m i n (s 550 to 900 pmol/g), cAMP was s p o n t a n e o u s l y r e l e a s e d into the medium. In the p r e s e n c e os t h e o p h y l l i n e (I0:-3M) h i s t a m i n e and p e n t a g a s t r i n i n c r e a s e d the cAMP content in the medium, e.g. h i s t a m i n e (I0-3M) p r o d u c e d an i n c r e a s e w i t h i n 10 min s 500 to 4800 pmol/g. A s s t i m u l a t i o n almost all os the cAMP f o r m e d was r e l e a s e d into the medium. A s i g n i s i n c r e a s e os cAMP levels in tissue and i n c u b a t i o n m e d i u m was only o b s e r v e d in the p r e s e n c e os theophylline. This indicates the presence os a high p h o s p h o d i e s t e r a s e a c t i v i t y in gastric tissue. Furthermore, it was shown that besides cAMP also p h o s p h o d i e s t e r a ~ e a c t i v i t y was r e l e a s e d into the i n c u b a t i o n medium. We suggest that the i n c r e a s e d p r o d u c t i o n os cAMP and its r e l e a s e from gastric cells may p l a y a role in HCI secretion. Pros s Pharmakologie,

M e d i z i n i s c h e H o c h s c h u l e Hannover, Institut K a r l - W i e c h e r t - A l l e e 9, 3000 H a n n o v e r - K l e e s

R 36 STRIATAL DOPAMINERGIC AND CHOLINERGIC MECHANISMS IN RATS WITH UNILATERAL SPREADING DEPRESSION (Striatale dopaminerge und cholinerge Mechanismen bei Ratten mit unilateraler "spreading depression") K.H. Keller, L. Pieri and G. Bartholini Unilateral application of a hyperosmolar solution of KCI on the dura of the rat cerebral cortex caused a functional inactivation of the ipsilateral cortex and striatum ("spreading depression", SD) without alterations in muscle tone and locomotor activity. Intraperitoneal pretreatment with drugs which enhance the dopaminergic function in the intact striatum (e.g. apomorphine or methamphetamine) lead to body torsion and rotational movements towards the side of SD. The apomorphine-induced functional changes were antagonized by arecoline and oxotremorine, but not by nicotine. Atropine, but not methylatropine, blocked the action of arecoline. It is concluded that in the regulation of muscle tone and locomotor activity, a dopaminergio and a cholinergio (probably muscarinic) system in the striatum, antagonistic to each other, are involved.

Dr. R.H. Keller, Medical Research Department, F. Hoffmann-La Roche & Co., Ltd. Basle, Switzerland

Dose-Effect-Relationship

of Tolbutamid at d i f f e r e n t b l o o d g l u c o s e l e v e l s in h e a l t h y v o l u n t e e r s (Dosis-Wirkungs-Beziehung yon Tolbutamid in Abh~ngigkeit vom Blutzucker bei gesunden Proband e n ) H . K e w i t z p U. S c h n a p p e r e l l e u. P. H e i n e A f t e r i.v. injection of 0 , 5 - 2 . 0 g T o l b u t a m i d in healthy humans with fasting blood glucose levels the plasma insulin (radioi m m u n e a s s a y e d ) i n c r e a s e s d e p e n d i n g on t h e d o s e s to m a x i m a l v a l u e s o f 62 - 94 u E / m l w i t h i n 2 , 5 - 5 min. Insulin declines t h e r e a f t e r b y a h a l f l i f e o f 15 - 20 min. B y i.v. i n f u s i o n o f 4 ml/min o f i0 % g l u c o s e s o l u t i o n t h e p l a s m a g l u c o s e l e v e l s w e r e b r o u g h t u p to 1 5 0 - 2 0 0 m g / 1 0 0 m l and i n s u l i n g r e w f r o m 15 - 2 0 u E / m l to 30 - 40 u E / m l . Under these conditions the activity of Tolbutamid is d o u b l e d as f a r as t h e i n c r e a s e o f i n s u l i n w i t h i n t h e f i r s t f e w m i n u t e s is c o n c e r n e d . B u t i n a d d i t i o n a s e c o n d p e a k a p p e a r s w h i c h at 1.5 a n d 2 . 0 Tolbutamld is h i g h e r t h a n t h e f i r s t o n e a n d w h i c h a l s o f o r m s a p l a t e a u o f 5 - 3 O" m i n u t e s d u r a t i o n . Most likely the second insu~ in p e a k m a k e s T o l b u t a m i d u s e f u l in d i a b e t i c s . S i n c e b o t h t h e s e p h a s e s o f i n s u l i n r e l e a s e a r e p r o b a b l y b a s e d on different mechanisms it is n o t s u i t a b l e to c o m p a r e t h e d o s e , effect-relationship at f a s t i n g g l u c o s e l e v e l w i t h t h a t at a higher glucose level in quantitative terms. Prof. Dr.H. Klinikum

K e w i t z , Institut S t e g l i t z d e r FU,

fur Klinische Pharmakologie 1 B e r l i n 45, H i n d e n b u r g d a m m

im 30.

R 37 UPTAKE OF CHOLINE ESTERS BY THE ISOLATED GUINEA-PIG ILEUM (Aufnahme von Cholinestern durch das isolierte Meerschweinchenileum) H. Kilbinger, G. Walter Pieces of gulnea-pig ileum were incubated in Tyrode solution containing elther DFP (lo -5 /V~ or eserine (lo -4 M). After a 30 rain preincubation period the ileum was exposed for 60 rain tc, 12.25 pM of either acetylcholine (ACh), propionylcholine (PCh) or butyrylcholine (BCh). At the end of the incubation period the ileum was washed once per rain for 5 min. The amounts of the choline esters in the tissue were estimated by gas chromatography (H. Kilbinger, Naunyn-Schmiedebergs Arch. Pharmak. 274, R 64, 1972). The ACh concentration of the ileum before incubation was 27.5 + 2.--'8 nmoles/g (mean + S.E.; n=13). During the incubation with DFP the ACh conten-i increased to 51.3 + 5".4 nmoles/g (n=5), but there was only a small additional increase to 57.6 + 5~5 nmoles/g (n=5) when the tissue was incubated with DFP plus ACh. After incubation with eserine the ACh concentration of the tissue was 85.2 + 5.0 nmoles/g (n=4). Incubation with ACh plus eserlne did not lead to a further increase of the ACh content of the ileum. In 3 experiments incubations with PCh or BCh in the presence of eserine did not significantly alter the ACh concentration of the tissue; additionally the ileum contained 1.5 + 0.1 nmoles PCh/g or 2.0 + 0.3 nmoles BCh/g, respectively. It is concluded that in the presence of a cholinesterase inhibitor the isolated guineapig ileum takes up only small amounts of choline esters. There is no evidence of an uptake mechanism for ACh analogous to that described for rat brain slices (R.L. Polak, Br.J.Pharmac. 36__, 144-152, 1969). Dr. H. Kilbinger, Pharmakologisches lnstitut der Universit~t D-65oo Mainz, Obere Zahlbacher Str. 67

THE INTERPRETATIONOF 131j EL[M[NAT[.ON UNDER VARIOUS DIETARY CONDITIONS (Die Beurteilung der 131j-Eliminatlon bei Ratten unter verschiedenen Ern~hrungsbedingun gen) M. Klett, A. Kaul Surgical thyroidectomy in conjunction with a thyroid gland test using a microradio-iodine-test (MRI) has proved to be a suitable method for evaluating completely thyroidectomized rats (SCHIMMELPFENNIG und KAUL, 1972) Qualitative and quantitative changes in diet alter, however the 131j-elimination -rate. We investigated the 131j-elimination with normal (Altromin CI00O 10 g/ day), protein deficient (Altromin CI030 10 g/day) and calory deficient diet (AItromin CIO00 5 g/day). Measurements of 131j retention were performed for the whole body and for the thyroid gland region (MRI) over a 161 hrs period. Euthyroid rats on a calory deficient diet show a higher 131j uptake in the thyroid gland region, while the 131j-elimination rate is decreased significantly when compared with controls. Euthyroid rats on a full calory but protein deficient diet show a normal 131j uptake and a decreased elimination rate. Completely thyroidectomized ra~s on a full calory but protein deficient diet show also a decreased elimination rate. The decrease of 131.l-elimination rates are to be explained partly as a disturbance of the endocrine system (synthesis and secretion), partly as a result of morphological alterations in the kidneys which show nephrotic changes with animals on protein deficient diets. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 29). Dr. Martin Klett, PharmakolOgisches Institut der Freien Universit~t Berlin, Abt. "Embryonal-Pharmakologie",~D-IOOO Berlin 33, Thielallee 69/?3

R 38

PRESSURE-VELOCITY-CURVES FOR EVALUATION OF MYOCARDIAL CONTRACTILITY (Druck-Geschwindigkeitskurven zur Beurteilung der myokardialen Kontraktilit~t) E. KShler

An narkotisierten Katzen wurde mit einem Kathetertipmanometer der Druck in der linken Herzkammer gemessen und ,nit einem Analogrechner (dp/dt)/p bzw. (dp/dt)/(p-edp) ermittelt; ventrikul~re Druck-Geschwindigkeitskurven erhielten wir durch Auftragen dieser Quotienten gegen p bzw. (p-edp) im xy-Speicheroszillographen. Druck-Geschvcindigkeitskurven, die sich aus dem entwickelten Druck (p-edp) errechnen, werden durch .~nderungen des p r e und Afterload nicht beeinflusst. Druck-Geschwindigkeitskurven, die aus dem Gesamtdruck ermittelt werden, zeigen bei Erh~hung des Preload ein niedrlgeres Maximum sowie eine Linksverschiebung. Acetylcholin senkt dp/dtmax w~hrend ((dp/dt)/p) max und Druck-Gesehwindigkeitskurven nieht ver~[ndert werden. Nitroglycerin verschiebt Druck-Geschwindigkeitskurven bei nicht signifikant ver~_ndertem dp/dtmax nach rechts. pentobarbital bewirkt eine Links-, g-S%rophanthin eine Rechtsverschiebung der Druck -Ges chwindigkeitskurven. Die Versuche zeigen, class Druck-Gesch~vindigkeitskurven ein sehr empfindlicher parameter fiir Kontraldilit~ts~nderungen sind; die erforderliche Interpolation der Verldirzungsgeschwindigkeit auf den Druck Null erschwert eine quantitative Kontraktilit~[tsbewertung. E. K6hler, pharmakologisches Institut der Universit~[t IMisseldorf D-4ooo Diisseldorf, Moorenstrasse 5

VERANDERUNGEN DER TRANSMEMBRANAREN CA- UND NA-KONDUKTIVIT~T DER WARMBLOTER-MYOKARDFASER BEI ERSATZ VON CA- DURCH SR-IONEN. (Changes in transmembrane Ca and Na conductance of mammalian myocardial fibres following substitution of Ca by Sr ions). M. Kohlhardt; A. Herdey und H. Krause Der langsame Membrankanal des WarmblGter-Myokards kann neben Ca ++ auch von Sr ++, Ba ++ und Mg ++ mitbenutzt werden (1). Wie VoltageClamp-Experimente an isolierten Trabekeln yon Katzen jetzt zeigten, wird der dutch Ca++-Entzug induzierte Verlust des langsamen Einwartsstromes dutch Zugabe von Sr ++ zur TyrodelSsung verhindert. Dieser Sr++-Strom ist dutch eine stmrk verzSgerte Inaktivierung charakterisiert, die auch am schnellen Na+-Strom auftritt. D 600 und Verapamil hemmen den transmembranMren Sr++-Strom ebenso wie den physiologischen Ca++-Strom. Dabei wird die verz6gerte Inaktivierung des Sr++-Stromes und des schnellen Na+-Stromes nicht beeinfluSt. Erst Ca++-Zusatz zur Sr++-haltigen Tyrodel6sung normalisiert die Inaktivierungskinetik beider Str6me. Offensichtlich hangt das normals "gating" in beiden Kan~len entscheidend yon der Anwesenheit yon Ca ++ ab. Die Befunde zeigen, dab Ca ++ als physiologischer Ladungstr~ger des langsamen Einw~rtsstromes komplett dutch Sr ++ ersetzt werden kann. Inhibitoren des langsamen Membrankanals sind auch dann roll wirksam, wenn dieses Transportsystem den Einstrom anderer bivalenter Kationen anstelle von Ca ++ vermittelt. (I) Haastert, H.-P., M. Kohlhardt und H. Krause: Arch. exp. Path. Pharmak. 274 , R 47 (1972) Anschrift der Verfasser: Physiologisches Institut der Universitat Freiburg i. Br.

R 39 NORADRENERGIC DEPRESSION OF SYMPATHETIC VASOMOTOR CENTERS. EXPERIMENTS WITH DOPS IN THE CIRCULATION OF THE DOG. (Noradrenerge D~mplung sympathlscher Krelslaufzentren. Untersuchungen mit DOPS am Krelslauf des Hundes). D, K rause Der Einsatz yon ~hreo-d,i-3,4-Dihydroxyphenylserin (DOPS) ale Noradrenalin-Precursor sollte eine direkte BeweisfGhrung fGr die angenommene noradrenerge D~mpfung sympathischer Kreislaufzentren erm5gllchen, Versuche mit iv. InJektion yon Dops zeitigten keine bewertbaren Resultate. Bei "gezielter" intraarterieller InJektion in die Art. vertebralis oder die Art. carotis (bei Unterbindung der A. carotis externa) wurden stets reproduzierbare Effekte erzielt. Die abgeschlossene Versuchsserie umfaSt Versuche mit intraarterieller InJektion an vagotomierten Hunden in Chloralose-Narkose. Folgende Resultate sind gesichert: I. DOPS fGhrt in Dosierungen von 10-30 mg/kg zu Blutdrucksenkung, Verlangsamung der Herzfrequenz und Reduktion des Effektes der C.S.E.. Wiederholung der InJektion zeltigt nut eine geringfGgige Wirkung, gelegentlich eine mlt "Weckwirkung" verbundene "Umkehr". 2. Hemmung der Decarboxylase durch Ro 4/4602 verhindert die Wirkung von DOPS vollst~ndlg. 3. Teilweise Hemmung der MAO dutch Tranylcypromin, Phenelzin oder Harmalin verst~rkt die Wirkung yon DOPS und ergibt eindeutige Versuchsprotokolle. 4. Atropin und Haloperidol haben keinen Einflu~ auf die Effekte von DOPS. 5. Yohimbin hebt die Wirkungen yon DOPS auf. Die Versuchsergebnisse stGtzen die Theorie einer noradrenergen Hemmung des zentralen Vasomotoren- und Acceleranstonus. Prof.Dr.D.Krause,

F.U. Berlin,

FB8, 1 Berlin

33, Eoserstr.

20

FAILURE OF ISOPROTERENOL TO RELAX RAT AORTA DEPOLARIZED BY K + AND RELATIONS TO CELLULAR CYCLIC AMP LEVELS (Ausbleiben der Iso~roterenolinduzlerten Relaxation des Aortenstrelfens der Ratte nach K -Depolarisation und Beziehungen zum intrazellul~ren cAMP-Spiegel) V.A.W. Ere[e and H. Schmidt-GaFk In organbath experiments, helical strips of rat aorta contracted with phenylephrine (PE, I0-7M) are relaxed by isoproterenol (ISO, 10-5M) by 70 to 100 %, but strips depolarized by K + (30 mM) are contracted by ISO. This contraction consists of an initial rapid and a following slowly progressive phase. In the presence of phentolamine (2.65 x I0-5M), however, ISO also relaxes K+-depolarized rat aorta. Thus, the ratio of ~ to 8-adrenergic activity of IS0 is increased after depolarization with K. Cyclic AM]~ levels under the various experimental conditions were measured in homogenates of rat aorta by the protein kinase method of Wastila. Basal cyclic AMP levels in contro~ preparations and in rat aortae preincubated with PE (I0-7M) or with K (50 mM) are similar (about 200 pmoles/ g wet weight). Incubation with ISO (I0-5M, 3 min) results in a pronounced increase in cyclic AMP concentration (303 pmolee/g w.w.) in control strips. After addition of ISO to strips preincubated with PE or K +, cyclic AMP levels are moderately elevated and similar in quantity (220 and 222 pmoles/g w.w., resp.), although ISO has opposite effects on muscular tone of rat aorta under these conditions. The results demonstrate that the increase in cyclic AMP induced by ISO and its relaxant power are not correlated in a simple manner. Dr. V. Kreye, Phaz.makologisches Institut der Universit~t, D-6900 Heidelberg, Hauptstr. 47 - 51

R 40 EFFECT OF FLUORODEOXYCYTIDINE(FCdr) ON DNA SYNTHESIS IN MAMMALIANEMBRYOSDURING ORGANOGENESIS (EinfluB yon FCdr auf die DNA-Synthese lm WarmblUterembryo wShrend der Organogenese). R. Krowke, G. Bochert, P. Mewes FCdr was shown to be a potent embryotoxic drug In mlce (DEGENHARDTet a l . , Teratology ~, 311, 1968). We therefore t r i e d to get a deeper insight into Its mode of action by using our bJochemlcal test system (KROWKE et a l . , Naun. Schm. Arch. Pharm. 271, 274, 1971; BOCHERTet e l . , to be published In t h i s journal 1973). I) Pregnant mice (day 1o of gestation) received an Intraperitoneal injection of the drug (o,I - 5 mg/kg) and an Intravenous mixture of IWC-u-glucose and 32_p_ phosphate (3 mCi/kg of each isotope) 90 min. prior to sacrifice. Using our screening method, reduced radioactivity was found only In the DNA fraction. The minimal teratogenic dose is about 3 mg/kg. DNA synthesis was already inhibited to more than 5o % at a dose of o,I mg/kg (30 mln. Incorporation tlme of the drug and precursors). 2) Highly purified DNA was hydrolized to nucleotldes and bases and the specific a c t i v i t i e s determined In the separated compounds. Data found wlth dosages of 3 mg/kg show a nearly complete block of DNA synthesis even after 3h. T G A C

hosph.

FCdr

32p

1 mcl/kq

3ontrol 3.12 lo5 min. 0.3 80 mln. 2.0 Data ;dpm/nmol;

12.o 0.7 6.9

13.2 1.9 12.9

3.25 0.57 2.1

21.6 3.8 11.6

lo.2 2.4 9.2

1.76 0.24 1.22

13.3 1.4 7.1

6.5 0.9 5.3

1.95 0.28 1.61

lo.6 1,2 8.9

4.7 0.7 6.0

n = at least 4 d i f f e r e n t estimations; SD. < • 4 %.

Or. R. Krowke, Pharmakologisches I n s t i t u t der Freien Universit~t Berlin, Abt. "EmbryonaI-Pharmakologle ", D-looo Berlin 33, T h l e l a l l e e 69/73

INHIBITION BY DDT AND RELATED COMPOUNDS OF CARDIAC MEMBRANEOUS ATPase (Hemmung der Membran-ATPase (E.C.3.6.1.3.)aus K~ilberherzen durch DDT und verwandte Verblndungen) V.v. KUgelgen und F. K. Ohnesorge The pH-optimum of ATPase of calf-heart cell membranes ranged from 7.6 to 8.2. The substrate-optimum was 1 rnM (Mg++-ATPase) and 2 mM (Na+, K+-ATPase). The activity increased nearly linear between 30 and 40oc (Q10 about 2). Different preparations differed slightly. Experimental conditions: 2 mM ATP, 37~ pH 7.4. p,p'--DDT, p,p'-DDD, p,p'-DDE and Kelthane (KEL) in concentrations ranging from 0.1 to 10pM inhibited both the Mg"H'- and the Na+, K+-ATPase. Inhibition of the Na+, K+ATPase by the pesticides was more pronounced than the inhibition of the Mg++-ATPase. The maximum degree of inhibition of the Na+, K+-ATPase was: 78% (KEL), 72% (ODD), 50% (DDT) and 51% (DDE), and of Mg'H'-ATPase: 67% (KEL), 39% (DDD), 35% (ODT) and 23% (DDE). None of the compounds completely inhibited the ATPase. The ID50-values (concentration of inhibitor to produce half-maximal effect) ranged between 1 and 3/uM for both ATPases. Combinations of DDT/DDE, DDD/DDE, KEL/DDE and KEL,/DDD in equimolar concentrations were less effective on both the Mg++- and the Na+, K+-ATPase than the sum of the effects of each of the compounds on both ATPases. Only the combination KEL,/DDD completely inhibited the Na+, K+-ATPase at a concentration of 10pM. Prof. Dr. F. K. Ohnesorge, Abteilung fur Toxlkologie, Institut fur Pharmakologle, Christian-Albrechts-Universlt~t 2300 Kiel, Hospitalstr. 4-6 West Germany

R 41 PHARMA~OKINETICS AND METABOLISM OF DIGOXIGENINMONODIGITOX0SIDE IN MAN(Pharmakokinetik und S t o f f w e c h s e l y o n D i g o x i g e n i n m o n o -

digitoxosid

im m e n s c h l i c h e n

0rganismus)

J.Kuhlmann,U.Ahshagen

lmg3H-digoxigeninmonodigitoxoside(spee.aetivity 98,01uC/mg), d i l u t e d in 100 ml t e a , w a s a d m i n i s t e r e d o r a l l y to 5 h e a l t h y subjects of e i t h e r s e x , a g e d 20-30 y e a r s , a f t e r s t a n d a r d b r e a k f a s t . M a x i m u m p l a s m a levels of r a d i o a c t i v i t y are r e a c h e d w i t h i n 1-2 h o u r s . A f t e r e q u i l i b r i u m h a d b e e n e s t a b l i s h e d the d e g r e e of specific a c t i v i t y in p l a s m a and the b i o l o g i c a l h a l f life times of radioactivity(25-50h)show large i n d i v i d u a l d e v i a t i o n s . 10-30% and 5 0 - 7 0 % ~ef the dose are e l i m i n a t e d in u r i n e and feces r e s p . w i t h i n 7 d a y s . B r i e f l y a f t e r a d m i n i s t r a t i o n 7 0 - 9 0 % of the total r a d i o a c t i v i t y in p l a s m a and in u r i n e are to be f o u n d in the C H C l _ - i n s o l u b l e , c a r d i o i n a c t i v e fraction. In c o n t r a s t the m a i n p o r t i o n ~n feces is r e p r e s e n t e d by C H C l _ - s o l u b l e m a t e r i a l . The T L C - s e p a r a t i o n of the l i p o p h i l i c f r ~ c t i o n in feces y i e l d s m o r e t h a n 70% u n c h a n g e d m o n o g l y c o s i d e b e s i d e s small a m o u n t s of 3-epidigoxigenin,3-ketodigoxigenin,digoxigenin and one more p o l a r m e t a b o l i t e . A p a r t from m e n o g l y c o s i d e the C H C l _ - s o l u b l e p o r t i o n in u r i n e c o n 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 ~ - e p i d i g o x i g e n i n and i n c r e a s i n g q u a n t i t i e s of the same p o l a r m e t a b o l i t e as in feces. In a d d i t i o n traces of d i g o x i g e n i n , 3 - k e t o d i g e x i g e n i n and a n o t h e r , m o r e p o l a r m e t a b o l i t e are d e t a c t a b l e . T h e s e r e s u l t s i n d i c a t e an i n c o m p l e t e a b s o r p t i o n and a r a p i d m e t a b o l i c i n a c t i v a t i o n of the d i g o x i g e n i n m o n o d i g i t o x o s i d e in man. Institut

f.Klin. P h a r m a k o l o g i e , 1 B l n . - 4 5 , H i n d e n b u r g d a m m

30

S T I M U L A T I O N A N D I N H I B I T I O N OF P H O S P H O L I P A S E S IN S Y N A P T O S O M E S G U I N E A PIG B R A I N ( A k t i v i e r u n g und H e m m u n g yon P h o s p h o l i p a s e n S y n a p t o s o m e n vom M e e r s c h w e i n c h e n h i r n ) H. K u n z e

OF in

I s o l a t e d s y n a p t o s o m e s were p r e p a r e d from g u i n e a pig b r a i n (V.P. W h i t t a k e r et al., B i e c h e m . J. 90, 293, 1964) and o s m o t i c a l l y r u p t u r e d . T h e i r p u r i t y was d e t e r m i n e d by m e a n s of m a r k e r e n z y m e m P h o s p h o l i p a s e a c t i v i t i e s were m e a s u r e d at 37~ with 100 ~g syna p t o s o m a l p r o t e i n in a total v o l u m e of 250 bl. The i n c u b a t i o n m e d i u m c o n t a i n e d 50 m M Tris (pH 7.4), 2 m M GaOl 2 and 0.1 m M sen i c a t e d m i x e d r a d i o a c t i v e p h o s p h a t i d y l e t h a n o l ~ m i n e (PE) as substrate, p r e p a r e d by m i x i n g 1 - ( 9 , ] O - 3 H ) p a l m i t o y l - P E and 2 - ( I - ~ C ) l i n e l e o y l - P E . P h o s p h o l i p a s e a c t i v i t i e s were c a l c u l a t e d from 3H and 14C in the u n h y d r o l y z e d substrate, free fatty a c i d s (FFA), and l y s o p h o s p h a t i d y l e t h a n o l a m i n e (LPE) f r a c t i o n s . H y d r o l y s i s of PE was linear, for 30 min, with s y n a p t o s o m a l p r o tein from 50 to 300 ~g. P r o d u c t f o r m a t i o n was 3 H - F F A ~ 1 4 C - L P E 9 1 4 0 - F F A ~ 3H-LPE, i n d i c a t i n g the p r e s e n c e of m o r e p h o s p h o l i pase AI than A2, and also of a l y s o p h o s p h o l i p a s e . D e a o y l a t i o n of PE i n c r e a s e d in the p r e s e n c e of 0.1 mM a c e t y l c h o l i n e with 0.1 m M n e o s t i g m i n e , ] m M n o r a d r 4 e n a l i n e and I m M 5 - h y d r o x y t r y p t amine, a p p a r e n t l y by s t i m u l a t i n g both, p h o s p h o l i p a s e AI and A 2. I mM N 6 , 0 2 - d i b u t y r y l cyclic A M P d e c r e a s e d PE h y d r o l y s i s , pres u m a b l y b y i n h i b i t i n g m a i n l y p h o s p h o l i p a s e A 2. Dr. H. Kunze, Abt. B i o c h e m . P h a r m a k o l o g i e , M a x - P l a n c k - I n s t i t u t fHr exp. M e d i z i n , D - 3 4 0 0 G S t t i n g e n , H e r m a n n - R e i n - S t r . 3

R 42 ON THE INVOLVEMENT OF BRAIN C A T E C H O L A M I N E S IN MORPHINE-INDUCED RUNNING ACTIVITY IN MICE (Die Beteiligung zentraler Katecholamine an der dutch Morphin verursachten Motilit~tszunahme bei M~usen) K. Kuschinsky, 0. Hornykiewicz

In mice, increased locomotor activity is a typical symptom after morphine injection. Blockade of dopamine-~-hydroxylase with ~dium diethyldithiocarbamate (DDC) or of tyrosine hydroxylase with alpha-methyl-p-tyrosine (alpha-MT) inhibited the stimulating action of morphine on running activity completely. L-DOPA restored the effectiveness of morphine in mice pretreated with alpha-MT, but had only a weak effect in DDC-treated animals. In the latter animals, t h r e o - 3 , 4 - d i h y d r o x y p h e n y l s e r i n e , which was used as a precursor of norepinephrine, was more effective in restoring the morphine effect than L-DOPA. Similarly, clonidine, which stimulates central norepinephrine receptors, antagonized DDC-induced inhibition of locomotor activity. Doses of morphine which induced locomotor activity produced a slight but statistically significant increase in striatal homovanillic acid concentration. Furthermore, bilateral lesions in the head of the caudate nucleus attenuated the locomotor effect after morphine. Probably, morphine increases dopaminergic activity in striata of mice by increasing the dopamine concentration at its receptors. Norepinephrine seems to have an important auxiliary function in the development of morphine-induced locomotor activity. Dr. K. Kuschinsky, Abteilung Biochemische Pharmakologie, MaxPlanck-Institut f~r experimentelle Medizin, D-3400 G~ttingen, Hermann-Rein-Stra~e 3

A B S O R P T I O N OF DRUGS BY INTESTINAL F I L T R A T I O N (Resorption yon Pharmaka dutch enterale Filtration) F. Lauterbach~ E.-A. Witte In the jejunal epithelium of guinea pigs the absorption rates of inulin and of cardiac glycosides, quaternary ammonium compounds and sugars are correlated. To elucidate the role of filtration in intestinal absorptign, inulin-C14 was separated on Sephadex G 50 into fractions (I-V) of decreasing m o l e c u l a r weight. Average molecular weight of p e a k fraction (II) was 2600. Absorption rates (0.2 cm2; 0.2ml; 45 min; Mean • of 11-30 exp.) for inulin -C14 (X) and digoxin -H3 (Y), regression (B) and correlation coefficients (R) were as follows: 02:

I

II

III

V

N2:

I

II

III

V

n

x Y

0.375

0.107

0.126 0.181 0.240 ~0.019 ~0.030 +0.028

•

0.299

0.278 0.352 0.353 ~0.o24 ~0.038 +0.036

•

• •

0.241 0.377 0.564 30.029 ~0.055 •

0.919

0.664 •

0.564 •

0.708 •

B

0.759

0.990

0.712

0.116

2.408

2.172

0.887

0.669

R

0.638

0.777

0.552

0.090

0.900

0.878

0.700

0.757

Thus it can be concluded that the pore size ranges in the order of magnitude of molecules of a few thousand M W and that passage through these pores (filtration) contributes considerably to the overall absorption of substances with low absorption rates. Dr. F. Lauterbach, Institut fur Pharmakologie und Toxikologie der R u h r - U n i v e r s i t ~ t Bochum, D-6300 Bochum, Im Lottental

R 43

EFFECTS OF MORPHINE, CHOLINERGIC AGENTS AND COLD EXPOSURE ON MIDBRAIN DOPAMINE (DA) NERVE CEILS OF MORPHINE-TOLERANT AND NON-TOLERANT MICE (Wirkung von Morphin, cholinergen Substanzen und K~lteexposition auf dopaminhaltige Neurone des Mittelhirns von toleranten und nicht-toleranten M~usen) W.Lichtenstei~er, Ruth Lienhart In microfluorimetric studies, morphine (40 mg/kg s.c.) was previously found to induce a rapid increase in the fluorescence intensity of DA neurons si~dlar to that seen after electrical stimulation or acute cold exposure. During tolerance development the fluorescence intensity of DA neurons in the substantia nigra of mice did not change, but the intensity response to a single dose of morphine disappeared. On the other hand, the reaction to acute cold exposure was still elicited, thus indicating that the capacity of the neurons to respond to some functional changes was in itself maintained Therefore, possible changes in the input to the DA neurons were investigated, especially an interaction with cholinergic systems. These DA nerve cells also exhibited a typical biphasic response of their fluorescence intensity to physos~igmine which resembled the one induced in tuberal DA neurons by transsynaptic stimulation. In early tolerance, this response was not completely suppressed but showed a clearly protracted course. This appears to indicate that an input to the DA neurons resulting from cholinergic activity might change during the development of tolerance. Experiments with atropine and nicotine did not yet provide unequivocal information, et, it may be mentioned that nicotine in a relatively high dose 1 mg/kg s.c.) induced a rapid increase in fluorescence intensity. Dr~ W. Lichtensteiger~ Pharmakologisches Institut der Universit~t, Gloriastrasse 32, CH-8006 Z~rich, Schweiz

~

SEX-SPECIFIC DIFFERENCES IN EXPERIMENTS WITH FLUFENAMIC ACID AND SOME DERIVATIVES AT LABORATORYANIMALS AND HUMAN BEINGS (Geschlechtsspezifische Unterschiede bei Untersuchungen mit Flufenamins~ure und einigen Derivaten an Versuchstieren und am Menschen) D. Lorenz, H.-D. Dell Bei Untersuchungen mit Flufenamins~ure ( I ) und ihren Derivaten N-(~,~,~-Trifluor-m-tolyl)-anthranils~ure-2-(2-hydroxy~thoxy)-~thylester ( I I ) und N-(mTrifluor-methylphenyl)-anthraniloyl-glycols~ureamid ( I I I ) wurden erhebliche Toxizit~tsunterschiede an m~nnlichen und weiblichen Versuchstieren gefunden. So betrugen z.B. bei der Ratte die DL5n-Werte yon I I bei m~nnlichen Tieren p.o. 292 mg/kg, bei weiblichen Tieren 47o m~/kg, von I I I bei m~nnlichen Tieren 532 mg/kg, bei weiblichen Tieren 1466 mg/kg. Auch am Kaninchen war I I I bei m~nnlichen Tieren doppelt so toxisch wie bei weiblichen. - Blutspiegelbestimmungen und die Untersuchung der Harnausscheidung an Ratte, Kaninchen, Schweinen und Menschen ergaben, dab weibliche Tiere und Menschen die Verbindungen offenbar anders metabolisieren als m~nnliche. Zum Teil waren die Blutspiegel der unver~nderten Substanzen bei weiblichen Individuen niedriger als bei m~nnlichen, zum Teil eliminierten weibliche Individuen aber auch h~here Mengen unver~nderter Substanzen als m~nnliche. Die Ergebnisse unterstreichen,da~ Toxizit~tsprUfungen und klinisch-pharmakologische Untersuchungen grunds~tzlich an m~nnlichen und weiblichen Versuchstieren oder Menschen durchgefUhrt werden sollten.

Dr. D. Lorenz, Experimentelle Forschung der Troponwerke, D-5ooo K~In 80, Berliner Str. 22o

R44

HISTAMINE, METHYLATEDHISTAMINESAND PARASYMPATHOMIMETIC STIMULATION OF SALIVARY SECRETION (Histamin, methylierte Histamine und die parasympathlsche Stimulierung der Speichelsekretion) W. Lor enz~ D. Maroske, A. Huhndt-Relmann r H. Sihlbom and. P. Keck Exogenous histamine (40Hg/kg i . a . ) only weakly stimulated the s a l i v a t i o n from submandibular and parotid glands of dogs (0.5 • 0.2 ml s a l i v a / ~ secretion period, n=lO, determination according to Lorenz et a l . , Arch. Pharmakol.exp.Pathol. 260, 416 (1968)). In glands prestlmulated by p i l o carpine ( 4 0 # g / k g i . a . ) , however, histamine showed a marked e f f e c t (6-15 ml secretion period, n=5). N'-methylhistamine and N ' , N ' - d i m e t h y l histamine (1-200 ~g/kg (n=5)) did not stimulate the s a l i v a t i o n , but i n h i b i t e d the p i l o c a r p i n e induced secretion of the submandibular gland, both to the same extent i n r e l a t i v e l y low doses (I.D.50 3 0 ~ g / k g , n=5). A f t e r p i l o c a r p i n e (40Mg/kg i . a . ) and a c e t y l c h o l i n e ( 4 0 # g / k g i . a . ) , histamine was found i n the submandibular s a l i v a i n concentrations of 9.0 • 4.3 ng/ml and 6.0 • 2.4 ng/ml (n=24) r e s p e c t i v e l y , determined according to Lorenz et a l . , Z.anal.Chem. 252, 94 (1970) (combined method)) By a n t i h i s t a m i n i c drugs (antazoline, dlmethinden maleate) acting on the Hi-receptor , but also by burimamide (H2-receptor blocker), both the p i l o carpine and carbachol (2.5 #g/kg) induced secretion of the submandibular gland could be i n h i b i t e d . The I.D.50 for burimamide, however, was 10 mg/ kg. I t i s concluded, that histamine, but not the side chain methylated histamines may be involved d i r e c t l y in the parasympathetic s t i m u l a t i o n of s a l i v a r y secretion i n dogs. Prof.Dr. W. Lo:enz, Abt.Exp.Chirurgie Pathol.Biochemle der U n i v e r s i t ~ t D-3550 Marburg/Lahn, Rober~-Koch-S~raBe 8 DRUG INDUCED CYTOPLASMIC INCLUSIONS, INDICATING A DISTURBANCE OF PHOSPHOLI PID METABOLISM (Arzneimittel-i nduzierte cytoplasmatische Einschlussk~rper als Ausdruck einer Lipidspeicherkrankheit) H. LUIImann, R. LuIImann-Rauch and G.-H. Reil Recently the anorectic drug chlorphentermine has been demonstrated to induce severe morphological alterations in rat pulmonary tissue: An augmentation of alveolar macrophages and a formation of lamellated cytoplasmic inclusions was induced in nearly all pulmonary cell types. The alterations were interpreted to reflect a drug-induced lipldosis.In the present communication evidence is to be given that these alterations are restricted neither to lung tissue, nor to the species of the rat, nor to the drug chlorphentermlne. Rats, mice, gulnea-pigs, and rabbits were chronically treated with chlorphentermine; a separate group of rats was treated with triparanol, an inhibitor of cholesterol biosynthesis, which is known to induce cytoplasmic inclusions in steroid producing cells. Inclusions of lamellated or crystalloid pattern were found in lungs, liver, adrenal glands, spleen, and testes of mice, guinea-pigs and rabbits; identical inclusions were observed in lungs, adrenal glands, spleen, testes and ovaries of rats after treatment either with chlorphentermlne or with triparanol. - It is concluded that a more general type of side reaction has been demonstrated, inducible by drugs of highly amphiphilic nature. Prof. Dr.med. H. LUIImann, Institut fur Pharmakologie, Christian-Albrechts-Universitdt 2300 Kiel, Hospitalstr. 4-6 West Germany

R 45 INCREASED INTESTINAL ABSORPTION OF E T H Y L E N E G L Y C O L IN T H E P R E S E N C E OF S A L I C Y L I C A C I D ( S t e i g e r u n g d e r e m t e r a l e n R e s o r p t i o n yon Athylenglykol in G e g e n w a r t y o n S a l i c y l s i u r e ) R. L y n c h , J. M i c k a n S i n c e s a l i c y l i c a c i d is m u c h b e t t e r a b s o r b e d b y the i n t e s t i n e t h a n w o u l d be e x p e c t e d f r o m its h i g h d e g r e e of i o n i z a t i o n at pH a b o v e 6.0, it s e e m e d c o n c e i v a b l e t h a t it m i g h t e n h a n c e the intestinal absorption of other hydrophilic compounds, too. This. h a s n o w b e e n e x a m i n e d in tied l o o p s of rat j e j u n u m in situ. ]dCl a b e l l e d e t h y l e n e g l y c o l was a p p l i e d at 20 m M c o n c e n t r a t i o n . All s o l u t i o n s i n j e c t e d into the l o o p s w e r e m a d e i s o t o n i c by a d d i t i o n o f N a C l and c o n t a i n e d 0.067 M p h o s p h a t e b u f f e r pH 6.5. T h e p e r c e n t a g e of r a d i o a c t i v i t y remaining in the l o o p s a f t e r 5 m i n u t e s absorption was d e t e r m i n e d . E t h y l e n e g l y c o l was r e a d i l y a b s o r b e d ; 52.5% • 5.5% disappeared w i t h i n 5 m i n u t e s . In the p r e s e n c e of 50 m M s a l i c y l i c a c i d the a b s o r p t i o n of e t h y l e n e g l y c o l r o s e to 62..6% • 5 . 0 % (p~=.O.O01). B e n z o i c acid, and p- or m - h y d r o x y b e n = i c acids were ineffective at the same c o n c e n t r a t i o n . 2:4-Dinitrop h e n o l (I mM) did l i k e w i s e not i n c r e a s e the a b s o r p t i o n of e t h y ~ glycol. EDTA even decreased the a b s o r p t i o n of e t h y l e n e g l y c o l to 4 3 . 9 % ! 7 . 4 % (p~=.O.O05). T h u s it is u n l i k e l y that the e f f e c t of s a l i c y l i c a c i d is b r o u g h t a b o u t b y u n c o u p l i n g of o x i d a t i v e p h o s phorylation or b y c h e l a t i o n of Me ++ . T h e e n h a n c e m e n t of the a b s o r p t i o n of e t h y l e n e g l y c o l s e e m s to be a r a t h e r s p e c i f i c e f f e c t of s a l i c y l i c acid. Dr. R. L y n e n , M a x - P l a n c k - l n s t i t u t fur Abteilung Biochemische Pharmakologie, Hermann-Rein-Stra~e 3

experimentelle Medizin, D-3400 Gbttingen,

BROMODEOXYURIDINE TREATMENT IN TISSUE CULTURE : LOSS OF MALIGNANCY IN A METHYLCHOLANTHRENE-TRANSF01hMED CELL CLONE (Bromodeoxyuridin-Behandlung in Zellkultur: Verlust der Malignit,~t in einem Methylcholanthren-transformlerten Zell-Klon). H.Marquardt ; Sloan-Kettering Inst.Cancer Research ; New York,New York, USA . Die Untersuchung der Stabilit~t des zu Neoplasie f6"nrenden Schadens ist nieht nur u~ yon klinischer Bedeutung, sondern auch interessant hinsiehtlich des Wirkungsmechanismus' der malignen Transformation. Es ist vielfach berichtet worden, dass transformierte Zellen sowohl in vivo als auch in vitro (in Zellkultur) Malignit~t verlieren kdnnen. So f{{hrt Z.Bo die Behandlung (spontan, mittels Viren oder mittels chemischer Carcinogene) transformierter Zellen mit zytotoxischen Substa~lzen (wie Fluordeoxyuridin) -vermutlich infolge Selektion pr~existenter weniger maligner Zellen- zu verminderter Malignit~t in diesen Zellen~ Es wird jetzt {~oer einen andersartigen Weg zur Erzeugung yon "Reversion" berichtet. Die Behandlung eines Methylcholanthren-transformierten Klons yon Maus-Fibroblasten mit nlcht-letalen Konzentratlonen (0,1/Zg/ml) yon Bromodeoxyuridin "de-transformiert" diese Zellen : die Kolonie-Anordnung und die "Saturation Density" derartig behandelter transformierter Zellen gleicht der nicht-transformierte1: Zellen und die Malignit~t der Zellen (= Wachstum als Sarkom naeh s,c. Injektion in isologen M~usen) geht verloren, Zellteilung zur Zeit der Bromodeoxyurldin-Behandlung ist notwendig zur Erzielung dieser Wirkung : die Behandlung eonfluenter Zell-Monolayer (keine Zellteilung infolge Kontaktinhibition) f~hrt nieht zu '%leversion". Die '~eversion" mittels Bromodeoxyuridin ist stabil : die Zellen bleiben nicht-maligne naeh Transfer in Bromodeoxyuridin- freie N~hr fl~ssigkeit. Der Wirkungsmechanismus dieser Bromodeoxyuridin-Wirkung ist unbekannt. Es ist jedoeh interessant in diesem Zusa~enhang, dass k@rzlich gezeigt werden konnte, dass Bromodeoxyuridln auch Differentiation in Zellkultur induziert.

R 46

INFLUENCE OF CATECHOLAMINEUPTAKEBLOCKING DRU~SON THE INCREASE IN WATER INTAKE AND PLAS~RENINCONCENTRATION INDUCED BY SOME HYPOTEN$1VEDRUGS (Wirkung yon neuronalen Catecholaminaufnahmeblockern auf die dutch blutdrucksenkende Pharmaka ausgelSste Stsigerung von Wasseraufnshme und Plaamareninkonzentration) DpK.Meyer. G.Herttin~ The ~-sympathomimetic drug isoproterenol (100 ~g/kg) (IS) and the s-receptor blocker phentolamine (2 m~kg) (PH) induce an increase in water intake and in plasma renin concentration (PRC), when given to rats. The influence of drugs which block the neuronal uptake on these actions of PHand IS was studied. Desmethylimipramine (DMI) 8.0 m~kg increased the water intake induced by PH significantly from 2.59 ml/rat to 5.43 ml/rat (~=0.05). The increase in PRC induced by PH was further raised from 129.28 ~ 12.36 to 175.7 ! 16.11 ng AI/ml Plasma (p 0.05). The actions of IS on both parameters remained unaffected by DMI. These results were confirmed by another uptake blocker study with amitryptiline. These results substantiate the hypothesis (Meyer et al., Europ. J. Pharmacol. 16, 1971, 278) that the increase in PRC induced by hypotensive drugs is mediated by a stimulation of 6-receptors and that this rise in PRC causes the increase in water intake by releasing angiotensinwhich is known as a dipscgenic agent (~itzsimons et al., J. Physiol. 20~, 1969, 45). PH stimulates these ~-receptorsbya reflexlyinduced catecholamine-release and is therefore potentiated in its action on PRC and drinking by a reuptake blocker. IS, on the other hand, directly stimulates these 6-receptors and cannot be potentiated by a NA-reuptake blocker. Dr. D. K. Meyer, Pharmakologisches Institut der Universit~t Wien, A-I090 Wien, ~ahringerstra~e 13a

H Y P O X I C AND P H A R M A C O L O G I C A L D I L A T A T I O N OF THE VESSELS OF THE M. G A S T R O C N E M I U S AT D I F F E R E N T C A L C I U E C O N C E N T R A T I O N S (Zur Beeinf l u s s u n g h y p o x i s c h e r und p h a r m a k o l o g i s c h e r M e h r d u r c h b l u t u n g des N. g a s t r o c n e m i u s d u r c h Calcium) U. Meyer, W. Marten, F.W. Schulz und W.K. Raff FGr N i f e d i p i n e und I s o p t i n ist eine B l o c k u n g der G e f ~ B w i r k u n g dutch Ca ++ am i s o l i e r t e n mit K r e b s - H e n s e l e i t l S s u n g p e r f u n d i e r ten H e r z e n n a c h g e w i e s e n (F. Kosche, W.K. Raff und W. Lochner, P f l ~ g e r s Arch. 335, 46-57, 1972). In V e r s u c h e n am M. g a s t r o c n e m i u s des Hundes in situ konnte die M e h r d u r c h b l u t u n g n a c h N i f e d i p i n e und Isoptin d u r c h intraarterielle Ca++ - Infusionen, die die C a l c i u m k o n z e n t r a t i o n im Perfusionsblut um 5 bzw. 50 m ~ % erhShten, nicht v S l l i g a u f g e h o b e n wetden. L e d i g l i c h die D a u e r der M e h r d u r c h b l u t u n g wurde signifikant verkGrzt. Eine dutch A d e n o s i n bedingte M e h r d u r c h b l u t u n g war ebenso durch Ca++ - I n f u s i o n e n g l e i c h e r HShe zu hemmen, w~hrend reaktive H y p e r ~ m i e und A r b e i t s m e h r d u r c h b l u t u n g dutch Ca ++ nicht zu b e e i n f l u s s e n waren. I. Calcium und A d e n o s i n h a b e n n a c h u n s e r e n V e r s u c h e n auf die Arbeitsmehrdurchblutung und die reaktive H y p e r ~ m i e k e i n e n EinfluB. 2. Die G e f ~ w i r k u n g v o n C o r o n a r d i l a t a t o r e n mit c a l c i u m a n t a g o n i s t i s c h e n E i g e n s c h a f t e n wird bei mit Blut p e r f u n d i e r t e n Organen auch bei hohen C a l c i u m k o n z e n t r a t i o n e n • Blut n u t zum Tell aufgehoben. U. Neyer, P h y s i o l o g i s c h e s Institut, Lehrstuhl I, D-4000 DGsseldorf, Moorenstr. 5

R 47 THE METABOLISM OF p-NITROANISOLE IN THE ISOLATED RAT LIVER [Metabollsmus v0n p-Nitroanlsol an der isollerten Rattenleber) K . Minckr R.R. Schupp, H.P.A. ILLing, G.F. Kahl

The metabolism of p-Nitroanisole (pNA) and p-Nitrophenol (pNP) in isolated livers, perfused with 100 ml of a haemoglobln-free medium, was studied to investigate the coordination of pNA oxidation and the conjugation reactions of the pNP formed. The interaction of pNA an glucuronide conjugation was studied in vitro. pNA (50 ~uM) is converted completely to pNP~ pNP is present as sulphate (3.5 + 0.2 nmoles/ml x g), glucuronlde (0.4 + 0.1 nrnoles/ml x g), and glucoside (0.3 + 0--~1 nmoles/ml x g) at 90 rain, all of ~,hich are hydrolysed by acid and each hydrolysed specifically by enzyme. The glucoside has been found as a metabolhe of pNP before (Gessner, T. and Hamada, N . , J.pharm. Sci. 59, 1528 (1970))~ If the concentration of pNA is increased to 100 juM then there is no change in the ratio glucuronide/ sulphate. When pNP is added at 25, 50, and 100 juM to the perfuslon medium there is a large increase in the ratio glucuronide/sulphate between 50 and 100,oM. pNA (0.5 mM) does not inhiMt UDPglucose dehydrogenase or UDPglucuronyltransferase in vitro, thus this may represent negative cooperativity of the UDPglucuronyltransferase -~/insnes, A., Biochlm.biophys.Acta (Amst~ 284, 394 (1972)). (Supported by a grant from the Deutsche Forschungsgemeinschaft~ H.P.A.I. is a Wellcome Trust (UK) Anglo-German Fellow.) Pharmakologisches Institut der Universitt~t, D 6500 Mainz, Obere Zahlbacher Str. 67

RELATION OF CYCLIC 3-5 AN~ TO RADIOPROTECTIVE EFFECTS INDUCED BY CYSTEAYfKNE.(Beziehungen des cyclischen 3,5 AFfP zur Strahlenschutzwirkung yon Cysteamin.) P. Fiitzne~ The i.p. administration of 450 mg/kg oysteamine leads to a transient inhibition of JH-thymidine incorporation into D~A of liver cells in 6-week-old white mice. This functional suspension of DNA synthesis seems likely to be responsible for cysteamine-induced radioprotective effects, i.e. the acceleration of DNA restoration and the prevention of loss in liver weight and in the total liver contents of DNA~ RNA~ and proteins ~ and 8 days after a total body gamma-irradiation with 500 fads. In two-year-old animals where cystesmine in the contraz7 to the you_ug mice increases DNA synthesis, no radioprotection occurs after pre-irradiation treatment with cysteamine. Similar effects are shown after the i.p. administration of 200 m~/kg dibutyryl cyclic 3-5 AFfP. Furthermore, cysteamine itself initiates an elevation of endogenous cyclic 3.5 AFfP in the liver cells during irradiation. Therefore it is assumed that cysteamine-induced radioprotective effects might mediated by cyclic 3-5 AFfP as intracellular second messenger. Priv.-Doz. Dr. ~. Fiitznegg, ~harmakologisches Institut der Universit[t Erlangen-NHrnberg, D-8520 Erlangen, Universit~tsstr. 22

R 48 THE INHIBITION OF PREMATURE LABOR BY 8-ADRENERGIC SYMPATHOMIMETICS AND PROSTAGLANDIN-ANTAGONISTS IN MANAGING PREMATURE ONSET OF LABOR (Die Hemmung vorzeltlger Wehen durch 13-adrenerge Sympathomimetlka, Spasmolytika und Prostaglandln-Antagonlsten bel drohender Fr0hgeburt) K.H. Mosler, W. Dornh~fer Under certain clinical conditions a pharmacological inhibition of premature labor can be achieved. During the acute phase the application of the 8-stlmulator Th 1165a ( 1 - (3, 5-dlhydroxyphenyl)- 2 - 1 -(4-hydroxyphenyl) - isopropyl - amlnoethanol ) combined with the spasmolytlc verapamil (iproveratril) has been successful~ For long term treatment an additional support with acetylsallcylic acid is indicated which most likely decreases the excessive production of prostaglandln in the uterus~ The mode of application and the dosage for this treatment of premature labor will be described. Prof. Dr. K.H. Mosler, Abteilung fur Experimentelle Gyn~kologie an der Universlt~tsFrauenklinik W0rzburg, 87 W0rzburg, Josef-Schneider-StraSe 4

PHARMACOKINETICS OF TRICHLOROETHYLENE METABOLITES IN MAN (Pharmakokinetik der Trichlor~thanol-Metaboliten im Menschen) G.MHIIer, M.Spassowski Trichloroethylene (Tri) inhalation by 5 volunteers (50 ppm, 6 h/ day, 5 days) results in high plasma levels for trichloroethanol (TCE) of 2-2.5 ug/ml with only slight accumulation from one day to the next, whilst trichloroacetic acid (TCA) levels rise from day to day to reach a level of 52 ~g/ml at the end of the 5th exposure. Urinary excretion of the metabolites (TCE mostly in the glucuronidized form) reveals the well-known reverse ratio: TCE/ TCA falls from i0 to 1-2 (day 1 to 5). In pharmacokinetic studies with ingested TCE (iO mg/kg) and TCA (4.5 mg/kg and 3.0 mg/kg) half lives of 17.5 and 52 h, respectively were obtained. The slow excretion of TCA is due, at least in part, to a high plasma protein binding (91-78% for 10-50 ~g/ml) as compared to that of TCE (approx. 50%). Thus, calculation of the magnitude of Tri exposure from the urinary excretion of TCE and/or TCA is only justified under steady state conditions; however, these are not representative for most types of exposure in working places. G.M~ller, Institut fur Pharmak01ogie und Toxikologie der Universit~t, D-87OO W~rzburg, KoellikerstraBe 2

R 49 THE INTERACTION OF SOME BENZODIAZEPINE DERIVATIVES WITH HUMAN SERUM ALBUMIN, CIRCULAR DICHROISM STUDIES ((Jber die Wechselwirkungen von Benzodlazepinderlvaten mit Humanserumalbumin, untersucht mit Hi lfe des Zirkulardichroismus) W. MUller1 U. Wollert The interactions of some benzodiazepine derivatives (BD, lo -6 M - 5 9 lo -5 M) with human serum albumin (HSA, 1.31 9 lo -5 M) in o.o7 M phosphate buffer pH 7.4o were investigated by measuring the circular dichrolsm (CD). All BD investigated showed extrlnslc Cotton effects between 25o nm and 4oo nm. Nearly all BD gave weak negative CD bands near 3oo nm and strong positive bands near 26o nm with very different intensities, e.g. the [ e ] of chlordiazepoxld (5 .31o -5 M) near 26o nm is 215 . lo -3 and of clonazepam (5 9 lo -5 M) is 4.4 9 lo . For six BD the dependence of [ e ] from the concentration of the BD was investigated. In all cases no further increase of [ e l was seen when the concentration of the BD exceeded that of HSA threetirnes. With eight of the substances we found a correlation of the anisotropy factor with the partition coefficient P (n--octanol/buffer). It is suggested that for the optically active binding of the BD hydrophobic bonds may be important bu~" the interaction can strongly be influenced by other factors e.g. steric factors.

W. MUIlerr Pharrnakologisches Institut der Universit~t D-65oo Mainz, Obere Zahlbacher Str. 67

FURTHER STUDIES ON THE EFFECTS OF ADRENALINE AND DIBUTYRYL-c-A/V~~ ON MYOCARDIAL 45Ca UPTAKE (Weitere Untersuchungen zur Wirkung von Adrenalln und Dibutyryl-c-AMP auf die myokardiale 45Ca-Aufnahme) H. Nawrath, T. Meinertz~ H. Scholz 1 In a previous study (l'4awrath et al.I Arch.Pharmacol. 274, R 811 1972) no influence of adrenaline (5.5 x lo -6 M) and cyclic N6-21-0-dibu-~yryl adenosine 3~15'-mono phosphate (DB-ANP; ]0 -3 M) on 45Ca uptake could be detected in isolated electrically driven left rat aurlcles. The present s_tud~ has been performed at the same experimental conditions but (1) at reduced [Ca]e (0.45 raM) and (2) at lower frequencies of stimulation (0, 15, 301 601 120 beats/rain). 45Ca incubation time was 5 min. Under these conditions where 45Ca uptake of the control preparations could be expected to be markedly reduced according to Grossman and Furchgott (J.Pharmacol.exp.Ther. 1431 1201 1964) both drugs significantly (p< 0.01) increased 45Ca uptake by about 30% (DB-AMP) and 4 0 ~ (adrenaline) in beating aurlclesl total tissue Ca concentration remained unchanged. In resting auricles 45Ca uptake was not altered. The results show that a DB-AMP or adrenaline induced increase in 45Ca uptake can indeed be detected even in rat auricles. It is concluded that DB-AMP can mimic the effects of adrenaline on myocardial Ca exchange under the conditions used. (1 Supported by a grant from the Deutsche ForschungsgemeinschaR). Dr. H. Nawrathr Pharmakologisches Institut der Unlversit~t I D-65oo Mainz, Obere Zahlbacher Str. 67

R 50 EFFECTS OF ADRENALIN, NORADRENALIN AND ISOPRENALIN ON VENOUS VOLUME AND VENOUS RESISTANCE N. Nedopil, J. Remien, W. Felix An den hinteren Extremit~ten yon 15 Katzen in Chloralosenarkose wurden die Wirkungen von Katecholaminen auf Druckvolumenkurven bei Dehnung und Entdehnung und der Widerstand der groSen Venen bestimmt. Das intravasale Volumen wurde bei verschiedenen hydrostatischen Drucken ermittelt; der Venenwiderstand wurde yon der V. metaxtarsalis superf, zur V. femoralis errechnet. Adrenalin, Noradrenalin und Isoprenalin wurden in Dosen yon 0,8 ~g/min intraarteriell als Dauerinfusion gegeben. Aus den Druckvolumenkurven ging hervor, dab Adrenalin die Venen in allen Versuchen kontrahierte, Noradrenalin dagegen nur in 7 yon i0 Versuchen, w~hrend Isoprenalin keine signifikante Wirkung hatte. Das Venenvolumen wurde unter Adrenalin um 25-30% verringert, unter Noradrenalin nut um 15-20%. Der Widerstand der Venen nahm unter Adrenalin um durchschnittlich 400% zu, unter Noradrenalin um 150%. Isoprenalin hatte auch hier keine signifikante Wirkung. Durch unsere Versuche lie~en sich keine konstringierenden oder dilatierenden $-Rezeptoren nachweisen. Die relativ st~rkere ~-kontrahierende Wirkung des Adrenalins gegenGber Noradrenalin erh~lt besondere Bedeutung beim Schock, da dadurch das Verh~ltnis yon pr~kapill~rem zu postkapill~rem Widerstand erheblich ver~ndert wird. Prof. W. Felix, Pharmakologisches Institut 8000 MGnchen 2, NuSbaumstrasse 26

der Universit~t,

CALCIUM DISPLACEMENT FROM SUPERFICIAL TO DEEP COMPARTMENTS: A POSSIBLE EXPLANATION FOR DIFFERENT CONTRACTILE STATES OF CARDIAC MUSCLE (Die Plasmamembran der Herzmuskelzelle als Sitz eines pharmakologisch beelnflussbaren Calcium-Kompartiments) L. Neubauer and Th. Peters Ca-exchange kinetics were established in isolated atria of gulnea-plgs. Under the experimental conditions applied the beat frequency did not change. The experiments were designed to 1) measure total tissue Ca and Ca-45 uptake in freshly dissected and aged atria, 2) determine the influence of pentobarbital on Ca-metabolism in freshly dissected atria and 3) investigate the effect of ouabain on Ca-metabolism in freshly dissected, aged, and pentobarbital treated preparations. A change in total tissue Ca did not occur under the conditions applied. However, the rate of exchange decreased considerably during aging or under pentobarbital. This decrease in rates of exchange paralleled the simultaneously occurring decline of contractile force. Ouabaln did not display any influence on Ca-metabolism of freshly dissected atria, but increased the rate of exchange in aged and in pentobarbital treated preparations. A kinetic analysis demonstrated the existence of a particular fast exchanging Ca-pool located at membranes facing the ECS. This pool forms a link between extracellular and intracellular Ca-concentrations. The lability of Cabinding at these sites is responsible for the coupling between membrane depolarisation and contraction. It can be influenced by drugs and ageing. Dr. Th. Peters, Institut fur Pharmakologie, Christlan-Albrechts-Universit~t 2300 Kiel, Hospitalstr. 4-6 West Germany

R 51

THE EFFECT OF B-RECEPTOR BLOCKING AGENTS ON THE VELOCITY OF Ca 2+UPTAKE. IN ISOLATED HEART AND LIVER MITOCHONDRIA (Die Beeinflussung der Calciumaufnahmegeschwindigkeit isolierter Herz- und Lebermitochondrien durch B-Sympathicolytica) E. Noack In a previous report we have shown the inhibition of the velocity of oxidative p~osphorylation by certain B-blockers. Indirect measurements of Ca~+uptake by means of the Ca-induced oxygen consumption demonstrated a corresponding decrease (E.Noack and K.Greeff: J.Nolec. Cell.Cardiol.~,145,1971 and Experientia27,810,1971). As our results indicate that heart mitochondria might have a regulatory function in excitation-contraction coupling we comoared the energy-dependent uptake of l~u~ol Ca ~+ (+ 5 ~Ci 45Ca) in the presence of 9 B-blockers in heart and liver mitochondria. Passive binding and active uptake os Ca 2+ were blocked immediately by means of our new method injecting rapidly the mitochondrial suspension into a solutiQn of 27~iM ruthenium red. We found that mitochondria take 1~p~Ca~+ very quickly (heart: 2.3 • 0.1; liver: 11o6 • 0.6 nMol OaL+/sec/mg prot.) There was a direct correlation between the concentration of each B-blocker and the log of time of halfmaximal uptake of Ca 2+. Comparing the ED 25 s the negative inotropic effect in guinea-pig isolated atria and the ED 25 for the delay os the uptake time of Ca ~+ in heart mitochondria there was a high significant correlation for all B-blockers tested (r~0.9652; N~9; P
INFLUENCE OF RARE EARTHS ON RNA AND PROTEIIN SYNTHESIS IN RAT LIVER (Einfluss Seltener Erden auf die RNA- und Proteinsynthese der Rattenleber) E. Oberdisse, R. Winkler, E. KShler, R. S churig, Ho -J. Stolpmann Die Salze Seltener Erden (Lanthan, Cer, Praseodym, l~eodym) besitzen nach i.v. Applikation eine ausgepr~gte Lebertoxizit~t. Es sollte daher gepriift werden, ob diese Lebertoxizit~t m6glicherweise Folge friihzeiiig gest6rter Transkriptionsund Translationsprozesse sein k6nnte. Dazu wurden die RNAPolymerase-Aktivititten in isolierten Leberzellkernen und die Aminos~ureinkorporierende Kapazit~i yon Mikrosornen und Ribosomen nach Applikation yon io mg Praseody:mnitrat (Pr) /kg KG bestimrnt. 12 h nach Applikation yon Pr ist die Aktivi• der Mn++-slimulierbaren RiNAPolymerase um 50% vermindert, w~hrend die Mg++-abh~ngige Polymerase unver~ndert bleibt. Nach weiteren 12 h f~llt die Aktivit~t dann auf 23% (Mn ++) bzw. 31% (Mg ++) ab. Die Proteinsynthese ist naeh 12 h unbeeinflusst und zeigt nach 24 heine Hemmung um etwa 5o%, die dutch Zugabe yon PolyU weitgehend aufgehoben werden kann. Dies sprieht fiir eine zun~chst Gberwiegende Hemmung der mRNA-Synthese, wobei der Angriffspunkl yon Pr unklar bleibt. Untersuehungen mit isoliertem Chromatin und exogener Polymerase zeigen, dass Pr nicht fiber einen Angriffspunkt an der Matrize wirkt. M6glicherweise wird die Affinit~i der Polymerase zur DNA vermindert. Das morphologische Bild entspricht den biochernischen Befunden: Segregation der DIqA-Strukturen des lqucleolus in die Peripherie, wabige Auflockerung des l~ucleohs und Abl6sung der segregierlen DIqA vorn Rest des Nucleolus. Eckard OberdiSse, Pharmakologisches Institut der Freien Universit~t Berlin, i Berlin 33, Thiela]lee 69/73

R 52

EFFECT OF THIAMPHENICOL (TAP) - AN ANALOGUE OF CHLORAMPHENICOL (CAP) - ON THE RATE OF SYNTHESIS OF CYTOCHROME OXIDASE AND ON RESPIRATION OF BONE MARROW HOM~3GENATES (GUINEA-PIG) (EinfluB des Chloramphenicolderivates Thiamphenicol auf die Cytochromexidase-Synthese und Atmun 0 im Knochenmark yon Meerschweinchen) D. Oerter~ H.-J. Kirstaedter, H. von Keyserlinck~ M. Stracke, I. LOcke (Berlin) CAP is known to cause two different hematotoxic effects: dose-independent, irreversible pancytopenia (I), dgse-dependent, reversible cytostatic effects on the peripheral blood cell count (II). The chemotherapeutic effect of CAP results from a selective repression of bacterial protein synthesis. CAP sensitivity of mammalian mitochondrial protein synthesis represents one of the similarities between bacteria and mammalian mitochondria. Cytochrome oxidase is a typical mitochondrial membrane bound enzyme. We investigated whether the reversible hematotoxicity (II) can be explained by the inhibition of cytochrome oxidase synthesis and respiratory rate. Since CAP is eliminated from the rat at a very fast rate, we used the methylsulfonyl derivative TAP. A 12 day treatment with 100 mg TAP per kg and day (s.c.) reduces cytochrome oxidase activity of bone marrow homogenates by 50 % (p = 0.0005) and the rate of respiration (WARBURG method) by 33 % (p > 0.005). A 6 day treatment already produces a pronounced reduction of lymphocytes and reticulocytes only, whereas the other blood cell components remain unaffected. The results obtained demonstrate the reversible hematoxicity (ll) as a consequence of the chemotherapeutic principle. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 29). Detlef Oerter, Pharmakologisches Institut der Freien Universit~t Berlin, Abt. "Embryonal-Pharmakologie", D-IO00 Berlin 33, Thielallee 69/73

E P O X I D E H Y D R A S E A C T I V I T Y IN H U M A N L I V E R

F.Oesch

R e c e n t e v i d e n c e s t r o n g l y suggests i n t e r m e d i a t e e p o x i d e s as the c a u s a t i v e a g e n t s for c a r c i n o g e n i c e f f e c t s of several p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s and for liver n e c r o t i c effects of h a l o b e n zenes. E p o x i d e h y d r a s e (EH) t r a n s f o r m s such e p o x i d e s to less reactive v i c - d i o l s and p r e v i o u s studies showed a r o u g h l y inverse r e l a t i o n s h i p b e t w e e n s u s c e p t i b i l i t y to liver cancer and levels of h e p a t i c EH in some animal species and strains. It was therefore of p a r t i c u l a r i n t e r e s t to d e t e r m i n e the p r e s e n c e and levels of E H in h u m a n liver. An a s s a y has now b e e n d e v e l o p p e d w h i c h allows the d e t e r m i n a t i o n of E H a c t i v i t y in 10-20 mg h u m a n liver b i o p s y specimens. Formation of 3 H - s t y r e n e glycol is taken as a m e a s u r e of enzyme activity. In h u m a n liver h o m o g e n a t e s EH a c t i v i t y is 7.50 ~ 0.84 ~moles styrene g l y c o l / h / m g protein, c o m p a r a b l e to rodents (rat: 4.44 + 0.51; rabbit: 5.20 ! 0.42; guinea pig: 9.84 ~ 0.80), b u t s u r p r T s i n g l y m u c h lower than in Rhesus m o n k e y (25.8 ~ 1.4). The e n z y m e is found e x c l u s i v e l y in the m i c r o s o m a l fraction. The p H o p t i m u m is 8-9. No low m o l e c u l a r c o f a c t o r is r e q u i r e d and no inh i b i t i o n is e x e r t e d b y c h e l a t i n g agents or p r o d u c t diols. The a c t i v i t y is fully a b o l i s h e d b y b o i l i n g the p r e p a r a t i o n for iO min w h e r e a s after h e a t i n g at 60 ~ for 5 min > 50 % of the initial a c t i v i t y is still present. F.Oesch, bergstr.

Abt. Pharmakologie, 70, CH-4056 Basel,

B i o z e n t r u m der Universit~t, Schweiz

Klingel-

R 53

EFFECTS OF ADENOSINE AND THEOPHYLLINE ON RENAL HAEMODYNAMICS IN DOGS (Wirkungen yon Adenosin und Theophyllin auf die H~mo dynamik der Hundeniere) H. OBwald The effects of injections of adenosine into the renal artery with and without simultaneous infusions of theophylline were investigated on renal blood flow (RBF), glomerular filtration rate (GFR) and electrolyte excretion. 12 mogrel dogs were anaesthetized wi~1 pentobarbital (15-20 mg/kg). If necessary anaesthesia was continued with 1 % chloralose. After preparation of the left renal artery a needle was inserted there. RBF was measured with an electromagnetic flow meter. O~2 ml of isotonic saline containing 0.15-18 /ug adenosine, injected into the renal artery, caused a dose dependent decrease of RBF from 5 to 70 % of control flow. After 10-30 sec the RBF turned back to the preinjection level. Theophylline infusions (i - 5 x 10 -6 mol/min) which did not affect the renal function by itself shifted the dose-response curve of adenosine to the right in a parallel manner. A competetive antagonism between adenosine and theophylline is assumed since pA2 - PAIO was 0.92. From measurements of the GFR during adenosine infusions it is concluded, that adenosine acts on the vas afferens. Dr. H. OBwald, Abt. Pharmakologie RWTH Aachen, 51 Aachen, Melatener

der Medizinischen Str. 213, MTI II

Fakult~t

der

ANGIOTENSIN II AI~D RENIN IN NEPHRECTOMIZED RATS (Angiotensin II und Benin bei nephrektomierten Hatten) P. 0ster, U. Miksche, H. 0rth In a first series of experiments, plasma renin concentration was estimated in 15 animals by the mioromethod of Boucher st al. (1967) having a sensitivity limit of 2 ng.ml-l.hour -I of incubation Inormal range 5.210.7). In another series of 14 rats, angiotensin II (AII) was extracted from plasma by meaas of a cation exchange resin (recovery 81.9%) and determined by radioimmunoassay; the sensitivity limit of this method is 12 pg/ml (normal range 62-142). In the latter animals, 3 ml of blood were taken under other anaesthesia 24 hours after nephrectomy and again 1 hour later. In none of the plasma samples, renin was detectable, whereas A I I was found in six samples of the second series, ranging from 12 to 32.6 pg/ml. In six other samples, A I I was below the sensitivity limit of 12 pg, and in two samples, concentration was virtually zero. One hour after taking 3 ml of blood, A I I concentration rose in one animal from 12 to 14.9 Pg/ ml, was lower in five of the six rats with demonstrable A I I , and remained below 12 pg or at zero in seven animals. No correlation of plasma A I I levels to food and water intake was found. These findings implicate that plasma of nephrectomized rats cannot be used as "angiotensin-free" plasma in the radioimmunoassay. The observed A I I is either formed by unmeasurable amounts of renin or may be due to the action of other proteolytic enzymes, such as pepsin. However, the plasma concentration of "residual" angiotensin does not increase after bleeding or anaesthesia as it is observed in the presence of the kidneys. Dr. P. Oster, Pharmakologisches Institut der Universit~t, D-6900 Heidelberg, Hauptstr. 47-51

R 54

R E G U L A T I O N OF M U C O P O L Y S A C C H A R I D E SYNTHESIS IN F I B R O B L A S T TISSUE CULTURES (Regulation der M u c o p o l y s a c c h a r i d b i l d u n g in F i b r o b l a s t e n kulturen) H.D. Peters t P. W e s t h o f e n r K. Karzel M u c o p o l y s a c c h a r i d e (MPS) release into m e d i u m was determined in p r i m a r y fibroblast tissue cultures of embryonic mice (17-18 days) in presence of 0.5-5.0 mM d i b u t y r y l - c y c l i c 3',5'-AMP (cAMP). The cyclic n u c l e o t i d e increased MPS formation per cell, but also decreased cell number. When the exposure of cells to d i b u t y r y l - c A M P was reduced to 2 hrs, the inhibition of m u l t i p l i c a t i o n d i s a p p e a r e d while the increase in MPS secretion, m e a s u r e d after 24 or 48 hrs, persisted. PGE 1 at 1-50 ug/ml stimulated MPS formation under identical conditions w i t h o u t a f f e c t i n g cell numbers. Exposure of iO min was sufficient to achieve a d o s e - d e p e n d e n t increase in MPS formation after 24 or 48 hrs, showing a maximal response at 5-10 ug/ml. In the same cultures PGE 1 also increased cAMP levels w h i c h were maximal after iO min exposure. Similar to results with steroid synthesis in adrenals (Grahame-Smith et al., 1967), cAMP levels continued to rise up to 50 ug/ml PGEI, while MPS formation was already maximal at 5-10 ug/ml. These results indicate that PGE 1 stimulates MPS formation in fibroblast tissue cultures and s this process is m e d i a t e d via cyclic AMP. H.D. Peters, Institut fHr Pharmakologie, M e d i z i n i s c h e H o c h s c h u l e Hannover, D-3OOO Hannover-Kleefeld, K a r l - W i e c h e r t - A l l e e 9

THE SUBCELLULAR DISTRIBUTION OF INTRAVENTRICULARY ADMINISTEREO 3H-METARAMINOL

IN INTACT AND IN RAT BRAIN PRETREATED WITH p-CHLOROAMPHETAMINE. A.K. Pfeifer, L. Cs~ki, M. Kom16s and A. Schaefer In previous experiments we have demonstrated that p-chloreamphetamine diminished the catecholamine depletion produced by alpha-methyl-m-tyrosine (MMT). (A.K. Pfeifer and E. Galambos J.Pharrn.Pharrnaeol. 19, 4oo, 1967). To study the mechanism of this phenomenon we investigated the influence of p-chloreamphetamine on the subcellular distribution of metareminol (MA). Io/uC 3H-MA were administered through a chronically implanted microeannule into the left ventricle and the rats were decapitated after one hour. Io mg/kg p-chloreanIohetamine was injected i.p. two and a half hours before the decapitation. In an other series the Pats recevied 5o mg/kg MMT i.p. simultaneously with 3H-MA. We prepered primer and submitochondrial fractions of brain according to Kataoka and Be Rober~is (J.Pharmacol.Exp.Ther. 156, 114, 1967). Our results show that the subcellular distribution of MA is different when only 3H-MA was administered on one hand or when the rats were injected with MMT on the other hand. In the first case the amount of MA is considerably higher in the soluble fractions both in the primer and in the submitochondrial fractions than in the paniculate fractions. In the second case significantly higher amounts of MA are accumulated in the particulate fractions and less in the soluble fractions. P-chloreamphetamine did not influence the subcellular distribution of MA. A.K. PlaiTer, Institute of Experimental Medical Research, Hungarian Academy of Sciences, Budapest.

R 55

PRESSORRESPONSES TO HYPOTHALAMIC STIMULATION AS INFLUENCED BY DRUGS AFFECTING ADRENERGIC RECEPTORS (Effekt yon alpha-Rezeptoren-Blockern und Clonidin auf die Blutdrucksteigerung w~hrend hypothalamischer Stlmulierung) A. Philippu~ W. Roensber$, H. Przuntek Cats were anaesthetized with pentobarbital sodium and the hypothalamic posterior nucleus (HPN) superfused with artificial cerebrospinal fluid through a push-pull cannula. Electrical stimulation (ES) was carried out with the noninsulated tip of the cannula. ES of the NFN evoked a rise of the arterial blood pressure. Superfusion of the HPN with piperoxan (IxlO-2M) or tolazoline(5xlO -2 or ixlO-IM) inhibited the pressor responses to ES of the HPN. Labelling of the HPN with 3H-noradrenaline two hours before superfusion revealed that the inhibition of the pressor responses caused by tolazoline was accompanied by an increased release of the total radioactivity and an increased per cent release of 3H-noradrenallne. Superfusion with clonidine (IxlO-4 to IxlO-iM) did not influence the pressor responses to ES of the HPN. Superfusion of the nucleus of the solitary tract (NST) with clonidine (IxlO-3 to IxlO-iM)inhibited, while superfusion with tolazoline (IxlO-IM) potentiated the pressor responses to ES of the NFN. It is concluded that alpha-adrenerglc receptors are present in the HPN and are involved I) in the pressor responses evoked by ES, 2) in the regulation of the release of the neurotransmitter. Superfusion of the NST with clonidine inhibits, that with tclazoline potentiates the pressor responses to ES of the HPN by affecting inhibitory neurones of the NST. Prof. Dr. A. Philippu, Institut f~r Pharmakologie und Toxikologie der Universit't, D-8700 W~rzburg, Koellikerstr. 2

T E C H N I Q U E OF A N E R V E M U S C L E PREPARATION ON THE RAT (Herstellung eines N e r v - M u s k e l - P r ~ p a r a t e s bei der Ratte) J.PlStz For a series of experiments w i t h muscle r e l a x a n t s on the w h o l e animal rats w e r e an obvious choice.The p r o c e d u r e of this method will be described. A f t e r the rat is a n a e s t h e t i z e d three successive o p e r a t i n g steps are performed: after exposure of the tibial nerve of the left hind leg,two w i r e electrodes are w o u n d around it ;the Achilles tendon of the same leg is detached from its insertion and connected to a force t r a n s d u c e r ; f o l l o w i n g tracheotomy,the animal is attached to a r e s p i r a t o r . T h e rat is joined on its b a c k to a p l e x i g l a s s plate. The left lower leg is anchored to a system of rods w h i c h are i m m o v a b l y connected to the p l a t e . S t i m u l a t i o n of the tibial nerve is c o n d u c t e d over the electrodes with w e l l - d e f i n e d electrical i m p u l s e s . T h e ensuing twitches of the triceps surae muscle are r e g i s t e r e d via the Achilles tendon on a d i r e c t - w r i t i n g polygraph. An initial muscle tension is preset. The p r e p a r a t i o n has p r o v e d itself resistant and v i a b l e , t h e technique is simple and lessens traumatization.By choosing an isometric r e g i s t r a t i o n m e t h o d over an i s o t o n i c , m a j o r inertial effects were avoided(REICHEL,H.:Muskelphysiologie,in L e h r b u c h der Physiologie,ed.by W . T R E N D E L E N B U R G and E . S C H ~ T Z , S p r i n g e r - V e r l a g , B e r l i n G 6 t t i n g e n - H e i d e l b e r g , 1 9 6 o ) . A pO~, pCO2, and pH in the physiological range w e r e assured by using the respirator for small animals, d e s c r i b e d by C U N I T Z et a l . ~ a u n y n - S c h m i e d e b e r g ' s A r c h . o f P h a r m a c o l . S u p p l . V o l . 2 7 4 , 1972). D r . J . P l ~ t z , A b t e i l u n g f~r A n a e s t h e s i o l o g i e D-87oo W ~ r z b u r g , J o s e f - S c h n e i d e r - S t r . 2

der Universit~t,

R 56 I N H I B I T I O N O F T H E C A R D I A C E F F E C T S O F H I S T A M I N E ON C O N T R A C T I O N A N D C Y C L I C AMP B Y B U R I M A M I D E ( H e m m u n 9 d e r H e r z w i r k u n 9 von H i s t a m i n auf z y k l o - A M P und K o n l r a k t i o n d u t c h B u r i m a m i d ) G. PSch I N. Scholz~ W . R . K u k o v e t z In the i s o l a t e d g u i n e a - p i g h e a r t ( L a n g e n d o r f f ) h i s t a m i n e i n c r e a s e s i s o t o n i c c o n t r a c t i o n and c y c l i c @ (cAIvP) l i k e i s o p r e n a l i n e . S i n c e these e f f e c t s of h i s t a m i n e a r e r e s i s t a n t to b e t a b l o c k e r s and c l a s s i c a l (H1) a n t i h i s t a m i n i c s , a s p e c i a l r e c e p t o r (H2) w a s p r o p o s e d . A c o m p e t i t i v e H 2 - a n t a g o n i s t ( b u r i m a m i d e ) w a s r e c e n t l y d e s c r i b e d by B l a c k et al. ( N a t u r e 2 3 6 : 3 8 5 ~ 1972). We 9 t h e r e f o r e ~ c o m p a r e d the i n f l u e n c e of b u r i m a m i d e on the r i s e s in c o n t r a c t i l e a m p l i t u d e and cAJvP~ p r o d u c e d by e q u i e f f e c t i v e d o s e s of 2~,9 h i s t a m i n e and O. 1 1.1,9 i s o p r e n a * line. B e f o r e b u r i m a m i d e , h i s t a m i n e i n c r e a s e d c o n t r a c t i o n (ram) by 30+2.14. ( S . E . M . ; N = 2 0 ) and c A t v ~ ( p m o l e s / m g w. w . ) by 1.37_+O. 0 9 9 (N =5) a b o v e c o n t r o l s . 2 rain a f t e r s i n g l e i n j e c t i o n s of 2, mg b u r i m a m i d e ~ w h i c h had no e f f e c t on these p a r a m e t e r s ~ the e f f e c t s of h i s t a m i n e on c o n t r a c t i o n ( + 5 + 2 . 6 a b o v e 1 8 + 2 . 9 m m ; N = 4 ) and cAMP (+O. 14_+O. O33 a b o v e 0 . 5 2 + 0 . 0 2 9 pmoles; N=9) were signif i c a n t l y r e d u c e d and a l m o s t c o m p l e t e l y abolished~ w h e r e a s the a c t i o n s of i s o p r e n a l i n e (Zlmm: 3 0 + l . 8 ; N = 4 ; Z l p m o l e s : 1.69_+O. 101; N = 9 ) r e m a i n e d un changed. T h e r e s u l t s s u p p o r t a) the e x i s t e n c e of a s p e c i f i c H 2 - r e c e p t o r in the h e a r t w h i c h is b l o c k e d by b u r i m a m i d e , and b) the r o l e of cAIvP as m e d i a t o r of 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 h i s t a m i n e . Doz. Dr.G.

PSch, Pharmakologisches

Institut der Universit~t

A-8010

Graz

EFFECT OF PROPRANOLOL AND TETRACAINE ON THE CATION PERMEABILITY OF HUMAN ERYTHROCYTE GHOSTS ~ e b e r den Einfluss yon Propranolol und Tetracain auf die Kationenpermeabilit~t menschlicher Erythrocytenschatten) H. Porzig It was tested whether the propranolol(P)-induced increase in red cell K-permeability might be related to the local anaestetic properties of P.Therefore the effects of P(10-S-10-3M/I) and tetracaine (T, 5x10-4-10-2M/l)on cation permeability of red cell ghosts were compared. The P-induced increase in K+-permeability is completely blocked by intracellular, but not by extracellular EGTA. It could be demonstrated that P raises the intracellular free Ca concentration ~ a + + ] i ) b Y releasing an amount of membrane-bound Ca to the inside which is sufficient to explain the K-loss. Intracellular T up to 5x10-3M/l reduces the K-permeability of red cell ghosts and inhibits the action of P. Higher concentrations of T lead to a nonselective increase in cation permeability, which is not blocked by EGTA. The action of T resembles the biphasic action of Ca on cation permeability: I f ~ a + + ] i in Mg-containing ghosts is increased gradually from 10 -8 to 1O-2M/I the K-permeability has a minimum at about 10-6M, while the membrane becomes increasingly leaky for cations with higher concentrations. It is concluded that both,P and T probably decrease membrane Ca binding. However, it seems that T in contrast to P is able to replace Ca functionally at permeabilitycontrolling sites on the inside of the red cell membrane. Dr. H. Porzig, Pharmakologisches Institut der Universit~t, CH 3008 Bern, Friedbfihlstr. 49 Supported by SNSF grant Nr. 3.734.72

R 57 SULFONYLUREAS: EFFECTS ON GLUCONEOGENESIS AND SURFACE FLUORESCENCE IN ISOLATED PERFUSED LIVERS (Gluconeogenese und Oberfl~chenfluoreszenz isoliert perfundlerter Lebern unter Sulfonylharnstoffen) W.PoseriJ.Sch~nborn#A.FriedrlchtU.Panten The inhibition of hepatic glueoneogenesls by sulfonylureas has previously been described. Since gluconeogenesis depends on the redox state of the liver cell, surface fluorescence of reduced pyridlne nueleotides(RPN) and of oxidized flavoproteins(OFP) has been recorded. Surface fluorescence of RPN is known to be produced both in the oytosol and the mltochondrlal compartment, while surface fluorescence of OFP seems to be specific for mitochondria. Rat livers were perfused without hemoglobin. In order to enable rapid changes of substrates and drugs, the livers were perfused without reeirculation. Glucose output and ketogenesis were monitored. Even in therapeutic concentrations, sulfonylureas inhibit gluconeogenesls and ketogenesis in the presence of pyruvate, alanine or fructose. This iz~ibltlon is accompanied by a rapid decrease of fluorescence of RPN and by an increase of fluorescence of OFP, indloatlng a more oxidized state of the mitochondrial compartment. The known inhibition of intrahepatic lipolysis does not seem to be the only reason of the fluorescence effects, since they can be observed even in the presence of oleate(0.8 mM) or octanoate(l mM). It cannot be decided from the present results to which extent the inhibition of hepatic gluconeogenesis participates in the hypoglycemic response to sulfonylureas. Or.W.Poser, Instltut f. Pharmakologie u. Toxikologie d. Universit,t, 34 GGttingen, Geiststr. 9

STUDIES CONCERNING THE APPLICABILITY OF THE MICHAELIS-ME NTEN KINETIC TO THE INHIBITORY EFFECT OF D-TUBOCURARINE (Unfersuchung iJber die Anwendbarke]t der Michaells-Menten Kinetic auf den Hemmeffekt von d-Tubocurarln) J. Preuner and H. Schaube In chronically denervated rat diaphragm, under isotonic conditions, ACh produces a biphasic response. The iniltlal fast phasic contracture reaches its maximum in 20-4 sec depending on the concentration, and the rate of ACh application. Due to the isotonic recording, already the excitation of a superficial muscle layer will be sufficient to produce a maximum response. The common interpretation of dose-response-curves is based on the Michaelis-Menten kinetics, which involves both equilibrium conditions and proportionality between receptor occupation and the size of effect. None of these postulations are fulfilled: 1) an equilibrium of diffusion has not been attained at the maximum of the phasic contracture, and 2) the size of the effects depends on the concentration of the agonist as well as on the rate of access. The presence of d-TC retarded the time course of the development of the phasic contracture and decreased the amplitude. Evaluations of the effects yielded dose-response-curves which apparently demonstrated a non-competitive mechanism. The evaluation, however, implies the applicability of the Michaelis-Menten kinetics, which is not the case under the present conditions. The slowed response of the organ to ACh in the presence of d-TC is considered to be induced by the dissociation rate of the d-TC-receptor complex. Dr.J. Preuner, Institut fur Pharmakologie, Chrlstian-Albrechts-Universit~t 2300 Kiel, Hospitalstr. 4-6 West Germany

R 58 CATECHOLAMINE-INACTIVATING ENZYMES IN RAT ERYTHROCYTES (Catecholamininaktivierende Enzyme in Rattenerythrozyten) K. quiring, G. Kaiser Previous investigations have shown that a B-receptor system is present in rat erythrocytes; this receptor system seems to be localized mainly in premature red blood cells, i.e. in the reticulocytes (Gauger et al., J. de Pharmacol. (3), 25, 1972). With regard to the functional role of this receptor, one should expect that there exist catecholamine-inactivating mechanisms which also disappear during the maturation process of the red cells. In membrane fractions of reticulocyte-rich blood samples from rats pretreated with 1-acetyl-2-phenyl hydrazine (APH), the presence of a monoamine oxidase activity (substrate: 14 C-tryptamine) has been d e monstrated for the first time. The enzyme is presumably localized in mitochondria from reticulocytes and is specifically inhibited by pargyline. Negligible MAO activity was found in blood samples from untreated rats~ Moreover, it could be shown that the activity of a membrane-bound catecholO-methyltransferase (substrate:14C-adrenaline) inhibited by tropolone is several-fold higher in ghost preparations from APH-treated animals than in ghosts from untreated control rats. From our investigations - which have shown that both catecholamine-stimulated and catecholamine-inactivating enzymes are present in premature erythrocytes - it can be concluded that these cells contain a complete adrenergic receptor system. Zentrum der Pharmakologie, Theodor-Stern-Kai 7

Universit~t

Frankfurt,

D - 6000 Frankfurt

70,

COMPARISON OF OUABAIN AND CASSAINE WITH THEIR ALKYLATING DERIVATIVES REGARDING THE CONTRACTILITY AND Na-K-ATPase OF HEART MUSCLE (Wirkung yon g-Strophanthin, Cassain und alkylierender Derivate auf Kontraktionskraft und Na-KATPase des Herzmuskels) R.H. Raben and O. Wassermann Recently we synthesized a new, alkylating derivative of cassaine (C) replacing one CH3 at the nitrogen of C by a -CH2-CH2-CI group (CSL). Due to its alkylating potency CSL was supposed to be an irreversible inhibitor of Na-K-ATPase. - Its efficiency both on contractility (isol. guinea-pig atria) and on Na-K-ATPase (calf heart muscle) was compared with C and ouabain (S). - Atria, ED50 (x 10-7M): CSL 2.7, C 0.9 and S 1.1; ATPase, 150 (x 10-6M): CSL 24, C 1.0 and S 0.25 (in low concentration ranges CSL, C and S were activators of Na-K-ATPase). - CSL and C showed a faster onset of action in atria and a higher rate of enzyme inhibition as compared with S.-T1/2 of the wash-out of a positive inotropic (p.i.) effect of 80% (min): CSL 8.3, C 2.8 and S 4.4. The reversibility of the drug-enzyme complex was determined by a dilution technique at 37~ For comparison strophanthidin3-bromoacetate was included in this study, which proved to be a completely reversible inhibitor of Na-K-ATPase (see also Fricke and Klaus, Arch. Pharmacol. 268, 200, 1971). Essentially the same was observed for the alkylating CSL. In contrast, the ouabaln-enzyme complex was remarkably stable, whereas C took an intermediate position. - These results question again a direct relation between inhibition of Na-K-ATPase and the p.i. effect. They rather suggest that an activation of Na-K-ATPase by CSL, C and S might be involved in the development of the positive inotropic effect. Doz. Dr. O.Wassermann, Institut fur Pharmakologle, Christian-Albrechts-Universb~t 2300 Kiel, Hospitalstr. 4-6 West Germany

R 59 THALLIUM PHARMACOKI~NETICS AND ITS MODIFICATION BY ORAL ADMINISTRATION OF PRUSSIAN BLUE (Pharmakokinetik des Thalliums und ihre Beeinflussung dutch die orale Gabe yon Berliner Blau) A.G.Rauws In 1969 Heydlauf showed the effectiveness of Prussian Blue (PB) in increasing the elimination rate of thallium in rats. PB exerts its influence by binding Tl-ions in the intestine. Kamerbeek and coworkers extended these observations to the central nervous system. To gain more insight in the possibilities of PB therapy in thallotoxicosis a pharmacokinetie study was undertaken. The investigation was carried out with radioactive TI-204. Rats were given PBsuspension (treated) or only solvent (controls) twice daily for six days. On the third day TI was given intravenously. The time course of Tl-load, tissue concentrations and excretion was followed over 3 days. Thallium biological half-life was halved to 2 days. The dominant fecal excretion was doubled, the urinary excretion was reduced by one third in accordance with the findings of Heydlauf. The maximal cerebral Tl-concentration was also reduced by one third. A three compartment open model for TI was programmed on an EAI-I80 analog computer, and pharmacokinetie parameters were optimized. The influence o# PB was first simulated by reducing intestinal reabsorption to zero. A satisfactory fit of model to experimental data however, was obtained by reducing intestinal reabsorption to only 30~ of its original value, instead of to 0~. It is concluded that the hospital practice of giving PB twice daily by garage is not optimal and that it should be extended to slow intraduodenal infusion. Dr.A.d.Rauws,

National

Institute

of Public Health,

P.O.Box i, Bilthoven, the Netherlands

THE TIME COURSES OF THE INOTROPIC EFFECTS AND OF THE UPTAKE OF 3H-OUABAIN IN ISOLATED PAPILLARY MUSCLES OF THE GUINEA-PIG (Die Zeitverl~ufe der positiv inotropen Wirkung und der Aufnahme von 3H-Strophanthin an isolierten Meerschweinchen Papillarmuskeln) U. Ravens The time courses of the uptake and wash-out of 3H-ouabain (1 x 10-7, 7.7 x 10-7, 5 x 10-6M) in papillary muscles of the guinea-plg were compared with the time courses of the effect of similar concentrations on the force of contraction. The extracellular space was estimated from the uptake curves of 3H-saccharose. For each concentration used, an equilibrium of uptake was obtained at a different T/M ratio. In the presence of 1 x I0-7M ouabain the ratio was 3.4,; with increasing concentrations it approached the T/M ratio of the exfracellular space (0.5). Furthermore, the equilibrium level was reached faster at high concentrations than at low ones, which correlates well with the different time courses of the positive inotropic effect of various ouabain concentrations. From the uptake curves of 3H-saccharose and 3H-ouabain, the concentration of ouabain bound specifically at any particular time during the course of the experiment was calculated and compared to the corresponding increase in force of contraction: A linear relationship between these two parameters was obtained for the small range of therapeutic concentrations. Thus, in papillary muscle, a specific compartment is present which can be saturated with ouabain. At lower concentrations, the increase in force of contraction seems to be directly proportional to the amount of ouabaln present in [hls specific compartment. This does, however, not imply a similar relationship for high concentrations. Dr. Ursula Ravens, Insfitut fur Pharmakologie, Christlan-Albrechts-Universitdt 2300 Kiel, Hospitalstr. 4-6 West Germany

R 60

B-ADRENOLYTIC AND ANTIARRHYTHMIC PROPERTIES OF PHENYLEPHRINE D. Reinhardt Experiments with phenylephrine (PE) on the isolated electrically driven atrium and on the tracheal chain of the guinearpig were carried out in Krebs-Henseleit solution at 32 ~ and 37vC, resp. The dose-response curves of PE for the positive inotropic action on atrium - characterized by the B-adrenolytic drug pindolol as a B-sympathomimetic effect - showed an intrinsic activity of 0.3 compared with isoprenaline (IPN). On tracheal chain the intrinsic activity for the B-mimetic effect of PE amounted to 0.7 of that of IPN. These findings suggested PE to be a partial B-agonist exerting a competitive dualism in action to IPN. This dualism could be confirmed by dose-response curves determined for PE in the presence of IPN and vice versa. After an equilibration time of one hour for PE its intrinsic activity had ~anished. Under these conditions PE in concentrations from lO- to 3 x lO-5M behaved as a competitive B-adrenolytic drug: the dose-response curves for IPN on atrium and tracheal chain were shifted to the right in a parallel fashion without reducing the maximal response. On atrium, suspended in Krebs-Henseleit solution containing 0.5 x normal KC1, PE proved to be effective against BaC12-, IPN-induced or spontaneous arrhythmias. This action may be due to a specific B-adrenolytic as well as to an unspecific quinidine-like activit~ Dr. D. Reinhardt, Pharmakologisches Universit~tsinstitut, Klinikum Essen, HufelandstraBe 55

METABOLISM OF PRENYLAMINE (SEGONTIN R) IN MAN (Metabolismus yon Prenylamin beim Menschen) G. Remberg, G. Spiteller~ M. Schomerus~ und H. J. Den~ler The metabolism of C-14-prenylamine has been studied in man after i.v. and oral administration of 0-S4-prenylamine. 41,3% of the 0-14-activity was excreted in the urine after i.v. and 4o.7% after oral administration. Using the cumulative urinary excretion of C-14-activity an absorption rate of 97% was calculated. After extracting the freeze dried urine at different pH's before and after enzymatic or acidic hydrolysis with organic solvents of increasing polarity and submitting the obtained extracts to thin-layer chromatography, gas-liquid chromatography and massspectrometry more than 5o metabolites could be isolated. The fractions of the urine showing the extraction charseteristlcs of amines consisted Of at least 25 metabolites. These fractions accounted for approximately 45 - 48 % of the C-14-activity excreted in the urine. In these fractions the following metabo!ires have been identified: prenylamine, amphetamine,norephedrine, diphenylpropylamine, N-aeetyldiphenylpropylamine, 4-OHdiphenylpropylsmine, 4-OE-prenylamine, 4,4-Di-OH-prenylamine, benzophenone, and diphenylpropylethylene. About 15 ~ of the excreted a-14-activity showed the extraction behaviour of neutral compounds. 3o% of the excreted O-14-activity exhibited the extraction characteristics of organic acids. The isolation and structural elucidation of these metabolites are under progress. Dr. G. Remberg, Windsusweg 2

Organisch-C"nemisches

Institut D 54oo GSttingen,

R 61

LINEAR PFLARMACQIq~ET:[CMODEL FOR ~ALYSES ~ T H FEW DATA (Ermittlung pharmakokinetischer Verteilung in Experimenten mit wenig Messdate~ G. Rentsch It is generally accepted that early studies of a new drug will be done in species which have a similar or the same pattern of distribution and metabolism as man. Most of these e.g. marmosets, squirrel monkeys, rhesus monkeys, rats or guinea pigs are very s~all, and only a few blood samples can be taken from one animal. This shortage of experimental data renders kinetic interpretation difficult. Acceptable solutions can, however, be obtained if the following assumptions are made: i. The start of the kinetic is given by the time of administration. 2. The distribution can be described in first approximation by HAT,MAN' s equations. 3. There is no special influence on the linearity of the ascending and descending part of the blood curve. 4. The available blcxx~ samples are taken at critical points of the distribution curve. F = A 1 ~ [exp(-Pl(T-A2) )- A 3 - exp (-P2 (T-A2))3 allows the data to be. fitted optimally. The equation can be solved by means of a cc~puter program. Fourier- or Laplacetransformation of the results enables further mathematical interpretation and the use of sophisticated pharmacokinetic mode'Ls. Gunter Rentsch, Biological and Medical Research Division, Sandoz Ltd., CH 4002 Basel, Switzerland.

D O P A M I N E , N O R E P I N E P H R I N E AND N O R E P I N E P H R O N E : C A R D I O V A S C U L A R A C T I O N S IN A N E S T H E T I Z E D CATS BEFORE AND A F T E R BETA A D R E N E R G I C B L O C K A D E ( D o p a m i n , N o r a d r e n a l i n und N o r a d r e n a l o n : H e r z - K r e i s l a u f w i r k u n g vor und nach b e t a - a d r e n e r g e r Blockade) N.Reuter,E.Heeg Es w u r d e n drei K a t e c h o l a m i n e mit u n t e r s c h i e d l i c h e n S u b s t i t u e n t e n am 8 - C - A t o m der S e i t e n k e t t e v e r g l e i c h e n d u n t e r s u c h t . F o l g e n d e P a r a m e t e r w u r d e n b e s t i m m t : H e r z f r e q u e n z , A n s p a n n u n g s - und A u s t r e i b u n g s z e i t , D r u c k und D r u c k a n s t i e g s s t e i l h e i t ( d D / d t . )im linken ~ m ~ V e n t r i k e l und a r t _ r i e l l e r B l u t d r u c k . D i e drel S u b s ~ a n z e n w u r d e n n a r k o t i s i e r t e n Katzen in g e o m e t r i s c h s t e i g e n d e n Dosen solange i n f u n d i e r t , b i s fHr dn/dtma x vor und nach b e t a - a d r e n e r g e r Blockad e ( P i n d o l o l o,32uM/kg) das M a x i m u m der Zunahme ~ b e r s c h r i t t e n war. Durch den Blocker P i n d o l o l nimmt der B l u t d r u c k s v s t o l i s c h und dias t o l i s c h a b ; H e r z f r e q u e n z und dp/dt w e r d e n nicht verMndert. Fax .. Die D o s i s w i r k u n g s k u r v e n for D o D a m i n und fur N o r a d r e n a l o n slnd gegen~ber N o r a d r e n a l i n um eine ZehnerDotenz nach rechts verschoben. Vor b e t a - a d r e n e r g e r B l o c k a d e steigern alle drei S u b s t a n z e n Herzf r e q u e n z , B l u t d r u c k und dn/dt ..Nach beta-adrenerger Blockade ma . ist bei allen drei S u b s t a n z e n ~le A f f i n i t ~ t zu den R e z e D t o r e n v e r m i n d e r t ( I / 2 Z e h n e r n o t e n z ) , b e z o g e n auf dn/dtmax. Die m a x i m a l e W i r k u n g ist nur bei N o r a d r e n a l o n a b g e s c h w ~ c h t , u . z w . f ~ r dn/dt und die H e r z f r e q u e n z . D i e B l u t d r u c k s t e i g e r u n g durch N o r a d r e n a ~ ist nach B l o c k a d e gr~Ser als v o r h e r , n i c h t aber bei Dopamin und Noradrenalon. Institut f~r P h a r m a k o l o g i e und T o x i k o l o g i e der U n i v e r s i t ~ t D - 33oo B r a u n s c h w e i g , B ~ i t e n w e g 17

R 62 METABOLISM AND ELIMINATION OF D I G I T O X I N I N T H E R A T ( M e t a b o l i s m u s und Aussebeidung yon Digitoxin bei der Ratte) N.RietbrockIH.-F. V~hrin~ertL.Weller After oral administration o f 40 ~ C i 3 H - l a b e l l e d D i g i t o x i n ( D T ) in female SD-rats(n=10)CHCl~-soluble and -insoluble metabolites can be separated.The p o l a ~ f r a c t i o n is i n c r e a s i n g o v e r 5 d a y s i n u r i n e f r o m 20 to 5 0 % , i n the f e c e s f r o m 5 to 15% o f the e l i m i n a ted amount.The CHClq-solubles derivates have been determined by thin-layer chromatography as D T , D i g i t o x i g e n i n - b i s - d i g i t o x 0 s i d e

(DT-bis),Digitoxi~enin-mono-digitoxosi~T-mono),Digoxin(D),Digoxigenin-bis-digitoxosi~-bis) and Digoxigenin-mono-digitoxoside (D-mono).DT d e c r e a s e i n t h e u r i n e from 35% at t h e b e g i n n i n g to 12% at the e n d o f the i n v e s t i g a t i o n . T h e main metabolite determ i n e d as D i n c r e a s e i n a c o r r e s p o n d i n g w a y f r o m 30 to 5 2 % . N e a r l y the s a m e a m o u n t s o f D - b i s a n d D T - b i s ( 1 0 - 2 0 % ) and only tracers of the m o n o - g l y c o s i d e s were detected. 10-20% of DT are eliminated in the CHCl3-fraction of the f e c e s , the r e l a t i v e a m o u n t s of D - b i s a r e t w i c e as h i g h as i n u r i n e . D, DT-bis and DT-mono are detectable to a s m a l l e x t e n t . T h e m a i n m e t a b o l i c p a t h w a y o f D T i n r a t s is t~e h y d r o x i l a t i o n to D.However,it cannot be decided,if a hydroxilation also occurs on the s t e p o f D T - b i s a n d D T - m o n o . T h e h i g h p e r c e n t a g e of DT-bis in the f e c e s is i n t e r p r e t e d as the r e s u l t of t h e i n t e n s i v e e n t e r o hepatic circulation of the h i s - g l y c o s i d e as s h o w n b y A B S H A G E N et a l . ( N a u n y n - S c h m i e d e b e r g ' s Arch.Pharmacol. 275,1,(1972). Inst.f. Klin.

Pharmakologie,

I Berlin-45,Hindenburgdamm

30

E F F E C T S OF R E S E R P I N E , 6-HYDROXYDOPAMINE, P-CHLOROPHENYLALANINE A N D A C O M B I N A T I O N OF T H E S E S U B S T A N C E S O N T H E G R O O M I N G B E H A V I O U R OF M I C E (Die W i r k u n g e n y o n R e s e r p i n , 6-Hydroxydopamin, p-Chlorophenylalanin und einer Kombination d i e s e r S u b s t a n z e n a u f das KSrperpflegeverhalten y o n M ~ u s e n ) 0. R o h t e ? J. M ~ n t z i n ~ T h e e x p e r i m e n t s w e r e c o n d u c t e d to c o n t r i b u t e to t h e u n d e r s t a n d i n g of the mechanisms underlying the effects of reserpine. Reserpine h a s b e e n s h o w n to m a r k e d l y r e d u c e t h e g r o o m i n g b e h a v i o u r a n d b o d y t e m p e r a t u r e of m i c e . T h e s e e f f e c t s w e r e a c c o m p a n i e d b y a profound reduction of the transmitter content of central and peripheral serotonergic a n d adrenergic n e u r o n s . T r e a t m e n t of m i c e with 6-hydroxydopamine a n d p-chlorophenylalanine, w h i c h r e d u c e d the transmitter c o n t e n t to t h e s a m e e x t e n t as t r e a t m e n t w i t h r e s e r p i n e , h a d n o e f f e c t o n t h e g r o o m i n g b e h a v i o u r or o n t h e b o d y temperature. It is t e n t a t i v e l y suggested that the effect of reserpine on the grooming behaviour and body temperature has no r e l a t i o n to t h e r e d u c t i o n of t h e t r a n s m i t t e r c o n t e n t of c e n t r a l and peripheral serotonergic an4 a d r e n e r g i c neurons. Dr. O. R o h t e , P h a r m a k o l o g i s c h - t o x i k o l o g i s c h J o h a n n A. W d l f i n g - B a u e r u. C i e . -, 3 2 1 2

e A b t e i l u n g d e r Fa. Gronau(Leine)

R 63

ON THE BIOTRANSFORMATION RATS (Zur Pharmakokinetik Ratten) H. Rommelspacher~

OF AMPHETAMINE IN ADULT AND OLD von Amphetarnin bet erwachsenen und alien H. Honecker

Using tritium labelled amphetamine the transformation of this drug to p-OHAmphetamine (p-OHA), p-OHNorephedrine (p-OHNe), conjugated pOHA and hippuric acid was studied in the liver, cerebral cortex, brain-stem, hypothalamns and piasrna of adult and old rats. The time course was examined during 4 hours after i.p. injection. Differences between the two groups in half-times were only found for cortex, stem and hypothalamus (2 p-~ 0. 01). The concentrations of amphetamine were elevated in the older group (e.g. in the liver 4 h p.i.: 2,0 pg/g w.w. (adult), 3,28 pg/ g w.w. (old); brainstem 4 h p.i.: 0,28 ~g/g w.w. (adult), 0,82 ~g/g w.w. (old)). The turnover of amphetamine to p-OHA was slowed in the old animals only in the brain, concerning the turnover of p-OHA to p-OHNe no age differences eo.lld be demonstrated. From 60 rnin, in the older group from 90 rain a correlation was found between the decline of the hypert h e r m i a and the c o n c e n t r a t i o n 2a =<0, 01; r o - 0, 9971, 2~ ~ t r a t i o n of a m p h e t a m i n e h a s a r a t s than on that of the o l d e r

of a m p h e t a m i n e in p l a s m a (r a = 0j 9890, 0, 01). D u r i n g t h i s p e r i o d a c e r t a i n c o n c e n s t r o n g e r e f f e c t on body t e m p e r a t u r e of adult g r o u p (2 p of th e r e g r e s s i o n l i n e s < 0,01).

D r . H. R o m n m e l s p a c h e r , I n s t i t u t fiir N e u r o p s y c h o p h a r m a k o l o g i e deE F r e i e n U n i v e r s i t ~ t B e r l i n , D - 1 0 0 0 B e r l i n 19, U l m e n a l l e e 30

I N F L U E N C E OF P R E G N E N O L O N E - 1 6 ~ - C A R B O N I T R I L E ( P C N ) P R E T R E A T M E N T ON MICROSOMAL CYTOCHROME P-450 DEPENDENT N-DEMETHYLASE ACTIVITY (EinfluS y o n P r e g n e n o l o n - 1 6 ~ - c a r b o n i t r i l ( P C N ) - V o r b e h a n d l u n g auf die m i k r o s o m a l e C y t o o h r o m P - 4 5 0 a b h ~ n g i g e N - D e m e t h y l i e r u n g ) I. R o o t s P r e t r e a t m e n t of male S p r a g u e D a w l e y rats (150g) w i t h PCN (3mg/rat p.o. at 2 days) r e s u l t s in a 1.4 fold i n c r e a s e on h e m e c o n t e n t of the h e p a t i c m i c r o s o m a l 6 f r a c t i o n . The f o l l o w i n g k i n e t i c p a r a m e t e r s are a s s e s s e d u s i n g 10- M m e t y r a p o n e ( 2 - m e t h y l - l . 2 - d i - 3 - p y r i d y l - 1 p ~ o p a n o n e ) as a tight b i n d i n g i n h i b i t o r and e t h y l m o r p h i n e as substrata (Roots and H i l d e b r a n d t , N a u n y n S c h m i e d e b e r ~ s Arch. Pharn~col., 1973, in press): The c o n c e n t r a t i o n of c a t a l y t i c a l l y a c t i v e centers (Et) , t h e i r t u r n o v e r n u m b e r (TN), Vmax, and the true K ivalue. TN Ki Vma x ~moles per (min -I) I0-6M (nmoles per mg p r o t e i n ) m g p r o t e i n and min) Control 3.1 1.94 2.4 6.0 PB 7.3 2.0 2.2 14.6 PCN 4.4 4.6 I. 9 20.2 The i n c r e a s e of Vma x a f t e r PCN t r e a t m e n t is m a i n l y a c h i e v e d by a h i g h e r t u r n o v e r of E t. This is c o n t r a r y to p h e n o b a r b i t a l (PB) treatment, w h i c h o n l y e n h a n c e s E t. F r o m c o m p a r a b l e e x p e r i m e n t s w i t h a m i n o p y r i n e as s u b s t r a t e it is s u g g e s t e d that a) d i f f e r e n t rate l i m i t i n g steps are i n v o l v e d in e t h y l m o r p h i n e m e t a b o l i s m as c o m p a r e d to a m i n o p y r i n e and b) that these steps are a f f e c t e d by PB, or PCN t r e a t m e n t , r e s p e c t i v e l y , in a d i f f e r e n t manner. I. Roots, Inst. f. Klin. Pharm., I B e r l i n 45, H i n d e n b u r g d a m m 30

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KINETIC STUDIES WITH LH-RH (Zur Wirkungskinetik des Luteinizing Hormone-Releasing Hormone) J.Sandow~W.Heptner~E.Niemann Extending previous studies on the inactivation of LH-RH by organ homogenates to neural tissues, rapid inactivation was also found in hypothalamic and cerebral cortical tissue homogenates. These findings correlate well with the observation of Redding (I972), that Tritium-labeled LH-RH is accumulated in neural tissues. Localization of the in vivo site of inactivation was attempted by surgical isolation of organs. Renal or hepatic circulation was blocked by occlusion of arterial and venous supply. Plasma disappearance time (T I/2) was significantly prolonged after isolation of the liver. From the results obtained independently in two animal species, it would appear, that the major site of inactivation is located in the liver. T I/2 of LH-RH from plasma, as measured by bioassay for LH-releasing activity in several species, was compared with the disappearance time of I25-I-LH-RH. LH-RH was iodinated by a modification of the method of Greenwood and Hunter (I962). Biological activity of (mono-I25-I-Tyrosine)-LH-RH was 80-90 % of uniodinated LH-RH. T I/2 was determined in rats and rabbits. The result obtained by Redding (I972) in rats using tritiated LH-RH was confirmed with I25-I-LH-RH. However, a different pattern of organ distribution of radioavtivity was observed one hour after injection of I25-I-LH-RH. Dr. J. Sandow, Abt.f.Pharmakologie, 623 Frankfurt/~. 80

Farbwerke Hoechst A G ,

DIFFERENT PHARMACOKINETICS IN RAT TISSUES OF LIPOPHILIC 3H-LABELLED AROMATIC AMINE METABOLITES ~Unterschiede in der Pharmakokinetik der lipophilen Metaboliten ~H-markierter aromatischer Amine in verschiedenen Geweben der Ratte) J.Schenk, H.-G.Neumann The qualitatively similar metabolism in the rat of carcinogenic trans-4-dimethylaminostilbene, inactive cis-4-dimethylaminostilbene and 4-dimethylaminobibenzyl proceed via various lipophilic, i.e. nonhydroxylated and unconjugated intermediates yielding hydrophilic, i.e. hydroxylated and conjugated excretion products. The pattern of these metabolites was analysed in lung, spleen and adrenals, brain and fat 1.5, 5 and 24 h after oral administration of the highly 3H-labelled compounds (I mg). Sulphates and glucuronides were less than 15% of the unbound metabolites in spleen and lung, and below 10% in adrenals, brain and fat. This is far less than that found in liver and kidney(Metzler and Neumann, 1971). It is therefore concluded that hydrophilic conjugates essentially are not taken up from the blood stream by peripheral tissues. In these tissues 70-98% of the lipophilic metabolites could be radio gas chromatographically determined (in lung, spleen and brain after 24 h only 52-82%). Elimination rates of total radioactivity vary among tissues and do not reflect elimination rates of individual metabolites. In some tissues though, significant differences were observed for certain metabolites with respect to the elimination rates of unbound radioactivity. Dr.J.Schenk, Institut f~r Pharmakologie und Toxikologie der Universit~t, D-8700 WHrzburg, KoellikerstraBe 2

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N-DEMETMYLASE AND BENZO(a)PYRENE HYDROXYLASE ACTIVITY IN THE ADULT, FETAL AND NEONATAL LIVER OF THE RAT AFTER PHENOBARBITAL TREATMENT (N-Demethylase und Benzo(~)pyren Hydroxylase Aktivit~t in der erwachsenen, foetalen und neonatalen Leber der Ratte nach Phenobarbital Behandlun 9) Eva Schlede~ C. Kasper, R. Borowski In the liver of pregnant rats (day 14 and day 21 of gestation) the N-Demethylase and Benzo(a)pyrene(BP)-Hydroxylase activity is significantly lower when compared to the enzyme activities in the liver of the non-pregnant rat. Treatment with Phenobarbital (PB, 75 mg/kg body wt. from day 10-13 and from day 17-20 resp.) results in an increase of the enzyme activities in the liver of the pregnant and non-pregnant rats but being significantly lower in the liver of the pregnant rats. - In the fetal livers investigated on day 14 and on day 21 of gestation the N-Demethylase activity can neither be measured in the control nor in the PB pretreated groups. The BP-Hydroxylase activity is detectable in fetal liver homo genates of control and PB treated rats only on day 21 of gestation. PB treatment results in a significant increase of the BP-Hydroxylase activity. The electromicroscopic examination of the fetal livers does not show any alterations of the endoplssmic reticulum (ER) after PB treatment. - Five days after delivery in the neonatal liver homogenates the two enzyme activities are detectable. When the newborn rats are treated with various doses of PB from day I to day 4 of age the highest enzyme induction is already achieved with 40 mg PB/kg body wt. which is in contrast to results obtained with adult animals. After this treatment the fine structure of the hepatocyte is characterized by an increase of the smooth ER,also in addition by a loss of the parallel arrays and a dilatation of the vesicles of the rough ER. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 29). Dr. Eva Schlede, Pharmakologisches Institut der Freien UniversitSt Berlin, Abt. "Embryonal-Pharmakologie", D-~O00 Berlin 33, Thielallee 69/73

E F F E C T OF I S O P R O T E R E N O L ON THE M Y O C A R D I A L E N E R G E T I C M E T A B O L I S M , H E A R T R A T E A N D ECG IN THE RAT (Einflu~ yon I s o p r o t e r e n o l auf den E n e r g i e - S t o f f w e c h s e l des H e r z m u s k e l s , H e r z f r e q u e n z und EKG bei der Ratte) K. S c h l o s s m a n n , S. K a z d a Rats were t r e a t e d w i t h i s o p r o t e r e n o l in the dose range 0.002 200 mg/kg. At d i f f e r e n t times after single i n j e c t i o n s m y o c a r d i a l c o n c e n t r a t i o n s of ATP, c r e a t i n e p h o s p h a t e ( C P ) , glycogen, lactate and free g l u c o s e were estimated. Heart rate and ECG were also measured. I s o p r o t e r e n o l - i n d u c e d t a c h y c a r d i a lasted a p p r o x i m a t e l y one hour, the i s o p r o t e r e n o l - i n d u c e d EOG changes were n o r m a l i z e d five hours a f t e r the injection. D u r i n g the time of i s o p r o t e r e n o l i n d u c e d ECG changes and t a c h y c a r d i a the c o n c e n t r a t i o n s of ATP, CP and g l y c o g e n were reduced; the m a x i m a l effect was o b s e r v e d 30 m i n u t e s a f t e r the injection. At the same time lactate and free g l u c o s e c o n c e n t r a t i o n s increased. A f t e r five hours the conc e n t r a t i o n s of ATP, lactate and the ECG r e t u r n e d to normal. At this time CP levels r e m a i n e d still diminished. The g l y c o g e n conc e n t r a t i o n was o b s e r v e d to change in a b i p h a s i c manner. Being reduced d u r i n g i s o p r o t e r e n o l - i n d u c e d t a c h y c a r d i a the g l y c o g e n c o n c e n t r a t i o n i n c r e a s e d r a p i d l y a f t e r one hour and r e a c h e d a peak of 60 ~ M o l / g a f t e r two hours (normal: 24 ~Mol/g). Therea f t e r the g l y c o g e n c o n c e n t r a t i o n a g a i n g r a d u a l l y d e c r e a s e d but 48 hours after i n j e c t i o n it was still s i g n i f i c a n t l y h i g h e r than control values. All the i s o p r o t e r e n o l effects d e s c r i b e d were d o s e - d e p e n d e n t and could be p r e v e n t e d or at least d i m i n i s h e d by p r o p r a n o l o l . Dr. K. S c h l o s s m a n n ,

I n s t i t u t f~r P h a r m a k o l o g i e , B A Y E R AG, D-56 W u p p e r t a l I, P o s t f a c h 130105

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STUDIES ON CHRONIC COPPER POISONING OF RUMINANTS: INFLUENCE O~ THIOLS AND REDUCING AGENTS ON THE HEMOLYTIC AND METHEMOGLOBINGENERATING EFFECT OF Cu ++ (Untersuchungen Gber die chronische Kupfervergiftung der Wiederk~uer: Beeinflussung der h~molytischen und ferrih~moglobinbildenden Wirkung von Cu ++ durch Thiole und Reduktionsmittel) A. Sohmid Bei 0-240 Minuten (min) langer temperaturkonstanter (37 eC) Exposition yon heparinlsiertem und verdGnntem (1/12,2; 0,9 %ige NaC1-LSsung) Rattenblut in 1,259.10 -4 molaren LSsgngen von Natrium- bzw. Cupfer(II)-acetat (I) sowie 2,518.!O---molaren L~sungen yon L-Alanin-HC1 (II), L-Cystein-HOl (III), Glutathion (reduziert)(IV), 2,3-Dimercaptopropanol-(1) (V) und L-Asoorbins~ure (VI) verursaoht I nach 180 (p ~ 0,02) und 240 min (p m 0,002) H~molyse, welche durch II, IV und V verhindert wird (p~ 0,001). VI und III verkGrzen den Eintritt der H~molyse auf 60 bzw. 120 min (p ~ 0,001) und verst~rken den H~molysegrad nach 240 min um mehr als den Faktor 4. Gleichzeitig fGhrt I naoh 240 min zur Bildung yon Ferrih~moglobin (Hb(III)) (p< 0,01), welohe durch II und IV (p
DOSE DEPENDENT INDUCTION OF MICROSONAL LIVER ENZYMES OF RATS BY POLYCHLORINATED BIPHENYLS (PCBs) Dosisabhingige Induktion mikrosomaler Leberenzyme yon Ratten nach Vorbehandlung mit polychlorierten Biphenylen (PCB)oAoSchmoldt~RoFrtthlin~t H.FoBenthe Polychlorinated biphenyls (PCBs) are widespread environmental pollutants and found in animal and human adipose tissue. They are potent inducers of microsomal liver enzymes (Benthe et al. Arch. toxicol. 29,97 (1972). For studying the effects of different chlorine contents of these compounds male Wistar rats were treated once with increasing doses of the commercial PCBmixtures: di-,tetra- and hexachlorobiphenyl (Arochlor )o Noting the doses for max. microsomal stimulation the resulting dose response curves showed parallel shifting from high to low chlorinated compounds. Average max.effects (% of controls) were: cytochrom P-450 280%, NADPH dependent cytochrome c reductase 218%, 0-demethylation of p-nitroanisole 603%, anilinehydroxylase 435%. The threshold dose of hexachlorobiphenyl is about 5 umoles /kg b.wo This high chlorinated PCB is 3 and 10 times more effective than tetra- and dichlorobiphenyl respectively. The k~values are altered:O,O9mM (control O,05mM) for p-nitroani~ole O-demethylase and 0,17mM (O,03mM) for anilinehydroxylase. Discussing the results GLC-analyses of induced livers are presented showing the accumulation of preferable high chlorinated biphenyls. Dr~ A. Schmoldt,Pharmakologisches Institut der Universitit D 2000 Hamburg 20 Martinistrasse 52

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THE EFFECTS OF CATECHOLAMINES AND OF DISODIUM CROMOGLYCATE (DSCG) ON THE CYCLIC A~P AND THE ANAPHYLACTIC RELEASE OF HISTAMINE IN THE GUINEA PIG LUNG (Wirkung yon Catecholaminen und Dinatrium cromoglycat auf den Gehalt an cyclischem AMP und die anaphylaktische Histaminfreisetzung in der Meerschweinchenlunge) W. Schmutzler, R. Derwall, G. Poblete Freundt Lung pieces from actively (ovalbumin) sensitized as well as from non-sensitized guinea pigs were incubated with various sympathomimetic amines, DSCG and histamine in the presence and the absence of the antigen. The lung c-AMP levels were determined using KRISHNA's method, the histamine release by bioassay. In the sensitized lungs (figures for non-sensitized lungs in brackets) isoprenaline I0-6 M increased the c-AMP to 150 % (148%) of the control sample, adrenaline I0-5 M to 155 % (180%), noradrenaline iO -5 M to 140 % (128%), DSCG 10 -3 M to 125 % (100%), histamine I0-5 M to 1 2 1 % (101%), and ovalbumin i0 /ug/ml to 120 % (i00%). In the presence of isoprenaline, adrenaline and ncradrenaline the anaphylactic release of histamine from the lung was clearly, in the presence of DSCG only slightly inhibited~ the degree of inhibition being inversely related to the changes in the tissue c-AMP levels. The results show that active sensitization induces changes in the reactivity of the adenylcyclase system in the guinea pig lung and that DSCG is not a specific inhibitor of IgE mediated allergic reactions. Prof. Dr. W. Schmutzler, Abteilung Pharmakologie, Med. Fakult~t der TH, D-5100 Aachen, Melatenerstr. 213.

STEREOSELECTIVE TISSUE DISTRIBUTION OF (*)- AND (-)-N-METHYL-SPHENYL-S-PROPYL-BARBITURATE EN THE RAT (Stereoselektive Gewebsverteilung des (§ und (-)-N-Methyl-5-phenyl-5-propyl-barbiturates bei der Ratte) F. Schneider-Affeld, H. P. Bfich The optical isomers of N-methyl-5-phenyl-S-propyl-barbiturate (I) have diametrically opposite CNS activity: (+)-I causes convulsions like pentetrazol, whereas (-)-I causes anesthesia (Bfich et al., Abstracts Fifth International Congress on Pharmacology,p.33,1972). With exception of the rotation of the plane polarized light all physicochemical properties of the isomers are identical. Nevertheless further biological differences may exist. Therefore, tissue distribution of the isomers of I was investigated. After i.v. administration of 75 mg/kg of the isomers concentration of I was measured in several tissues (Tab.) during the CNS effect (3 min.), immediately afterwards (10 min.) and later (180 min.). serum (pg/ml) brain (pg/g) liver (pg/g) fat (pg/g) min. (+) (-) (+) (-) (§ (-) (+) (-) 3 72+11 Q 8 3 + 6 1 5 0 + 1 3 0 1 3 4 + 5 1 1 3 + 2 9 0 1 8 5 § 39§ 50§ 8 10 597 7 0 6 6 7 7 95u 847 9 142u 6 0 1 5 5 7 1 5 63~16 0101719 180 19u I Q 277 4 -~-~277 S 9 557 8 245764 0328759 (~ ! SD; n = 6 - ]!7; p (§

O <

O.05; @

<

O.O];]

Differences in the tissue concentration between (+)- and (-)-I exist: at 3 min. different binding to tissue constituents might be the cause, at 180 min. mainly metabolism is responsible. Dr. F. Schneider-Affeld, Pharmakologie und Toxikologie, Universit&t des Saarlandes, D-6650 Homburg/S. Supported by DFG

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INFLUENCE OF DRUGS ON MUCOPOLYSACCHARIDE SYNTHESIS IN FIBROBLAST TISSUE CULTURES (Beeinflussung der Mucopolysaccharidbildung in Fibroblastenkulturen durch Pharmaka) P.S. Sch~nhSfer r Vt Dinne ndahl~ D. Padber~ Stimulation of cyclic 3',5'-AMP (cAMP) formation and mucopolysaccharide (MPS) synthesis was determined in primary fibroblast tissue cultures of embryonic mice. Norepinephrine, isoproterenol, histamine and serotonin had no or minimal effects on cAMP levels or MPS release into medium. Bradykinine at 0.i-i.O ug/ml increased both cAMP levels and MPS formation. Of the steroids tested, S-oestradiol at O.i-i.0 ug/ml stimulated both cAMP and MPS formation. Similar to PGEI, MPS formation was already maximal at 0.5 ug/ml, while cAMP levels rose up to I.O ug/ml. However, stimulation by B-oestradiol was less than that by PGE 1 . Prednisolone at O.3-10.0 ug/ml decreased both cAMP levels and MPS formation even in presence of stimulatory concentrations of PGE . Since prednisolone appears to be also effective in reducing MPS 1 synthesis when cells are treated with dibutyryl-cAMP, an inhibition of MPS formation beyond adenyl cyclase has to be discussed. These results with prednisolone are compared to effects of nonsteroidal anti-inflammatory drugs on the adenyl cyclase system. P.S. SchSnh~fer, schule Hannover,

Institut f~r Pharmakologie, Medizinische HochD-3000 Hannover-Kleefeld, Karl-Wiechert-Allee 9

THE CHOROID PLEXUS UPTAKE OF GLUCOSE AND OTHER COMPOUNDS UNDER HYPEROXYGENATION (Die Choroid Plexus Aufnahme yon Glukose und anderen Komponenten w~hrend Hyperoxygenation). J. Sehou~ L. Proekop, C. Puglia~ C. Lambertsen Because the choroid plexi serve as the site of bidirectional movement of substances across the blood-CSF interface, we utilized choroid plexus incubations to investigate whether 0 2 toxicity convulsions are associated with altered CNS glucose uptake or damage to the blood-CSF barrier. Albino rabbits were exposed to 100% 0 2 at 3 or 4 atmospheres for a period of i, 2 or 3 hours, i, e., just short of the convulsion threshold. Choroid plexi (T) were removed and incubated in artificial CSF (M) eontain in.g Na~O~I, 3-Omethyl-d-glucose-14C (3 MG), eholine-14C, or glucose-14C for 5 or 30 minutes under identical hyperbaric 0 2 conditions. Results are expressed as a Tissue : Medium ratio (T/M). Although there was a tendency for OHP to depress choroid plexus uptake of 1311, this effect was not significant, e.g., the T/M for 1311 was 12.2 +_ 1.9 S.D. in controls, and i0.0 +. 3.9 in animals after 4 arm 0 2 for 2 hours. Plexus uptake of 3 MG and choline was not affected, i.e., variation of T/M for either was less than 5% between control and OHP conditions. Contrariwise, choroid plexus uptake of glucose-14C was significantly decreased, e.g., T/M of 31.5 +_ 4. 1 in controls, T/M of 22.5 _+ 2.4 after 3 arm 0 2 for 2 hours. Evidently, OHP affects ehoroid plexus glucose uptake specifically. These studies supply the first evidence that 0 2 toxicity convulsions may be associated with altered CNS glucose transport or metabolism. Professor J. Schou, Department 20 Juliane Maries Vej, DK-2100

of Pharmacology, University Copenhagen, Denmark

of Copenhagen,

R 69 E F F E C T OF D R U G S W H I C H R E D U C E T H E O S M O T I C F R A G I L I T Y , ON T H E H A E M 0 L Y T I C A C T I V I T Y OF P H O S P H O L I P A S E A F R O M B E E V E N O M (Wirknng v o n l ~ r m a k a , die d i e o s m o t i s c h e R e s i s t e n z steigeru, auf die haemolyti~he A k t i v i t ~ t y o n P t l o s p h o l i p a s e A aus Biene gift) R. S c h r o e t e r , A . A n s a r i Phospholipase A, t h o u g h not a b l e to d i r e c t l y h a e m o l y s e e r y ~ r o c y ~ s in i s o t o n i c salt s o l u t i o n , e f f e c t i v e l y splits membrane-bound pho~ pholipids in the p r e s e n c e of v a r i o u s p e p t i d e s (e.g. D i r e c t L y t i c F a c t o r of c o b r a v e n o m ) , or in h y p o t o n i c s o l u t i o n , thus c a u s i n g haemolysis. Several anesthetics are k n o w n to s t a b i l i z e e r y t h r o c y t e s against o s m o t i c s t r e s s at l o w c o n c e n t r a t i o n s . T h e y are i n c o r p o r a t e d into the e r y t h r o c y t e membrane, e x p a n d i n g the s u r f a c e area. It was c o n f i r m e d that p r e i n c u b a t i o n with 0.1M l - b u t a n o l in b u f ~ ed s a l i n e p r o t e c t s g u i n e a p i g e r y t h r o c y t e s against hypotonic ha~ molysis. Such treatment, however, increases their susceptibility to the h a e m o l y t i c a c t i o n of p h o s p h o l i p a s e A. In h y p o t o n i c solutkxl phosphlipase A-induced l y s i s p r o c e e d e d m u c h f a s t e r in the p r e s ence, t h a n in the a b s e n c e of b u t a n o l . E v e n in i s o t o n i c s o l u t i o n phospholipase A a c t e d as a d i r e c t h a e m o l y s i n , w h e n b u t a n o l was present. Qualitatively, the same r e s u l t s w e r e o b t a i n e d w i t h 1-pent a n o l ( 3 x 1 0 - 2 M ) , c h l o r p r o m a z i n e ( S x l O - 5 M ) , a n d pen~bsrbital ( 5 x 1 0 - 4 ~ T h e r e s u l t s s u g g e s t that m e m b r a n e p h o s p h o l i p i d s a r e e x p o s e d to attack by phospholipase A not o n l y b y an i n c r e a s e in t e n s i o n of the e r y t h r o c y t e m e m b r a n e , as p r o d u c e d in h y p o t o n i c m e d i u m , b u t a l s o b y c h a n g e s in the m e m b r a n e a r r a n g e m e n t (surface enlargement), d u e to i n c o r p o r a t i o n of a n e s t h e t i c drugs. Dr. R. S c h r o e t e r , I n s t i t u t fGr exp.

Abt. B i o c h e m i s c h e Pharmakologie, Max-PlanckMedizin, D-3400 G~ttingen, Hermann-Rein-Str.

3

PHARMACOKINETICS 0F PHENAZONE IN MAN AS AFFECTED BY A C ~ ETHANOL LOAD (Zum pharmakokinetischen Verhalten yon Phenazon beim Menschen unter akuter ~thanolbelastung) R. Sch~ppel~ E. Steinhilber Microsomal drug hydroxylation was studied in man under the influence of a single moderate dose of ethanol (I.$ ml/kg as 25 % -V/V-solution). Phenazone was used as a model substance, given together with water or ethanol in an intraindividual trial (n = 6). Both, #-H0-phenazone and l-phenyl-3-methylpyrazolone-5 (norphenazone), were determined in the urine over 24 hours after dosing with phenazone (i~ mg/kg p.o.) in four sampling periods of six hours duration. Unchanged phenazone and creatinine were also measured in the urine, to assess complete absorption of the drug and normal renal function. Elimination of phenazone from the blood was followed by determination of phenazone in plasma. Elimination of hydroxylated metabolites in the urine was markedly decreased under acute ethanol load for 6 hours and increased later. In contrast, phenazone excretion was increased in all sampling periods compared to controls. Thus, ethanol brings about a dissociation of excretion pattern of parent drug and hydroxylated metabolites,indicating a selective inhibition of mlcrosomal hydroxylases in the liver by ethanol. In addition, elimination of phenazone from the circulati(m was significantly delayed in the ethanol group. The results obtained a~,e interpreted to show a reversible inhibition of microsomal drug hydroxylatlon in the liver by ethanol to occur also in man. PD Dr. Dr. R. Sch•ppel, Institut fGr Toxikologie der Universit~t, D-7~O0 TUbingen, WilhelmstraBe 56

R 70 THE INFLUENCE OF A SMBPARALYZING DOSE OF d-TUBOCURARINEON T H E A C T I O N O F S U X A M E T H O N I U M IN M A N ( D e r E i n f l u 6 einer" l i s s i v e n ' D o s i s C u r a r e a u f d i e W i r k u n 9 von S u x a m e t h o n i u m am Menschen) F. T. Schuh In c l i n i c a l a n e s t h e s i a u n w a n t e d s i d e - e f f e c t s of s u x a m e t h o n i u m (SUM) a r e a t t e n u a t e d o r p r e v e n t e d by a p r i o r i n j e c t i o n of a s m a l l dose of d - t u b o c u r a r i n e ( d - T C ) . T o a s c e r t a i n the i n f l u e n c e of d - T C on the m u s c l e r e l a x i n g a c t i o n of SuM, d o s e - r e s p o n s e c u r v e s of SuM w e r e r e c o r d e d fn 74 a n e s t h e t i z e d p a t i e n t s 15 rain b e f o r e as w e l l as 15, 30, and 45 min a f t e r a d m i n i s t r a t i o n of d - T O 0. 0 4 m g / k 9 . S u p r a m a x i m a l e l e c t r i c a l s t i m u l a t i o n of the u l n a r - n e r v e r e s u l t i n g in a f l e x i o n - g r i p r e s p o n s e of the hand w i t h c o m p r e s s i o n of an a i r b l a d d e r w a s used to m e a s u r e q u a n t i t a t i v e l y muscular" p a r " a l y s i s and w a s r e c o r " d e d v i a S t a t h a m p r e s s u r e t r " a n s d u c e r on a H o n e y w e l l 1508 O s c i l l o g r a p h . P r e t r e a t m e n t w i t h d - T O r"esulted in a s h i f t of the SuM d o s e - r " e s p o n s e c u r v e s to h i g h e r c o n c e n t r a t i o n s in compar"ison w i t h c o n t r o l s w i t h o u t d - T C a d m i n i s t r a t i o n ; EDS0 of SuM (0. 12 m g / k g , 15 rain b e f o r e d - T C ) w a s incr"eased to 0. 3 m g / k 9 (15 min a f t e r d - T C ) , 0 . 2 6 m g / k g (after" 30 min), and 0. 2,3 m g / k 9 ( a f t e r 45 rain). In a d d i t i o n , the d u r a t i o n of SuM p a r ` a l y s i s w a s shor`tened. Thus, t h e a n t a g o n i z i n 9 e f f e c t of a s u b t h r " e s h o l d dose of d - T C on i n t e n s i t y and dur"ation of SuM i n d u c e d n e u r o m u s c u l a r b l o c k r e q u i r e s a l a r g e r dose of SuM to e n s u r e a d e q u a t e relaxation. F . T . Schuh, Dept. of A n e s t h e s i o l o g y , B u f f a l o Gen. H o s p . , S t a t e U n i v e r " s ] t y of N e w Yor"k, B u f f a l o , N . Y . 14203; p r e s e n t a d r ' e s s : Z e n t r a l e A b t e i l u n g f{Jr A n a e s t h e s [ e d e r U n i v e r s i t ~ t , 2300 K i e [ I, H o s p i t a l s t r . /40

ADENOSINE-31,51-MONOPHOSPHATE IN CEREBRALCORTICALSLICES FROM GUINEA PIG AND RAT: EFFECT OF ~ - AND H-ADRENERGIC COMPOUNDS (Die W i r k u n g v o n ~ - u n d B-Rezeptorenstimulierung auf die Bildung yon cyclischem Adenosin-3',5'-monophosphat in Gehirnrindenschnitten yon Meerschweinchen und Ratte) J. Schultz, J. W. Daly In cerebral cortical slices from guinea pig the formation of adenosine-31,5 tmonophosphate (cyclic AMP) is not enhanced by the addition of norepinephrine or isoproterenol (0. I ~ ) to the incubation medium (Krebs Ringer bicarbonate). In rat, however, under the same conditions cyclic AMP levels are elevated 6-8 and 2-3 fold, respectively, over control levels (10-15 pMol/mg protein). Adenosine stimulates formation of cyclic AMP in cerebral cortical slices from guinea pig and rat. In guinea pig, in the copresence of adenosine and norepinephrine a potentiation of the adenosine effect is observed (Sattin and Hall, 1970). This synergism can be blocked by the~-blocker phentolamine but not by the H-blocker propranolol. As well, the copresence of isoproterenol and adenosine has only a slightly better effect as adenosine alone on cyclic AMP levels in cerebral cortical slices from guinea pig. In rat, a synergistic action is observed between norepinephrlne and adenosine as well as between isoproterenol and adenosine. Use of phentolamine and propranolol a s ~ - and B-blocking agents reveals the presence of ~ - and B-receptors in cerebral cortical slices from rat. We conclude from our results that in cerebral cortical slices from guinea pig the formation of cyclic AMP is stimulated via classical~-receptor. This receptor is sensitive to norepinephrine only in the presence of adenosine. In cerebral cortical slices from rat, ~ - and H-receptors are interacting with the adenyl cyclase system. In addition, adenosine synergistically enhances both, ~ - and ~-adrenergic stimulation of cyclic AMP formation. Dr. J. Schultz Institut fur Toxikologie der Unlversit~t~ D-74OO T~bingen, WilhelmstraHe 56

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ADENOSINE AS MEDIATOR OF INSULIN EFFECTS ON FAT CELLS (Adenosin als Mediator yon Insulinwirkungen aus Fettzellen) U. Schwabe The well known ability of insulin to stimulate Glucose transport and to inhibit hormone-induced lipolysis is dependent on the concentration of fat cells in the incubation medium. At high concentrations (100 000 cells/ml) of rat fat cells insulin (10-100 ~E/m~ strongly depressed lipolysis induced by noradrenaline (I ~M), but failed to inhibit lipolysis at low cell concentrations (20 000 cells/ml). Addition of 0.1 bM adenosine to dilute cell suspensions completely restored the antilipolytie action of insulin. At 5000 cells per ml adenosine (I MM) in presence of insulin potentiated C-14-02 production from glucose-l-C-14, whereas adenosine itself had only small effects on glucose oxidation. The mise Of cyclic AMP levels induced by noradrenaline (I ~M) Was partly antagonized by insulin in concentrated cell suspensions. In dilute cell suspensions which exhibit a I0-s higher response to lipolytic hormones, no inhibitory effect was obtained with insulin. In the presence of adenosine which itself strongly depressed the cyclic AMP content, insulin was able to produce an additional inhibitory effect. Insulin did not enhance the uptake os adenosine into fat cells. These results suggest that adenosine is involved in the processes concerned with insulin effects on the fat cell membrane. U. Schwabe, Institut fur Pharmakologie, Medizinische Hochschule Hannover, 3000 Hannover-Kleefeld, Karl-Wiechert-Allee 9

THE EFFECT OF TESTOSTERONE ON NITROGEN METABOLISM IN WHITE NICE. (Der Testosteroneffekt auf den Stickstoff-Stoffwechsel in weiBen M~usen.) W. Schwarzlose In the liver of female white mice the enzymes GPT, GOT and GLDH, also alanine and glutamate and ammonia increased #8 hours after subcutaneous application of q00 mg/kg testosterone propionate. But there was a decrease of aspartate and urea in the liver. A decrease of aspartate in the liver could be seen already 12 and very intensive 24 hours after testosterone application. A decrease of urea excretion in urine became definitely evident 48 hours after testosterone injection, Nhile ammonia excretion increased. Whereas nitrogen uptake by food, measured over 4 days, did not changed, the retention of urea was higher than the excretion of smmonia resulting in a p o s i t i v e n i t r o g e n balance. From these metabolism changes observed after testosterone it can be concluded that the hormone action at first leads to a decrease of aspartate concentration in the liver. The lower level of aspartate then leads to a diminished synthesis and excretion of urea. Since not all ammonia can be utilized in liver for the urea synthesis, its concentration in the liver and excretion by the kidneys increased. The resulting positive nitrogen balance implicates that free ammonia in liver and kidneys is used for synthesis of glutamate and other amino acids by increased activities of GLDH and transaminase. Dr. W. Schwarzlose~ Pharmakologisches Institut der Universit~t Erlangen-NGrnberg, D-8520 Erlangen, Universit~tsstraBe 22

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STUDIES ON KINETICS AND MECHANISM OF PHALLOLYSIN-HEMOLYSIS (Untersuchungen zur Kinetik und zumMechanismus der PhallolysinH~molyse) R. Seeger, M. Burkhardt, G. Neeb Phallolysin, a toxic hemolysin of high molecular weight, was isolated from Amanita phalloides (R. Seeger, H. Scharrer, NaunynSchmiedeberg's Arch. Pharmacol. 274, R 106, 1972). It had a direct hemolytic effect on washed ~ t h r o c y t e s of various species: Mouse ~ rabbit = guinea pig ~ rat ) man ~ dog ~ pig ~ sheep = cattle. Red blood cells of sheep and cattle were highly resistan~ Dose-response curves for rabbit and human cells were steep. Hemolytic activity was optimal at pH 6.0-7.0. Hemolysis was not osmotic in nature. Hemolysis proceeded more slowly than saponin hemolysis did. In response to a just fully e{fective dose of phallolysin, hemolysis of rabbit cells at 25 C began after 4 min and w~s nearly complete after 60 min. Hemolysis proceeded faster at 37 C. The dagree of hemolysis increased with decreasing temperature (to 2 C). Very little phallolysin was used up during hemolysis: when incubated with increasing numbers of cells, any given concentration of phallolysin liberated increasing amounts of hemoglobin. Hemolytic activity was only slightly reduced by addition of erythrocyte ghosts. It also was not inhibited by cholesterol or human serum. Phallolysin lacked phospholipase C activity against lecithin and sphingomyelin. Supported by the Deutsche Forschungsgemeinschaft. Priv.-Doz. Dr. Ruth Seeger, Institut fttr 2harmakologie der Universit~t D-8700 ~ttrzburg, Koellikerstrasse 2.

BLOOD-BRAIN BARRIER OF RATS WITH LYMPHOSTATIC ENCEPHALOPATHY (Die Bluthirnschranke be• Ratten mit lymphostatischer Enzephalopath• G. Seidel~ G. Back~ technical assistance: M. Welge In rats a specific disease, the so-called lymphostatic encephalopathy (LE) is induced 3 days after surgical blockade of the cervical lymph vessels. Total brain of LE-rats contained significantly more dyepost injection of Evans blue than brain of shamoperated animals. This was interpreted as an increase in permeability of the blood-brain barrier. However, doubts arose when it was found that after rinsing with 0.9% NaC1, brain of LE-rats did not contain more Evans blue than brain of controls. Tremor setting in 5 min post injection of harm• (lO mg/kg i.p.) lasts longer in LE-rats than in sham-operated animals, i.e. 31 min vs. 24 min post injection. Prolongation of the harmine action is combined with significantly elevated contents of harmine in brain of LE-rats (values in ~g/g tissue, means • s.e.m.; 24 min: 3.6 • 0.i vs. 2.8 • 0.2; 51 min: 3-7 • 0.2 vs. 2.7 • 0.2). Treatment of LE-rats with cellulose sulphate for 4 days (lO mg/kg i.p. each time), beginning with the day of operation, inhibits the harmine tremor and lowers the hsrmine content in brain tissue,although it is of no effect on harmine blood concentration. These results clearly show that the blood-brain barrier is more permeable to harmine in LE-rats than in sham-operated controls. The "therapeutic" effect of cellulose sulphate may be connected with its kininogen-depleting effect. Dr. G. Seidel, Institut fGr Pharmakologie der Nedizinischen Akademie, D-2~O0 LGbeck, Ratzeburger Allee 160

R 73 METABOLISM OF HISTAMINE IN THE RAT STOMACHAND DUODENUM (Histaminstoffwechsel im Magen und Duodenum der Ratte) K.-Fr. Sewing: Barbara Binder and Gerwit Frisch The a c t i v i t y of the i~istamine catabolizing enzyme diamine oxidase (DAO) was found to be very low in the rat gastric ~1.86 + 0.61 nMoles.g-~.min -~) and very high in the duodenal mucosa (2.43 + 0.11 ~Moles.g-~.min -~) when ~C-putrescine was used as substrate. The questio~was whether in both organs endogenous h i s t amine is broken down to the same highly d i f f e r e n t degree as the DAO a c t i v i t i e s r e f l e c t . Therefore the histamine breakdown rate (HBR) was determined by measuring the histamine f l u o r o m e t r i c a l l y (Shore et a l . , J.Pharmac.exp.Ther. 127~ 182186, 1959) remaining in gastric and duodenal mucosal homogenates a f t e r ~ c u b a tion for i and 2 hr in 0.1 M phosphate buffer (pH 7.4) at 37~ C without or in the presence of i n h i b i t o r s of histamine catabolism (10 -~ M aminoguanidine, 10-~ M chlorpromazine, 2.10 -2 M nicotinamide). In the duodenal mucosa HBR was reduced only in those incubation media containing aminoguanidine alone or in combination with others. In the stomach the e f f e c t of i n h i b i t o r s on HBR was less uniform. However, aminoguanidine s i g n i f i c a n t l y (p < 0.01) i n h i b i t e d HBR. In the stomach the DAO a c t i v i t y was only s l i g h t l y higher than the HBR due to oxidative deamination (both expressed as nMoles.g-l.min-~). In contrast, in the duodenum the DAO a c t i v i t y was about 4700 times higher than HBR by DAO. Although the DAO a c t i v i t y in the duodenum was 1300 times of that in the stomach the HBR due to oxidative deamination was only about 1/3 of the stomach. The results indicate that a least in the duodenum a high DAO a c t i v i t y does not necessarily r e f l e c t a high oxidative deamination rate of endogenous histamine. This may be d i f f e r e n t at high levels of endogenous histamine. Prof. Dr. K.-Fr. Sewing, Pharmakologisches I n s t i t u t der U n i v e r s i t ~ t , D-7400 TUbingen, Wilhelmstrasse 56.

IS OYTOCHROME C ~ N A N T I D O T E AGAINST AMANITA PHALLOIDES (DEATH CAP) POISONING? (Cytochrom C - ein Antidot gegen die Knollenbl~tterpilzvergiftung?) C.-P. Siegers Recently, Floersheim reported on curative effects of cytochrome C against a-amanitin poisoning in female mice (Schweiz.med.Wschr. 102, 901, 1972). We now tested antidotal properties of cytochrome C against poisoning induced by the whole mushroom Amanita phalloides. The freeze-dried mushrooms were suspended in a 1% Tylose solution and injected i.p. in doses of lO, 15, 20, 40, and 60 mg/ kg, respectively. The LD50 of A.phalloides in male control mice was 26.5 (22.7-30.9) mg/kg. Four preparations of cytochrome C (Boehringer, Calbiochem, Mack, Serva), injected i.p. 30 min after the mushroom in doses of 30, 60, and 120 mg/kg, respectively, did not change significantly the LD50 of Aophalloides. In rats, the i~ LD50 of the lyophilised mushroom was 41.7 (35.0-49.6~ mg/kg in control experiments and 37.0 (31.4-43.7) mg/kg in animals treated with cytochrome C (60 mg/kg i.p. 30 min later). Furthermore, increase of serum enzyme activities (SGOT, SGPT, SDH, and GLDH) in rats poisoned with A.phalloides (p.o. or i.p.) were not influenced by treatment with cytochrome C. Thus, cytochrome C is not an antidote to poisoning with A.phalloides. Furthermore, there was no curative effect of cytochrome C even to a-amanitin poisoning in our experiments: In male NMRI-mice, LD50 of ~-amanitin i.p. was 0.55 (0.46-0.66) mg/kg in controls and 0.61 (0.50-0.74) mg/kg in mice treated with 120 mg/kg cytochrome C i.p. 30 min after a-amanitin. Dr. C.-P. Siegers, Institut fGr Pharmakologie der Medizinischen Akademie LGbeck, D-2400 LGbeck, Germany.

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REDUCTION OF PATHOLOGICALLY INCREASED MOTOR ACTIVITY IN INTERCOLLICULARLY DECEREBRATEO CATS BY DIMETHYLAMINOAOAMANTAN (DMAA) (Abnahme patholozisch gesteigerter motorlechsr Aktivit~t intercoliicul~r dezerebrierter Katzen nach Oimethylaminoadamantan) K.-H. Sontag, P. Wand 7 - 14 mg/kg i.p. (DMAA] lowers the tone of the gastrocnemiuscoleus (GS) of free moving cats, and soon the cats sit down. Greater quantities provoke massive, often convulsive motor disturbance. The decerebrate rigidity after intercollicular transection of the brain stem is very much reduced or even stopped under the influence of 7 - 10 mg/kg i.v.. The spontaneous discharge rate of motor units in GS of intercollicularly decerebrated cats decreases up to 30 % after injection of 10 mg/kg i.v. and the active, reflectory tension on stretch of GS decreases considerably with 7 mz/k Z i.v. in both dynamic and static periods of stretch. The decrease of GS cizidity we account for at the moment ae a reduction of the hyperactive a- end y-systems. ~he decrease of the GG tone in free moving cats could also be explained as a reduced output of ~-motoneurons. Max-Planck-lnstitut Hermann-Rein-StrsBs

for experimentelle 3

Medizin,

0-3400 G6ttinzen,

EFFECTS OF CATECHOLAMINES AND PROSTAGLANDINS ON CYCLIC AMP LEVELS IN BRAIN IN VIVO (Wirkungen yon Catecholaminen und Prostaglandinen auf den cAMP-Spiegel des Hirns in vivo) T. Stamm, W. Wellmann Microwave irradiation was used as method of s~crifice to estimate cyclic AMP levels in brains of rats and mice in vivo. Following intravenous injection os isoprenaline (I mg/kg) and dopamine (20 mg/kg) cyclic AMP levels in whole brains of mice were elevated by 70 Z and 40 Z respectively within one minute and had declined to control values after 3 minutes. Noradrenaline (I mg/kg and phenylephrine (I mg/kg) had no effect. The increase os cyclic AMP induced by isoprenaline was completely prevented by pretreatment with propranolol (20 mg/kg i.p.). This B-receptor-blocking agent had an effect of its own as a 30 Z decrease of cyclic AMP levels was observed 30 minutes after intraperitoneal injection. PGE1 and PGE 2 (I mg/kg i.v.) led to a 60 - 70 Z increase of the cyclic AMP content, whereas PGF2~ was much less effective. The estimation os cyclic AMP levels in seven discrete areas of rat brains showed that the increase of the cyclic nucleotide in response to PGE2 was highest in the cerebral cortex, intermediate in the thalamus, and lowest in the cerebellum and brain stem. The cyclic AMP increase in brain was associated with opposite effects on the behaviour os the animals, isoprenaline inducing central excitation and the Eprostaglandins inducing sedation. W. Wellmann, Institut s Pharmakologie, Medizinische Hochschule Hannover, 3000 Hannover-Kleefeld, Zarl-Wiechert-Allee 9

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ON THE I N H I B I T I O N OF P E R I P H E R A L AND C E N T R A L N O R A D R E N E R G I C N E U R O T R A N S M I S S I O N BY C L O N I D I N E (Zur H e m m u n g p e r i p h e r e r u n d z e n t r a l e r n o r a d r e n e r g e r Neurone durch Clonidin) K. Starke, H_~. Montel, K.P. A l t m a n n The effect of c l o n i d i n e (C) on the release of n o r a d r e n a l i n e (NA) in r e s p o n s e to e l e c t r i c a l s t i m u l a t i o n of n o r a d r e n e r g i c n e r v e s was studied in i s o l a t e d rabbit hearts (endogenous~NA) and in slices of rat cerebral c o r t e x (previously stored ~H-NA). At a s t i m u l a t i o n f r e q u e n c y of 5 Hz, C d i m i n i s h e d ~he stimulation-induced o v e r f l o w of NA a l r e a d y at 10M. C o m p l e t e i n h i b i t i o n could not be obtained: the c o n c e n t r a t i o n - r e s p o n s e curves l e v e l l e d off at 68 and 35% i n h i b i t i o n in hearts and brain slices, respectively. - The effect of C was i n v e r s e l y r e l a t e d to the o v e r f l o w of NA u n d e r control conditions. Thus, the inh i b i t i o n was reduced, if the control NA o v e r f l o w was i n c r e a s e d by r a i s i n g the f r e q u e n c y of stimulation~ or by adding cocaine to the medium. - In the ~ e s e n c e of l0 ~ M C, the increase of NA o v e r f l o w c a u s e d by l0 "5 M p h e n o x y b e n z a m i n e or phentclamine, but not that c a u s e d by 10M cocaine, was blocked. The results c o n f i r m the idea that the i n h i b i t o r y effect of C as well as that of other s y m p a t h o m i m e t i c drugs is m e d i a t e d by mr e c e p t o r s n o r m a l l y i n v o l v e d in a f e e d - b a c k i n h i b i t i o n of NA release (Farnebo and Hamberger: Acta. physiol, scand. Suppl. 371, 35, 1971; Starke: N a t u r w i s s e n s c h a f t e n 5_~8, 420, 1971). Doz. Dr. Klaus Starke, P h a r m a k o l o g i s c h e s Institut Gesamthochschule, D - 4 3 0 0 Essen, Hufelandstr. 55

der

EFFECTS OF OXPRENOLOL AND PROPRANOLOL ON PUIMONARY FUNCTION AND BLOOD GASES IN THE DOG (Der Einfluss yon 0xprenolol und Propranolol auf Atemfunktionsgr~ssen und art. Blutgase am Hund) J. Stepanek Anaesthetized, intubated dogs showing an 02 saturation (S02) of 90% were given 0.1, 0.3, 1 and 3 mg/kg 0xprenolol or Propranolol i.v. (n=8 per dose). Before and 5, 20, 40 and 60 min. after the injection, respiration rate (RR), tidal volume (TV), minute volume (MV), S02 and heart rate (HR) were determined; pH, P02 and PC02 were only measured 5 and 40 min. after the injection. MV was generally increased by 0xprenolol and reduced by Propranolol in comparison with the controls; changes in TV were slight and bidirectional. MV, RR and HR were slightly (<10%) modified by 0.i and 0.3 mg/kg 0xprenolol; only MV increased by 10% after 0.3 mg/kg. After i and 3 mg/kg the respiratory changes (MV + 25%, RR + 30%) were slightly more marked than the change in heart rate (+ 20%). 0.i mg/kg Propranolol diminished MV and HR by 10-15%. The higher doses caused mean reductions of 20% in MV, 30-35% in RR and 25% in HR. The decrease in MV could be corrected with Aminophylline. Distinct changes in the blood gases were noted 5 rain. after the injection of the higher doses. 3 mg/kg 0xprenolol increased P02 by 12% and S02 by 0.5%; PC02 decreased by 2%. 0.3, 1 and 3 mg/kg Propranolol led to mean decrease of 5% in P02 and 1.2% in SO2; PC02 increased by 4.2%. Quantitatively, the changes in HR and respiration provoked by the two 8blockers were the same. Dr. J. Stepanek, Biological Research Laboratories of the Pharmaceuticals Division of CIBA-GE]GY Limited, Basle

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THE METABOLISM OF GLYCINE IN ACUTELYAND CHRONICALLY MORPHINIZED MICE /Glyzinmetabolismus bei akut und chronisch morphinisierten M~usen/ P. Stern Glycine /G/ inhibits the activity o f ~ - m o t o r cells in medulla spinalis. Its decrease causes rigor in rats. Since m o r p h i n e / M o / and all its derivates cause: rigor~ it is possible that G also inhibits certain functions at supraspinal level. For these reasons~ w e examined the effect of M o upon the quantity of G in the brain of acutely and chronically morphinized mice. The results showed that M o decreases G in brain significantly~ both in acutely and in chronically m o r phinized mice~ while nalorphine increases G in both groups of mice i.e. has the opposite effect. Our experiments showed that aminopterine~ which decreases G in CNS~ causes analgezia but does not tranquilize mice. Also~ the application of G to chronically morphinized mice doesn,t cause abstinential symptoms. The question is whether the decrease or increase of G is related to the sedative and abstinential s y m p t o m s in morphinized mice. G increases significantly the level of acetylcholine in the brain. Dr. P. Stern~ InstitutfiirPharmakologie und Toxikologie der Medizinischen Fakult~t ~ Sarajevo ~ Jugoslawien.

THE ROLE OF THE M I C R O S O M A L M E M B R A N E IN THE B I O A C T I V A T I O N OF C A N C E R O G E N S (Die Rolle der m i k r o s o m a l e n M e m b r a n bei der B i o a k t i vierung von Cancerogenen) A. Stier, I. Reitz, E. S a c k m a n n Radicals have been d e t e c t e d by E S R - s p e c t r o s c o p y in rabbit liver m i c r o s o m a l i n c u b a t i o n s of 8 c a r c i n o g e n i c aromatic amines and of several n o n c a r c i n o g e n i c a r o m a t i c amines, for instance, of p h e n a c e tin and of benzpyrene. In the case of N - m e t h y l a n i l i n e N - d e m e t h y l a tion via N - o x i d a t i o n was observed. The radicals p r o d u c e d accumulate in the m i c r o s o m a l membrane, those o r i g i n a t i n g from n o n c a r c i nogens at a lower rate than those formed from carcinogens. The o b s e r v e d s u r p r i s i n g l y long lifetime of the radicals is caused by the p h y s i c a l state in w h i c h they are stored in the membrane: E x p e r i m e n t s in m o d e l m e m b r a n e s c o n s i s t e n t with the findings in the m i c r o s o m e s show f o r m a t i o n of clusters and/or strong immobilization in the quasi c r y s t a l l i n e phase of the lipid. By a p p l i c a t i o n of a new v e r s i o n of the s p i n l a b e l t e c h n i q u e evidence for a m o s a i q u e - s t r u c t u r e of the lipid layer of the m i c r o s o m a l m e m b r a n e w i t h a quasi c r y s t a l l i n e area around the m i c r o s o m a l e l e c t r o n transfer chain is presented. The implications of these findings for the p o s s i b l e m e c h a n i s m s of tumor i n i t i a t i o n are discussed. PD Dr. Dr. A. Stier, M a x - P l a n c k - I n s t i t u t fHr B i o p h y s i k a l i s c h e Chemie, D-3400 G ~ t t i n g e n - N i k o l a u s b e r g , A m FaBberg

R 77 EFFECTS OF PHENYLISOPROPYLADENOSINE AND PGE ON cAMP-CONTENT AND GLYCEROL RELEASE OF RAT FAT CELLS. Quantitative Analyse der Wirkung von Phenylisopropyladenosin(PIA)und PGE auf cAMP-Gehalt und Glycerinfreisetzung isolierter Fettzellen. K. Stock u. M. Prilop. In fat cell suspensions(50 x 103/ml) of rat epididymal fat tissue glycerol release (60 min.incubation), total cAMP(5 min. incubation) and 3H-cAMP, after labelling intracellular nucleotides by preincubation with 3H-adenine, were determined. Norepinephrine(NE) increased, both cAMP and 3H-cAMP dose-dependently. Emax was obtained at I-3 ~M NE: cAMP rose from 4 to appr. 100 pmoles/105 cells, 70 % of intracellular label was converted to 3H-cAMP~glycerol release at 3 ~M NE increased from 20 to 260 nmoles/10 cells/hr. Emax of NE on cAMP and 3H-cAMP was reduced dose-dependently by both PIA and PGE at 0.003-0.3 ~M, the levo-isomer of PIA being 50 to 100 times more active than dextro-PIA. In contrast, the b-receptor blocking drug K1255 (0.01-1.0 ~M) shifted dose response curves of NE to the right without affecting Emax. NE stimulated glycerol release was partly inhibited by PIA or PGE at less than 0.5-I ~M NE. Above I ~M NE dose response curves of NE in the absence and presence of PIA or PGE (0.01-I ~M) joined to reach equal Emax. NE stimulated increase of cAMP could be reduced drastically by inhibitor concentrations which had no or only small effects on the release of glycerol. (Supported by the Deutsche Forschungsgemeinschaft) K. Stock, Institut ffir Pharmakologie, Medizinische Hochschule Hannover, D-3 Hannover-Kleefeld, Karl-Wiechert-Allee 9, BRD

THE EFFECTS OF IMIPRAMINE AND PROMAZINE ON ENERGY METABOLISM AND EEG OF THE ISOLATED PERFUSED RAT BRAIN (Die Wirkung von Imipramln und Promazin auf den Energiestoffwechsel und das EEG des isoliert perfundierten Rattenhirns) R. Stock, J. Krieglstein, H. Rieger Imipramine and promazine were studied in regard to their effects on energy metabolism and the EEG of the isolated perfused rat brain prepared according to Andjus et al. (J.appl.Physiol. 2._22, lo33, 1967). As described in a previous paper (Krleglstein et al., Naunyn--SchmiedebergJs Arch.Pharmacol. 275, 124, 1972), high energy phosphates as well as substrates and metabolites of the glycolytic pathway were determined and bipolar •EGs were recorded by two symmetrical leads from the parietal regions. The drugs were added to the perfusion medium to give concentrations of 5 x lo -6 M, Io -5 M,, and lo -4 M, respectively. During the perfusion different EEG patterns were observed: modifications of the dominant frequency, spiking and a flat EEG with bursts of sp'~kes ("secondary discharges'~. The EEG modifications seemed to depend on drug concentration. In contrast to these effects on the EEG, the lower drug concentrations did not demonstrably affect cerebral metabolism. Only, when drug concentrations were raised to a toxic range (lo -4 ~ , reflected in the EEG by the pattern of secondary discharges, higher levels of P-creatine and glucose and lower levels of glucose-6-P were detectable. The lactate/pyruvate ratio remained unchanged. No major differences in their effect on energy status and EEG were observed between imlpramine and promazine, when equimolar drug concentrations were administered. R. Stock, Pharrnakologisches Institut der Universit~t D-65oo Mainz, Obere Zahlbacher Str. 67

R 78

D I F F E R E N C E S B E T W E E N G U I N E A - P I G S AND RATS IN THE A B S O R P T I O N AND IiEPATOTOXICITY OF E T H A ~ O L ( U n t e r s c h i e d e z w i s c h e n M e e r s c h w e i n c h e n und R a t t e n in d e r R e s o r p t i o n und H e p a t o t o x i z i t i t von ~ t h a n o l ) O. Strubelt~ H. B r e i n i ~ In fed g u i n e a - p i g s , an oral dose of 6.A g/kg of ethanol p r o d u c e d a m a x i m a l b l o o d a l c o h o l level of 6.8 • O.3 mg/ml, w h e r e a s in fed rats, b l o o d a l c o h o l levels after the same dose did not exceed 2.1 • 0.2 mg/ml. M a x i m a l b l o o d alcohol levels in f a s t e d animals after an oral load of 4.8 g / k g ethanol were 6.3 • 0.2 mg/ml f o r g u i n e a pi~s and 3-7 • 0.3 for rats. However, i.v. i n j e c t e d ethanol (1 g/ kg~ was e l i m i n a t e d at the same rate in b o t h species (275 mg/kg/h), and A D H a c t i v i t y of the liver r e l a t e d to b o d y w e i g h t was by 20% g r e a t e r in g u i n e a - p i g s t h a n in rats. Therefore, a b s o r p t i o n of ethanol occurs at a much s l o w e r rate in rats t h a n in g u i n e a - p i g s , m a k i n g the l a t t e r more c o m p a r a b l e to m a n in this respect. The slow a l c o h o l a b s o r p t i o n by rats is p r o b a b l y the cause for t h e i r high r e s i s t a n c e to the toxic actions of ethanol, e s p e c i a l l y to the h e p a t o t o x i c a c t i o n (c.f. S t r u b e l t et al., A r c h . T o x i k o l . 2 9 , 129, 1972). Not ~ guinea-pigs: H e p a t o t o x i c i t y (increased serum'-" a c t i v i t i e s of GOT, GPT, and GLDH) was evident 16 h after an oral dose of 4.8 g/kg ethanol. SGOT i n c r e a s e d a l r e a d y after 1.6 g/kg of ethanol p.o. D o s e - d e p e n d e n t v a c u o l a r d e g e n e r a t i o n was the m o r p h o l o g i c s u b s t r a t e of e t h a n o l - i n d u c e d liver d a m a g e in guineapigs. In conclusion, the g u i n e a - p i g is far b e t t e r suited for research on alcohol t o x i c i t y than the rat. Prof. Dr. med. O. Strubelt, Institut fGr P h a r m a k o l o g i e H e d i z i n i s c h e n A k a d e m i e Ldbeck, D - 2 4 0 0 Libeck, Germany.

der

THE CORRELATION OF Ca2+ UPTAKE AND Ca2+- DEPF2~DENT ATP HYDROLYSIS BY CARDIAC SARCO. PLASF~C RETICULUM ( Uber die Korrelation yon Ca2+ Aufnahme und Ca2+- aktivierter ATP Spaltung durch Membranen des cardialen sarkoplasmatischen Retikulums) J,Suko Cardiac sarcoplasmic reticulum (SR) fractions were prepared from dogsoCa2+uptake (Ca-upt) and Ca2+- dependent ATP hydrolysis (Ca-ATP-hydr) by SR fractions (measured by Mill• technique and as creatine liberated, respectively) were investigated under various conditionso Ca-upt and Ca-ATP-hydr increase proportionally to the amount of SR protein in the medium ( range tested: o,o25-o,15 mJml)o The rate of Ca-upt and the rate of Ca-* ATP-hydr have the same dependence on the ionized Ca2+ concentration. Both processes follow Michaelis-Menten kinetics. The Michaelis constant (KCa) is about 5 x lo-7 M. The rate of Ca-upt and the rate of Cs,-ATP-hydr increase parallel but in two steps when the ATP concentration is raised ( range tested: 3 x 19 to 3 x lo-~ M). The Michaelis constants ( KATP) are 1-2 x lo-5 M and 2-3 x lo"~ M, respectively. The increase in the rate of Ca-upt at higher temperatures (range tested: io~ - 35~ C) correlates with the increment in Ca_ATP-hydra The energies of activation were 16,65 ~ o,87 for Ca-upt and 17,93 • 0,49 Kcal/mole-1 for Ca-ATP-hydr ( means ~ SE. n = 4). The rate of Ca-upt and the rate of Ca-ATP-hydr. is inhibited by about 5o %' by tee following drugs: Chlorpromazine ( 5 x lo-5 M), reserpine ( 2 x lo"4 M), streptomycine ( 5 x lo-4 M) and quiz• ( 2-3 x lo-4 M)o A strong correlation between the rate of Ca-upt and the rate of the Ca-ATP-hydr was found at all experimental conditions. Thus the transport ratio ( rate of Caupt divided by the rate of Ca-ATP-hydr) which was about 1.o in a large number of different SR preparations, remained unchanged. Dr. J.$uko, 9harmakologieches Institut der Universit~t Wien, A-lo9o Wien, ~ihringerstr. 13a

R 79 TRANSPHOSPHORYLASE A C ~ l ~ OF ISOLATED MEMBRANES OF CATECHOLAMINE STORAGE VESICLES (Transphospho~jlase Aktivit~t isolierter Membranen yon CatecholaminSpeichervesikeln) Go Taugner The isolated membrane of catecholamine storage vesicles from adrenal medulla binds ATP, 32p labelled in y'-position and - at lower rate - adenosine-8-1qC l~belled ATP in presence of Mg ++ at 31 ~ The difference between 32P-ATP and 14C-ATP represents a P-protein compound. The 14C-ATP fraction consists of ATP + ADP which have combined with membrane structures. Also at 0 ~ when the ATPase activity bec(~nes unmeasureable low, mentionable amounts of ATP are bound to the membrane. Preincubation with ATP at 0 ~ inhibits ATPase activity and catecholamine uptake. ADP does not inhibit ATPase activity and catecholamine uptake, provided ATP is present. The membrane transfers the tezTninal phosphate group from ATP to ADP according to the reaction (!) ATP + E. "E ~ P + ADP (nucleoside-diphosphate kinase), an~2from ITP to ATP according to (2) iTp32 + E + ATP2+ E . ~IDP~ E + ADP ~E + P P + P. In a back reaction ATP32 is formed when ~ PO 4 is added. un!y reaction (2) can directly be related to the ATPase activity, since extraction of the membrane with acetone inhibits ATPase activity and 32p04 incorporation into ATP completely and the transfer of y -phosphate from ITP to ATP (2) partially, whereas reaction (I) proceeds 2 - 3 times faster after acetone treatment of the membrane. From these results follows that two enzyme systems transferring y -P must be present in the membrane of catecholamine storage vesicles. G. Taugner, Max-Planck-lnstitut fCtr medizinische Forschung, Abt. Physiologic D-69oo Heidelberg, Jahnstrasse 29

INFLUENCE OF POLYA~ION5 ON INDUCTION PHENOMENA (Zur Wirkung yon Polyanionen auf Induktionevorg~nge) K.Tempell R.Hollatz Biochemical reactions, depending on the presence of nucleic acids, are influenced in vitro by other polyanionic compounds. Some of these wellknown in-vitro-effects of polyanions may also occur under in-vivo-conditions: I. The induction of DNBBe I (EC 3.1.4.5) within kidneys of mice by total-body X-irradiation (Tel,420R) and/or 2,4,6-triethyleneimino1,3,5-triazine (TEm, 1,2 mg/kg i.v.), measured in vitro 72 hours later,

has been i n h i b i t e d , depending on the dose, by p o l y ( v i n y l s u l p h a t e ) ( I , 1 , 5 6 - 2 5 , 0 mg/kg) and/or p e n t o s a n e p o l y s u l f u r i c acid ( I I , 25,0-200 mg/kg), when given O, 24, and 48 hours after Tel or TEN. DNA as well as RNA (12,5-50,0 mg/kg i.v.) enhanced DNase I-induction under the same conditions. - 2. Proliferation of kidneys of mice after i.v. injections of folic acid (FA, 160 mg/kg i.v.) was lower by about 30-50~, when I (25 and 50 mg/kg rasp.) has bean given some hours before treatment with FA. Weaker effects have been produced by II and nucleic acids. When incorporation of 14C-oretic acid into RNA was measured 24 hours after treatment with FA, only II showed some inhibitory activity, which was dosedependent (25-200 mg/kg). Other polyanions had no effect. - 3. I (12,5, 25,0, and 50,0 mg/k9 i.v.) inhibited the in-vitro-activity of the aniline-hydroxylase-system within liver of mice 24 hours after i.v.injections of phenobarbital (PH, lO0 mg/k9 i.v.) by 30, 40, and 60~ resp., when given 1 hour before treatment with PH. II (50-400 mg/kg) and nucleic acids (12,5-i00 mg/kg) remained without any effect. - Mechanisms of the observed effects of polyanions ere under investigation. Univ.-Doz. Or.K.Tempel, Dr.R.Hollatz, Institut for Pharmakologie, Toxikologie und Phermazie der Universit~t, 8 ~Onchen 22, KSniginstra~e 15

R 80 THE IMPORTANCE OF AN ACTIVE TRANSPORT THROUGH THE CHOROID PLEXUS FOR THE PERMEATION OF MORPHINE INT0 THE CENTRAL NERVOUS SYSTEM (Die Bedeutung des aktiven Transports dutch den Plexus chcrioidens fGr die Permeation yon Morphin in das Zentralnervensystem) H~. Teschemacher IN VITRO investigations had shown an accumulation of morphine in choroid plexus tissue, which seemed to be due to an energy-dependent process. The significance of an active transport through the choroid plexus IN VIVO to be d e r i ~ d from these results was investigated in rabbits: Concentrations of ~C-morphine in blood, blood ultrafiltrate, cerebrospinal fluid(CSF), brain and spinal cord after i.v. injection were determined by means of liquid scintillation counting. In consequence of very high concentrations of morphine in the blood immediately after injection, the concentration in the central nervous system(CNS) increased very quickly. However, since the concentration in the blood decreased very rapidly, the concentration in the CNS reached its maximum already about 10 minutes after injection, subsequently lying above its equilibrium level. Equilibrium concentrations in the CSF were not reached within an observation period of two hours after injection. This points to a very slow diffusion through the blood-brain barrier as corresponding to a low partition coefficient heptane/water at pH 7.4 (<0.00001); in addition, elimination of morphine from CSF as due to other mechanisms like active transport should be unimportant, too. Accordingly, from a plot of concentrations of morphine in CSF versus its concentrations in blood ultrafiltrate no significant evidence was obtained for the existence of an active transport of morphine from CSF into blood or vice versa. Dr. Hi. Teschemacher, Max-Planck-Institut D-8000 MGnchen 40, Eraepelinstr. 2

fGr Psychiatrie,

THE EFFECT OF TWO BARBITURATE ANTIPODES ON S P I N A L REFLEXES AND SINGLE MOTONEURONES ( D i e W i r k u n g z w e i e r Barbiturs~ure-Antipoden auf spinale Reflexe und einzelne Motoneurone) * C. T h e r e s The stereospecificity of the effect of 1-methyl-5-phenyl-5-propyl-barbituric acid ( (+)- and (-)-MPPB, 50 mg/kg intra venously) on mono- and polysynaptie reflexes was studied in spinal rats. Reflex acitivity was elicited by electrical stimulation of the sciatic nerve and recorded from the ventral roots of L 6 of S 1. (+)-MPPB increased and (-)-MPPB reduced the amplitude of mono- and polysynaptic reflexes. Both antipodes did not influence the amplitude and the conduction velocity of the early component of the afferent volley which was recorded from the dorsal roots. - In cats, which were intercollicularly decerebrated and spinalized at the lower thoracic level, recordings were made from single motoneurones with microelectrodes. Both antipodes (50 mg/kg i.v.) did not change significantly the amplitude of the action potentials elicited by antidromic stimulation.(+)MPPB r e d u c e d the firing threshold to intracellular stimulation, whereas (-)-MPPB increased the threshold. (+)-MPPB increased and (-)-MPPB decreased mono- and polysynaptic exitatory postsynaptic potentials produced by stimulation of dorsal roots or of the gastrocnemius nerves. The results indicate that the antipodes of MPPB e x e r t an opposite effect on the motoneurone membrane. D r . C. T h e r e s , Institut ffir Pharmakologie und Toxikologie der Universit~t des Saarlandes, 665 Homburg/Saar. 9 Supported by the Sonderforschungsbereich 38 Membrane Research and a grant of the Stiftung Yolkswagenwerk.

R 81 EFFLUX OF 3H-(z)-NO~ADRENALINE

(NA) AND ITS METABOLITES FROM RABBIT AORTIC STRIPS (Efflux von "H-Noradrenalin und seinen Metaboliten aus Aortenstreifen des Kaninchens) U. Trendelenbur~

Aortic str%ps were obtained from reserpine-pretreated rabbits and exposed to 200 ng/ml ~H-NA for 30 min. During subsequent washing with amine-free solution efflux was measured for up to 240 mln. After block of MAO (pargyline) and COMT (U-O521) efflux came from 3 compartments: t/2 = 5 min or less for 35 % of the labelled amine (presumably extracellular and superficially bound), t/2 = 21 min for 26 % (presumably extraneuronal), and t/2 = 120 min for 39 % (presumably neuronal). After pargyline only, the neuronal efflux consisted of NA and normetanephrine (NM) (ratio 3:1). After U-O521 only, neuronal efflux consisted of NA and deaminated catechols (DC)(ratio I:I0). When no enzymes were blocked, neuronal efflux consisted mainly of DC and deaminated-O-methylated metabolites; NA amounted to about 15 % of the efflux, whereas NM was not detected during neuronal efflux. When rabbits were not pretreated with reserpine, neuronal efflux had a longer t/2, possibly because of vesicular retention with a t/2 of > 120 min, After U-O521 only, the neuronal efflux of DC had a t/2 of > 120 min. Otherwise the pattern of the efflux of the amine and its metabolites was similar to that seen after pretreatment with reserpine. The neuronal origin of the late phase of efflux was ascertained with cocaine. When cocaine was added to the incubation medium, late efflux was nearly abolish ed. When cocaine was present during efflux only, the t/2 of neuronal efflux was reduced. U. Trendelenburg, Institut fHr Pharmakologie und Toxikologie der Unlversitgt, 87 W~rzburg, Koellikerstr. 2

A NEW MECHANISM TO POTENTIATE THE PENTAGASTRZNAND HISTAMINE STIMULATED GASTRIC SECRETION (Ein neuer Mechanlsmus zur Potenzierung der pentag a s t r i n - und hlstaminstimulierten Magensaftsekretion) H.. Tro,idlrH. Rohde, K. Goecke t and M. Spandau Many mechanisms are known, by which g a s t r o i n t e s t i n a l hormones potentiate eacn other. Studies with the antimalarial drug amodiaquine possibly i n dicate a new way to enhance the histamine and gastrin stimulated acid secretion. Amodiaquine strongly inhlbi~ed gastric histamine methyltransferase in man, dog and pig (I.D.50=10- H) using the isotope assay of Snyder and Axelrod (Biochim. Biophys. Acta 111, 4 (1965). In 13 dogs with Heidenhain pouches the maximum secretory response to pentagastrin and histamine was determined. Then, amodiaquine (0.25-3.0 mg/kg i . m . ) was administered 30 min. before the i.m. i n j e c t i o n of the stimulants. In two tests a week, one with stimulants alone was followed by one with stimulants + amodiaquine and then again by one with stimulants alone. The maximum secretory response (2.8 and 5.2 mval/h) to pentagastrin(6 #g/kg) and histamine (40pg/kg) was enhanced by various doses of amodiaquine, the optimum of which (2mg/kg) increased the acid secretion by 60 and 80 respectively. A parasympathomimetic mechanism seemed unlikely, since amodiaquine i t s e l f did not stimulate gastric secretion. Probably amodiaqulne potentiated the secretory response by i n h i b i t i n g histamine methyltransferase. Since 1-methylhistamine, which also i n h i b i t s t h i s enzyme, is present in the gastric mucosa, t h i s mechanism possibly has a physiological significance. Dr. H. T r o i d l , Chirurglsche K l i n i k der U n i v e r s i t ~ t , D-3550 Harburg/Lahn, Robert-Koch-Stra~e: 8

R 82 INHIBITION OF DRUG METABOLISM IN RAT LIVER MICROSOMES BY PROGESTATIONAL STEROIDS (Hemmung des ArzneimltteJsfoffwechseJs in Rattenlebermikrosomen durch Gesfagene) K.H. TLittenberg, B. HUthwohl, R. Kahl The inhibitory effect of synthetic progestational steroids on microsomal mixed function oxidase was investigated. Norethisterone acetate, d-norgestrel, lynestrenol, and atlylestrenol inhibit p-nitroanisole demethyJation and aniline hydroxylation in liver microsomes from phenobarbital pretreated male rats. Inhibitor constants K i derived from Dixon plots were 48 isM for norethisterone acetate, 21 ~M for d-norgestrel, 17 #M for lynestrenol, and 44 pM for allylestrenol ~n p-nltroanisole demethylatlon. Inhibitor constants for aniline hydroxylation were in the range 5 x 10-5 to 5 x 10-4 M. The binding of the progestagens to cytochrome P-450 was determined by recording difference spectra in microsomes under aerobic conditions. All progestagens exhibited a type I binding spectrum. Apparent spectral dissociation constants K S calculated from double reciprocal plots were 36 + 10 pM for norethisterone acetate, 46 + 7 pM for d-norgestrel, 22 + 2 pM for lynesTrenol, and 17 + 1 pM for allylestrenol.(Supported by a grant from the Deutsche Forschungsgemeinschaft) Pharmakologisches Institut der Universit~it, D 6500 Mainz, Obere Zahlbacher Str. 67

STUDIES ON THE CELLULAR LOCALIZATION OF THE INTESTINAL TRANSPORT PROCESS FOR QUATERNARY A ~ O N I U M COMPOUNDS (Studien zur zellul~ren Lokalisation des intestinalen Transportprozesses fGr quatern&re Ammoniumbasen) K.Turnheim Active intestinal secretion of monoquaternary ammonium compounds has been shown in vitro (Acta Pharmacol.Toxicol.29,Suppl.4:60,197 I) and in vivo (Naunyn-Schmiedeberg's Arch.Pharmacol.274,Suppl.:R 118,1972). In the present study it was attempted to localize this carrier in either the basal or the luminal cell membranes of the intestinal epithelium by use of the 'accelerated exchange diffusion' phenomenon. The intracellular uptake within 10 min of I /uM 3H-N-methylscopolamine (3H-NMScop) through the basal cell membrane~ of isolated jejunal epithelia of guinea pigs was significantly increased after preloading the mucosal preparations 45 min with I mM unlabeled NMScop in comparison to non-preloaded controls, whereas the permeation of 3H-NMScop to the luminal side of the mucosa was significantly decreased. The intracellular uptake (10 min) of I/uM NMScop through the luminal cell membrane was only slightly and insignis higher in preloaded mucosae (I mM NMScop, 45 min) than in controls. The efflux (10 min) from mucosal preparations, preloaded with I /uM NMScop (45 min),through the basal cell membrane was significantly a~celerated, when 1 mM NMScop was added to the blood side of the mucosa. The efflux to the lumen side from equally preloaded epithelia was less markedly increased by the luminal offering of I m~ N~Scop. These experiments are interpreted in terms of the existence of a carrier for monoquaternary ammonium compounds in the basal ~embrane of the intestinal cell. In addition there is some evidence for a luminal pump, so that the resulting cell model includes two carriers in series. Dr. K. Turnheim, Pharmakologisches A-I090 Wien, WM,hringerstra~e I 3 a

Institut

der Universit~t Wien,

R 83 INFLUENCE OF CHLORAMPHENICOL (CAP) AND THIAMPHENICOL (TAP) ON IN VITRO PROTEIN SYNTHESI S I N MICROSOMAL, MITOCHONDRIAL AND NUCLEAR FRACTIONS FROM RAT-L I VER AND -EMBRYOS ( E i n f l u 6 van CAP und TAP auf d i e in v i t r o P r o t e i n s y n t h e s e in Mikrosomen Mitochondrien und Zel I kernen aus Ratten-Leber und -Embryonen). F__. U e h l l n , E. J@ger, R. BoB CAP is known t o I n t e r f e r e w i t h p r o t e i n s y n t h e s i s r e a c t i o n s from mammalian m l t o c h o n d r i a . I t s analogue TAP t i l l now could not be proved t o induce i r r e v e r s i b l e pancytopenia. A p p l y l n 9 equal amounts TAP more o f t e n shows c y t o s t a t i c i n h i b i t i o n o f t h e p e r i p h e r a l blood c e l l count. Since t h e serum h a l f - t i m e of TAP is not cons i d e r a b l y longer than t h a t of CAP we were i n t e r e s t e d t o learn whether the s t r o n ger e f f e c t s o f TAP might depend on a more pronounced i n h i b i t i o n of mammalian m i t o c h o n d r l a l p r o t e i n s y n t h e s i s . Moreover we t e s t e d t h e e f f e c t s o f TAP on embryon i c s u b c e l l u l a r f r a c t i o n s ( a l l data given in t h e t a b l e are f o r a d u l t r a t l i v e r , and embryos - day 14!) as t h e t h e r a p e u t i c doses of TAP i n h i b i t embryonic c y t o chromeox,,dase and o v e r a l l @rowth r a t e .

TABLE control 3AP x) rap x) .....

a) M I C R O S O M E S I i v e r I e m b r y o ~ poly U + I ~ poty U + 8~637 I 21~42 11rg4 ~ 26t63 1oo % 62 % 1oo % I 60 % loo % 40 % loo % I 45 % x) 200 ~9/ml x) 2oo ug/ml

I

b) N U C L E I

c) MITOCHONDRIA

liver embryo o~647 o~7o3 72 % 33 % 67 % 36 % x) 60 ~9/ml

liver embryo 1,174 2,o3o 15 % 1o % 12 % 12 % x) 60 ~g/ml

C o n t r o l s : lo "12 moles l~C-Phe i n c o r p o r a t e d / m i n I n c u b a t i o n , a) per mg RNA, b) per m9 DNA, c) per mg p r o t e i n . CAP and TAP given as % o f c o n t r o l s . R. BASS, G. WlEDEMANN: AEP 272, 234 (1972); D. OERTER, R. BASS: 5th I n t e r n a t i o nal Congress of Pharmacology, San Francisco 1972, ~ lo19. Supported by 9 r a n t s from t h e DFG, given t o SFB 29. Dr. R. BaB, Pharmakologisch. I n s t i t u t , Abt. Embryonalpharmak., FU, 1 B e r l i n 33, T h i e l a l l e e 69/73

METABOLIC EFFECTS OF HALOTHANE AND METHOXYFLURANE IN RATS (Metobolieche Wirkungen van Halothan und Methoxyfluran bei Ratter) H. Vapaatalo T A. M~kel~inen and P. Nikki Halothone-enaeathesia induces in rata lipolysis which con be blocked by propranolol (Vmpaatalo at el., Naunyn-Schmiedeberg's Arch. Pharmacol. 274, suppl., R 119, 1972). In the present study, we hove analyzed further m e t a b ~ o effects of halothane and compared them with those of methoxyflurene. Halothane-induced lipolysis and hyperglycBemia could not be abolished by adrenal demedullation and syrosingopine-treatment even in rate which did not develop hypsthermio during mnmesthesia. We 8uppome that lipolysis is due to direct stimulation of sympathetic ~-reoeptors by halothane. This is further supported by the finding that holothane acts lipolytically also in vitro. The K A value was about 10 ~M of halothone. A slight adenyl cyclaee stimulating ability of hmlothone in adipose tissue (40~ stimulation with 100 MM of halothane) also suggests direct ~-sympathomimetic action. Halothane did not affect phosphodieetarase activity. Hyperglycaemia seems to be due to the interference of halothane with insulin action, because phenoxybenzamine did not inhibit the elevation of blood glucose and holothane did not enhance hyperglycaemia in alloxan-diabetic rats. Methoxyfluters, another halogenated inhalation anaesthetic (0.9 and 1.2 ~), increased also plasma FFA and blood glucose levels during anaesthesia end raised plasma glycerol duming recovery period. Propranolol blocked its lipolytic action. Sympathetic deprim~bion reduced the increase of FFA during anaesthesia, but did not modify the postanaeethetic elevation of plasma glycerol or hyperglysaemia. Methoxyflurane (10 pM - I mM) had no clear effect on lipolysis in vitro. The metabolic effects of methoxyflurane are mostly secondary to the sympathetic stimulationby anaesthesia thus differing from those of halothane. Dr.H. Vapaotalo, Dept.Pharmacol. University of Oulu, SF-90100 Oulu 10, Finland. Grants: Leiraa, Research Council for Medical Sciences, Finland

R 84

RADIOIMMUNOASSAY OF OUABAIN AND DIGITOXIN (Radioimmunologische Bestimmung von g-Strophanthin und Digitoxin) E. Verspohl Mit arts g-Strophanthin und Digitoxin dargestellte mAntigen wurden bei Kaninchen AntikSrper erzeugt und hiermit quantitative Bestimmungen im Serum und Harn vorgenommen. Nach i. v. -Gabe von o, 25 bzw. o, 5 mg g-Strophanthin (purostrophan ~) liegt der Serumspiegel nach 2 Stunden bei o, 33 bzw. o, 91 ng/ml und betr~igt die Halbwertszeit in der anschliessenden langsamen phase 4, 7 Stunden. Im Ham finden sich am 1. Tag 28 ~c, in 4 Tagen 6o ~c der gegebenen Dosis. Nach oraler Gabe von 4 bzw. 8 mg g-Strophanthin erreicht der Serumspiegel nach 4 Stunden ein Maximum mit o, 27 bzw. o, 31 n g / m l ,nit einer anschliessenden Halbwertszeit von 4, o Stunden. Im Ham finden sich am 1. Tag 1, 2 ~c und in 4 Tagen 2, 4 ~c der verabreichten Dosis. Nach oraler Gabe von o, 5 mg Digitoxin (DigimerckP~ liegt der Serums]0iegel nach 2 Stunden bei 8, 6 n g / m l und die Halbwertszeit in der anschliessenden langsamen phase bei 6, 5 Tagen. Im Ham finden sich am 1. Tag 4, 8 ~c, in 3 Tagen 13, 1 gc der verabreichten Dosis. Bei ~atienten mit Herzinsuffizienz liegt der therapeutische Serurnspiegel zwischen 11 und 21 ng/ml, im Mittel bei 16 n g / m l und bei patienten mit Intoxikationserscheinungen zwischen 27 und 42 ng/rnl, im Mittel bei 35 ng/ml. E. Verspohl, pharmakologisches Institut der Universit~it Diisseldorf D-4ooo D[isseldorf, Moorenstrasse 5

THE INFLUENCE OF SEVERAL DOPAMINERGIC STIMULANTS ON THE ANTINOCICEPTIVE ACTIVITY OF MORPHINE (Der Einfluss verschiedener dopaminerger Stimulantien auf die analgetische Wirkung von Morphin) J.M. Viqouret, A.M. Johnson, D.M. Loew In the rabbit, the hind leg flexion response, a polysynaptic reflex, is totally inhibited 15 minutes after an intravenous injection of 5 mg/kg morphine HCl; frequency of respiration is also markedly depressed. In our experiments this response was induced by applying radiant heat to the shaved lower surface of the rabbit's hind foot. Response latency was measured automatically by means of an electric counter (Vigouret et al, 1971). The dopaminergic stimulants apomorphine (O.i - 1 mg/kg i.m.), d-amphetamine (0.5 - i mg/kg i.v.) and I-DOPA (25 mg/kg i.v.), by themselves had no pronounced activity in this test. When administered at peak effect of morphine, i.e. after 30 minutes,antagonistic effects were observed. Apomorphine, a specific stimulant of dopaminergic receptors, antagonised the anti-nociceptive effects of morphine (80 - 100% antagonism at 1 mg/kg i.m.) and reduced morphine-induced respiratory depression. The effects observed were dose-dependent. Both, d-amphetamine (i mg/kg i.v.) and I-DOPA (25 mg/kg i.v.) caused similar reductions in the anti-nociceptive effects of morphine (70 and 4~%, respectively) but were less potent than apomorphine. These results are consistent with the notion that the mechanisms responsible for the anti-nociceptive effects of morphine in the rabbit are in some way linked with the central dopaminergic system. J.M. Vigouret,

Medizinisch-Biologische

Forschung,

Sandoz AG, Basel

R 85

THE RELEVANCE OF THE 0SMOLARITY OF THE INSTILLATION FLUID TO THE "EFFECTIVENESS" AND "TOXICITY" OF DRUGS GIVEN BY THE INTRADUODENAL ROUTE (Die Bedeutung der 0smolarit~t der InstillationsflGssigkeit fGr "Wirksamkeit" und "Toxizit~t" Intraduodenal applizierter Pharmaka) G. Vo~el~ U. Becker~ M. Ulbrich Previous investigations have shown that the instillation volume has an important bearing on the "effectiveness" and "toxicity" of drugs. The relevance of the osmolarity of the instillation fluid has now been studied. The following drugs were investigated: sodium phenobarbital, (n)-nicotine, azo~Liaspiro[}a-benziloyloxy-nortropan-8,1'-pyrrolidine]chloride and atropine sulphate. The drugs were dissolved in normotonic or hypertonic solutions (mannitol) and given intraduodenally. For sodium phenobarbital the time elapsing before the onset of sleep was measured, while for the other drugs survival time and death rate were the criteria. As the osmolarity of the instillation fluid was raised there was a steady increase in the time to induce phenobarbitone-sleep. In the case of the other drugs, as tonicity increased, so the death rate decreased and survival time lengthened. The findings are explained as follows: The introduction of hypertonic mannite solutions into the gut is followed by enterosorption of fluid. The drugs contained in the solution pass from the gut into the blood in accordance with their concentration gradients. When the passage of the drug has to take place against the osmotically induced fluid inflow less of the drug will be absorbed in unit time. This is reflected in a diminution of the "effectiveness" and "toxicity" of the drug. Prof. Dr. G. Vogel, Biologisches Institut Dr. Madaus, 5 KSln 91, Ostmerheimer Str. 198

THE INHIBITION OF CA-DEPENDENT ACTION POTENTIALS IN M A M M A L I A N MYOCARDIUM BY C A - A N T A G O N I S T I C COMPOUNDS (VERAPAMIL, D 600).(Ca++-ab hangige A k t i o n s p o t e n t i a l e im W a r m b l O t e r m y o k a r d und deren Blockade dutch organische C a + + - A n t a g o n i s t e n (Verapamil, D 800)). R. Volkmann, H. Tritthart and A. F l e c k e n s t e i n P o t a s s i u m - i n d u c e d d e p o l a r i z a t i o n of the cardiac membrane from -80 to about -50 mV causes an inactivation of the N a - s y s t e m in the membrane. In this situation stimuli with higher intensity than normal (frequency 60/min or less) elicited action potentials with a reduced rate of rise but an augmented overshoot. In accordance to (1) these action potentials depended mainly on the extracellufar Ca-concentration. The specific Ca-antagonistic compounds verapamil and D 600 (2) impaired the ability o ~ the K - d e p o l a r i z e d cardiac fibres to respond to stimulation with action potentials. This inhibitory effect of verapamil or D 600 was similar to the effect of Ca-withdrawal in the e x t r a c e l l u l a r fluid and could, accordingly, be fully a n t a g o n i z e d by addition of extra Ca. Changing to T y r o d e - s o l u t i o n with normal potassium concentration restored resting potential[ and availability of the N a - s y s t e m w i t h i n about I min. By this, the b l o c k i n g effect of Ca-antagonistic compounds on action potential production was eliminated. i) Mascher, D., PflNgers Arch. 317, 359-372 (1970) 2) Fleckenstein, A., In: "Calcium and the Heart" ed. by P. Harris and L.H. Opie, A c a d e m i c Press London, New York, pp. 135-188

(1970/71) Anschrift Freiburg,

der Verfasser: Physiologisches Instituz der Universit~t D-78 F r e i b u r g i . B r . , H e r m a n n - H e r d e r - S t r . 7

R 86

DIFFERENTIATION OF THE CARDIOSTIMULATING EFFECT OF PHENYLEPHRINE BY ADRENOLYTIC DRUGS J. Wagner and H.J. Sc~ttmann According to Govier a-receptors are mediating the positive inotropic effect of phenylephrine (PE) on atria. This postulate is still under discussion. Therefore experiments using the ~- and Badrenolytic drugs phentolamine and pindolol, resp., were performed on isolated electrically driven (1 or 2/sec) and spontaneously beating atria of guinea-pigs and rabbits. On preparations of reserpine-pretreated animals the indirect action of PE was evaluated. All experiments were carried out in Krebs-Henseleit solution at 32~ The e-adrenolytic drug phentolamine (10-6; 3 x 10-6M) proved to be without any influence on the dose-response curves for positive ino- and chronotropic action of PE in both atrial preparations of guinea-pigs or rabbits.- On the other hand A the B-a~renolytic drug pindolol in concentrations of 3 x l0 -~ and 10-~ competitively antagonized both effects in all preparations tested. In reserpine-pretreated stimulated atria of guinea-pigs the pD 2value of P E w a s only slightly, but significantly lowered when compared with that of untreated controls indicating a small indirect action of PE.- By increase of temperature from 25 ~ to 42oC the dose-response curve for the positive inotropic effect of PE was shifted to the right as were those of B-sympathomimetics. The results indicate that the cardiostimulating effect of PE on atria is mediated by B-receptors. Doz. Dr. J. Wagner, Pharmakologisches Klinikum Essen, Hufelandstr. 55

Institut,

Universit~ts-

STUDIES ON ~-METHYIDOPA: REIATIONSHIPBETWEENMETABOLISM, BIOGENIC AMINES AND ANTIHYPERTENSIVE EFFECT (Studien t~oer G-Methyldopa: Zusammenh~ zwischen Metabolismus, biogenen Aminen und antihypertensiver Wirkung) P.C. Waldmeier, L. Maitre und P.R. Hedwall In renal hypertensive rats the cerebral contents of G-methyldopa, S-methyldopamine, G-methylnoradrenaline, dopamine and noradrenaline as well as blood pressure were determined simultaneously. The estimations of a-methyldopa and the amines were carried out by combining ion-exchange separation and differential fluorometric determination. The antihypertensive effect showed a time course closely associated with an increase in the content of cerebral @-methyldopamine and a decrease in dopamine, whereas lowering of blood pressure and changes in the levels of ~-methylnoradrenaline and noradrenaline were dissociated. The results suggest that non-B-hydroxylated catecholamines play a major role in mediating the antihypertensive effect of G-methyldopa. Dr. P.C. Waldmeier, Biological Research Laboratories of the Pharmaceuticals Division of CIBA-GEIGY Limited, Basle

R 87

I N V E S T I G A T I O N S U P O N THE V A G A L R E G U L A T I O N OF THE HEART RATE IN C O N S C I O U S DOGS (Untersuchungen Hber die v a g a l e H e r z f r e q u e n z s t e u e r u n g am w a c h e n Hund) A. W g l l a n d t W. K o b i n g e r P r e v i o u s i n v e s t i g a t i o n s in a n a e s t h e t i z e d dogs d e m o n s t r a t e d the f a c i l i t a t o r y e f f e c t of c l o n i d i n e on v a g a l l y m e d i a t e d reflex b r a d ~ cardia, an e f f e c t w h i c h was l o c a l i z e d w i t h i n the CNS. The p r e s e n t e x p e r i m e n t s w e r e done in c o n s c i o u s dogs; s t i m u l a t i o n of b a r o r e ceptors was a c c o m p l i s h e d by i n c r e a s e of b l o o d p r e s s u r e (BP) in the upper hart of the body: a rubber cuff was p l a c e d around the A o r t a d e s c e n d e n s just b e l o w the d i a p h r a m a , 1-2 week~ b e f o r e the e x p e r i m e n t and could be i n f l a t e d t h r o u g h a p l a s t i c tube w h i c h was a c c e s s i b l e in local a n a e s t h e s i a t h r o u g h the skin of the back. 5 m g / k q d o b e r o l (adrenergic B - r e c e p t o r b l o c k i n g agent) w e r e injected i.v. I n f l a t i o n of the cuff fer 30 seconds r e s u l t e d in BP rise and b r a d y c a r d i a . BP was p l o t t e d against h e a r t rate and a n e g a t i v e c o r r e l a t i o n was found w h i c h a n p e a r e d to be linear. After i . v . - i n j e c t i o n of 30 ~g/kg c l o n i d i n e the curve was shifted towards the origin of the coordinates. In another series of conscious dogs t r e a t e d w i t h doberol the m a x i m u m of the reflex bradyc a r d i a e l i c i t e d b v a n ~ i o t e n s i n was n o t e n t i a t e d by 30 ~g/kq clonidine i.v. This e f f e c t was a n t a g o n i z e d by 1 m ~ / k q p i o e r o x a n e i.v. (a-receptor b l o c k i n g agent). These e x p e r i m e n t s d e m o n s t r a t e that c l o n i d i n e i n d e o e n d e n t l v of a n a e s t h e s i a f a c i l i t a t e s vaqus a c t i v i t y over a w i d e BP range, an effect w h i c h can be a n t a g o n i z e d bv ~r e c e p t o r blockade. Dr. A. W a l l a n d , A r z n e i m i t t e l f o r s c h u n q

GmbH, A - I f 2 1 W i e n ,

Laskeq.5

THE INFLUENCE OF DEOXYCHOLATE ON THE UNIDIRECTIONAL SODIUM FLUXES I N RAT COLON IN VITRO ( U n i d i r e k t i o n a l e Natriumbewegungen am C o l o n d e r R a t t e in vitro unter dem EinfluB yon Desoxycholat) R.

Wanitschke~

G.

Nell~

W. R u m m e l

Forth et al. showed that Deoxyeholate (DC) n o t o n l y i n h i b i t s the absorption of sodium and fluid in tied off segments of rat colon. In addition, DC p r o d u c e s a net transfer of sodium and water into the colonic lumen(Naunyn-Schmiedebergs Areh.Pharmak. u. exp.Path.254,364-380;i966). In order to exclude an influence o f DC o n t h e m i e r o e i r c u l a t i o n we e x a m i n e d the effects o f DC o n the transfer of fluid and electrolytes in the isolated colonic m u c o s a u s i n g an e v e r t e d sac p r e p a r a t i o n a c c o r d i n g to P a r s o n s and P a t e r s o n (Quart. J.of P h y s i o l . 5 0 , 2 2 0 - 2 3 i ; 1 9 6 5 ) . 3 . I 0 - 4 M DC on the m u e o s a l side a b o l i s h e s the net t r a n s f e r of s o d i u m and fluid. The m u c o s a is h i s t o l o g i c a l l y intact. 3 . 1 0 - 4 M DC i n c r e a s e s the u n i d i r e c t i o n a l s o d i u m f l u x m e a s u r e d by m e a n s of 2 2 N a + from s e r o s a to m u e o s a fourfold, w h e r e a s the f l u x from m u c o s a to s e r o s a r e m a i n s n e a r l y constant. The i n c r e a s e of the s o d i u m f l u x from s e r o s a to m u c o s a u n d e r the i n f l u e n c e of DC is the cause for the a b o l i t i o n of the s o d i u m and, c o n s e q u e n t l y , also of w a t e r net transfer. The net t r a n s f e r of s o d i u m c a l c u l a t e d from the t r a c e r m e a s u r e m e n t s agree w i t h the v a l u e s b a s e d on f l a m e p h o t o m e t r y . The e f f e c t of 3 . 1 0 - 4 M DC on the u n i d i r e c t i o n a l f l u x from s e r o s a to m u e o s a is r e v e r s i b l e . D r . R . W a n i t s c h k e , I n s t i t u t f~r P h a r m a k o l o g i e und T o x i k o l o g i e U n i v e r s i t i t des S a a r l a n d e s , D 665 H o m b u r g / S a a r

der

R88

Ca-ANTAGONISTISCHE ELEKTROMECHANISCHE ENTKOPPELUNG DER GLATTEN GEFASSMUSKULATUR ALS WIRKUNGSPRINZIP VASODILATATORISCHER NITROVERBINDUNGEN. U. Weder~ G. Grfin Untersuehungen Ober den Wirkungsmeehanismus muskulotroper Relaxantien (1) haben gezeigt, da~ aueh die vasodilatatorisehen Effekte yon Nitroglycerin und Isosorbitdinitrat an peripheren G e f ~ e n und Coronararterien (2,3,4) auf Ca++-antagonistischen Wirkungen beruhen. Dieses Ergebnis wurde nunmehr dutch Einbeziehung yon Nitrit und anderen organisehen Nitroverbindungen sowie m~glichen Abbauprodukten (Hydroxylamin) wetter gesichert. Nitroverbindungen scheinen ~hnlich wie andere Ca++-antagonistische Vasodilatatoren (Verapamil, D 600~ Nifedipine, Papaverin) die transmembran~re Ca++-Versorgung des kontraktilen Systems zu reduzieren. Nach funktioneller ZerstSrung der Zellmembranen ("chemically skinned fibres" nach WINEGRAD) ist daher in keinem Fall ein direkter Hemmeffekt auf die dutch A T ~ und Ca++-Zusatz induzierte Kontraktion nachweisbar. Trotzdem u n t e ~ scheidet sich die Kinetik der Relaxation dutch NitrokSrper yon der dutch Verapamil, D 600 und Nifedipine in mehrfaeher Hinsieht. 1) Fleckenstein, 49,

32

A., G. Gr0n, H. Tritthart u. K. Byon: Klin. W s e h n

(1971)

2) Gr~n~ G. u. A. Fleckenstein: Arzneim. Forsch.22~ 33q (1972) 3) GrNn~ G., M. Bayer u. A. Fleckenstein: Naunyn--Sehmiedeberg's Arch. exper. Path. u. Pharmakol. Suppl. to Vol. 274,R 43 (1972) 4) Gr0n, G., U. Weder u. A. Fleekenstein: PflNgefs Arch. ges. Physiol. 335~ R 10 (1972) Anschrift der Verfasser: Physiologisches Institut der Universit~t Freiburg, Freiburg i. Br., Hermann-Herder-Str. 7

THE EFFECTS OF ADENOSINE ON CORONARY CONDUCTANCE,CARDIAC MECHANICS AND METABOLISM OF THE ISOLATED PERFUSED CAT HEART(Die Wirkung yon Adenosin auf die coronare Leitf~higkeit~sowie die Mechanik und den Stoffwechsel isoliert durchstrSmter Katzenherzen) MoWeissel~GoRaberqer It has been shown in experiments on dogs in situ that the ioarto application of adenosine (ASN) results in enhanced glucose uptake and H+-release,accompanied by a significant fall in Op-consumption in heart and skeletal muscleoin order to study t~e influence of the perfusion conditions on the effects of ASN on Cardiac mechanics and metabolism the following experiments were carried out:l)the perfusion volume;2)the perfusion pressure was held constantoThe hearts of both groups were perfused with Ringer's solution~containing 5mM glucoseo!n the Ist group ASN,infused over a 5-minoperiod,increased coronary conductance 2-fold and decreased max~ left ventricular systolic pressure(LVSPmax),dp/dt and heart rate as a consequence of the fall in perfusion pressure(down to 9 + 50% ofcontrol)~ and free COn-release fell signiz ficantly~wheras Iactate release and glucose uptake increased. In the 2nd group ASN also increased the coroconductance 2-fold,but LVSPmax,dp/dt and heart rate changed concomitantlyoAlSo Or-con9 sumptlon ~H + -and free CO2-release and glucose uptake wer e z augmented - lactate release remained nearly unchangedoThUs it can be concluded that the effects of ASN on cardiac mechanics and metabolism are largely influenced by the perfusion pressureoA decrease in perfo pressure apparently leads to a decrease in nutritional flow,as indicated by the increase in lactate release~ DroMoWeissel,Pharmakologisches Institut der U n i v e r s i t ~ t Wien A-10909Wien , W[hringerstraBe 13A- AUSTRIA

R 89 INTRACELLULAR INVESTIGATIONS ON THE EFFECT OF MICROELECTROPHORETICALLY APPLIED GLUTAMATE ANTAGONISTS UPON SPINAL NEURONES OF THE CAT.(Untersuchungen Gber d i e W i r k u n g mikroelektrophoretisch applizierter Glutamins~ureantagonisten auf spinale Neurone der Kmtze). W. Zieglg~nsberger, E.A. Puil Our recent data on the action of microelectrophoretically applied glutamic acid (GLUT) speak in favour of this amiuo acid being an excitatory transmitter in the spinal cord of the cat. In accordance with others we found that both~-methyl-glutamic acid end the diethylester of GLUT block GLUT-induced cell activity. To study their action in more detail the compounds were applied extrscellularly while recording intracellu18fly from cat spinal neurones. It was found that low doses of both drugs hyperpolarize the cells (5-15 mV) and that this hyperpolarizatiou was associated with slight conductance increase. Simultaneously applied GLUT depolarized the cell membrane ss before but the cell reached firing level only when the dose was increased. Such hyperpolarization can explain the blocking of GLUT action observed in extracellular recording. There remains the unanswered question as to why the depolarization induced by acetylcholine (Ach) is not blocked that readily in most neuroues. We studied the actions of Ach in similar experiments and found in accordance with E~IIVJEVIC et al. (1971) that the depolarizing motion of this compound is most frequently associated with no change or decrease in membrane conductance. This different mechanism might account for the observed effects. KRNJEVIC, K., PUNAIN, R., RENAUD, L., J. Physiol. 215, pp 247-268 (I 971) Dr~ W. Zieglg~nsberger, Max-Planck-lnstitut fGr Psychiatrie, 8000 MGnchen 40, KraepelinstrsBe 2

EPFECTS OF FUROSEMIDE ON RENAL FUNCTION AND R E N I N R E L E A S E IN THE IS0LATED RAT KIDNEY (Wirkung von Furosemid auf Nierenfunktion und Reninfreisetzung in der isolierten Rattenniere) H. Zschiedrich, K.G. Hofbauer, W. Rauh Isolated rat kidneys were perfused in an "open" system with a 38% suspension of washed bovine red blood cells in an electrolyte solution containing 3.5 g%Haemaceel. In control studies renal plasma flow (RPF), glomerular filtration rate (GFR), urine flow (V), and sodium excretion (UNaV) remained constant for two hours of perfusion. Renin release (RR) from the kidneys was 950• ng AI-min-1/12 hours of incubation, with little variation during the course of the perfusion. Renin content of the perfused kidneys was equal to that of the oontralateral non-perfused kidneys. Furosemide infused at a rate of 0.5 mg-m1-1.min -1 for 30 min markedly increased V and UNa V. RPF rose, GFR remained constant, and a two to sevenfold increase in RR was seen. After stopping the infusion, RPF, V, UNaV, and RR returned to control values. When furosemide was infused at a rate of 0.05 mg.ml-l-min-lfor 15 min, the diuretic and natriuretic effects were smaller, and no elevation in RR could be observed. From these results it is concluded that furosemide stimulates RR by an intrarenal mechanism. This effect may be explained by a change in distal tubular sodium load or a direct effect of furosemide on the juxtaglomerular cells. Dr. H. Zschiedrich, Pharmakologisohes Institut der Universit~t, D-6900 Heidelberg, Hauptstr. 47-51.