Naunyn-Schmiedeberg's Naunyn-Schmiedeberg's Arch Pharmacol (1983) 323:149-154 Archives of Pharmacology Springer-Verlag 1983

Yohimbine and Rauwolscine Inhibit 5-Hydroxytryptamine-Induced Contraction of Large Coronary Arteries of Calf Through Blockade of 5 HT2 Receptors

A. J. Kaumann Lehrstuhl ftir Klinische Physiologie~ Physiologisches Institut der Universit~it Dtisseldorf, Universit/itsstrasse 1, D-4000 Dfisseldorf 1, Federal Republic of Germany

Summary. 5-Hydroxytryptamine (5 HT)-induced contrac- ) antagonize competitively the effects of 5 HT on tions were investigated on -treated strips of bovine strips of bovine large coronary arteries. Moreover, evidence large coronary arteries. is presented consistent with the idea that both and 1. The a2-adrenoceptor blockers rauwolscine and yohim- rauwolscine block responses to 5 HT by a Competitive bine antagonized competitively 5 HT-induced contractions. interaction at postsynaptic 5 HT2 receptors. Part of the The estimated equilibrium dissociation constants KB (--log results were recently reported (Kaumann 1982). tool/l) were 7.1 for rauwolscine and 7.3 for yohimbine. The affinity of yohimbine for the receptors mediating the re- Methods sponse to 5 HT appears to be 10 times higher than for postsynaptic el-adrenoceptors but 10 times lower than for Experiments were carried out at 32.5~ on large coronary postsynaptic vascular c~2-adrenoceptors. arteries. The arteries were dissected in the slaughterhouse at 2. (-)-Noradrenaline and the c~2-adrenoceptor-selective room temperature. Arteries obtained from freshly excised agonist B-HT 920 caused maximum contractions amounting hearts were rinsed and cut out in a modified Krebs solution to only 20 % and 2 %, respectively, of the maximum 5 HT containing (mmol/1): Na + 120, K + 5, Ca 2+ 2.25, Mg 2+ 1, effects. Neither 60 gmol/1 B-HT 920 nor 1 gmol/1 prazosin C1- 98.5, SO42- 1, HCO~- 29, HPO~- 1, EDTA 0.04, antagonized the 5 HT effect. equilibrated with 95 % O2 and 5 % CO2. The water was 3. was a competitive antagonist (KB=9.2 deionized and redistilled in glass. The tissues were transpor- (--log mol/1)) of the effects of 5 HT. Combinations of ted to the laboratory in a sealed flask containing above rauwolscine or yohimbine with ketanserin antagonized the solution. Helicoids were cut and then 8 to 10 strips prepared 5 HT effects as expected from competition of the 4 drugs for as roughly isosceles triangles (approximately 4 x 8 ram) a single class of receptor. which were set up (usually in pairs) in an apparatus with a 50 4. The evidence is consistent with an interaction of 5 HT, ml organ bath (Blinks 1965) containing the solution de- ketanserin, rauwolscine and yohimbine with 5 HT2 re- scribed above supplemented with (mmol/1): Na + 20, fu- ceptors, c~-Adrenoceptors only play a minor role in large marate 10, pyruvate 5, L-glutamate 5, glucose 10, ascorbate coronary arteries and appear not to be involved in the 5 HT- 0.1, cocaine 0.006 (which causes a five-fold potentiation of induced contractions. A possible clinical involvement of the effects of 5 HT (Kaumann et al. 1982) probably due to 5 HT in coronary artery spasm is discussed. blockade (Thoa et al. 1969) of its neuronal uptake (Snipes et al. 1968)), 0.001 (-)- (used in experiments with Key words: 5 HT2 receptors - Large coronary arteries - (-)-noradrenaline and B-HT 920 to block/~-adrenoceptors). Yohimbine - Rauwolscine - Ketanserin Each strip was tied on one end to a thread (Flexafil 6/0, J. Pfrimmer & Co., Erlangen, FRG) and clamped on the other end. The strips were attached to strain gauge transducers connected via amplifiers to polygraphs. Resting force was Introduction adjusted to approximately 20 mN and the strips left at the 5-Hydroxytryptamine (5 HT) is a powerful constrictor of corresponding length throughout the experiment. large coronary arteries (Brazenor and Angus 1981 ; Porquet Up to 4 successive concentration-effect curves of 5 HT et al. 1982). Using as a tool to block were determined on each arterial strip. Curves were nearly 5 HT D-receptors (Gaddum and Picarelli 1957), a low- superimposable and desensitization was always smaller than affinity but heterogeneous receptor population was found in 0.3 log units. To estimate desensitization, 4 arterial strips large coronary arteries of calf (Kaumann et al. 1982). One from each heart were routinely used as controls. The reason for heterogeneity may be the contribution of e- equilibrium dissociation constant KB for an antagonist- adrenoceptors to the effect of 5 HT (Innes 1962), because receptor complex was calculated from phenoxybenzamine also blocks c~-adrenoceptors (Nickerson 1970). The blocking effects of the cq-selective antagonist CRd prazosin and ez-selective antagonists yohimbine and rauwol- KB=[~] CRB-CR~ (1) scine (Starke 1981) were therefore investigated. It is now reported that both yohimbine and rauwolscine (but not where [B] is the concentration of the antagonist, CRB the concentration-ratio of 5 HT (i.e. ECso ratio) in the presence Send offprint requests to the author at the above address and absence of [B] and CRd the concentration-ratio caused 150 by desensitization. Simple competitive inhibition was reproducible concentration-effect curves for the ~- checked with the slope of Schild plots (Arunlakshana and adrenoceptor agonists. Up to 60 drool/1 B-HT 920 did not Schild 1959) modified for desensitization: modify the concentration-effect curve of 5 HT (n = 8 strips of ( CRB- '~ 3 hearts) (not shown). In some arteries the 5 HT effect was log \C~-d ) = log [B]--log KB. (2) approximately as strong as the contractions caused by 137 mmol/l potassium (Fig. 1); in others (not shown) the 5 HT Simple competition between 5 HT and an antagonist for a effect was more pronounced than the potassium-contraction. receptor predicts a slope of 1.0 from Eq. (2). The Effects of Selective ct-Adrenoceptor Blockers. The cq- Drugs. (-)-Noradrenaline (-)-tartrate was from Merck- adrenoceptor blocker prazosin did not modify the effect of Schuchardt (Hohenbrunn b. Miinchen, FRG), (-)-bupra- 5 HT at a concentration (1 drool/l) (n = 14 strips of 3 hearts) nolol hydrochloride from Sanol (Monheim, FRG), phe- (not shown) saturating el-adrenoceptors (Starke 1981). noxybenzamine hydrochloride from Smith, Kline & French Figure 2 shows that rauwolscine and yohimbine cause Ltd. (Philadelphia, PA, USA), rauwolscine hydrochloride parallel shifts of the concentration-effect curves for 5 HT. from Roth (Karlsruhe, FRG), prazosin hydrochloride and With both antagonists the blockade was completely sur- 5-hydroxytryptamine hydrochloride () from Sigma mounted by 5 HT, consistent with simple competitive inhibi- (St. Louis, MO, USA), B-HT 920 [6-allyl-2-amino-5,6,7,8- tion. This mode of blockade is supported by slopes of Schild tetrahydro-4H-thiazolo-(4,5-d)-azepine] dihydrochloride from plots (Fig. 2D) not significantly different from 1.0. Equil- Thomae (Biberach, FRG), and cocaine hydrochloride. ibrium dissociation constants for rauwolscine and yohimbine are listed in Table 1. Statistics. Equilibrium effects of 5 HT, slopes of Schild plots, log values of equilibrium concentration-ratios of 5 HT and Competitive Antagonism by Ketanserin. The 5 HTz receptor- log values of equilibrium dissociation constants are ex- selective blocker ketanserin antagonized competitively the pressed as mean _+ S.E.M. effects of 5 HT (Fig. and D). The blocking potency of ketanserin was approximately 100 times higher than the blocking potency of rauwolscine and yohimbine (compare Results KB values of Table 1). Comparison of the Effects of 5 HT and c~-Adrenoceptor Agonists Antagonism of the 5 HT Effect by Blocker Combinations. Are the c~2-adrenoceptor blocker and ketanserin antagonizing the Figure 1 shows the effects of maximal effective concen- effects of 5 HT through the same receptor class or through trations of 5 HT and (-)-noradrenaline. Maximum 5 HT- different receptor classes? If an ~z-adrenoceptor antagonist induced contractions were 5 times greater in magnitude than and ketanserin both block the same receptor, then the those of (-)-noradrenaline. B-HT920 (0.2-200 Ixmol/1) antagonism in the presence of both antagonists should be reputed to be an ez-selective agonist in other systems nearly additive (Paton and Rang 1965). On the other hand, if (Timmermans et al. 1981; van Meel et al. 1981; Lues and one blocks a different receptor than the other, then the Schfimann 1982), only caused marginal contractions amount- antagonism caused by the two blockers combined should be ing to 1.8 • 0.6 % (n = 9 strips of 3 hearts) of the maximum equivalent to the product of the blockade of the same contraction induced by 5 HT. Due to the small effects of(-)- concentrations investigated separately (Paton and Rang noradrenaline and B-HT 920, it was difficult to determine 1965). Figure 3 and Table 2 show the results of two

I ! I ! I [J llll I I I I I ;I 1 I I I I I N =.

20 pmol/I 5HT ', .... ~ E V tti o 200 pmol/I 0 (-)-noradrenaline l'o~ II. Fig. 1 Comparison of the effects of 5HT, (-)-noradrenaline and high potassium on a Hlll 1,[' Ilil:11rl:::l i I'll'L L:I Ul-t] ]40 strip of bovine left anterior descending 137 mmol/l coronary artery incubated with 6 gmol/l I~1I 1t4 I t:LI I.:r;:lLlr!~l1:1~[~11:i'j 200 cocaine and 1 lamol/l (-)-bupranolol. To KCI achieve 137 mmol/1 K +, 132 mmol/1 Na + was replaced by K +. Histograms represent mean t _+ SEM of 6 strips; w, washes 151

A Iscine olO f ./../ / / ~ ,~mo,/, 20 -- 0 '/"'~' "~"~'/ , 30 , Z E 3~10 I _f /. .auo,scoe B 3O(z UJ 0 I I ~ I I I ua 20 ine (.9 rr" ,o O O 0 I r~q II I I I ii 2O LL <3 < C 10 20 serin 0 i I M I I I I10 9 8 7 6 5 4 ~1~f J.~ Z % ~o .r.o,/h -LOG. [5HT] mol/I 9 8 7 6 5 4 3 2 Fig. 3. The effect of combinations of ketanserin with rauwolscine or - LOG. [5HT] mol/I yohimbine on the concentration-effect curve for 5HT. Two successive curves for 5HT were determined, the 1st in the absence of blocker ( and "6 4 _~ D the second (I, A) after lh of incubation with the indicated blocker ~" ~ 3 / YOHIMBINE combinations, n=4 strips from 2 hearts for each group SLOPE 1.00*-0.03

.~~' Z KETANSEFIIN ~// 1//I SLOPE 104"-0.03 /w ;.'. Table 2. Concentration-ratios (CR) of 5 HT with 20 nmol/1ketaserin (K), OCC ~ RAUWOLSClNEO O 03UJ 1 93+-0.04 3 gmol/1 yohimbine (Y) and 3 gmol/1 rauwolscine (R) and blocker combinations o, 0 d'" I ,.~1" I I I 10 9 8 7 6 5 4 K Y R K+Y K§ -LOG. CONCENTRATION mol/I Observed 34 63 47 99 91 Fig. 2. Competitive inhibition of the 5HT effects by rauwolscine (A), Expected from same receptors" 96 80 yohimbine (B) and ketanserin (C). Four successiveconcentration-effect Expected from different receptorsb 2142 1598 curves of 5HT were determined on each arterial strip. The 1st curve (O) was in the absence of antagonist; the following curves were in the Mean values were estimated from the experiments of Figs. 2 and 3 presence of the indicated blocker concentrations. The antagonists were " Calculatedfrom: CRK+CRy-1 and CRK+CRR--1 (Paton and incubated 1 h before a curve for 5HT was begun. (D) Schild plots from Rang 1965) data of above experiments. For number of strips see Table 1. Arteries b Calculatedfrom: CRK" CRy and CRK" CRR (Paton and Rang 1965) were from 6 hearts

Table 1. Equilibrium dissociation constants for rauwolscine, yohimbine and ketanserin

Antagonist n - log KB" approximately 99.8 %, 98.7 % and 98.7 % fractional receptor mol/1 occupancy in the rabbit pulmonary artery (calculated from KB-values for prazosin of Starke (1981)), canine femoral Rauwolscine 8 7.10 _+0.18 artery and splenic artery (calculated from data of De Mey Yohimbine 8 7.21 +_0.11 and Vanhoutte (1981)), respectively. (Fractional receptor Ketanserin 15 9.18 _+0.14 occupancy = [B]/([B] + KB), where B is the antagonist.) Thus, a contribution of el-adrenoceptors to the 5 HT effect can be " Calculated using Eq. (2) from the data of Fig. 2 n Number of coronary artery helicoids ruled out. The blocking potency of yohimbine of e-adrenoceptor mediated effects has been found to vary more than 100-fold combination experiments. The data are consistent with the as a function of both agonist and vascular tissue. Starke assumption that 5 HT, ketanserin, rauwolscine and yohim- (1981) estimated KB-values (--log mol/1) of 6.4 for the bine interact with the same receptors. postsynaptic-adrenoceptor-yohimbinecomplex in the rabbit pulmonary artery. De Mey and Vanhoutte (1981) reported pAz-values of 6.5 and 6.6 for yohimbine as antagonist of the Discussion contractile effects of (-)-noradrenalinein canine femoral and splenic artery, respectively. Shepperson and Langer (1981) 5 HT-Induced Contractions of Large Coronary Arteries estimated pA 2 values of 8.6 and 7.5 for yohimbine anta- are not Mediated Through ~-Adrenoceptors gonism of the contractile effects of the ez-selective agonist The effect of 5 HT was not antagonized by 1 pmol/1 prazosin. M 7 and (-)-noradrenaline, respectively, in dog saphenous This concentration saturates ~l-adrenoceptors by causing vein. In the same tissue, but treated with phenoxybenzamine, ]52

Constantine et al. (1982) estimated a pA2-value of 8.2 for receptor population for 5 HT (Kaumann et al. 1982) appears yohimbine from the antagonism of the effects of . It to be unrelated to c~-adrenoceptors. The slopes of Schild plots should be pointed out, however, that in most of these reports were found not to be significantly different from 1.0 for only a 10- to 30-fold concentration-range of yohimbine was ketanserin, rauwolscine and yohimbine, consistent with a used, and that the slope of the Schild plot [Eq. (2)] was often single class of antagonist-5 HIT2receptor complex. However, different from 1.0. The low affinity estimates of Starke (1981 ; receptor heterogeneity can be concealed even in results with see also Weitzell et al. 1979) and De Mey and Vanhoutte slopes of 1.0 of Schild plots (Lemoine and Kaumann 1983). (1981) probably represent rough approximations of the Further work with 5 HT-like agonists (Feniuk et al. 1981) is equilibrium dissociation constant of yohimbine at ~l- necessary in order to search for receptor heterogeneity. A adrenoceptors. On the other hand, the high affinity estimates plausible agonist-dependence of the antagonism by ketan- of Shepperson and Langer (1981) and of Constantine et al. serin, yohimbine and rauwolscine would be an indication for (1982) probably represent a rough approximation to the 5 HT receptor heterogeneity. The question of 5 HT receptor equilibrium dissociation constant of yohimbine for postsyn- subtypes in large coronary arteries (Kaumann et al. 1982) aptic ez-adrenoceptors of dog saphenous vein. The pAz of 7.5 could also be clarified by using at least 9 blocker con- for yohimbine against (-)-noradrenaline (Shepperson and centrations per Schild plot as in a recent analysis of /~- Langer 1981) perhaps represents just an average affinity for adrenoceptor heterogeneity (Lemoine and Kaumann 1983). both subtypes of e-adrenoceptors. The antagonism by rauwolscine and yohimbine of the 5 HT effect in bovine large coronary arteries may suggest an involvment of c~2-adrenoceptors. For three reasons, Antagonism of 5 HT Effects by Ketanserin, Rauwolscine however, this appears unlikely: 1) In large coronary arteries and Yohimbine Through 5 HTz Receptors the affinity of yohimbine appears to be one order of Ketanserin is a high affinity ligand (Leysen et at. 1981, magnitude lower for the receptors mediating the response to 1982; Van Nueten et al. 1981) for 5 HT2 receptors (Pe- 5 HT for arterial postsynaptic ez-adrenoceptors (vide supra). routka and Snyder 1979). Ketanserin was a simple competi- 2) (-)-Noradrenaline and the e2-adrenoceptor selective tive antagonist in bovine large coronary arteries. For ligand B-I-IT 920 exhibit only small or marginal agonistic unknown reasons Brazenor and Angus (1982) found that activity in large coronary arteries. 3) Both rauwolscine and ketanserin was a non-competitive antagonist of the effects of yohimbine appear to antagonize the 5 HT effects through 5 HT in dog circumflex arteries. The KB value estimated for 5 HT2 receptors (as discussed in the following section). the ketanserin-5 HT receptor complex of bovine large coro- The small effects found for (-)-noradrenaline nary arteries agrees (Table 1) well with similar affinity values and B-HT920 suggest that postsynaptic e-adrenoceptors estimated for a variety of arteries (Van Nueten et al. 1981). play a minor role in calf large coronary arteries. The The evidence is, therefore, consistent with the existence of stimulant effects of (-)-noradrenaline and B-HT 920 cannot 5 HT2 receptors in large coronary arteries. Furthermore, the be due to the simultaneous occurrence of/%adrenoceptor- experiments with combinations of antagonists (Fig. 3) and mediated relaxation, because the concentration of (-)- the analysis of Table 2 suggest that ketanserin and rauwol- bupranolol used (1 gmol/1) causes 99.9 ~ /?-adrenoceptor scine or yohimbine act additively through 5 HT2 receptors. occupancy (calculated from data in heart (Morris et al. 1981) Interestingly, Lambert et al. (1978) found that both and smooth muscle (Lemoine and Kaumann 1982)). Because yohimbine and rauwolscine antagonized 5 HT-induced con- of the minimal stimulant activity of B-HT 920, this ez- tractions in the rat fundus. They estimated -log KB values adrenoceptor selective ligand was used as a putative an- (tool/l,)7 _+ SEM) of 6.77 ___0.13 for yohimbine and 7.48 + 0.45 tagonist. However, even the rather high concentration of 60 for rauwolscine. The data have to be interpreted with some gmol/1 B-HT 920 did not block the 5 HT effect, indicating no caution because Lambert et al. (1978) reported neither the involvement of c~2-adrenoceptors. concentration-range used with yohimbine and rauwolscine Evidence from in vivo experiments in canine left circum- nor the slopes of the corresponding Schild plots. Never- flex artery indicates that c~2-adrenoceptors mediate coronary theless, the similarity of these affinity estimates with the artery constriction (Holtz et al. 1982). These authors found corresponding affinity estimates for yohimbine and rauwol- that the constrictor effects of noradrenaline and azepexole, scine on bovine large coronary arteries suggests at first glance an e2-adrenoceptor selective agonist, are preferentially an- that the 5 HT receptors of both tissues are similar. However, tagonized by rauwolscine. Holtz et al. (1982) also speculated the affinity of yohimbine appears to be at least 3 times lower that c~2-adrenoceptors might be clinically involved in spasms for 5 HT receptors of rat fundus than for 5 HT receptors of of coronary arteries because e2-adrenoceptor-mediated ef- bovine large coronary arteries. Furthermore, and more fects are particularly easily antagonized by calcium an- importantly, the effects of 5 HT on rat fundus are not tagonists (Van Meel et al. 1981). The present experiments on antagonized by concentrations of ketanserin up to 104 times bovine large coronary arteries do not support an important greater than the ECso for blockade of 5 HT contractions of role of ez-adrenoceptors because of the small effects of (-)- rat caudal arteries (Awouters et al. 1982). It is therefore noradrenaline and marginal effects of the e2-adrenoceptor concluded that 5 HT receptors of rat fundus and large bovine selective agonist B-HT 920. The results of Holtz et al. (1982) coronary arteries are different. may rather reflect a role of e2-adrenoceptors in small The affinities of yohimbine and rauwolscine were found coronary arteries or arterioles. In contrast to the effects seen to be approximately 1/100 of the affinity of ketanserin for with noradrenaline, small coronary arteries do not contract 5 HT receptors, as estimated from the blockade of the (Porquet et al. 1982) and may even dilate with 5 HT (Mena contractile effects of 5 HT in bovine large coronary arteries. and Vidrio 1976). Binding-inhibition data of Leysen et al. (1982) suggest that Because c~-adrenoceptors do not appear to mediate 5 HT- the affinity of yohimbine is only 1/1000 of the affinity of induced contractions, the heterogeneity of the low-affinity ketanserin for 5 HT2 receptors of rat brain membranes 153 labelled with 3H-ketanserin. A possible reason for the 10-fold Gaddum JH, Picarelli ZP (1957) Two kinds of receptor. Br affinity difference ofyohimbine could be due to the homoge- Pharmacol 12:323-328 nization procedure for preparing brain membrane particles. Holtz J, Saeed M, Sommer O, Bassenge E (1982) This procedure may decrease the affinity of yohimbine more constricts the canine coronary bed via postsynaptic e2-adrenocep- tors. Eur J Pharmacol 82:199-202 than that of ketanserin. Similar discrepancies between ligand Innes IR (1962) An action of 5-hydroxytryptamine on affinities obtained from antagonism and direct binding have receptors. Br J Pharmacol 19:427 - 442 been reviewed by Kaumann et al. (1980) for heart /~- Kaumann AJ (1982) Yohimbine, rauwolscine and ketanserin antagonize adrenoceptors. Thus, the 10-fold lower affinity of yohimbine 5HT contractures through the same receptors in large coronary found in brain by Leysen et al. (1982) may not necessarily arteries of calf. Pflfigers Arch 394:R89 imply a difference between 5 HT2 receptors of brain and Kaumann A J, Mc Inerny T, Gilmour DP, Blinks JR (1980) Comparative large coronary arteries. assessment of//-receptor blocking agents as simple competitive antagonists in isolated heart muscle. Similarity of inotropic and chronotropic blocking potencies against isoproterenol. Naunyn- Schmiedeberg's Arch Pharmacol 311:219 - 236 On the PathophysioIogicaI Role of 5 HT Kaumann AJ, Breuer H-WM, Arnold G (1982) Heterogeneous spare in Large Coronary Arteries receptors for serotonin (5HT) in bovine coronary arteries. Pflfigers Arch 392 :R 36 5 HT elicits marked contractions in bovine large coronary Lambert GA, Lang WJ, Friedman E, Meller E, Gershon S (1978) arteries. No regional differences of 5 HT potency were found Pharmacological and biochemical properties of isomeric yohimbine on the right coronary artery, and branches of the left . Eur J Pharmacol 49:39 - 48 coronary artery (posterior, circumflex, anterior) (Kaumann Lemoine H, Kaumann AJ (1982) A novel analysis of concentration- et al. 1982). A possible clinical role of 5 HT in spasms of large dependence of partial agonism. Ring-demethylation of bupranolol results in a high-affinity (K 105) for myocardial and coronary artery can thus be envisaged. How might 5 HT tracheal/~-adrenoceptors. Naunyn-Schmiedeberg's Arch Pharmacol reach the coronary arteries? The first possibility would be 320:130-144 following release from aggregating platelets. 5 HT may also Lemoine H, Kaumann AJ (1983) A model for the interaction of be released from mast cells. Using a monoclonal antibody antagonists with two receptor-subtypes characterized by a Schild- against 5 HT (Consolazione et al. 1981), Kaumann and plot with apparent slope unity. Agonist-dependent enantiomeric Cuello have detected 5 HT in mast cells of bovine large affinity ratios for bupranolol in trachae but not in right atria of coronary arteries (unpublished experiments). However, a guinea pigs. Naunyn-Schmiedeberg's Arch Pharmacol 322:111 - possible release of 5 HT from mast cells of large coronary 120 arteries awaits experimental verification. Leysen JE, Awouters F, Kenis L, Laduron PM, Vandenberg J, Janssen PAJ (1981) Receptor binding profile of R 41468, a novel antagonist Acknowledgements. The author would like to thank Dr. A. Zentai at 5HT2 receptors. Life Sci 28:1015-1022 (Janssen, Neuss, FRG) for gifts of 5HT (serotonin) and ketanserin, Dr. Leysen JE, Niemegeers CJE, van Nueten JM, Laduron PM (1982) G. Cordes (Sanol, Monheim, FRG) for a gift of (-)-bupranolol, Dr. I. [3H]Ketanserin (R 41468), a selective 3H-ligand for serotoninz Lues (University of Essen, FRG) for a gift of B-HT920, Dr. H.-W. M. receptor binding sites. Binding properties, brain distribution, and Breuer for help with some of the experiments, Sibille Holzmann for functional role. Mol Pharmacol 21:301-314 preparing the figures and typing the manuscript and the Deutsche Lnes I, Schfimann HJ (1982) Characterization of postsynaptic ~- Forschungsgemeinschaft (SFB 30, Kardiologie) for support. adrenoceptors in the isolated saphenous vein of the rabbit. Naunyn- Schmiedeberg's Arch Pharmacol 321 :R 65 Mena MA, Vidrio H (1976) On the mechanism of the coronary dilator References effect of serotonin in the dog. Eur J Pharmacol 36:1-5 Morris TH, Sandrock K, Kaumann AJ (1981) 3H-(-)-bupranolol Arunlakshana O, Schild HO (1959) Some quantitative uses of drug (3H-Bu), a new/~-adrenoceptor radioligand: Binding to cat heart antagonism. Br J Pharmacol 14:48-58 /3-adrenoceptors. Naunyn-Schmiedeberg's Arch Pharmacol 317: Awouters F, Leysen JE, De Clerck F, van Nueten JM (1982) General 19-25 pharmacological profile of ketanserin (R 41468), a selective 5-HT2 Nickerson M (1971) Drugs inhibiting nerves and structures . In: De Clerck F, Vanhoutte PM (eds) 5- innervated by them. In: Goodman LS, Gilman A (eds) The Hydroxytryptamine in peripheral reactions. Raven Press, New pharmacological basis of therapeutics, 4th edition. Collier-MacMil- York, pp. 193-197 lan, pp 549--584 Blinks JR (1965) Convenient apparatus for recording contractions of Paton WDM, Rand HP (1965) The uptake of atropine and related drugs isolated muscle. J Appl Physiol 20:755-757 by intestinal smooth muscle of the guinea-pig in relation to Brazenor RM, Angus JA (1981) contracts isolated canine receptors. Proc Roy Soc Ser B (London) 163:1- coronary arteries by a mechanism: No role for alpha 44 adrenoceptors. J Pharmaeol Exp Ther 218:530-536 Peroutka ST, Snyder SH (1979) Multiple serotonin receptors: Dif- Brazenor RM, Angus JA (1982) Actions of serotonin antagonists on dog ferential binding of (3H)5-hydroxytryptamine, (3H)lysergic acid coronary artery. Eur J Pharmacol 81:569-576 diethylamine and (3H)spiroperidol. Mol Pharmacol 16:687- Consolazione A, Milstein C, Wright B, Cuello AC (1981) Immunocyto- 699 chemical detection of serotonin with monoclonal antibodies. J Porquet M-F, Pourrias B, Santamaria R (1982) Effects of 5-hydroxy- Histochem Cytochem 29:1425 - 1430 tryptamine on canine isolated coronary arteries. Br J Pharmacol Constantine JW, Lebel W, Archer R (1982) Functional postsynaptic ez- 75:305-310 but not cq-adrenoceptors in dog saphenous vein exposed to phenoxy- Shepperson NB, Langer SZ (1981) The effects of the 2-amino-tetrahydro- benzamine. Eur J Pharmacol 85:325- 329 naphthalene derivative M7, a selective ~2-adrenoceptor in vitro. De Mey J, Vanhoutte PM (1981) Uneven distribution of postjunctional Naunyn-Schmiedeberg's Arch Pharmacol 318 : 10 - 13 alpha1- and alphaz-like adrenoceptors in canine arterial and venous Snipes RL, Thoenen H, Tanzer JP (1968) Fine structure localization of smooth muscle. Circ Res 48:875- 884 exogeneous 5-HT in vesicles of adrenergic nerve terminals. Ex- Feniuk W, Humphrey PPA, Watts AD (1981) Further characterization of perientia (Basel) 24:1026 - 1027 pre- and post-junctional receptor for 5-hydroxytryptamine in Starke K (1981) e-adrenoceptor subclassification. Rev Physiol Biochem isolated vasenlature. Br J Pharmacol 73:191P-192P Pharmaeol 88:199 - 235 154

Thoa NB, Eccleston D, Axelrod J (1969) The accumulation of 14C- Van Nueten JM, Janssen PAJ, van Beek J, Xhonneux R, Verbeuren T J, serotonin in the guinea-pig vas deferens. J Pharmacol Exp Ther Vanhoutte PM (1981) Vascular effects of ketanserin (R 41468), a 169:68 -73 novel antagonist of 5-HT2 serotonergic receptors. J Pharmacol Exp Timmermans PBMWM, de Jonge A, van Meel JCA0 Slothorst-Grisdijk Ther 218:217-230 FP, Lam E, van Zwieten PA (1981) Characterization of c~-adreno- Weitzell R, Tanaka T, Starke K (1979) Pre- and postsynaptic effects of ceptor populations. Quantitative relationships between cardiovas- yohimbine stereoisomers on noradrenergic transmission in the cular effects initiated at central and peripheral ~-adrenoceptors. J pulmonary artery of the rabbit. Naunyn-Schmiedeberg's Arch Med Chem 24:502-507 Pharmacol 308:127 - 136 Van Meel JCA, de Jonge A, Kalkman HO, Wilffert B, Timmermans PBMWM, van Zwieten PA (1981) Vascular smooth muscle contrac- tion initiated by postsynaptic c~2-adrenoceptor activations is induced by an influx of extracellular calcium. Eur J Pharmacol 69:205 -212 Received November 12, 1982/Accepted March 11, 1983