Characterization of an endothelial 5-hydroxytryptamine (5-HT) receptor mediating relaxation of the porcine coronary artery

Summary The pharmacological properties of the endothelial 5-hydroxytryptamine (5-HT) receptors involved in relaxation of vascular smooth muscle were determined in rings of pig coronary artery contracted with 10 nmol/1 of the thromboxane A₂ receptor agonist 9,11-dideoxy-11,9-epoxy-methano-prostaglandin F₂ (U 46619).(1) In the presence of 10 mol/l ketanserin, relaxation was obtained with: 5-HT (apparent pD₂ value 7.00), 5-carboxamidotryptamine (5-CONH₂-T; 6.42), 5-aminotryptamine (5-NH₂-T; 5.96), 5-methoxytryptamine (5-OCH₃-T; 5.92), tryptamine, 7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrrolo(1,2-a)quinoxaline maleate (CGS 12066 A) and 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole succinate (RU 24969). The maximum relaxation obtainable with the agonists was about 40–60% of the U 46619-induced contraction and the concentration-response curves for 5-HT, 5-NH₂-T and 5-OCH₃-T were bell-shaped. The endothelium-dependence of this effect (i. e. the failure to relax the artery in endothelium-denuded preparations) was demonstrated for 5-HT, 5-CONH₂-T, RU 24969, CGS 12066A and tryptamine.(2) 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), 4-hydroxytryptamine, quipazine and yohimbine were ineffective in decreasing the tension of arteries with or without endothelium. Ipsapirone elicited full relaxation of U 46619-induced contraction, but this effect was not endothelium-dependent.(3) Metitepine (0.03-1 mol/l), 6-chloro-2-(1-piperazinyl)pyrazine (MK 212; 10 mol/l), methysergide (1 gmol/l) and cyanopindolol (0.1 mol/l) antagonized the relaxing effect of 5-HT in a non-surmountable manner, whereas metergoline (0.1 mol/l), quipazine (10 mol/l), yohimbine (1 mol/l), propranolol (1 mol/l) and (3-tropanyl)-1H-indole-3-carboxylic acid ester (ICS 205-930; 0.1 mol/l) did not. However, spiroxatrine (0.1 mol/l) and mesulergine (10 mol/l) enhanced the 5-HT-induced relaxation. The endothelium-dependent relaxation induced by 5-CONH₂-T was also inhibited by metitepine 1 gmol/l.(4) The 5-HT-induced relaxation was probably mediated by release of an endothelium-derived relaxing factor (EDRF). Gossypol, an inhibitor of EDRF, virtually abolished the 5-HT-induced relaxation while indometacin, an inhibitor of cyclooxygenase and accordingly of PGI₂ formation, did not.In conclusion, the failure of ketanserin and ICS 205–930 to counteract the relaxant effect of 5-HT receptor agonists excludes the involvement of 5-HT₂ and 5-HT₃ receptors, respectively, in the endothelium-dependent relaxation of the porcine coronary artery. The rather high potency of 5-CONH₂-T and the ability of certain 5-HT receptor antagonists, such as metitepine, methysergide and cyanopindolol, to counteract the effect of 5-HT are compatible with a 5-HT₁ character of the endothelial receptor. However, on the basis of the present data, no final classification, in particular with respect to the known 5-HT₁ receptor subtypes, is possible. Classification is also hampered by the bell-shaped character of the concentration-response curves for 5-HT receptor agonists and by their property to produce only partial relaxation. Send offprint requests to M. Gothert at the above address     

5-Hydroxytryptamine 5-HT1D receptors mediating inhibition of cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells

5-Hydroxytryptamine 5-HT_1B/5-HT_1D receptors are members of the same receptor subfamily, but display a different pharmacology (Hartig et al. (1992) Trends Pharmacol Set 13:152–159). Whereas several cell lines have been reported to contain 5-HT_1B receptors, none has been described, however, that endogenously expresses well-characterized 5-HT_1D receptors. The present study deals with the identification of 5-HT_1D receptors inhibiting cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells. 5-HT (1 nM– 10 M) induced a concentration-dependent inhibition of the cyclic AMP accumulation stimulated by prostaglandin E₁ (1 M) in MDCK cells. The maximal effect of 5-HT averaged 50% inhibition and was abolished after a pre-treatment of the cells with pertussis toxin. Other agonists mimicked the effects of 5-HT, with the following rank order of potency (pEC₅₀ ± SEM, n 3): 5-carboxamidotryptamine (8.36 ± 0.48) > PAPP (p-aminophenylethyl-m-trifluoromethylphenyl piperazine, 7.89 ± 0.23) > 5-HT (7.35 ± 0.05) > sumatriptan (6.65 ± 0.27). PAPP behaved as a partial agonist. 8-OH-DPAT (8-hydroxy-2(di-n-propylamino)tetralin) was less potent, its maximal effect being not reached at 0.1 mM. Methiothepin, GR127935, (–)propranolol, rauwolscine and ketanserin were all devoid of intrinsic activity (up to 10 M or 0.1 mM). Methiothepin (10 nM, 0.1 M and 1 M) antagonized 5-HT effect (pA₂ 8.57 ± 0.44, Schild slope 1.17 ± 0.21, n = 3). GR127935 (1 nM, 10 nM and 0.1 M) shifted the curve of 5-HT to the right, but the antagonism was not fully surmountable (apparent pK_B value, 9.80 ± 0.16, n = 9). From the shifts obtained with rauwolscine (1 M) and (–)propranolol (10 M), respective pK_B values were estimated 6.68 ± 0.30 and 5.4 (n = 3 each). PAPP, when tested as an antagonist at 1 M, also shifted the curve of 5-HT to the right, with a pK_B of 8.27 ± 0.16 (n = 3). Finally, ketanserin (10 M) also antagonized the effects of 5-HT, the pK_B being 6.54 ± 0.16 (n = 9). The rank orders of agonist and antagonist potencies strongly suggest 5-HT receptors mediating inhibition of cyclic AMP accumulation in MDCK cells to be 5-HT_1D receptors. This is the first report of a cell line expressing endogenous, well-characterized, 5-HT_1D receptors. With regard to the 5-HT_1D receptor subtype involved, the relatively high potency of ketanserin would suggest it to be a 5-HT_1D subtype or a mixture of 5-HT_1D/5-HT_1D\ subtypes. However, caution must be exercised here, owing to the poor knowledge of canine 5-HT_1D receptor subtypes.     

5-Hydroxytryptamine-induced contractions of the human isolated saphenous vein: involvement of 5-HT2 and 5-HT1D-like receptors,and a comparison with grafted veins

Summary The receptors mediating the contractile effect of 5-hydroxytryptamine (5-HT) on the human isolated saphenous vein, obtained from 42 patients undergoing coronary bypass surgery, have been further characterized using a number of 5-HT-related drugs. The rank order of agonist potency was 5-carboxamidotryptamine (5-CT) 5-HT > methysergide sumatriptan -methyl-5-HT 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1-Hindolesuccinate (RU 24969) 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) > 2-methyl-5-HT > 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT). Flesinoxan was inactive as an agonist. Ketanserin (1 mol/l) hardly affected sumatriptan-induced contractions but it caused a rightward shift of the upper part of the concentration-response curve of 5-HT and 5-CT. The same concentration of ketanserin caused a parallel rightward shift of the concentration-response curves of -methyl-5-HT and DOI with pK_B values of 7. 1 and 7.1, respectively. The responses to sumatriptan were antagonized by methiothepin (0.1 mol/l), metergoline (0.1 and 1 mol/l), rauwolscine (1 mol/l) and cyanopindolol (1 mol/l); the calculated pK_B values were 7.3, 6.9, 7.3, 6.7 and 6.5, respectively. Contractions to 5-HT were antagonized by methysergide (1 mol/l), methiothepin (0.1 mol/l; pKB = 7.1), ICS 205-930 (1 mol/l; pK_B = 5.9) and flesinoxan (30 mol/l; pK_B = 5.3). Remarkably, the contractions elicited by 2-methyl-5-HT were not attenuated by ICS 205-930, but were antagonized by methiothepin (0.1 mol/l) and, more markedly, by ketanserin (1 mol/l).There was a high correlation between the functional pD₂ values of 5-HT₁-like receptor agonists (5-CT, 5-HT, methysergide, sumatriptan, RU 24969 and 8-OH-DPAT) and their reported binding affinities for the 5-HT_1D receptor in human or calf brain membranes. Such a correlation for the antagonism of sumatriptan-induced responses was less marked than for the agonists, but of the 5-HT₁-like receptor subtypes it was the highest for the 5-HT_1D receptor identified in human or calf brain membranes.In 3 patients, undergoing heart transplantation, saphenous vein which had previously functioned as a graft for 6–11 years, was dissected out from the heart. Though the contractions to potassium were significantly smaller in the grafted veins, the pD₂ and E_max values (calculated as percentage of potassium-induced contractions) for 5-HT and sumatriptan were similar to those found in the veins obtained directly from the lower leg.It is concluded that contractions in the human isolated saphenous vein induced by 5-HT are mediated by 5-HT₂ receptors as well as by a 5-HT₁-like receptor resembling the 5-HT_1D subtype found in brain membranes. It is also to be noted that 2-methyl-5-HT, considered selective for the 5-HT₃ receptor, contracts the saphenous vein mainly via 5-HT₂ receptors.This study was supported by the Netherlands Heart Foundation, grant 89.252 Send offprint requests to W. A. Bax at the above address     

Subclassification of presynaptic 5-HT autoreceptors in the human cerebral cortex as 5-HT1Dβ receptors

Human cerebral cortical synaptosomes were used to determine the 5-hydroxytryptamine (5-HT) receptor subtype to which the inhibitory presynaptic 5-HT autoreceptor belongs. The synaptosomes preincubated with [³H]5-HT were superfused and tritium overflow was stimulated by high K⁺. The K⁺-evoked tritium overflow, which was Ca²⁺-dependent but tetrodotoxin-resistant, was concentration-dependently inhibited by the nonselective 5-HTlD_1D/1D receptor agonist, 5-carboxamidotryptamine. Ketanserin at a concentration which should block the 5-HT_1D but not the 5-HT_1D receptor failed to antagonize the inhibitory effect of 5-carboxamidotryptamine. In contrast, the non-selective 5-HT_1D/1D receptor antagonist, methiothepin, at a concentration which should block both the 5-HT_1D and the 5-HT_1D receptor abolished the effect of 5-carboxamidotryptamine. It is concluded that the presynaptic 5-HT autoreceptor, which has previously been classified as 5-HT_1D, belongs to the 5-HT_1D subtype.     

Interconversion into a low active state protects vascular 5-HT2-receptors against irreversible antagonism by phenoxybenzamine

Summary Recently, Kaumann and Frenken (1985) proposed an allosteric model of vascular 5-HT₂-receptors. We now present experiments in both bovine coronary and pulmonary artery, using the method of irreversible receptor occlusion, that support and extend the model. 1 Phenoxybenzamine was found to cause irreversible antagonism of the effects of 5-hydroxytryptamine (5-HT). Maximal contractile effects induced by 5-HT were depressed and, with further receptor occlusion, concentration-effect curves for 5-HT became biphasic. The high-sensitivity and the low-sensitivity component of the curve for 5-HT consisted of quickly and slowly developing contractions, respectively. 2 Biphasic concentration-effect curves for 5-HT after receptor occlusion were shifted to the right in non-parallel manner by ketanserin and became monophasic with an unexpected partial restoration of maximal responses to 5-HT. The magnitude of the shift of the partially restored concentration-effect curve for 5-HT by ketanserin after receptor occlusion by phenoxybenzamine is consistent with an interaction of ketanserin with 5-HT₂-receptors. 3 Preincubation with methysergide before phenoxybenzamine-treatment followed by washout of both drugs, and subsequent incubation with ketanserin completely prevented a depression of 5-HT-induced effects by phenoxybenzamine. 4 Estimates for the equilibrium dissociation constant of 5-HT for the 5-HT₂-receptor derived from fast developing contractions range from 0.1 mol/l to 0.4 mol/l. 5 The results are consistent with a model of two interconvertible states of the 5-HT₂-receptor. Phenoxybenzamine occludes the 5-HT₂-receptor in the R-state but not in the R-state. The low active R-state of the 5-HT₂-receptor appears to pre-exist in the absence of drugs and is not affected by phenoxybenzamine. By converting R into R ketanserin restores partially the response to 5-HT after occlusion of the R-state by phenoxybenzamine. Methysergide prevents the 5-HT₂-receptor occlusion induced by phenoxybenzamine indirectly by favouring isomerisation into the R-state.This work was supported by grant SFB 30 Kardiologie 04 of the Deutsche Forschungsgesellschaft     

5-Methoxytryptamine and 2-methyl-5-hydroxytryptamine-induced desensitization as a discriminative tool for the 5-HT3 and putative 5-HT4 receptors in guinea pig ileum

Summary Agonist-induced desensitization has been utilized to discriminate and independently isolate the neuronal excitatory receptors to 5-hydroxytryptamine (5-HT) in the guinea pig ileum (5-HT₃ and putative 5-HT₄ receptors). Electrically stimulated longitudinal muscle myenteric plexus preparations, and non-stimulated segments of whole ileum were used. Exposure to 5-methoxytryptamine (10 mol/l) inhibited completely responses to 5-HT at the putative 5-HT₄ receptor without affecting 5-HT₃-mediated responses. Conversely, exposure to 2-methyl-5-HT (10 mol/l) inhibited completely responses to 5-HT at the 5-HT₃ receptor without affecting putative 5-HT₄-mediated responses. The inhibition with 5-methoxytryptamine and 2-methyl-5-HT, either alone or in combination, appeared selective as responses to KCI, DMPP, carbachol, histamine, and substance P were unaffected or only very slightly modified. Furthermore, the pA₂ values for ICS 205–930 at the putative 5-HT₄ (pA₂ = 6.2 to 6.5) and 5-HT₃ (pA₂ = 7.6 to 8.1) receptors (estimated in the presence of 2-methyl-5HT and 5-methoxytryptamine, respectively) were consistent with those estimated in the absence of desensitization.5-Methoxytryptamine, but not 2-methyl-5-HT, suppressed completely but reversibly the concentration-effect curve to renzapride, suggesting that responses to this agent are mediated exclusively via agonism at the putative 5-HT₄ receptor.It is concluded that 5-methoxytryptamine and 2-methyl-5-HT can be utilized as selective probes to discriminate the putative 5-HT₄ receptor from the 5-HT₃ receptor in guinea pig ileum. This finding is of importance as no selective antagonist exists for the putative 5-HT₄ receptor. Furthermore, the presently described method of agonist-induced desensitization and 5-HT receptor discrimination may be useful for the identification and characterization of the putative 5-HT₄ receptor in other tissues and species. Send offprint requests to D. E. Clarke at the above address     

5-Hydroxytryptamine receptors with a 5-HT6 receptor-like profile stimulating adenylyl cyclase activity in pig caudate membranes

This study deals with the characterization of 5-hydroxytryptamine (5-HT, serotonin) receptors positively linked to adenylyl cyclase in membranes from pig brain caudate. 5-HT and related agonists induced a concentration-dependent stimulation of adenylyl cyclase activity in pig caudate membranes, with the following rank order of potency (mean pEC₅₀ values): 5-HT (7.1) 5-methoxytryptamine (6.9) > 5-carboxamidotryptamine (5.6) > sumatriptan (<5). Maximal stimulation by 5-HT averaged 35 pmol cyclic AMP/min/mg protein over a basal activity of 159 pmol cyclic AMP/min/mg protein. 5-Methoxytryptamine and 5-carboxamidotryptamine had similar efficacies to that of 5-HT, whereas sumatriptan was about half efficacious. Other compounds known as agonists at some 5-HT receptors were weakly potent (mean pEC₅₀ values <5). They include the 5-HT_1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), the 5-HT₄ receptor agonist, renzapride and the 5-HT₂ receptor agonist, (1-(2,5-dimethoxy-4-iodophenyl)-2 aminopropane) (DOI). In antagonist studies, methiothepin (0.1 and 1 mol/l) shifted the 5-HT curve to the right with no depression of the E_max, yielding pK_B values of 7.4–8.0. Clozapine (1 mol/l) also produced surmountable antagonism of 5-HT-induced effects (pK_B 6.9). Ketanserin (10 mol/l) weakly antagonized 5-HT (pK_B 5.0). The 5-HT₄ receptor antagonists, tropisetron (ICS 205–930) and SDZ 205–557 (2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester), each at 1 mol/l, did not significantly alter the concentration-response curve of 5-HT. The present receptor shares some characteristics of the recently cloned 5-HT₆ receptor (Monsma et al. (1993) Mol Pharmacol 43:320–327): similar pharmacological profile, location (striatum) and ability to stimulate adenylyl cyclase. It may thus represent the functional 5-HT₆ receptor in its natural environment. Correspondence to: P. Schoeffter at the above address     

Analysis of the 5-HT receptor in rabbit saphenous vein examplifies the problems of using exclusion criteria for receptor classification

Summary 5-Hydroxytryptamine (5-HT) contracts ring preparations of rabbit saphenous vein via direct and indirect components, the latter being- compatible with a tyramine-like action at sympathetic nerve terminals. Here an attempt was made to establish the identity of the receptor mediating contraction directly, in terms of the currently accepted proposals (Bradley et al. 1986).Results with agonists suggested 5-HT₁-like receptor activation: methysergide behaved as a partial agonist with microcolar affinity and 5-HT effects were mimicked by 5-carboxamidotryptamine (5-CT) and GR43175. The agonist potency order was 5-CT > 5-HT > methysergide GR43175, the same as that reported at the 5-HT₁-like receptor in dog saphenous vein (Feniuk et al. 1985; Humphrey et al. 1988). Consistent with this, 5-HT effects were resistant to blockade by the selective 5-HT₃ receptor antagonist MDL72222 (1.0 mol/l). In contrast, methiothepin (0.01–0.3 mol/l), ketanserin (0.3–30.0 mol/l) and spiperone (0.3–30.0 mol/l) each produced surmountable antagonism which, although competitivv in nature only for methiothepin (pK_B = 9.45 ± 0.09, 17 d. f.), implied 5-HT₂ receptor involvement. The possibility that these discrepancies resulted from mixed populations of 5-HT₁-like and 5-HT₂ receptors can be excluded because; 1). Ketanserin and spiperone blocked the actions of 5-HT and the selective 5-HT₁-like receptor agonist GR43175 with equal facility and 2). Responses to all of the agonists studied were similarly antagonised by flesinoxan (pKB 6.4), a simple competitive antagonist at the receptor in rabbit saphenous vein. This novel result with flesinoxan demonstrates that the ligand displays affinity at 5-HT receptors other than the 5-HT_1A subtype.These data show that the 5-HT receptor in rabbit saphenous vein shares features in common with, and may be identical to, the 5-HT₁-like receptor in dog saphenous vein. However, unlike the latter it demonstrates qualities evident in both 5-HT₁-like and 5-HT₂ receptors and for this reason fails to meet the currently accepted criteria for admission into any of the recognised classes. It is suggested that this sort of problem reflects the generally unreliable behaviour of the available receptor antagonists and the emphasis which the Bradley et al. (1986) scheme places upon them for classification by exclusion. A complementary approach which provides a rigorous, quantitative basis for receptor differentiation uses finger-prints comprising affinity and relative efficacy estimates for a set of tryptamines. This study illustrates the power and economy of this approach by showing how affinity and relative efficacy finger-prints obtained using 5-HT, 5-CT, (±) -methyl-5-HT, 5-methyltryptamine and N,N-dimethyltryptamine establish a positive identity for the 5-HT receptor in rabbit saphenous vein and at the same time enable it to be distinguished from other 5-HT receptor types presently allocated to the 5-HT₁-like, 5-HT₂ and so-called orphan receptor classes.7Send offprint requests to G. R. Martin at the above address     

Inhibition of the 5-HT-induced cardiogenic hypertensive chemoreflex by the selective 5-HT3 receptor antagonist ICS 205-930

Summary We investigated the effect of ICS 205-930 [(3-tropanyl)-1H-indole-3-carboxylic acid ester], a selective antagonist at 5-HT₃ receptors, on the cardiogenic hypertensive chemoreflex in the anaesthetized dog. The reflex was elicited by injection of 5-HT (12.5–1600 g) into the left cardiac ventricle and consisted of a dose-dependent systemic hypertension associated with a decrease in heart rate. ICS 205-930 (10, 30, and 100 g/kg i.v.) caused a displacement to the right of both the dose-response curves of 5-HT-induced blood pressure increase and heart rate reduction. Its blocking effects upon the action of 5-HT could be surmounted by increasing the dose of the agonist. The selective 5-HT₂ receptor antagonist, ketanserin (0.1 mg/kg i.v.) and the combined 5-HT₁ and 5-HT₂ receptor antagonist, methiothepin (0.1 mg/kg i.v.) had no influence on the hypertensive reflex. When the reflex was elicited by the ganglionic stimulant, 1,1-dimethyl-4-phenyl-piperazinium (DMPP; 100–1600 g), ICS 205-930 had no blocking effect. The results suggest that the 5-HT-induced cardiogenic hypertensive chemoreflex is mediated by 5-HT₃ receptors. Send offprint requests to H. Berthold at the above address     

Functional 5-HT receptors in human occipital artery

5-HT receptors were studied in human occipital arteries, obtained from patients during neurosurgery. We detected mRNA for the following receptors (incidence): 5-HT_1B (14/18), 5-HT_1D (15/18), 5-HT_2A (16/18), 5-HT_2B (8/8), 5-HT_4(a) (13/18), 5-HT_4(b) (5/18), 5-HT_4(g) (7/18), 5-HT_4(i) (1/18), 5-HT_7(a/b) (10/18) and 5-HT_7(d) (12/18). 5-HT contracted and relaxed arterial rings at low (–logEC₅₀ M=7.0) and high (–logEC₅₀ M=4.2) concentrations, respectively. 5-HT-evoked contractions were antagonized partially by both 5-HT_1B-selective SB224289 (200 nM) and 5-HT_2A-selective ketanserin (1 M) but not by 5-HT_1D-selective BRL15572 (500 nM) or prazosin (1 M). Sumatriptan caused contractions (–logEC₅₀ M=6.8, intrinsic activity with respect to 5-HT=0.3). Sumatriptan-evoked contractions were antagonized by SB224289 with high potency (pK_B=9.4) but not by BRL15572. 5-HT-induced relaxations were resistant to blockade by 5-HT_1B-selective SB224289 (1 M), 5-HT_1D-selective BRL15572, 5-HT_2B-selective SB204741 (1 M), 5-HT₄-selective GR113808 (100 nM) and 5-HT₇-selective SB269970 (1 M), and a combination of SB204741 and SB269970, inconsistent with an involvement of 5-HT_1B, 5-HT_1D, 5-HT_2B, 5-HT₄ and 5-HT₇ receptors. Triton X-100 treatment of the arteries abolished acetylcholine-induced relaxations of rings precontracted by prostaglandin F₂, but a reduction of the relaxant effects of 5-HT did not reach significance. Nitro-L-arginine (1 mM) reduced 5-HT-induced relaxations, suggesting a contribution of nitric oxide released from endothelial cells. Ketanserin (1 M) prevented the relaxant effects of 5-HT. We conclude that 5-HT contracts human occipital artery through 5-HT_1B receptors at low concentrations and through 5-HT_2A receptors at high concentrations. Sumatriptan contracts mostly through 5-HT_1B receptors. These results are consistent with the 5-HT_1B and 5-HT_2A mRNA data. 5-HT-induced relaxation is mediated, in part, through ketanserin-sensitive receptors, but 5-HT_1B, 5-HT_1D, 5-HT_2B, 5-HT₄ and 5-HT₇ receptors appear not to be involved.     

5-Hydroxytryptamine causes rate-dependent arrhythmias through 5-HT4 receptors in human atrium: facilitation by chronic β-adrenoceptor blockade

We have investigated the ability of 5-hydroxytryptamine (5-HT) to elicit arrhythmic contractions in isolated human atrial strips as a function of pacing rate (0.1–2 Hz) using a method recently introduced by us (Kaumann and Sanders, this journal, 1993b) and examined the nature of the 5-HT receptors involved. Right atrial appendage tissue was obtained from 14 patients undergoing cardiac surgery. None of the patients had advanced heart failure. 5-HT (0.6–20 mol/l) induced arrhythmic contractions during pacing in 4/11 atrial strips from 3/4 patients who had not received blockers and in 21/27 atrial strips from 9/10 patients who had been chronically treated with blockers (primarily ₁-selective). The incidence of arrhythmic contractions evoked by 5-HT did not reach statistical significance in the atrial tissue from the non- blocked patients but was highly significant in the atrial tissue from the chronically blocked patients. The arrhythmic contractions usually occurred more frequently at low than at high pacing rates and were observed at the physiological frequency of 1 Hz in 1/4 atrial strips from 1/4 of the non- blocked patients and 6/11 strips from 5/10 of the blocked patients. The 5-HT-evoked arrhythmic contractions were observed during blockade of ₁-adrenoceptors, ₂-adrenoceptors and 5-HT₃ receptors, ruling out the participation of these receptors. The 5-HT-evoked arrhythmic contractions were totally inhibited within 30 min by the selective 5-HT₄ receptor antagonist SB 203186 ((1-piperidinyl)ethyl 1H-indole 3-carboxylate) 100 nmol/l whereas they persisted in time-matched controls. The blockade of 5-HT-evoked arrhythmic contractions by SB 203186 was surmounted by high concentrations (400–1800 mol/l) of 5-HT. Our results demonstrate that 5-HT elicits rate-dependent arrhythmic contractions in isolated human atrium through the 5-HT₄ receptor and that they are facilitated in atrial tissue from patients treated with blockers. Our results suggest that endogenous, platelet-derived 5-HT may cause atrial arrhythmias and that exogenous 5-HT₄ agonists/partial agonists may be arrhythmogenic. Correspondence to: A. J. Kaumann at the above address     

The actions of 5-hydroxytryptamine receptor agonists and antagonists at pre- and postjunctional level on the canine saphenous vein

Summary The prejunctional and postjunctional 5-HT receptors of the canine saphenous vein were studied. The release of ³H-noradrenaline (³H-NA) from incubated saphenous vein strips was inhibited by 5-hydroxytryptamine (5-HT) in a concentration-dependent way (5-HT concentrations: 0.01, 0.1 and 1.0 mol · l^–1), but not by the selective 5-HT_1A agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT; 1 and 10 mol · l^–1). The inhibitory effect of 5-HT was antagonized by metitepine and methysergide, but not by yohimbine, (–)-pindolol or ketanserin. In strips preincubated with 5-HT (1.2 mol · l^–1), the fractional release of ³H-NA was slightly reduced (paired experiments). 5-HT and 8-OH-DPAT caused concentration-dependent contractions of the saphenous smooth muscle. A parallel shift of the concentration-response curve for 8-OH-DPAT to the right was caused by metitepine and yohimbine, but not by ketanserin. The contractions caused by 5-HT were antagonized by metitepine and yohimbine (parallel displacement of the curves to the right), as well as by ketanserin and methysergide (with a depression of the upper part of the curve). Blockade of -adrenoceptors (due to prazosin plus a low concentration of yohimbine) also resulted in a weak antagonistic effect. Ketanserin and metitepine displaced the noradrenaline concentration-response curve to the right. We conclude that the saphenous vein of the dog is endowed with prejunctional receptors of the 5-HT₁ type which can not be classified as belonging either to the 1 A or 1 B subtype; and that at the postjunctional level 5-HT₁ (possibly of the 1D subtype) and 5-HT₂ receptors are present. 5-HT is able to activate all these receptors.Supported by INIC (Centro de Farmacologia e Biopatologia Química da Universidade do Porto) Send offprint requests to M. Q. Paiva at the above address     

5-HT1-like and 5-HT2A receptors mediate 5-hydroxytryptamine-induced contraction of rabbit isolated mesenteric artery

The contractions induced by 5-hydroxytryptamine (5-HT) and the 5-HT₁-like receptor agonist, sumatriptan, were investigated in the open ring preparations of rabbit mesenteric artery in order to characterize the 5-HT receptors. 5-HT induced concentration-dependent contractions. Sumatriptan did not induce any contraction of unstimulated rings, whereas it elicited concentration-dependent contractions in preparations given a moderate tone by a threshold concentration of prostaglandin F₂ (PGF₂). Pargyline, cocaine or normetanephrine were without significant effect on the contractions induced by 5-HT and sumatripan. The 5-HT concentration-effect curve was clearly biphasic. Methiothepin (0.01 M) shifted the both phases of the concentration-effect curve to the right. Ketanserin (0.1 M) shifted the second, low affinity, phase and prazosin did not alter concentration-effect curve to 5-HT. The sumatriptan concentration-effect curve was shifted by methiothepin (0.01 M) to the right (pK_B = 9.19) but not by ketanserin (1 M). Concentration-effect curves to 5-HT and sumatriptan were not affected by the 5-HT₃ receptor antagonist tropisetron (1 M). These results suggest that 5-HT₁-like type receptors are responsible for the first phase of 5-HT-induced contraction and 5-HT_2A receptor for the second phase, in rabbit mesenteric artery. Sumatriptan-induced contractions appear to be mediated by 5-HT₁-like type receptors in this artery. These results also suggest that this kind of amplification may be a common feature of vascular 5-HT₁-like type receptor as has been shown in other vascular segments such as rabbit femoral, iliac and renal arteries, and guinea-pig iliac artery.     

Effects of 5-HT4 receptor stimulation on basal and electrically evoked release of acetylcholine from guinea-pig myenteric plexus

Summary The effects of 5-methoxytryptamine and 5-hydroxytryptamine (5-HT) on both basal and electrically evoked outflow of tritium were studied in guinea-pig myenteric plexus preparations preincubated with [³H]-choline. Basal outflow. 5-Methoxytryptamine caused a transient and calcium-dependent increase in basal outflow of [³H]acetylcholine that was abolished by tetrodotoxin. Ondansetron (1 mol/1) did not affect the stimulatory response of 5-methoxytryptamine but ICS 205-930 (1 and 3 mol/1) produced parallel rightward displacements of the concentration-response curve to 5-methoxytryptamine. The PK_B value for ICS 205-930 was 6.6 suggesting an involvement of 5-HT₄ receptors. 5-HT caused an increase in basal outflow of [³H]acetylcholine and a biphasic concentration-response curve was obtained. The maximal response of the first phase to 5-HT (release of 0.98% of tissue tritium) and the maximal response to 5-methoxytryptamine (0.94% of tissue tritium) were similar but 5-methoxytryptamine (-log EC₅₀: 6.9) was less potent than 5-HT (-log EC₅₀ of the high affinity component: 7.9). ICS 205-930 (0.01–1.0 mol/1) acted as a competitive antagonist against the low affinity component of the 5-HT concentration-response curve with a pA₂ value of 8.0. It is concluded that stimulation of both 5-HT₄ receptors (by 5-methoxytryptamine and submicromolar concentrations of 5-HT) and 5-HT₃ receptors (by micromolar concentrations of 5-HT) causes a release of acetylcholine which in turn leads to smooth muscle contraction. Electrically evoked outflow. This outflow of [³H]acetylcholine was concentration-dependently inhibited by both 5-methoxytryptamine and 5-HT. ICS 205-930 (1 mol/1) reinforced the inhibitory effect of 5-methoxytryptamine but not that of 5-HT. In the presence of methiothepine (0.1 mol/1) 5-methoxytryptamine enhanced the evoked outflow of [³H]acetylcholine, an effect which was attenuated by 3 mol/1 ICS 205-930. These results suggest that 5-methoxytryptamine may both inhibit (via 5-HT₁ receptors) and facilitate (via 5-HT₄ receptors) the evoked release of acetylcholine from guinea-pig myenteric neurones. The facilitatory action is unmasked when the 5-HT₁ receptor is blocked by methiothepine. Send offprint requests to H. Kilbinger at the above address     

Evidence for presynaptic location of inhibitory 5-HT1D\-like autoreceptors in the guinea-pig brain cortex

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K⁺ were determined in superfused synaptosomes and slices, preincubated with [³H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [³H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT_1A and 5-HT_2C sites) to guinea-pig cortical synaptosomes and membranes.5-HT receptor agonists inhibited the K⁺-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine (L-694,247) >5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) 5-HT > sumatriptan 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT_1D, and 5-HT_1D receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA₂: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT_1D, but not the 5-HT_1D\, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [³H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [³H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin >5-CT >5-methoxytryptamine >5-HT sumatriptan oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [³H]5-HT binding to synaptosomes and membranes.In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [³H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT_1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT_1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT_1D\-like.     

5-Hydroxytryptamine4 receptors mediate relaxation of the rat oesophageal tunica muscularis mucosae

Summary The present study was designed to characterize an atypical 5-hydroxytryptamine (5-HT) receptor mediating relaxation of the rat oesophageal tunica muscularis mucosae. All experiments were performed under equilibrium conditions, using pargyline to inhibit the oxidative deamination of indoleamines, and cocaine and corticosterone to inhibit neuronal and extraneuronal uptake. Under these conditions 5-HT (0.3–1000 nmol/l) produced a concentration-dependent relaxation of carbachol-induced tension. The concentration-effect curve to 5-HT was unaffected by potent antagonists for 5-HT₁, 5-HT₂, 5-HT₃ and so called 5-HT_1P receptors (metergoline, methysergide, ketanserin, ondansetron, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide), but was antagonized competitively by ICS 205–930 (pA₂ = 6.7). Responses to 5-HT were mimicked by other indoleamines and substituted benzamides with the following order of potency: 5-HT 5-methoxytryptamine > cisapride = -methyl-5-HT = (S)-zacopride = renzapride > (RS)-zacopride > 5-carboxamido-tryptamine = metoclopramide = (R)-zacopride > tryptamine > 2-methyl-5-HT. ICS 205–930 afforded similar pA₂ values (6.0–6.7) against each agonist, indicating a common site of action. Concentration-effect curves to 5-HT were not affected by tetrodotoxin or indomethacin, sugesting that 5-HT-induced relaxation of the tunica muscularis mucosae was mediated via a postjunctional receptor, independent of endogenous prostanoids. The pharmacological profile of the 5-HT receptor in the rat oesophageal tunica muscularis mucosae correlates well with the 5-HT₄ receptor characterized recently in both the CNS and gastro-intestinal tract. Send offprint requests to D. E. Clarke at the above address     

5-Hydroxytryptamine 5-HT1B receptors inhibiting cyclic AMP accumulation in rat renal mesangial cells

A clonal cell line derived from rat renal mesangial cells was shown to express endogenous 5-hydroxytryptamine (serotonin, 5-HT) receptors that mediate inhibition of cyclic AMP accumulation. These receptors were characterized as being of the 5-HT_1B receptor subtype. 5-HT₁ receptor agonists inhibited forskolin-stimulated cyclic AMP accumulation in rat renal mesangial cells (60–70% maximal inhibition) with the following rank order of potency (mean pEC₅₀ values±SEM, n 3): ergotamine (9.58±0.51)>RU 24969 (8.67±0.23)5-CT (8.42±0.06)CP 93129 (8.15±0.27)>5-HT (7.75±0.11) > sumatriptan (6.29±0.30) > 8-OH-DPAT (4.32±0.15). 5-HT₂ and 5-HT₄ receptor agonists were without effect. 5-HT-induced inhibition of cyclic AMP accumulation was abolished by a pre-treatment of the cells with pertussis toxin. (-)Propranolol was a partial agonist (27% maximal inhibition, pEC₅₀ 7.19±0.24, n = 3); when used as an antagonist at 1 M, it shifted the concentration-response curve of 5-HT to the right (pKB 7.22±0.35, n = 3). Methiothepin was a competitive antagonist of 5-HT (pA₂ 8.04±0.10, Schild slope 0.87±0.21, n = 3). Rauwolscine (10 M) had no antagonist activity. There was a significant correlation (r = 0.98, P = 0.0001) between the cyclic AMP data obtained in rat mesangial cells and 5-HT_1B binding data reported in rat brain cortex. The same pattern of responses was observed in early passages of primary cultures of rat mesangial cells. This study shows that rat mesangial cells can be used as a convenient source of functional 5-HT_1B receptors. It also constitutes further evidence for the widespread distribution of 5-HT_1B receptors outside the brain.     

Evidence for 5-HT2B and 5-HT7 receptor-mediated relaxation in pulmonary arteries of weaned pigs

This study characterizes the relaxant response to 5-hydroxytryptamine (5-HT) in prostaglandin F₂ (PGF₂)-precontracted pulmonary arteries of weaned pigs. In arterial rings with intact endothelium, the relaxation to 5-HT was biphasic. The high affinity component of relaxation to 5-HT (0.1–10 nM) was abolished by mechanical removal of the endothelium or after the addition of l-NAME (200 M), and was inhibited by the 5-HT_2B/2C receptor antagonist SB 206553 (1 M), but not the 5-HT_2C receptor antagonist SB 242084 (0.1 M). Endothelium-intact arteries were also relaxed by the selective 5-HT_2B receptor agonist BW 723C86 (pD₂ 7.7). The relaxant response to BW 723C86 was inhibited by 1 M SB 206553 (pK_B 6.8). The low affinity component of relaxation to 5-HT (30 nM) remained unaffected after mechanical removal of the endothelium or the addition of l-NAME. In endothelium-denuded arterial rings, 5-HT, 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT), and frovatriptan produced monophasic relaxations with pD₂ values of 6.5, 7.5, 5.9, and 4.7 respectively. Relaxant responses to the agonists were antagonized by the selective 5-HT₇ receptor antagonist SB 269970 (pK_B 8.2–8.9). The relaxant response to the potent 5-HT₇ receptor agonist 5-CT was also antagonized by methiothepin (pK_B 9.6), pimozide (pK_B 8.2), mesulergine (pK_B 7.7), methysergide (pK_B 7.4), clozapine (pK_B 7.6), and spiperone (pK_B 7.4). The estimated pK_B values argue in favor of an involvement of 5-HT₇ receptors in the direct vasorelaxant action of 5-HT in the pulmonary arteries of weaned pigs. The relaxant response to 5-CT was associated with an increase in cAMP that was surmountably antagonized by SB 269970 (pK_B 8.6). The present in vitro bioassay can be used to characterize new drugs with potential agonist or antagonist properties at functional 5-HT₇ receptors.     

Comparative analysis of two types of 5-hydroxytryptamine receptor mediating vasorelaxation: differential classification using tryptamines

Summary Two receptors mediating relaxant responses to 5-hydroxytryptamine (5-HT) were studied comparatively in rings of rabbit jugular vein contracted with U-46619 (10 nmol/l). At low concentrations of 5-HT (0.001–0.1 mol/l) vascular relaxation was mediated indirectly by the endothelial 5-HT receptor previously described by Leff et al. (1987). In preparations denuded of endothelium, higher concentrations of the amine (0.03–30 mol/l) caused relaxation responses directly, presumably via a receptor located on the smooth muscle cells. Similarity between the receptors was evident in that both were susceptible to antagonism by methysergide, but resistant to blockade by ketanserin and MDL 72222. In these respects, the receptors qualified for a 5-HT₁-like classification. Consistent with this, 5-carboxamidotryptamine demonstrated a higher agonist potency than 5-HT at the receptor mediating relaxation directly. However, in endothelium-intact jugular vein rings this potency order was reversed, showing that the endothelial 5-HT receptor did not satisfy completely the criteria for a 5-HT₁-like designation. When the activities of a single set of tryptamines were compared in endothelium-intact and -denuded jugular vein rings, different affinity and relative efficacy estimates were obtained, confirming that two distinct 5-HT receptors mediate relaxation responses in this tissue. The most striking difference between these two receptor types was demonstrated by -methyl-5-HT since it expressed a high affinity comparable to 5-HT at the endothelial receptor, but was inactive at the receptor in endothelium-denuded preparations at concentrations up to 30 mol/l. In antagonist studies, the butyrophenone spiperone (1 ol/l) likewise distinguished between these two receptor types, since it failed to block endothelium-dependent relaxations, but caused an 8-fold parallel, rightward displacement of the 5-HT concentration-effect curve obtained in endothelium-denuded preparations. The possibility that one or both of these receptors might mediate vasodepressor responses to tryptamines in vivo was investigated in pentobarbitone-anaesthetised rats treated with ketanserin. The relative hypotensive potencies of the tryptamines showed a close agreement with their relative affinities for the receptor type mediating directly vascular relaxation, but not with their relative affinities for the receptor type located on the vascular endothelium. In agreement with this, spiperone (1 mg/kg, i. v.) antagonised the hypotensive response to 5-carboxamidotryptamine, causing a 10-fold right-shift of the dose-effect curve. These results further support the utility of tryptamines as probes for 5-HT receptor classification and, in addition, suggest they can be used for the differential classification of receptors in vivo as well as in vitro. Send offprint requests to G. R. Martin     

Sumatriptan contracts large coronary arteries of beagle dogs through 5-HT1-like receptors

Summary The effects of 5-hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), methysergide and sumatriptan were studied on endothelium-denuded rings of beagle dog large coronary arteries. Submicromolar concentrations of the compounds contracted the rings with the order of potency 5-CT > 5-HT >sumatriptan = methysergide. Concentrations greater than 2 M of both 5-HT and 5-CT, and 60 mol/l methysergide also caused concentration-dependent relaxation. Sumatriptan did not cause relaxation. Peak intrinsic activities relative to the plateau contraction to sumatriptan (1.00), were 5-CT 0.47, 5-HT 0.87 and methysergide 0.51. Ketanserin 1 mol/l affected neither contractile responses nor relaxant responses to 5-CT, methysergide and sumatriptan and only caused marginal blockade of the contractile effects of 5-HT. Methiothepin 200 nM shifted the concentration-contractile response curves by around 2 log units, as expected from its affinity for 5-HT₁-like receptors.The rank order of contractile potency of the agonists, the antagonism by methiothepin and the resistance to blockade by ketanserin are consistent with a nearly exclusive involvement of 5-HT₁-like receptors. Isolated large coronary arteries from beagle dogs may be a suitable model for the study of human coronary artery 5-HT₁-like receptors that are involved in the spasm observed with 5-HT and sumatriptan. Correspondence to A. J. Kaumann at the above address