A component of 5-HT-evoked depolarization of the rat isolated vagus nerve is mediated by a putative 5-HT4 receptor

Summary This study describes a component of 5-HT-evoked depolarization of the rat isolated vagus nerve which was unaffected by the 5-HT₃ receptor antagonist ondansetron. A grease-gap extracellular recording technique was used. Ondansetron (10–100 nmol/1) displaced the 5-HT concentration-response curve to the right yielding a pA₂ value of 8.6 (8.5–8.8), consistent with 5-HT₃ receptor antagonism, and revealing a component of the 5-HT response which was resistant to ondansetron blockade. In the presence of ondansetron (100 nmol/1) the maximum depolarization in the resistant phase was 15.5 (12.6–19.2)% of the initial maximum response to 5-HT and the pEC₅₀ value was 7.0 (6.7–7.3). The mechanism of the ondansetron-resistant component of the 5-HT response resembled a 5-HT₄ -receptor-effect in being absent in preparations equilibrated with 5-methoxytryptamine (10 mol/1) and antagonised by ICS 205930 (tropisetron, pA₂ 6.4). 5-Methoxytryptamine alone was an agonist in the vagus nerve with a maximum response similar to that of the ondansetron resistant phase of the 5-HT response. similarly renzapride alone evoked small depolarizations of this preparation but antagonized the ondansetron resistant phase of the 5-HT response (pA₂ 7.3–7.4). These effects of 5-methoxytryptamine and renzapride are also consistent with a 5-HT₄ receptor mechanism. Ketanserin (1 mol/1) and methysergide (1 mol/1) had little effect on responses to 5-HT. The depolarization evoked by this putative 5-HT₄ receptor mechanism was small but prolonged and appears to mask and after-hyperpolarizing phase of the 5-HT response in this tissue. Correspondence to: K. F. Rhodes at the above address     

Modulation by 5-HT3 and 5-HT4 receptors of the release of 5-hydroxytryptamine from the guinea-pig small intestine

Summary The effects of agonists and antagonists of 5-hydroxytryptamine (5-HT) receptors on the release of endogenous 5-HT from enterochromaffin cells were studied in the vascularly perfused isolated guinea-pig small intestine. The experiments were done in the presence of tetrodotoxin in order to exclude a neuronally mediated influence on 5-HT release.The 5-HT₃ receptor agonist 2-methyl-5-HT increased 5-HT release, and this effect was antagonized by 1 nmol/l tropisetron. Nanomolar concentrations of tropisetron, MDL 72 222 and granisetron decreased 5-HT release. Ondansetron (0.1 and 1 mol/1) did not modify 5-HT release.5-Methoxytryptamine, BIMU8 and cisapride concentration-dependently inhibited 5-HT release. BIMU8 was more potent than 5-methoxytryptamine. Micromolar concentrations of tropisetron (1 and 10 mol/1) enhanced the release, whilst methiothepine (0.1 mol/l) did not affect the release of 5-HT.The results suggest that enterochromaffin cells of the guinea-pig ileum do not contain 5-HT₁ and 5-HT₂ receptors, but are endowed with 5-HT₃ and 5-HT₄ autoreceptors. Activation of the 5-HT₃ receptors triggers a positive feedback mechanism leading to an increase of 5-HT release. The 5-HT₃ receptors on the enterochromaffin cell differ from neuronal 5-HT3 receptors on guinea-pig myenteric plexus by their high affinity for tropisetron and MDL 72 222, and their very low affinity for ondansetron. Stimulation of 5-HT₄ receptors causes inhibition of release; the inhibitory 5-HT₄ receptor mechanism appears to predominate.Correspondence to H. Kilbinger at the above address     

Endothelium-dependent relaxation of porcine pulmonary arteries via 5-HT1C-like receptors

Summary In PGF₂-precontracted pulmonary arteries with intact endothelium, 5-hydroxytryptamine (5-HT, 1.0-100 nmol/l) caused a concentration-dependent reversible relaxation, at higher concentrations the contractile response prevailed. In endothelium-denuded vessels relaxation was absent. 5-HT-induced relaxation of precontracted pulmonary arteries was probably mediated by release of an endothelium-derived relaxing factor (EDRF). Preincubation of the arteries with methylene blue or N^G-nitro-Lrarginine (200 mol/l) attenuated the relaxant effect. The 5-HT-induced relaxation was accompanied by an increase in cGMP. Indomethacin (3 mol/l) did not influence the 5-HT-induced relaxation indicating that eicosanoids are not involved in the relaxant response to 5-HT.The 5-HT_1C and 5-HT₂ receptor agonist -methyl-5HT was as potent as 5-HT in inducing relaxation. The rank order of relaxant potency of the agonists investigated was -methyl-5-HT > 5-HT > 5-methoxytryptamine > tryptamine > -methyl-5-HT > 5-carboxamidotryptamine >2-methyl-5-HT > 5,6-dihydroxytryptamine > m-chlorophenylpiperazine >sumatriptan > 8-OH-DPAT.Phentolamine, pindolol and ICS 205-930 did not interfere with the relaxant effect. The 5-HT₂ receptor antagonist ketanserin (1 mol/l) inhibited the contractile response but did not alter vasodilatation. Apart from the blockade of the contractile effects, mesulergine, cyproheptadine and mianserin (0.1-3.0 mol/l, each) induced a parallel shift to the right of the concentration-response curve for the relaxation induced by a-methyl-5-HT or 5-HT. Spiperone (0.3 mol/l) exerted weak inhibitory effects on relaxation and contraction. The most potent (noncompetitive) antagonist against relaxant responses was metitepine (0.1-1.0 mol/l) which markedly depressed the relaxant maximum effect of the agonists.The failure of ketanserin and ICS 205-930 to inhibit the relaxant effect of 5-HT receptor agonists suggests that classical 5-HT₂ and 5-HT₃ receptors are not involved in the endothelium-dependent relaxation. Comparison of the rank order of potencies of agonists and antagonists with their affinities for brain binding sites revealed that the endothelial 5-HT receptors are similar to the 5-HT_1C receptor subtype. Furthermore, the endothelial receptors exhibit marked similarity to the recently cloned 5-HT receptor mediating contraction of the rat stomach fundus. Correspondence to E. Glusa at the above address     

SDZ 205-557, a selective,surmountable antagonist for 5-HT4 receptors in the isolated guinea pig ileum

Summary A selective antagonist for the recently characterized 5-HT₄-receptor is lacking. The only surmountable antagonist available, ICS 205-930, is a weak antagonist and is far more potent at 5-HT₃-than at 5-HT₄ receptors. In this paper, SDZ 205-557 (2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester) is characterized as the first potent, selective and surmountable antagonist at 5-HT₄ receptors in the isolated guinea pig ileum.SDZ 205-557 was investigated in the non-stimulated and in the field-stimulated guinea pig ileum longitudinal muscle preparation for its affinity for 5-HT₄-, 5-HT₃-, muscarine-, nicotine- and histamine H₁ receptors. The affinity for 5-HT₁-, 5-HT₂-, ₁-, ₂- and opiate () receptors was determined by binding assays. SDZ 205-557 was devoid of substantial affinity (pK_D values below 5.6) for all receptors investigated except for 5-HT₃- and 5-HT₄ receptors. At these two receptors, SDZ 205-557 acted as an antagonist without measurable intrinsic activity.At the 5-HT₄ receptors of the non-stimulated guinea pig ileum, responses to 5-HT and 5-methoxytryptamine were antagonized by SDZ 205-557 with identical pA₂ values of 7.4. The effect of renzapride was also blocked with no significant change in the maximum response; Schild analysis, however, revealed that the interaction was not competitive with an apparent pA₂ value of 7.6. A pA₂ of 6.8 was obtained using zacopride as a contractile agent; this value differed significantly from 7.4, the value obtained for 5-HT and 5-methoxytryptamine. The effects of metoclopramide were inhibited non-competitively with a pD₂' value of 5.4. 5-HT₄ receptor mediated effects in the field-stimulated guinea pig ileum were antagonized in a similar way by SDZ 205-557. The resulting pA₂ values were not significantly different from those obtained in the non-stimulated preparation.In the non-stimulated preparation, contractions induced by 5-HT and 2-methyl-5-HT via activation of 5-HT₃ receptors were antagonized by SDZ 205-557 only at concentrations of 10^–6 mol/l and above yielding pA₂ values of 5.6 and 5.9, respectively, which were considerably lower than those obtained for the blockade of 5-HT₄ receptors. At concentrations greater than 10^–6 mol/l, SDZ 205-557 caused a significant reduction of the maximum responses, indicating non-competitive antagonism.It is concluded that SDZ 205-557 is a potent, selective and surmountable antagonist at 5-HT₄ receptors in the isolated guinea pig ileum, and is a more practical tool for the characterization of 5-HT₄ receptors than ICS 205-930.A part of these results was presented at the 32nd Spring Meeting of the German Society for Pharmacology and Toxicology, Mainz, March 12–15, 1991, and published as Rapid communication (Buchheit et al. 1991) Send offprint requests to K. H. Buchheit at the above address     

Serotonin autoreceptor in rat hippocampus: Pharmacological characterization as a subtype of the 5-HT1 receptor

Summary The 5-hydroxytryptamine (5-HT) autoreceptors mediating inhibition of [³H]5-HT release in rat hippocampus have been characterized pharmacologically in terms of 5-HT receptor subtype by using superfused synaptosomes depolarized with 15 mM KCl. Exogenous 5-HT inhibited in a concentration-dependent way (pEC₃₀=8.74) the K⁺-evoked release of [³H]5-HT. Methiothepin shifted the concentration-response curve of 5-HT to the right (pA₂=8.62). The 5-HT₂ receptor antagonists, ketanserin, methysergide or spiperone were ineffective against 5-HT. The 5-HT₁ receptor agonist, 5-methoxy-3-[1,2,3,6-tetra-hydropyridin-4-yl]-1H-indole (RU 24969) mimicked 5-HT and was equipotent as an inhibitor of the release of [³H]5-HT. In contrast, the putative 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was almost ineffective at 1 M. Finally, (–)propranolol, used as a non-selective 5-HT_1A/5-HT_1B receptor antagonist, shifted to the right (pA₂=7.91) the concentration-response curve of 5-HT whereas the 5-HT_1C receptor antagonist mesulergine was ineffective. In conclusion, 5-HT nerve terminals of rat hippocampus possess autoreceptors which appear to belong to the 5-HT_1B subtype.     

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     

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.     

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     

Characterization of the contraction to 5-HT in the canine colon longitudinal muscle

1. Although conscious dogs have often been used for colonic motility studies with 5-hydroxytryptamine (5-HT), the effects of 5-HT on the isolated colon have not been thoroughly characterized yet. The current study was undertaken to characterize the response to 5-HT of the canine isolated colon longitudinal muscle.
2. Longitudinal strips of canine midcolon deprived of (sub)mucosa were prepared for isotonic measurement. 5-HT induced contractions from 3 nM onwards, which were not affected by selective inhibition of 5-HT re-uptake, monoamine oxidase or blockade of α-adrenoceptors. Tetrodotoxin (0.3 μM) did not affect the responses to 5-HT, suggesting that smooth muscle 5-HT receptors are involved. The selective 5-HT₄ receptor antagonist SB 204070 (10 nM) slightly enhanced contractions to 5-HT and therefore it was included in the organ bath solution in all further experiments. The 5-HT₁ and 5-HT₂ receptor antagonist methysergide (0.1 μM) depressed the curve to 5-HT, but the selective 5-HT₃ receptor antagonist granisetron (0.3 μM) had no effect.
3. Besides 5-HT, α-methyl-5-HT (α-Me-5-HT), 5-methoxytryptamine (5-MeOT), 2-methyl-5-HT (2-Me-5-HT) and 5-carboxamidotryptamine (5-CT) also induced contractions, with the following rank order of potency (pEC₅₀ values in parentheses): 5-HT (6.9)=α-methyl-5-HT (6.9)>2-Me-5-HT (5.8)=5-MeOT (5.7)=5-CT (5.6), indicative of 5-HT₂ receptor involvement. α-Me-5-HT produced a bell-shaped curve, which was not affected by α-adrenoceptor blockade. 5-HT, 5-MeOT, 2-Me-5-HT and 5-CT produced a monophasic concentration-response curve, consistent with an interaction with a single receptor site. 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and tryptamine only induced contractions at a concentration exceeding 1 μM.
4. The selective 5-HT_2B receptor antagonist SB 204741 (0.3 μM) did not affect the curve to 5-HT. Ketanserin, cisapride and spiroxatrine behaved as competitive antagonists with pK_b values of, respectively, 8.4, 8.1 and 6.7. Spiroxatrine (1 μM) shifted the curve to 5-MeOT rightward yielding an apparent pA₂ of 7.1. Other antagonists at 5-HT_2A receptors also surmountably inhibited the contractions to 5-HT (apparent pA₂ value in parentheses): mesulergine (8.2), cinanserin (8.2), yohimbine (6.2) and mianserin (8.6). However, as well as a rightward shift, methiothepin (8.3), pizotifen (8.6) and spiperone (8.8) also caused a depression of the curve, indicative of ‘pseudo-irreversible'' antagonism. Taken together, the above mentioned affinity estimates most closely corresponded to literature affinity values for 5-HT_2A receptors.
5. It was concluded that 5-HT induces contractions of the canine midcolon longitudinal muscle primarily by stimulation of smooth muscle 5-HT_2A receptors. The presence of inhibitory 5-HT₄ receptors cannot be ruled out.

     

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     

5-Hydroxytryptamine-induced increase in left ventricular dP/dtmax does not suggest the presence of ventricular 5-HT4 receptors in the pig

Summary Although 5-hydroxytryptamine (5-HT) increases porcine atrial force and rate via 5-HT₄ receptors, its effect on left ventricular contractility is not known. Therefore, using the maximum rate of rise of left ventricular pressure (LVdP/dt_max) as an index of cardiac contractility, we have attempted to analyze the possible role of ventricular 5-HT₄ receptors in the anaesthetized pig. The full agonists at 5-HT₄ receptors, 5-HT and 5-methoxytryptamine (each 3, 10 and 30 g · kg^–1), and the -adrenoceptor agonist, isoprenaline (0.01, 0.03 and 0.1 g · kg^–1), increased heart rate, LVdP/dt_max and cardiac output. For a given degree of tachycardia, the increase in LVdP/dt_max by isoprenaline was substantially more than that observed with either 5-HT or 5-methoxytryptamine. The 5-HT₄ receptor partial agonist, renzapride (3, 10, 30, 100 and 300 g · kg^–1), also increased heart rate and LVdP/dt_max dose-dependently. When the heart was paced at 150 beats · min^–1, increases in LVdP/dt_max as well as cardiac output (except with the highest doses) by 5-HT, 5-methoxytryptamine and isoprenaline were clearly attenuated. However, the magnitude of attenuation of LVdP/dt_max responses by cardiac pacing was more marked in the case of 5-HT and 5-methoxytryptamine than with isoprenaline.The effects of renzapride (300 g · kg^–1) and tropisetron (0.3 and 3 mg · kg^–1) on increases in heart rate and LVdP/dt_max by 5-HT, 5-methoxytryptamine and isoprenaline were also studied. In the absence of atrial pacing, both renzapride and tropisetron (3 mg · kg^–1) effectively antagonized the responses to 5-HT and 5-methoxytryptamine; except for some decrease in the LVdP/dt_max response by tropisetron, the effect of isoprenaline remained essentially unchanged after the antagonists. During atrial pacing, renzapride significantly antagonized the responses to the first two doses of 5-HT, but the responses to the highest 5-HT dose and to 5-methoxytryptamine remained unaffected. Though, particularly after its higher dose, tropisetron reduced the responses to 5-HT and 5-methoxytryptamine, isoprenaline responses were also affected.The above results show that a significant part of the increase in LVdP/dt_max by 5-HT receptor agonists in the anaesthetized pig is a consequence of tachycardia elicited by these compounds via 5-HT₄ receptors. Since the increase in LVdP/dt_max, compared to tachycardia, was much less with 5-HT and 5-methoxytryptamine than with isoprenaline, and since the antagonism by renzapride and tropisetron against 5-HT and 5-methoxytryptamine during atrial pacing was relatively weaker and/or unspecific, it appears unlikely that the increase in LVdP/dt_max, during atria] pacing is mediated by ventricular 5-HT₄ receptors. This view is substantiated by our recent in vitro experiments where 5-HT (0.01 to 100 mol/l) failed to significantly increase contractions of porcine left ventricular trabeculae.Correspondence to P. R. Saxena at the above address     

Neuronal 5-HT receptors in the periphery

5-Hydroxytryptamine (5-HT) induces responses in neurones from all branches of the mammalian peripheral nervous system. Responses may be excitatory or inhibitory and are mediated through at least four distinct receptor sites.One receptor mediates excitation in motoneurones and preganglionic sympathetic neurones and can be designated a D (or possibly 5-HT₂) receptor since “classical” antagonists such as methysergide, metergoline or cinanserin are potent and selective antagonists at this site. A second receptor mediating neuronal excitation can be positively identified on the basis of susceptibility to blockade by small concentrations of 1αH,3α,5αH-tropan-3-yl-3,5-dichlorobenzoate (MDL 72222) and the weak or negligible affinity, relative to 5-HT, of certain agonists such as 5-methoxytryptamine. Such sites mediate depolarization of sympathetic and parasympathetic neurones and excitation of both the cell bodies and terminals of primary afferent fibres. A third receptor, mediating neuronal excitation, is the classical M-receptor of Gaddum and Picarelli, at this stage clearly identified only on postganglionic parasympathetic neurones of the guinea-pig myenteric plexus. These sites can be differentiated from other excitatory 5-HT receptors since MDL 72222 is neither potent nor selective as an antagonist and 5-methoxytryptamine approaches the potency of 5-HT as an agonist. (3α-Homotropanyl)-1-methyl-5-fluoro-indole-3-carboxylic acid is a potent, surmountable antagonist of 5-HT at the M-receptor of the ileum, but is non-selective.Neuronal inhibitory responses have been observed using electrophysiological techniques or by monitoring the decrease in depolarization-evoked release of transmitter in enteric, parasympathetic and sympathetic neurones. Largely negative results, using selective agonists and antagonists, allow the receptor(s) mediating inhibition to be clearly differentiated from the three neuronal excitatory receptors for 5-HT. Comparison of relative potencies of agonists suggests similarities with the 5-HT₁ recognition site of the central nervous system; no selective antagonist has yet emerged to permit their positive identification.     

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     

Pharmacology of cloned human 5-HT1D receptor-mediated functional responses in stably transfected rat C6-glial cell lines: further evidence differentiating human 5-HT1D and 5-HT1B receptors

This study was undertaken to investigate the pharmacology of human serotonin (5-HT)_1D receptor sites by measuring two functional cellular responses, inhibition of forskolin-stimulated cAMP formation and promotion of cell growth, using transfected rat C6-glial cell lines and a broad series of 5-HT receptor agonists. Stable and separate transfection of a pcDNA3 or pRcRSV plasmid, each containing a cloned human 5-HT_1D receptor gene, in rat C6-glial cells was confirmed with RT PCR of 5-HT_1D receptor mRNA and radioligand binding with [³H] 5-carboxamidotryptamine (5-CT) and [³H] sumatriptan. The 5-HT_1D receptor density was 350 and 1050 fmol/mg protein for the C6-glial/pcDNA3/5-HT_1D and C6-glial/pRcRSV/5-HT_1D cell line, and forskolin (100 M)-induced cAMP formation was inhibited by 45 and 78% in the presence of 1 M 5-HT, respectively. A comparison of the intrinsic agonist activities for sixteen 5-HT receptor ligands with their corresponding binding affinities for the human 5-HT_1D receptor site showed similar results for both cell lines with the exception of the partial agonist m-trifluoro-phenyl-piperazine (TFMPP). Three classes of compounds were observed: 1. efficacious agonists, such as 5-CT, 5-methoxytryptamine, 5-HT, sumatriptan, bufotenine, 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)1H-indole (RU 24,969), tryptamine and 8-hydroxy-2(di-n-propilamino)tetralin (8-OH-DPAT), with agonist potency close to their binding affinity; 2. the partial agonists metergoline, 7-trifluoromethyl-4(4-methyl-l-piperazinyl)-pyrolo-(1,2-a) quinoxaline (CGS 12066B), 1-naphthylpiperazine and 2-methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)-phenyl]-amide (GR 127,935) with marked intrinsic agonist activity but at concentrations higher than their binding affinity; and 3. the silent antagonists ritanserin, ketanserin and methiothepin, apparently free of intrinsic agonist activity, with antagonist potency close to their binding affinity. The cAMP data were further supported by the observed promotion of cell growth by stimulation of both transfected cell lines with sumatriptan under serum-free conditions; half-maximal stimulation was obtained at 4.4 nM (C6-glial/pcDNA3/5-HT_1D) fully in agreement with its EC₅₀-value (5.7 nM) for inhibition of cAMP formation. This growth promoting effect was antagonised by 1 M methiothepin and not observed in pcDNA3-plasmid-transfected and non-transfected C6-glial cells. A comparative study with a C6-glial/pcDNA3/5-HT_1B cell line expressing a similar amount of cloned human 5-HT_1B receptors (B _max: 360 fmol/mg protein) showed almost no intrinsic agonist activity for metergoline, 1-naphtylpiperazine and GR 127,935. Together with the 5-HT_1D receptor binding selectivity and antagonist activity of ketanserin and ritanserin, the findings define important pharmacological differences between cloned human 5-HT_1D and 5-HT_1B receptor sites.     

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.     

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.     

Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

1. It has been suggested that the tachycardic response to 5-hydroxytryptamine (5-HT) in the spinal-transected cat is mediated by ‘5-HT₁-like'' receptors since this effect, being mimicked by 5-carboxamidotryptamine (5-CT), is not modified by ketanserin or MDL 72222, but it is blocked by methiothepin, methysergide or mesulergine. The present study was set out to reanalyse this suggestion in terms of the IUPHAR 5-HT receptor classification schemes proposed in 1994 and 1996.
2. Intravenous (i.v.) bolus injections of the tryptamine derivatives, 5-CT (0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μg kg⁻¹), 5-HT (3, 10 and 30 μg kg⁻¹) and 5-methoxytryptamine (3, 10 and 30 μg kg⁻¹) as well as the atypical antipsychotic drug, clozapine (1000 and 3000 μg kg⁻¹) resulted in dose-dependent increases in heart rate, with a rank order of agonist potency of 5-CT >> 5-HT > 5-methoxytryptamine >> clozapine.
3. The tachycardic effects of 5-HT and 5-methoxytryptamine were dose-dependently antagonized by i.v. administration of lisuride (30 and 100 μg kg⁻¹), ergotamine (100 and 300 μg kg⁻¹) or mesulergine (100, 300 and 1000 μg kg⁻¹); the highest doses of these antagonists used also blocked the tachycardic effects of 5-CT. Clozapine (1000 and 3000 μg kg⁻¹) did not affect the 5-HT-induced tachycardia, but attenuated, with its highest dose, the responses to 5-methoxytryptamine and 5-CT. However, these doses of clozapine as well as the high doses of ergotamine (300 μg kg⁻¹) and mesulergine (300 and 1000 μg kg⁻¹) also attenuated the tachycardic effects of isoprenaline. In contrast, 5-HT-, 5-methoxytryptamine- and 5-CT-induced tachycardia were not significantly modified after i.v. administration of physiological saline (0.1 and 0.3 ml kg⁻¹), the 5-HT_1B/1D receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (500 μg kg⁻¹) or the 5-HT_3/4 receptor antagonist, tropisetron (3000 μg kg⁻¹).
4. Intravenous injections of the 5-HT₁ receptor agonists, sumatriptan (30, 100 and 300 μg kg⁻¹) and indorenate (300 and 1000 μg kg⁻¹) or the 5-HT₄ receptor (partial) agonist cisapride (300 and 1000 μg kg⁻¹) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses.
5. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht₇ receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT₇ receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht₇ gene product, the 5-HT₇ receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT₇ receptors.

     

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     

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     

The pharmacological properties of the presynaptic serotonin autoreceptor in the pig brain cortex conform to the 5-HT1D receptor subtype

Summary The effects of serotonin receptor agonists and antagonists on the electrically (3 Hz) evoked ³H overflow were determined on pig brain cortex slices preincubated with ³H-serotonin and superfused with physiological salt solution containing indalpine (an inhibitor of serotonin uptake) plus phentolamine. The potencies of the serotonin receptor agonists and antagonists were compared with their affinities for 5-HT_1A, 5-HT_1B, 5-HT_1c, and 5-HT_1D binding sites in pig or rat tissue membranes; in addition, the potencies of the agonists were compared to their potencies in inhibiting adenylate cyclase activity in membranes of calf substantia nigra. In the superfusion experiments on pig brain cortex slices the following rank orders of potencies were obtained: agonists, serotonin > 5-methoxytryptamine = 5-carboxamidotryptamine >R U 24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole) > SDZ 21009 (4(3-terbutylamino- 2-hydroxypropoxy)indol- 2-carbonic-acid-isopropylester) yohimbine cyanopindolol > 8-OHDPAT (8-hydroxy-2-(di-n-propylamino)tetralin) CGS 12066 B (7-trifluoromethyl-4(4-methyl-l-piperazinyl)-pyrrolo[1,2-a]quinoxaline); ipsapirone and urapidil were ineffective; antagonists (antagonism determined against 5methoxytryptamine as an agonist), metitepine > metergoline > mianserin. Propranolol, spiperone or mesulergine did not produce a shift of the concentration-response curve for 5-methoxytryptamine. The potencies of the serotonin receptor agonists in pig brain cortex slices were significantly correlated with their affinities for 5-HT_1c and 5-HT_1D binding sites in membranes of the pig choroid plexus and caudate nucleus, respectively, but not with their affinities for 5-HT_1A and 5-HT_1B sites in membranes of the cerebral cortex of pig and rat, respectively. The agonist potencies in decreasing ³H overflow were also significantly correlated with their potencies in inhibiting adenylate cylase activity in calf substantia nigra (i.e., a 5-HT_1D receptor-mediated effect). In conclusion, the pig brain cortical 5-HT autoreceptor probably belongs to the 5-HT_1D subtype. The involvement of 5-HT_1c recognition sites was excluded by the low potency of mianserin as an antagonist and, in particular, by the ineffectiveness of the 5-HT_1c receptor antagonist mesulergine.E. S. and M. G. were supported by grants of the Deutsche ForschungsgemeinschaftSend offprint requests to M. Göthert at the above address