A 5-HT4-like receptor in human right atrium

Summary The effects of 5-carboxamidotryptamine (5-CT) and the gastrokinetic benzamides renzapride and cisapride on contractile force were investigated using isolated paced right atrial appendages from patients treated with -adrenoceptor blocking agents who were undergoing open heart surgery. These effects were compared to those of 5-hydroxytryptamine (5-HT). The effects of the drugs on atrial cyclic AMP levels and cyclic AMP-dependent protein kinase ratios were also investigated.The drugs all increased contractile force rank order of potency was 5-HT > renzapride > cisapride > 5-CT. The maximum responses, expressed as a fraction of the response to 200 mol/l (–)-isoprenaline, were 5-HT 0.6, 5-CT 0.6, renzapride 0.4 and cisapride 0.2, suggesting that the latter two are partial agonists. 5-HT, 5-CT and renzapride but not cisapride caused significant shortening of time to peak force. The effects of the four drugs were blocked by molar concentrations of ICS 205-930, suggesting an involvement of 5-HT4 receptors. As expected of partial agonists both renzapride and cisapride caused simple competitive antagonism of the positive inotropic effects of 5-HT The estimated equilibrium dissociation constants pK _p (–log mol/l K _p ) were 6.7 for renzapride and 6.2 for cisapride. 5-CT at concentrations up to 10 mol/l did not antagonise the effects of 5-HT. In the presence of (±)-propranolol 0.4 mol/I, 5-HT 10 mol/l, 5-CT 100 mol/I, renzapride 10 mol/l and cisapride 40 mol/I significantly increased cyclic AMP levels. 5-HT and renzapride also significantly increased cyclic AMP-dependent protein kinase activity, whereas 5-CT caused only marginal stimulation and cisapride was ineffective.The results confirm the existence of a human right atrial 5-HT receptor that is similar in nature to, but not necessarily identical with, the 5-HT₄ receptor of mouse embryonic colliculi neurones. The main difference is that in human right atrium the benzamides are less potent and efficacious than 5-HT and that cisapride is less potent and less efficacious than renzapride while in mouse embryonic colliculi these two benzamides are equipotent with and more efficacious agonists than 5-HT We designate the human right atrial 5-HT receptor 5-HT₄-like. The human right atrial 5-HT₄-like receptor greatly resembles porcine sinoatrial and left atrial 5-HT₄-like receptors and also appears to be similar to 5-HT₄-like receptors of guinea-pig ileum and rat oesophagus. Send offprint requests to A. J. Kaumann at the above address     

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     

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     

5-Hydroxytryptamine (5-HT) contracts the guinea-pig isolated iliac artery via 5-HT1-like and 5-HT2 receptors

Summary The characterization of 5-hydroxytryptamine (5-HT) receptors mediating contractions of the guinea-pig isolated iliac artery was studied when the basal tone was slightly increased by prostaglandin F₂ (PGF₂).In the presence of ketanserin (1 mol/l), 5-HT and several 5-HT receptor agonists induced contractile responses with the rank order of agonist potency: 5-HT = 5-carboxamidotryptamine (5-CT) = lysergol > ergometrine = methylergometrine > RU 24969 5-methoxytryptamine (5-MeOT) > methysergide > sumatriptan > tryptamine. Concentration-effect curves to the ergot alkaloids, lysergol, ergometrine, methylergometrine and methysergide, were biphasic.In the presence of ketanserin (1 mol/l), contractile responses to 5-HT, 5-CT, RU 24969, 5-MeOT, sumatriptan and tryptamine were antagonized by methiothepin (30 nmol/l) and flesinoxan (3 mol/l) with approximate pK_B values of 8.5–9.0 and 6.0–6.3, respectively. The first phase of contraction produced by the ergot alkaloids, lysergol, ergometrine, methylergometrine and methysergide, were blocked by methiothepin (30 nmol/l) and flesinoxan (3 mol/l), respectively, with approximate pK_B values about 8.4–8.7 and 6.2–6.4, respectively. The mechanism underlying the second phase of contraction remains to be established.Maximum responses of the concentration-effect curves to 5-HT (1 nmol/l-1 mol/l) were concentration-dependently depressed by ketanserin (1 nmol/l-1 mol) and spiperone (30 nmol/l-0.3 mol/l) and reached approximately 60% of the 5-HT maximum response in the presence of ketanserin (1 mol/l) and spiperone (0.1 mol/l), respectively. Agonist potency of 5-HT was not affected by the antagonists. 5-HT (1 nmol/l-1 mmol) produced biphasic concentration-effect curves (first phase: 1 nmol/l-1 gmol/l; second phase: 1 mol/l-1 mmol/l) in the presence of ketanserin (100 and 300 nmol/l), spiperone (100 and 300 nmol/l), (R)--methylketanserin (3 mol/l) and (S)--methylketanserin (10 nmol/l). Contractions mediating the first phase of the effects of 5-HT accounted for approximately 60% of the 5-HT maximum response and were resistant to blockade by the antagonists. pK_B values at the receptor mediating the second phase of the effects of 5-HT were 9.2–9.3 for ketanserin, 9.2–9.6 for spiperone, 10.5 for (S)--methylketanserin and 7.2 for (R)--methylketanserin.It is concluded that 5-HT contracts the guinea-pig isolated iliac artery via a mixture of 5-HT₁-like receptors and 5-HT₂ receptors. At low concentrations contractions are mediated via 5-HT₁-like receptors which accounted for approximately 60% of the 5-HT maximum response. At higher concentrations 5-HT-induced contractions are mediated via 5-HT₂ receptors.     

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     

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     

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     

Inhibition of noradrenaline release via presynaptic 5-HT1Da receptors in human atrium

In segments of human right atrial appendages preincubated with [3^H]noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, we determined the effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by transmural electrical stimulation (2 Hz).Tritium overflow was inhibited by 5-HT, 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-McOT), 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)-1H indole succinate (RU 24969) and sumatriptan. Yohimbine and oxymetazoline (in the presence of idazoxan) also inhibited tritium overflow. The inhibitory potency of the drugs was significantly correlated with their affinity for 5-HT_ID receptors in human brain and for cloned human 5-HT_1D and 5-HT_1D receptors, but not with their affinity for 5HT_1B, 5-HT_1E, 5-HT_1F, 5-HT_2A, 5-HT_2B, 5-HT_2C, 5-HT₃, 5-HT_5A, 5-HT_5B and 5-HT₇ receptors. The potency order 5-CT >5-HT >5-MeOT is opposite to the order of affinities reported for 5-HT₆ binding sites. The preferential 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetraline (up to 0.3 M) and the selective 5-HT₄ receptor agonist cisapride (up to 1 M) failed to inhibit tritium overflow. L-694,247, a potent 5-HT_ID receptor agonist, did not inhibit tritium overflow, but counteracted the inhibitory effect of 5-HT. Ketanserin at a concentration which should block 5-HT_1Da but not 5-HT_1D receptors and methiothepin at a concentration which may be assumed to block both 5-HT_1D and 5-HT_1D receptors antagonized the inhibitory effect of 5-HT. Propranolol and ondansetron did not modify the 5-HT-induced inhibition of release. In conclusion, noradrenaline release in human right atrial appendages is inhibited via 5-HT receptors which are located on the noradrenergic axon terminals. These inhibitory presynaptic 5-HT receptors belong to the 5-HT_ID subfamily. The ability of ketanserin to antagonize the inhibitory effect induced by activation of these receptors suggests that they can be subclassified as 5-HT_1D.     

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     

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 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     

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     

O-Acylated lysergol and dihydrolysergol-I derivatives as competitive antagonists of 5-HT at 5-HT2 receptors of rat tail artery

Summary Twelve ergolines (O-Ayated lysergol and dihydrolysergol-I derivatives) were synthesized to study their antagonism of 5-HT responses in comparison with methysergide and LY 53857 [6-methyl-l-(1-methylethyl)-8-ergoline carboxylic acid 2-hydroxy-l-methyl-propylester hydrogen maleate] in cylindrical segments of the isolated rat tail artery. With regard to (9.10-didehydro-6-methyl-8-ergoline)methyl R,S-2-methylbutyrate, the most potent new ergoline derivative, we examined the phenomenon of insurmountable antagonism to 5-HT by methysergide.O-Acylated lysergol and dihydrolysergol-I derivatives competitively antagonized 5-HT-induced contractions with calculated pA₂. values of 7.30 ± 0.42 for the weakest and 8.42 ± 0.35 for the most potent ergoline derivative in this series. N¹-isopropyl substitution did not generally enhance 5-HT₂ receptor affinities but lowered affinities for ₁ adrenoceptors in rat aorta. Methysergide and LY 53857 were insurmountable, antagonists of 5-HT in rat tail artery.Preincubation with (9.10-didehydro-6-methyl-8-ergoline)methyl R,S-2-methylbutyrate (1 mol/l) partially prevented the depression of 5-HT-induced contractions caused by methysergide (1–10 nmol/l). Methysergide (100 nmol/l) abolished the protective effect of (9.10-didehydro-6-methyl-8-ergoline)methyl R,S-2-methylbutyrate. (9.10-Didehydro-6-methyl-8-ergoline)methyl R,S-2-methylbutyrate (1 mol/l), concomitantly incubated with methysergide (30 nmol/l), partially restored the maximum response to 5-HT that had been depressed by methysergide (30 nmol/l). Partial restoration could not be mimicked by washout of methysergide. In view of this result, it is suggested that insurmountable antagonism by methysergide of 5-HT responses in rat tail artery is due to allosteric modulation of 5-HT₂ receptors rather than pseudoirreversible inhibition. Send offprint requests to H. Pertz at the above address     

Actions of non-peptide ergot alkaloids at 5-HT1-like and 5-HT2 receptors mediating vascular smooth muscle contraction

Summary This report describes the actions of the non-peptide ergot alkaloids methysergide, methylergometrine and ergometrine at two types of 5-HT receptor mediating vascular contraction; the well established 5-HT₂ receptor in rabbit aorta and a non-5-HT₂ receptor in rabbit saphenous vein which resembles the 5-HT₁-like receptor in dog saphenous vein.In the rabbit aorta ergometrine (1 mol/l) and methylergometrine (0.3 mol/l), but not methysergide, produced small contractions (14% and 7% respectively of the maximal response to 5-HT). This contraction was not related to activation of 5-HT₂ receptors since it was resistant to blockade by ketanserin (0.3 mol/l). When examined as antagonists of 5-HT-induced contractions of rabbit aorta, each ergot displayed nanomolar affinity at the 5-HT₂ receptor but only methysergide behaved as a simple competitive antagonist (pK_B = 8.25). Methylergometrine and ergometrine produced surmountable blockade which was accompanied by a non-parallel displacement of the 5-HT concentration-effect curves. The selective 5-HT₁-like receptor agonist GR43175 ( 30 mol/l) was devoid of affinity at the 5-HT₂ receptor in rabbit aorta.In the rabbit saphenous vein each of the ergots produced concentration-dependent contractions which resulted in overtly biphasic concentration-effect curves. Only the first phase of contraction mimicked the effects of 5-HT and GR43175 since contractions were not blocked by MDL 72222 (1 mol/l), but were surmountably antagonised by methiothepin (10 nmol/1), ketanserin (0.3 mol/l) and spiperone (0.3 mol/l). These results are expected for interactions at the 5-HT₁-like receptor in this preparation (Martin and MacLennan 1990). The mechanism(s) underlying the second phase of contraction with the ergots remains to be established. Receptor inactivation studies using the alkylating agent benextramine tetrahydrochloride enabled each agonists' affinity and efficacy at the 5-HT₁-like receptor to be estimated. Affinity estimates (pK_A) decreased in the order: methylergo metrine (7.79), ergometrine (7.75), 5-HT (7.19), methysergide (6.76), GR43175 (6.20), whereas efficacies () decreased in the order: 5-HT (3.28), methylergometrine (2.24), GR43175 (2.14), ergometrine (1.94), methysergide (0.99). Of particular interest, methysergide was significantly lower in affinity and efficacy than its primary demethylated metabolite methylergometrine. Evidently, at the 5-HT₁-like receptor mediating vascular contraction the ergots ergometrine and methylergometrine are both higher in affinity than, and comparable in efficacy to, the natural receptor agonist 5-HT. This contrasts with their actions at the 5-HT₂ receptor in rabbit aorta where they demonstrated a higher affinity but much lower intrinsic efficacy than 5-HT. These results favour the view that vascular contraction induced by these ergots is more likely to be mediated by 5-HT₁-like, rather than 5-HT₂ receptors. These results are discussed in relation to the therapeutic applications of these ergots, particularly in obstetrics and in migraine, and to their utility as diagnostic agents in patients with Prinzmetal's variant form of angina.Send offprint requests to S. J. MacLennan at the above address     

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.     

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     

MDL 72222: a potent and highly selective antagonist at neuronal 5-hydroxytryptamine receptors

Summary The properties of MDL 72222 (1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate), a novel compound with potent and selective blocking actions at certain excitatory 5-hydroxytryptamine (5-HT) receptors on mammalian peripheral neurones, are described.On the rabbit isolated heart, MDL 72222 was a potent antagonist of responses mediated through the receptors for 5-HT present on the terminal sympathetic fibres. The threshold for antagonism was approximately 0.1 nM and the negative logarithm of the molar concentration of MDL 72222 which reduced the chronotropic response of the isolated rabbit heart to twice an ED₅₀ of 5-HT to that of the ED₅₀ was 9.27. MDL 72222 was also highly selective since responses to the nicotine receptor agonist, dimethylphenylpiperazinum iodine (DMPP), were inhibited only at concentrations more than 1000 times those necessary to inhibit 5-HT.In the anaesthetised rat, MDL 72222 produced marked blockade of the Bezold-Jarisch effect of 5-HT. Again, inhibition was selective since much higher doses of MDL 72222 failed to alter the response to electrical stimulation of the efferent vagus nerves. In contrast, MDL 72222 proved only a weak and essentially non-selective antagonist of responses mediated by the 5-HT M-receptor present on the cholinergic nerves of the guinea-pig ileum.MDL 72222 does not block smooth muscle contractile responses elicited by oxytocin or mediated through 5-HT D-receptors, muscarinic or nicotinic cholinoceptors or histamine H₁-receptors except at relatively high concentrations. Similarly, in a number of radioligand binding assays carried out using brain tissue membranes, the displacing effects of MDL 72222 were absent or weak at sites identifying compounds with activity at ₁, ₂ or -adrenoceptors, 5-HT₁ or 5-HT₂ receptors, benzodiazepine receptors or histamine H₁-receptors.MDL 72222 is the first reported selective and potent antagonist of responses mediated through the 5-HT receptors present on the terminal sympathetic neurones of the rabbit heart and on the neurones subserving the afferent limb of the Bezold-Jarisch reflex. The compound should provide a useful means by which responses mediated through such sites can be distinguished.     

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.     

The depolarizing action of 5-hydroxytryptamine on rabbit isolated preganglionic cervical sympathetic nerves

Summary This study describes a depolarizing action of 5-hydroxytryptamine (5-HT) on rabbit isolated preganglionic cervical sympathetic nerves using an extracellular recording technique. From cumulative concentration-response curves for 5-HT (1 mol/1-1 mmol/1), the mean maximal depolarization was shown to be 277 ± 32 V and EC₅₀ was 9.4 mol/l(6.5–13.6 mol/l, geometric mean, 95% confidence limits, n = 42). The responses to 5-HT displayed marked tachyphylaxis. When cumulative concentration-response curves to 5-HT and 2-methyl-5-HT were determined in the same preparations (n = 4), the mean maximal response to 5-HT was 519 ± 167 V, EC₅₀ 32.2 mol/l (8.8–118 mol/l) and the mean maximal response to 2-methyl-5-HT was 317 ± 63 V, EC₅₀ 35.1 mol/l (12.9–95.5 mol/l, geometric means, 95 % confidence limits). The action of selective 5-HT antagonists was tested on repeated cumulative concentration-response curves to 5-HT. Neither methiothepin (0.1–1 mol/l, _n = 3) nor ketanserin (0.1–1 mol/l, n = 3) had an action on 5-HT responses. The selective 5-HT₃ antagonists MDL 72222, ICS 205-930 and SDZ 206–830 were all potent antagonists of the 5-HT depolarizations. The action of these antagonists was quantified by determining the apparent pA₂ from the dose ratios and a Schild plot. For MDL 72222 (1 nmol/1-0.1 mol/l), the apparent pA₂ was 9.1 ± 0.1 (n = 12), Schild plot: 9.2; for ICS 205–930 (0.1 nmol/l–3 nmol/1), the apparent pA₂ was 10.4 ± 0.1 (n = 11), Schild plot 10.3, and for SDZ 206–830 (0.03 nmol/l-1 nmol/1), the apparent pA₂ was 11.2 ± 0.1 (n = 12), Schild plot 11.2. 5-HT depolarizations were unaffected by hexamethonium (0.5 mmol/1). 5-HT depolarizations were reduced by superfusion with both Na-free (42 ± 8% of controls, n = 4) and Na/Ca-free media (35 ± 7% of controls, n = 4). It is concluded that 5-HT depolarizations of rabbit preganglionic cervical sympathetic nerve are mediated by 5-HT₃ receptors. The data with selective 5-HT₃ receptor antagonists suggest that the receptor profile may be more like that for the 5-HT₃ receptor on the terminals of sympathetic nerves than that for the 5-HT₃ receptor on the soma of superior cervical ganglion cells or on vagal afferent neurones. Send offprint requests to D. I. Wallis at the above address