Benzimidazolones and renzapride facilitate acetylcholine release from guinea-pig myenteric plexus via 5-HT4 receptors

The effects of the 5-HT₄ receptor agonists BIMU 8, BIMU 1, renzapride and of the 5-HT_1p receptor agonist 5-hydroxyindalpine on basal and electrically evoked outflow of tritium were studied in guinea-pig longitudinal muscle myenteric plexus preparations preincubated with [³H]choline. Muscle contractions were recorded simultaneously.BIMU 8 caused a calcium dependent and tetrodotoxin sensitive increase in basal [³H]outflow that was assumed to represent release of [³H]acetylcholine. In addition, BIMU 8 enhanced the release of [³H]acetylcholine and twitch contractions evoked by submaximal electrical stimulation. Ondansetron (1 mol/l) did not change the effects of BIMU 8, but DAU 6285 and tropisetron (each 1 mol/l) competitively antagonized the various facilitatory effects of BIMU 8 with pA₂ values of 7.0–7.2 (DAU 6285) and 7.0–7.3 (tropisetron). The phosphodiesterase inhibitors IBMX and rolipram did not increase the effects of BIMU 8. BIMU 1 and renzapride also concentration-dependently increased basal release of acetylcholine, and release and contractions caused by submaximal stimulation. The effects of BIMU 1 and renzapride were competitively antagonized by 1 mol/l tropisetron (pA₂ 6.6–7.1). The EC₅₀ values for the increase in the evoked [³H]acetylcholine release and contractions were closely similar. 5-Hydroxyindalpine did not change basal release and slightly inhibited the evoked release of [³H]acetylcholine. Release of acetylcholine and contractions elicited by submaximal stimulation were strongly inhibited by ( + )-tubocurarine which indicates that nicotinic ganglionic transmission is involved in this kind of release.The results suggest that BIMU 8, BIMU 1 and renzapride stimulate 5-HT₄ receptors at cholinergic interneurones and thereby facilitate nicotinic ganglionic transmission in the myenteric plexus. Cyclic AMP is probably not involved in the 5-HT₄ receptor mediated facilitation of acetylcholine release.     

Characterization of a novel 5-HT4 receptor antagonist of the azabicycloalkyl benzimidazolone class: DAU 6285

Summary Three chemical classes of serotonin 5-HT₄ receptor agonists have been identified so far: 5-substituted indoles (e.g. 5-HT), benzamides (e.g. renzapride) and benzimidazolones (e.g. BIMU 8). In a search for 5-HT₄ receptor antagonists, we have discovered that the benzimidazolone derivative DAU 6285 (for structure see text), is 3–5 times more potent than tropisetron in blocking 5-HT, renzapride and BIMU 8 induced stimulation of adenylate cyclase activity in mouse embryo colliculi neurons. Schild plot analysis yielded K_i values of 220, 181 and 255 nmol/l, respectively. In addition, DAU 6285 showed poor activity as a 5-HT₃ receptor ligand with respect to tropisetron, as demonstrated by in vitro binding studies (K_i, 322 vs 2.8 nmol/l) and by its antagonistic activity in the Bezold-Jarisch reflex test (ID₅₀, 231 vs 0.5 g/kg, i.v.). No significant binding (K_i>10 mol/l) of DAU 6285 to serotonergic 5-HT_1A, 5-HT_1B, 5-HT_1C, 5-HT_1D, and 5-HT₂ receptors as well as to adrenergic ₁, ₂, dopaminergic D₁, D₂ or muscarinic M₁–M₃ receptor subtypes was found. The data indicate that DAU 6285 has a somewhat higher affinity than tropisetron for 5-HT₄ receptors, a property confirmed in functional tests, and much lower affinity than tropisetron for 5-HT₃ receptors. The compound represents a new interesting tool for investigating the pharmacological and physiological properties of 5-HT₄ receptors. Send offprint requests to A. Dumuis at the above address     

Cisplatin increases the release of 5-hydroxytryptamine (5-HT) from the isolated vascularly perfused small intestine of the guinea-pig: Involvement of 5-HT3 receptors

Summary Isolated segments of the guinea-pig small intestine were vascularly perfused and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) into the portal venous effluent determined by high pressure liquid chromatography with electrochemical detection. Release of acetylcholine from isolated superfused intestinal segments was determined as outflow of [³H]radioactivity from preparations preincubated with [³H]choline. Cisplatin (3 M) increased the outflow of 5-HT and 5-HIAA by about 90%. At 30 and 100 M cisplatin decreased the outflow of 5-HT and its metabolite by 40%–50%. The stimulatory effect of cisplatin was consistently observed only when the bicarbonate-phosphate buffer of the Tyrode's solution was replaced by HEPES-buffer. The stimulatory effect of cisplatin was abolished in the absence of extracellular calcium or presence of tetrodotoxin (1 M). The stimulatory effect of cisplatin was also prevented by hexamethonium (100 M) or scopolamine (100 nM). The 5-HT₃ receptor antagonists ondansetron and ICS 205-930 in concentrations as low as 1 pM also abolished the stimulatory effect of cisplatin. The 5-HT₃ receptor antagonist MDL 72222 prevented the stimulatory effect of cisplatin only at a concentration of 1 M. None of the 5-HT₃ receptor antagonists alone significantly altered the outflow of 5-HT and 5-HIAA.Cisplatin (3 M) enhanced the outflow of [³H]radioactivity from intestinal segments and caused longitudinal muscle contractions that were abolished by 100 nM scopolamine.In conclusion, cisplatin, at concentrations which occur during anti-cancer therapy in humans and induce emesis, increases the release of 5-HT from the enterochromaffin cells of the small intestine of the guinea-pig. This effect of cisplatin is mediated by a cascade of events which involves release of acetylcholine and stimulation of 5-HT₃ receptors. Send offprint requests to H. Schwörer at his present 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-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     

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     

Inhibition by 5-HT3 receptor antagonists of release of cholecystokinin-like immunoreactivity from the frontal cortex of freely moving rats

Summary Effects of the 5-HT₃ receptor antagonists, ondansetron and tropisetron, on the release of cholecystokipin-like immunoreactivity (CCK-LI) in rat frontal cortex were investigated in conscious, unrestrained rats using intracerebral microdialysis. The release of CCK-LI was augmented by perfusion with 100 g/ml veratrine and was fully Ca²⁺-dependent and tetrodotoxin-sensitive. Ondansetron and tropisetron, each at 0.1–1 mol/l, decreased concentration-dependently the veratrine-evoked efflux of CCK-LI. The reduction of CCK-LI output was approximately 30%u when the antagonists were infused at 0.1 mol/l.The data suggest that 5-HT₃ receptor antagonists prevent the release of CCK evoked by endogenous 5-hydroxytryptamine. These drugs may thus represent a novel therapeutic approach in disease states, like anxiety, in which an inappropriately high release of brain CCK or 5-hydroxytryptamine seems to be involved.Correspondence to M. Raiteri at the above address     

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     

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     

Piglet sinoatrial 5-HT receptors resemble human atrial 5-HT4-like receptors

Summary 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT) and the gastrointestinal kinetic benzamides renzapride and cisapride caused tachycardia in spontaneously beating right atria of piglet in the presence of 400 nmol/l(±)-propranolol and 6 mol/l cocaine. The maximum tachycardia caused by agonists, compared to that evoked by 200 mol/l(–)-isoprenaline, was 63% for 5-HT, 50% for 5-CT, 50% for renzapride and 28% for cisapride. The rank order of potency was 5-HT > renzapride > cisapride > 5-CT. The effects of the agonists, but not those of (–)-isoprenaline, were antagonised by 3-tropanyl-1H-indole-3-carboxylic acid (ICS 205930); the pK_B of ICS 205930 (vs 5-HT) was 6.9. These characteristics suggest that piglet sinoatrial 5-HT receptors are similar to so-called 5-HT₄ receptors previously described in mouse colliculi neurons. Piglet sinoatrial 5-HT₄-like receptors resemble the human atrial 5-HT receptors that mediate positive isotropic effects of 5-HT.Send of fprint requests to A. J. Kaumann at the above address     

Functional 5-HT receptors in human occipital artery

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

5-Hydroxytryptamine-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 (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.     

Autoreceptors can modulate 5-hydroxytryptamine release from porcine and human small intestine in vitro

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists were studied on the release of 5-HT from enterochromaffin cells of incubated strips of porcine and human small intestine. Tetrodotoxin (1 μmol/l) was present in the incubation medium to block neuronally mediated inputs to the enterochromaffin cells. The 5-HT_1A receptor agonist (+)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT, 1 μmol/l) and the 5-HT₂ receptor agonist α-methyl-5-HT (1 μmol/l) increased 5-HT release by 40% in about 60% of the human preparations.These agonists showed no effect on 5-HT release in porcine intestinal mucosa. The 5-HT₃ receptor agonist 2-methyl-5-HT (3–100 μmol/l) increased 5-HT release in both species by 60% (pig) and 90% (man), respectively. These stimulatory effects were antagonized by tropisetron (10 nmol/l). The 5-HT₄ receptor agonist 5-methoxytryptamine (0.3–30 μmol/l) reduced 5-HT release by about 50% in both species. These inhibitory effects were antagonized by tropisetron (3 μmol/l). The basal outflow of 5-HT from the intestinal mucosa was not significantly affected by tropisetron (10 nmol/l; 3 μmol/l). The specific 5-HT₄ receptor antagonist GR 113808 ((1-[2-methylsulphonyl)amino]ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate) (0.1 μmol/l) which by itself did not significantly affect 5-HT release from human duodenal specimens blocked the inhibitory effect of 5-methoxytryptamine (30 μmol/l). These findings indicate that stimulatory 5-HT₃ and inhibitory 5-HT₄ receptors are present on enterochromaffin cells of the porcine and human intestinal mucosa. Under the present experimental conditions endogenous 5-HT does not significantly activate these receptors. Stimulatory 5-HT_1A and 5-HT₂ receptors may additionally be present on human enterochromaffin cells. Received: 19 September 1997/ Accepted: 29 January 1998     

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-HT1C receptor-mediated stimulation of inositol phosphate production in pig choroid plexus

Summary 1) 5-HT (5-hydroxytryptamine, serotonin) induces inositol phosphate production in a pig choroid plexus preparation. This effect has been pharmacologically characterized and the data compared to those obtained from radioligand binding studies performed with [³H]mesulergine to 5-HT_1C sites in pig choroid plexus membranes. 2) The rank order of potency of agonists stimulating inositol phosphate production was: -methyl-5-HT > 1-methyl-5-HT > DOI > bufotenine = SKF 83566 = 5-HT > 5-MeO-DMT > 5-MeOT = RU 24969> SCH 23390> 5-CT. 8-OH-DPAT was virtually devoid of activity at 100 mol/l. 3) The increase in inositol phosphate production induced by 5-HT and other agonists was surmountably antagonised by mesulergine, ketanserin and spiperone with pK_B values of 8.7, 6.7 and 5.3, respectively. 4) The rank order of potency of antagonists was: metergoline > mesulergine > LY 53857 > ritanserin > methiothepin > mianserin > cyproheptadine > pirenperone > cinanserin > ketanserin > spiperone. The following antagonists were virtually devoid of activity at 100 mol/l; pindolol, 21-009 and yohimbine. 5) The results obtained both with agonists and antagonists strongly support the view that 5-HT_1C receptors mediate agonist induced production of inositol phosphates in pig choroid plexus. This is illustrated by the close similarity between 5-HT_1C binding and stimulation of inositol phospholipid turnover in this preparation. 6) The present data also show that compounds believed to be selective for dopamine D1 receptors (SKF 83566, SCH 23390) or 5-HT₂ receptors (DOI, -methyl-5-HT, LY 53857, ritanserin, cyproheptadine) also interact with 5-HT_1C receptors. 7) A case can be made for the 5-HT_1C receptor, with its similarities to the 5-HT₂ receptor in terms of pharmacology and second messenger coupling, being a 5-HT₂ receptor subtype.These data have been presented in part at the Spring Meeting of the German Pharmacological Society, March 1987 (Hoyer et al. 1987) Send offprint requests to D. Hoyer at the above address     

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

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

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     

BIMT 17, a 5-HT1A receptor agonist/5-HT2A receptor antagonist,directly activates postsynaptic 5-HT inhibitory responses in the rat cerebral cortex

BIMT 17 (1-[2-[4-(3-trifluoromethyl phenyl) piperazin-1-yl] ethyl] benzimidazol- [1H]-2-one), a 5-HT_1A receptor agonist/5-HT_2A receptor antagonist (see Borsini et al., accompanying paper), in a dose range of 1–10 mg/kg i.v., dose-dependently inhibited the electrical activity of rat medial prefronto-cortical neurons, whereas buspirone, in a dose range of 0.1–1000 g/kg, increased it. 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and 1-[2-(2-thenoylamino)ethyl]-4[1-(7-methoxynaphthyl)] piperazine (S 14671) presented biphasic patterns of response; they increased electrical activity at doses in the range of 0.1–10 g/kg and 0.1–3 g/kg i.v. respectively, and reduced it at higher doses, 30–300 g/kg and 10–30 g/kg i.v., respectively.The inhibitory effect of BIMT 17 on the firing rate of neurons in the frontal cortex was antagonized by the 5-HT_1A antagonists tertatolol and WAY 100135, and was still present after destruction of serotonin (5-HT) containing neuronal endings by the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT; 150 g/rat, given intraventricularly), which reduced the cortical 5-HT content by 85%. This destruction of 5-HT neurons, while suppressing the ability of 8-OH-DPAT to inhibit the firing rate at high doses, did not change the excitatory action of this compound at low doses. The addition of ritanserin, a 5-HT2A receptor antagonist, potentiated both the excitatory and inhibitory effects of 8-OHDPAT on neuronal electrical activity. Direct microiontophoretic application (100 nA/20 s) of 5-HT and BIMT 17, but not that of 8-OH-DPAT, onto medial prefronto-cortical neurons, decreased the firing rate of these neurons.These findings suggest that BIMT 17 directly inhibits the electrical activity of medial prefronto-cortical neurons through its dual mode of receptor interaction.     

Blockade of human and porcine myocardial 5-HT4 receptors by SB 203186

We investigated the blockade of the positive inotropic effects of 5-hydroxytryptamine (5-HT) by SB 203 186 (piperidinoethyl-indole-3-carboxylate hydrochloride) and its affinity for 5-HT₄ receptors of human right atrium and piglet left atrium. We also compared the blocking effects of SB 203 186 against 5-HT-evoked tachycardia in anaesthetised adult Yucatan minipigs as well as new-born Camborough piglets.SB 203 186 caused competitive antagonism of the positive inotropic effects of 5-HT in electrically paced atrial preparations of man (pK _B = 8.9) and piglet (pK _B = 8.5) at concentrations (up to 0.3 mo]/l) which were devoid of depressant or stimulant effects. The affinity of SB 203 186 for atrial 5-HT₄ receptors was 30–160 times higher than that of tropisetron. 5-HT caused tachycardia with similar potency and efficacy in Yucatan minipigs and new-born Camborough piglets. SB 203 186 (0.1–3 mg/kg, i.v.) surmountably antagonised 5-HT-evoked tachycardia in anaesthetised Yucatan minipigs or new-born Camborough piglets with similar potency. The blocking potency of SB 203 186 in Yucatan minipigs was 17 times higher than that of tropisetron. Intraduodenally administered SB 203 186 (0.3–3 mg/kg) to new-born Camborough piglets produced blockade of 5-HT-evoked tachycardia which was maximal after 20 min and lasted for more than 3 h with 0.3 mg/kg. The antagonism produced by the SB 203 186 administration in new-born Camborough piglets was dose-related and threefold greater through the intravenous route than through the intraduodenal route.We conclude that SB 203 186 is an antagonist with nanomolar affinity for both human and porcine atrial 5-HT₄ receptor. The in vivo results demonstrate that the sinoatrial 5-HT₄ receptors function is similar in new-born Camborough piglets and adult Yucatan minipigs. Both porcine breeds are valid models for human atrial 5-HT₄ receptors as demonstrated with the antagonist SB 203186.