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-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.     

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     

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-HT3 receptor ligands lack modulatory influence on acetycholine release in rat entorhinal cortex

Summary The objective of this study was to explore the role of 5-HT₃ receptors in modulating potassium (K⁺)-evoked release of [³H]-acetylcholine ([³H]-ACh) from superfused slices of rat entorhinal cortex previously loaded with [³H]-choline. Rat entorhinal cortices were cross-chopped into 300 m slices, superfused with oxygenated Krebs buffer containing 2.5 mmol/1 Ca²⁺ and stimulated with two consecutive exposures of 20 mmol/l K⁺ for 4 min (S₁ and S₂, respectively). Compounds were added 20 min before S₂ stimulation and remained in the superfusion buffer for the duration of the experiment. The S₂/S₁ ratio was then calculated.Stimulated release of [³H]-ACh was dependent on extracellular Ca²⁺ and K⁺ concentration. In Sprague Dawley rats, 2-methyl-5-HT (10^-9–10^-6 mol/l), in the presence of 1 mol/l ritanserin or 1 gmmol/l ondansetron, had no influence on K⁺-evoked release of [³H]-ACh. In slices prepared from Hooded Lister rats, 2 mol/l 5-HT but not 2-Me-5-HT significantly (P<0.05) inhibited K⁺-evoked [³H]-ACh release only 17% in the presence of 1 mol/l ritanserin. However, 2 mol/l 2-Me-5-HT plus 1 nmol/l ondansetron had no effect. High performance liquid chromatography coupled to electrochemical detection (HPLC-ECD) was used to monitor endogenous release of ACh in the above conditions to confirm data from the radiolabelled experiments. No significant inhibition or increase in K⁺-evoked ACh release was observed with either 5-HT₃ receptor agonists or antagonists. 2-Me-5-HT (10^–9 – 10^–5 mol/l) or 1-(m-chlorophenyl)-biguanide (10^–9 – 10^–5 mol/l), when added simultaneously at the S₂ stimulation, in the presence of 1 l/l methysergide, also showed no effect on [³H]ACh release.In entorhinal cortex slices from aged Wistar rats, neither 1-(m-chlorophenyl)-biguanide (2 or 10 ol/l) nor 2-Me-5-HT (2 mol/l) in combination with ritanserin (1 mol/l) or ondansetron (1 nmol/l) elicited any effect on K⁺-evoked [³H]-ACh release. However, release of [³H]-ACh was inhibited by carbachol (10 mol/l) and adenosine (10 mol/l). DuP 996 (3,3-bis(4- pyridinyl-methyl)-1-phenylindolin-2-one) (10^–7 – 10^–5 mol/l), a known releaser of ACh, markedly augmented K⁺-evoked [³H]-ACh release.These studies have failed to confirm the postulated role of 5-HT₃ receptors in modulating cortical ACh release in rat entorhinal cortex slices and suggest that a critical reexamination of the interaction of 5-HT₃ receptor and cortical cholinergic function needs to be addressed.Abbreviations 5-HT serotonin - ACh acetylcholine - HPLC-ECD high performance liquid chromatography - electrical chemical detection - EGTA ethylene glycol bis(-aminoethyl ether)-N,N-tetraacetic acid - 2-ME-5-HT 2-methyl-5-hydroxytryptamine - DuP 996 (3,3-bis(4pyrindinylmethyl)-1-phenylindolin-2-one) A preliminary report of this work was presented at the 1992 Federation of American Societies for Experimental Biology, April 6–9, Anaheim, California, USA (The FASEB J 6A1559) Correspondence to R. M. Johnson at the above address     

Mu and kappa opioid receptor modulation of 5-HT3 and NK-3 receptor-evoked release of acetylcholine from the guinea-pig ileum myenteric plexus

Summary The effects of three different opioid agonists on contractions and [³H]-acetylcholine (ACh) release evoked by 5-hydroxytryptamine₃ (5-HT₃) and neurokinin-3 (NK-3) receptor activation were examined in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. The selective mu ()-opioid receptor agonist (d-Ala²,NMe-Phe⁴,Gly-ol]-enkephalin) (DAMGO; 1 nM–100 nM) and the selective kappa ()-opioid receptor agonist U50488 (10 nM -1 M) inhibited contractile responses to 5-HT and to the selective NK-3 receptor agonist senktide, producing a concentration-related progressive flattening of their concentration-response curves. IC₅₀ estimates for DAMGO and U50488 were somewhat higher for inhibition of 5-HT-evoked as compared to senktide-evoked contractions, and overall lay in the range 6 nM – 51 nM. The selective delta ()-opioid receptor agonist [d-Pen^2,5]-enkephalin (DPDPE) inhibited contractile responses only at the highest concentration used (1 M). ³H-overflow from LMMP preparations preincubated with [³H]-choline was measured as an indicator of [³H]-ACh release. DAMGO (1 nM –100 nM) and U50488 (10 nM -1 M) inhibited the increases in release of [³H]-ACh evoked by 5-HT (10 M) and by senktide (10 nM) in a concentration-dependant manner. IC₅₀ estimates for DAMGO and U50488 were not significantly different for inhibition of 5-HT as compared to senktide-evoked increases in [³H]-ACh release and lay in the range 6 nM –23 nM. DPDPE again only inhibited these responses at the maximum concentration used (1 M). The inhibitory effects of DAMGO, U50488 and DPDPE on contractions and [³H]-ACh release evoked by 5-HT and senktide were completely reversed by naloxone (10 M).These results show that ACh release in the guinea-pig ileum evoked by 5-HT and senktide can be modulated to a similar extent by the opioid agonists DAMGO and U50488, but not by DPDPE. This suggests that the pathways of excitation for 5-HT₃ and NK-3 receptors converge at some level susceptible to opioid inhibition, which may be mediated by - and -, but not -, opioid receptors.     

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     

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     

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     

Further characterization of the putative 5-HT4 receptor mediating depolarization of the rat isolated vagus nerve

A putative 5-HT₄ receptor-mediated depolarization of the rat isolated vagus nerve has been studied using a grease-gap extracellular recording technique. Ondansetron (1 M) was used to block the predominant 5-HT₃ receptor mediated depolarization in this preparation and the effects of the 5-HT₄ receptor antagonists DAU 6285 (endo-8-methyl-8-azabicyclo [3.2.1] oct-3-y1-2,3-dihydro-6-methoxy-2-oxo-1H-benzimidazole-1-carboxylate HCl); 0.3, 1.0 or 3.0 M and SDZ 205–557 (2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino)-ethyl ester HCl); 0.1, 0.3 or 1.0 M were studied on the residual, ondansetron-resistant, component of the response. The effects of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) and of forskolin on the ondansetron-resistant response were also studied.Both DAU 6285 and SDZ 205–557 acted as competitive antagonists of the ondansetron-resistant response to 5-HT with pA₂ values of 6.8 (6.7–7.1, n = 12) and 7.1 (6.9–7.5, n = 12) respectively. The vagus nerve was depolarized by IBMX (100 M) or forskolin (10 M), the effects being similar to the maximum response to 5-HT. In the presence of IBMX (100 M) or forskolin (10 M) the ondansetron-resistant component of the response to 5-HT was enhanced and the 5-HT₃ receptor-mediated component reduced.These results with DAU 6285 and SDZ 205-557 are consistent with a 5-HT₄ receptor-mediated mechanism of the ondansetron-resistant depolarizing response to 5-HT.     

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     

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     

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     

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     

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.     

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.     

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.     

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.     

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

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