NK1- and NK3-receptor mediated inhibition of 5-hydroxytryptamine release from the vascularly perfused small intestine of the guinea-pig

The effects of tachykinins on the spontaneous release of 5-hydroxytryptamine (5-HT) from the enterochromaffin cells into the portal circulation was investigated in vitro using the vascularly perfused isolated guinea-pig small intestine. 5-HT was determined by HPLC with electrochemical detection. Test substances were applied intraarterially. Substance P (SP) caused a concentration-dependent decrease in 5-HT outflow with an EC50 of 50pmol/l. Similarly, the selective NK1 receptor agonist SP methyl ester (1nmol/l) significantly inhibited 5-HT outflow (to 51±3%). When tetrodotoxin (1μmol/l) was added to the arterial perfusion medium, the inhibition by SP of 5-HT outflow was not affected. The selective NK1 receptor antagonist CP 99994 [(+)-(2S,3S)-3-(2-methoxyben-zylamino)-2-phenylpiperidine] (0.1μmol/l) prevented the inhibitory effect of SP (0.1μmol/l). Neither GR 94800 (PhCO-Ala-Ala-DTrp-Phe-DPro-Pro-NleNH₂) (0.1μmol/l) nor SR 142801 [(S)-(N)-(1-(3-(1-benzoyl-3-(3, 4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenyl-piperidin-4-yl)-N-methylacetamide] (10nmol/l), which are selective NK2 and NK3 receptor antagonists, changed the SP-mediated inhibition. The selektive NK3 receptor agonist senktide (10nmol/l) also decreased the 5-HT outflow (to 57±5%). This inhibition was prevented by SR 142801 (10nmol/l) and by tetrodotoxin. CP 99994 (0.1μmol/l) significantly antagonized the senktide-mediated inhibition of 5-HT outflow. The outflow of 5-HT was unaffected when CP 99994, GR 94800 or SR 142801 alone were added to the perfusion medium. It is concluded that the release of 5-HT from enterochromaffin cells is directly inhibited by NK1 receptors, and indirectly by neuronal NK3 receptors whose stimulation leads to the release of SP. Received: 11 June 1997 / Accepted: 28 July 1997     

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     

Effects of the benzodiazepine receptor agonist midazolam and antagonist flumazenil on 5-hydroxytryptamine release from guinea-pig intestine in vitro

Summary Isolated segments of the guinea-pig small intestine and the guinea-pig stomach were vascularly perfused and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxy-indoleacetic acid into the portal venous effluent determined by high pressure liquid chromatography with electrochemical detection. Test substances were applied intraarterially.The benzodiazepine receptor agonist, midazolam, concentration-dependently increased (by 58%, at 1 nmol/l) and decreased (by 32%, at 100 nmol/l) the release of 5-HT from small intestine preparations. Both effects were blocked by the benzodiazepine receptor antagonist flumazenil (10 nmol/l) The stimulatory effect of midazolam was also abolished in the presence of tetrodotoxin (1 mol/l) or scopolamine (100 nmol/l). In the absence of tetrodotoxin, flumazenil (10 nmol/l) alone decreased the release of 5-HT from the small intestine by 41%, but it increased the release of 5-HT by 50% in the presence of tetrodotoxin. Both effects of flumazenil were abolished in the presence of bicuculline (50 mol/l). In the absence of tetrodotoxin, flumazenil (10 nmol/l) decreased also the release of 5-HT and its metabolite from the perfused stomach by about 40%, whereas midazolam (1 nmol/l) caused an increase by about 60%. In conclusion, benzodiazepine receptors modulate the previously described intrinsic GABAergic regulation of 5-HT release from enterochromaffm cells in the guinea-pig intestine. It is suggested that an endogenous benzodiazepine-like substance of non-neuronal origin is present in the small intestine and stomach of the guinea-pig. Send offprint requests to K. Racke at the above address     

Effect of vasoactive intestinal polypeptide on the release of serotonin from the in vitro vascularly perfused small intestine of guinea pig

Summary Isolated segments of the guinea pig small intestine were vascularly perfused and the release of endogenous serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) into the portal vein was measured. All test substances were intraarterially perfused. Vasoactive intestinal polypeptide (VIP, 1 pmol/l — 100 nmol/1) inhibited the spontaneous release of 5-HT and 5-HIAA. The maximal inhibitory effect (about 60%) was seen at 100 pmol/1. The effect of VIP on the spontaneous release of 5-HT and 5-HIAA was not changed in the presence of 1 ol/l tetrodotoxin (TTX).Raising intraluminal pressure by 500 Pa for 5 min increased the release of 5-HT and 5-HIAA by about 25%. Raising the intraluminal pressure in the presence of VIP reduced the release of 5-HT and 5-HIAA by about 75%. In the presence of TTX (1 gmol/l), raising intraluminal pressure also caused a decrease of the release of 5-HT and 5-HIAA which was unaffected by the additional presence of VIP. The fluid volume expelled during peristaltic activity was not affected by VIP, but reduced by about 90% in the presence of TTX.In conclusion the results demonstrate a direct inhibitory effect of VIP on the release of 5-HT from the enterochromaffin cells. In addition, VIP appears to interfere with the neuronally mediated stimulation of 5-HT release during peristaltic activity. Send offprint request to H. Schwörer 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     

Cholinergic modulation of the release of 5-hydroxytryptamine from the guinea pig ileum

Summary Isolated segements of the guinea pig ileum were vascularly perfused and the release of 5-HT and its metabolite 5-HIAA into the portal venous effluent determined by HPLC with electrochemical detection. Test substances were applied via the arterial perfusion medium. Oxotremorine inhibited concentration-dependently the release of 5-HT and 5-HIAA (by 47% at 1 mol/l). Scopolamine (0.1 mol/1) did not affect the release of 5-HT and 5-HIAA, but antagonized the effect of oxotremorine. In the presence of TTX (1 mol/1) oxotremorine (1 pmol/1) increased the release of 5-HT by 150% and that of 5-HIAA by 220%. This increase was completely blocked by scopolamine. Hexamethonium (100 pmol/1) and TTX (1 pmol/1) reduced the release of 5-HT by 32 and 40%, respectively. DMPP (10 pmol/1) increased the release of 5-HT by 57%, and this effect was prevented by hexamethonium. Neither DMPP nor hexamethonium significantly affected the release of 5-HIAA. The enhancing effect of DMPP on 5-HT release was increased and prolonged in the presence of TTX or scopolamine. Nicotine (1, 10 or 30 mol/l) alone did not cause a consistent increase in the release of 5-HT. However, in the presence of scopolamine nicotine increased the release of 5-HT by 57%. In conclusion, the release of intestinal 5-HT is facilitated via muscarine and nicotine receptors located on the enterochromaffin cells. Indirect evidence suggests that the release of 5-HT is additionally modulated by an as yet unknown inhibitory neurotransmitter released by muscarine receptor activation.Abbreviations DMPP 1,1-dimethyl-4-phenylpiperazinium - 5-HT 5-hydroxytryptamine - 5-HIAA 5-hydroxyindoleacetic acid - TTX tetrodotoxin Send offprint requests to H. S. at the above address     

Peripheral 5-HT1B and 5-HT2A receptors mediate the nociceptive response induced by 5-hydroxytryptamine in mice

While the role of 5-hydroxytryptamine (5-HT, serotonin) in the nociceptive processing has been widely investigated in the central nervous system, information regarding its role in peripheral tissues is still lacking. Noteworthy, 5-HT induces phenotypic changes of nociceptors and peripheral injection induces pain in humans and nociceptive response in rodents. However, local receptors involved in 5-HT effects are not well characterized. Thus, we aimed to investigate the role of 5-HT and some of its receptors in the peripheral nociceptive processing in mice. Intraplantar injection of 5-HT (10, 20 or 40 μg) into the hind-paw of mice induced paw licking behavior, which was inhibited by previous intraplantar treatment with cyproheptadine (5-HT₁ and 5-HT₂ antagonist; 0.5 or 5 μg), mianserin (5-HT₂ and 5-HT₆ antagonist; 0.1 μg), isamoltane (5-HT_1B antagonist; 0.5 or 5 μg) and ketanserin (5-HT_2A antagonist; 0.1 or 1 μg), but not by BRL 15572 (5-HT_1D antagonist; 1 or 10 μg), ondansetron (5-HT₃ antagonist; 1, 5, 10 or 20 μg) and SB 269970 (5-HT₇ antagonist; 2.5 and 25 μg). Altogether, these results indicate the local involvement of 5-HT₁, 5-HT₂ and 5-HT₆, especially 5-HT_1B and 5-HT_2A, in the nociceptive response induced by 5-HT in mice, thus contributing to a better understanding of 5-HT role in the peripheral nociceptive processing. In addition, they also point to important species differences and the need of a wide evaluation of the peripheral nociceptive processing in mice as these animals have been increasingly used in studies investigating the cellular and molecular mechanisms mediating the nociceptive response.     

5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes

The effect of the selective r5-HT_1B agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo [3,2-b] pyril-5-one (CP93,129) on the K⁺-evoked overflow of [³H]dopamine was studied in rat striatal synaptosomes loaded with [³H]dopamine. The aim of the study was to investigate the participation of 5-HT_1B receptors in the serotonergic modulation of striatal dopaminergic transmission. The Ca²⁺-dependent, tetrodotoxin-resistant K⁺-evoked overflow of [³H]dopamine was inhibited by CP93,129 (0.01–100 μM) in a concentration-dependent manner (IC₅₀=1.8 μM; maximal inhibition by 35.5% of control). [±]8-OH-DPAT, a 5-HT_1A receptor agonist, [+/–]DOI, a 5-HT₂ receptor agonist, and 2-methyl-5-hydroxytryptamine, a 5-HT₃ receptor agonist, at concentrations ranging from 0.01 μM to 100 μM did not show any significant effect. Neither ketanserin (1 μM and 5 μM), a selective 5-HT₂/5-HT_1D receptor antagonist, nor ondansetron (1 μM), a 5-HT₃ receptor antagonist, changed the inhibitory effect of CP93,129. SB224289, GR55562, GR127935, isamoltane and metergoline, selective and non-selective 5-HT_1B receptor antagonists, in contrast, used at a concentration of 1 μM, antagonized the inhibitory effect of CP93,129 (3 μM and 10 μM). SB224289, a selective 5-HT_1B receptor antagonist, inhibited the effect of CP93,129 in a concentration-dependent manner; the calculated K _i value was 1.8 nM. Our results indicate that in rat striatal axon terminals the K⁺-evoked release of dopamine is regulated by the presynaptic 5-HT_1B heteroreceptors. Received: 7 September 1998 / Accepted: 2 November 1998     

[3H]Rauwolscine: an antagonist radioligand for the cloned human 5-hydroxytryptamine2B (5-HT2B) receptor

In previous reports, [³H]5-HT has been used to characterize the pharmacology of the rat and human 5-HT_2B receptors. 5-HT, the native agonist for the 5-HT_2B receptor, has a limitation in its usefulness as a radioligand since it is difficult to study the agonist low-affinity state of a G protein-coupled receptor using an agonist radioligand. When using [³H]5-HT as a radioligand, rauwolscine was determined to have relatively high affinity for the human receptor (K_i human = 14.3 ± 1.2 nM, compared to K_i rat = 35.8 ± 3.8 nM). Since no known high affinity antagonist was available as a radioligand, these studies were performed to characterize [³H]rauwolscine as a radioligand for the cloned human 5-HT_2B receptor expressed in AV12 cells. When [³H]rauwolscine was initially tested for its usefulness as a radioligand, complex competition curves were obtained. After testing several α₂-adrenergic ligands, it was determined that there was a component of [³H]rauwolscine binding in the AV12 cell that was due to the presence of an endogenous α₂-adrenergic receptor. The α₂-adrenergic ligand efaroxan was found to block [³H]rauwolscine binding to the α₂-adrenergic receptor without significantly affecting binding to the 5-HT_2B receptor and was therefore included in all subsequent studies. In saturation studies at 37° C, [³H]rauwolscine labeled a single population of binding sites, K_d = 3.75 ± 0.23 nM. In simultaneous experiments using identical tissue samples, [³H]rauwolscine labeled 783 ± 10 fmol of 5-HT_2B receptors/mg of protein, as compared to 733 ± 14 fmol of 5-HT_2B receptors/mg of protein for [³H]5-HT binding. At 0° C, where the conditions for [³H]5-HT binding should label mostly the agonist high affinity state of the human 5-HT_2B receptor, [³H]rauwolscine (B_max = 951 ± 136 fmol/ mg), again labeled significantly more receptors than [³H]5-HT (B_max = 615 ± 34 fmol/mg). The affinity of [³H]rauwolscine for the human 5-HT_2B receptor at 0° C did not change, K_d = 4.93 ± 1.27 nM, while that for [³H]5-HT increased greatly (K_d at 37° C = 7.76 ± 1.06 nM; K_d at 0° C = 0.0735 ± 0.0081 nM). When using [³H]rauwolscine as the radioligand, competition curves for antagonist structures modeled to a single binding site, while agonist competition typically resulted in curves that best fit a two site binding model. In addition, many of the compounds with antagonist structures displayed higher affinity for the 5-HT_2B receptor when [³H]rauwolscine was the radioligand. Typically, ∼ 85% of [³H]rauwolscine binding was specific binding. These studies display the usefulness of [³H]rauwolscine as an antagonist radioligand for the cloned human 5-HT_2B receptor. This should provide a good tool for the study of both the agonist high- and low-affinity states of the human cloned 5-HT_2B receptor. Received: 26 June 1997 / Accepted: 30 August 1997     

Effect of ergot drugs on central 5-hydroxytryptamine neurons: Evidence for 5-hydroxytryptamine release or 5-hydroxytryptamine receptor stimulation

In combined biochemical and functional studies it has been possible to show that ergocornine (0.5-5 mg/kg) and the ergolene derivative (5R,8R)-8-(4-p-methoxyphenyl-1-piperazinylmethyl)-6-methylergolene (PTR 17402; MPME) (0.25-5 mg/kg) reduce in a dose-dependent way brain 5-hydroxytryptamine (5-HT) turnover in rat as evaluated with the tryptophan hydroxylase inhibitor, alpha-propyl-dopacetamide (H 22/54), whereas 2-Br-alpha-ergocryptine (CB 154; Br-EC) had no effect on brain 5-HT turnover. Effects on 5-HT receptor activity were evaluated using the extensor hindlimb reflex of acutely spinalized rats. It was found that ergocornine increased the 5-HT receptor activity independent of presynaptic 5-HT stores and that it didnot have any effects on uptake, retention and spontaneous overflow of 3-H-5-HT in vitro but reduced the fiedl stimulation-induced release of 3-H-5-HT in vitro. Therefore, it is suggested that ergocornine is a 5-HT recpetor-stimulating agent, an effect which may lead to reduction of nervous impulse flow in the 5-HT neurons and subsequently of 5-HT release and turnover. MPME, on the other hand, seems to increase 5-HT receptor release of 5-HT stores, mainly from extragranular sites. Thus, the increase in extensor reflex activity found after MPME was reduced by reserpine and H 22/54 and enhanced by nialamide and in vitro MPME markedly increased 3-H-5-HT overflow in cortical slices of nialamide-pretreated rats and inhibited uptake and retention of 3-H-5-HT (EC50 equals 1.6 times 10-minus 6 M) in cortical slices of normal rats. Inhibition of the 5-HT membrane pump does not seem to be of any major importance, since chlorimipramine was only weakly active on the extensor reflex in the pharmacological models used and since MPME did not block but rather enhanced the 5-HT depletion caused by 4-methyl-alpha-ethyl-m-tyramine. It is suggested that MPME is a releaser of extragranular 5-HT stores leading to increased 5-HT receptor activity and reduction of 5-HT turnover in the same way as indicated for ergocornine. This new ergolene derivative may represent a new class of antidepressant drugs acting via release of extragranular 5-HT stores.     

Structure-activity relationships at 5-HT1A receptors: binding profiles and intrinsic activity.

The 5-HT_1A receptor has been one of the most studied 5-HT receptor subtypes in terms of its pharmacologic profile. Comparisons of various studies of structure-activity relationships (SAR) at this receptor shows an emerging profile for this receptor's pharmacophore. The present discussion focuses on the findings generated with relatively small molecules that can be considered as analogs of serotonin itself and that illustrate some of the structural properties that are important for high-affinity recognition by the receptor. Most of the SAR work has been based on the affinities of compounds for the receptor as determined by the radioligand-binding technique, which has a significant limitation in that it cannot define the intrinsic activity of compounds at the receptor. This problem can be addressed by functional assays, and an example of SAR at the 5-HT_1A receptor-coupled adenylate cyclase system is provided.     

Comparisons of hallucinogenic phenylisopropylamine binding affinities at cloned human 5-HT2A, 5-HT2B and 5-HT2C receptors

Since the classical hallucinogens were initially reported to produce their behavioral effects via a 5-HT₂ agonist mechanism (i.e., the 5-HT₂ hypothesis of hallucinogen action), 5-HT₂ receptors have been demonstrated to represent a family of receptors that consists of three distinct subpopulations: 5-HT_2A, 5-HT_2B, and 5-HT_2C receptors. Today, there is greater support for 5-HT_2A than for 5-HT_2C receptor involvement in the behavioral effects evoked by these agents. However, with the recent discovery of 5-HT_2B receptors, a new question arises: do classical hallucinogens bind at 5-HT_2B receptors? In the present study we examined and compared the binding of 17 phenylisopropylamines at human 5-HT_2A, 5-HT_2B, and 5-HT_2C receptors. Although there was a notable positive correlation (r>0.9) between the affinities of the agents at all three populations of 5-HT₂ receptors, structural modification resulted only in small differences in 5-HT_2B receptor affinity such that the range of affinities was only about 50-fold. As with 5-HT_2A and 5-HT_2C receptor affinity, there is a significant correlation (r>0.9, n=8) between 5-HT_2B receptor affinity and human hallucinogenic potency. Nevertheless, given that 5-HT_2A and 5-HT_2A/2C antagonists – antagonists with low affinity for 5-HT_2B receptors – have been previously shown to block the stimulus effects of phenylisopropylamine hallucinogens, it is likely that 5-HT_2A receptors play a more prominent role than 5-HT_2B and 5-HT_2C receptors in mediating such effects despite the affinity of these agents for all three 5-HT₂ receptor subpopulations.     

Pharmacological analysis of the myoclonus induced by 5-hydroxytryptophan in the guinea pig suggests the presence of multiple 5-hydroxytryptamine receptors in the brain

Myoclonus induced in guinea pigs by administration of l-5-hydroxytryptophan (5-HTP) appears to be due to stimulation of central 5-HT receptors. Dose-dependent myoclonus was induced by administration of 5-HTP (with carbidopa pretreatment), l-tryptophan and tryptamine (both with pargyline pretreatment). and also by N,N-dimethyltryptamine, 5-metoxy-N,N-dimethyltryptamine and d-lysergic acid diethylamide (LSD). The synthetic 5-HT agonists quipazine, MK-212 and 1-(m-trifluoromethylphenyl) piperazine, however, evoked only occasional myoclonus at toxic doses. Antagonists of 5-HT receptors differed markedly in their ability to inhibit 5-HTP-induced myoclonus; while methergoline and cyproheptadine were potent inhibitors, mianserin, methysergide and BW 501C67 only caused effective inhibition in large doses. Similarly, 5-HT re-uptake blockers showed different activities in potentiating the effects of a threshold dose of 5-HTP; myoclonus was greatly potentiated by chlorimipramine, paroxetine and Org 6582, while femoxetine, fluoxetine and desmethylimipramine were only weakly active.The lack of uniform effects of the 5-HT agonists, antagonists and re-uptake blockers studied is not attributable entirely to their variable actions on central 5-HT mechanisms, to their effects on other brain neuronal pathways, or to differences in cerebral penetration. The data may provide functional evidence for a multiplicity of cerebral 5-HT receptors.     

Panuramine, a selective inhibitor of uptake of 5-hydroxytryptamine in the brain of the rat

The neurochemical profile of the novel inhibitor of uptake of 5-hydroxytryptamine (5-HT) panuramine (Wy 26002) has been investigated in the rat. In vitro, panuramine was found to be a potent and selective inhibitor of uptake of 5-HT with an IC50 of 22 +/- 4 nM. The IC50 for inhibition of uptake of noradrenaline was 848 nM and that for uptake of dopamine greater than 10 micron. Panuramine, in concentrations up to 10 micron did not displace the specific binding of either [3H]spiroperidol or [3H]5-HT and had no effect on the spontaneous or potassium-evoked release of 5-HT, suggesting that the compound had little effect on serotonergic transmission other than the inhibition of uptake of 5-HT. Panuramine also produced a dose-related antagonism of the depletion of 5-HT in brain induced by p-chloroamphetamine, confirming the ability of the drug to inhibit uptake of 5-HT in vivo.     

Investigations of cardiovascular 5-hydroxytryptamine receptor subtypes in the rat

Summary Peripheral 5-HT receptor-mediated responses were examined in pithed spontaneously hypertensive rats and normotensive wistar rats. Responses examined were: Pressor and depressor responses, tachycardia and inhibition of stimulation-evoked tachycardia. In pithed spontaneously hypertensive rats, 5-HT, but not the 5-HT₁-selective agonist 5-carboxamidotryptamine, produced pressor responses, and these were potently antagonised by the 5-HT₂-selective antagonists ketanserin and LY 53857. In pithed spontaneously hypertensive rats, the tachycardia to 5-HT was abolished by a combination of the 5-HT₂ receptor antagonist LY 53857 and propranolol, suggesting that the tachycardia is mediated by 5-HT₂ receptors and by release of noradrenaline. In pithed spontaneously hypertensive rats, 5-carboxamidotryptamine, 5-HT, and to a lesser extent the 5-HT₁ receptor agonist RU 24969, but not the 5-HT_1A receptor agonist 8-OH-DPAT, produced depressor responses which were antagonised by methysergide and metitepin, but which do not clearly fit with any of the 5-HT, ligand binding sites. In pithed normotensive wistar rat, 5-carboxamidotryptamine was approximately 100 times more potent than 5-HT and 8-OH-DPAT at inhibiting the cardio-acceleration produced by single pulse electrical stimulation and this inhibition was antagonised by metitepin, so that the response is mediated by 5-HT₁ receptors.     

Relative activities of substances related to 5-hydroxytryptamine as depolarizing agents of superior cervical ganglion cells

Membrane potential changes evoked by 5-HT and related substances were recorded by the sucrose-gap method from rabbit ganglia superfused with Krebs solution at 20°C. A solution of the substance under test was injected into the superfusion stream. The activity of 23 substances was compared to that of 5-HT in respect of depolarizing capacity. 0.01 μmol 5-HT produced a near-threshold depolarization, while 0.6–0.8 μmol induced a maximal one. Some 5-HT analogues evoked prolonged responses distinctly different from the rapid depolarization and repolarization characteristic of 5-HT, while others were inactive. Compounds di- or trimethylated at the side-chain nitrogen atom were capable in addition of activating nicotinic receptors. The results suggest that: (1) the optimal requirements for activating ganglionic 5-HT receptors are a hydroxyl group at position 5 on the indole nucleus and a side-chain bearing an ethylamine amino group; (2) methyl substituents around the terminal nitrogen atom are well tolerated and a quaternary nitrogen may increase activity at the 5-HT receptor; and (3) substitution of a methyl group at carbon atom 2 of the indole nucleus reduces activity. a limitation of the technique is the difficulty of obtaining more than one dose-response curve from a particular preparation; a reduction in potency due to lower affinity cannot be readily distinguished from one due to lower intrinsic activity.     

Metabotropic glutamate receptors modulate serotonin release in the rat periaqueductal gray matter

The role of metabotropic (mGluRs) and N-methyl-D-aspartate (NMDA) glutamate receptors on 5-hydroxytryptamine (5-HT) release has been studied in rat periaqueductal gray (PAG) matter by using in vivo microdialysis. (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD; 0.5 or 1 mM], a group I/group II mGluRs agonist, increased the dialysate 5-HT concentration. (2S)-α-ethylglutamic acid (EGlu; 1 mM), an antagonist of group II mGluRs, but not (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), an antagonist of group I mGluRs, antagonized the 1S,3R-ACPD-induced effect. (S)-3,5-dihydroxyphenylglycine (DHPG; 0.5 and 1 mM), an agonist of group I mGluRs, did not modify dialysate 5-HT. (2S, 3S, 4S)-α-(carboxycyclopropyl)-glycine (L-CCG-I; 0.5 and 1 mM), an agonist of group II mGluRs, increased extracellular 5-HT. This effect was antagonized by EGlu. Similarly, L-serine-O-phosphate (L-SOP; 1 and 10 mM), an agonist of group III mGluRs, increased extracellular 5-HT and this effect was antagonized by (RS)-α-methylserine O-phosphate (M-SOP; 1 mM), an antagonist of group III mGluRs. Out of the several N-methyl-D-aspartate concentrations used (NMDA; 10, 50, 100, 500 and 1000 μM) only the 50 μM infusion significantly decreased dialysate 5-HT. The GABA_A receptor agonist, bicuculline (30 μM), increased 5-HT release on its own and antagonized the decrease caused by the opiate antagonist, naloxone (2 mM), as well as the increases caused by CCG-I or L-SOP. These data show that stimulation of PAG’s group II/group III mGluRs increases 5-HT release, while stimulation of NMDA glutamate receptors may decrease it. We speculate that glutamate does not modulate 5-HT release in the PAG directly, but via activation of tonically active GABAergic interneurons. Received: 15 January 1998 / Accepted: 22 July 1998     

In vivo control of 5-hydroxytryptamine release by terminal autoreceptors in rat brain areas differentially innervated by the dorsal and median raphe nuclei

Selective serotonin reuptake inhibitors (SSRIs) reduce the 5-HT release in vivo. This effect is due to the activation of somatodendritic 5-HT_1A receptors and it displays a regional pattern comparable to that of selective 5-HT_1A agonists, i.e., preferentially in forebrain areas innervated by the dorsal raphe nucleus (DRN). However, despite a comparatively lower 5-HT_1A-mediated inhibition of 5-HT release and a greater density of serotonergic uptake sites in hippocampus, the net elevation produced by the systemic administration of SSRIs is similar in various forebrain areas, regardless of the origin of serotonergic fibres. As terminal autoreceptors may also limit the SSRI-induced elevations of 5-HT in the extracellular brain space, we reasoned that a differential control of 5-HT release by terminal autoreceptors in DRN- and median raphe-innervated areas might be accountable. To examine this possibility, we have conducted a regional microdialysis study in the DRN, MRN and four forebrain regions preferentially innervated either by the DRN (frontal cortex, striatum) or the median raphe nucleus (MRN; dorsal and ventral hippocampus) using freely moving rats. Dialysis probes were perfused with 1 μM of the SSRI citalopram to augment the endogenous tone on terminal 5-HT autoreceptors. The non-selective 5-HT₁ antagonist methiothepin (10 and 100 μM, dissolved in the dialysis fluid) increased extracellular 5-HT in frontal cortex and dorsal hippocampus in a concentration-dependent manner. The 5-HT_1B/1D antagonist GR 127935 was ineffective at 10 μM and tended to reduce 5-HT in dorsal hippocampus at 100 μM. The local infusion of 100 μM methiothepin significantly elevated the extracellular 5-HT concentration to 142–173% of baseline (mean values of 260 min post-administration) in the DRN, MRN, frontal cortex, striatum and hippocampus (dorsal and ventral). Comparable elevations were noted in the four forebrain regions examined. As observed in frontal cortex and dorsal hippocampus, the perfusion of 10 μM GR 127935 did not elevate 5-HT in DRN, MRN, striatum or ventral hippocampus. Because the stimulated 5-HT release in the DRN has been suggested to be under control of 5-HT_1B/1D receptors, we examined the possible contribution of these receptor subtypes to the effects of methiothepin in the DRN. The perfusion of sumatriptan (0.01–10 μM) or GR 127935 (0.01–10 μM) did not significantly modify the 5-HT concentration in dialysates from the DRN. Thus, the present data suggest that the comparable effects of SSRIs in DRN- and MRN-innervated forebrain regions are not explained by a preferential attenuation of 5-HT release by terminal 5-HT_1B autoreceptors in hippocampus, an area with a low inhibitory influence of somatodendritic 5-HT_1A receptors. Methiothepin-sensitive autoreceptors (possibly 5-HT_1B) appear to play an important role not only in the projection areas but also with respect to the control of 5-HT release in the DRN and MRN. In addition, our findings indicate that GR 127935 is not an effective antagonist of the actions of 5-HT at rat terminal autoreceptors. Received: 27 February 1998 / Accepted: 12 June 1998     

Involvement of 5-hydroxytryptamine7 receptors in inhibition of porcine myometrial contractility by 5-hydroxytryptamine

1. 5-Hydroxytryptamine (5-HT; 1 nM–100 μM) concentration-dependently inhibited the amplitude and frequency of spontaneous contractions in longitudinal and circular muscles of the porcine myometrium. The circular muscle (EC₅₀; 68–84 nM) was more sensitive than the longitudinal muscle (EC₅₀; 1.3–1.44 μM) to 5-HT. To characterize the 5-HT receptor subtype responsible for inhibition of myometrial contractility, the effects of 5-HT receptor agonists on spontaneous contractions and of 5-HT receptor antagonists on inhibition by 5-HT were examined in circular muscle preparations.
2. Pretreatment with tetrodotoxin (1 μM), propranolol (1 μM), atropine (1 μM), guanethidine (10 μM) or L-NAME (100 μM) failed to change the inhibition by 5-HT, indicating that the inhibition was due to a direct action of 5-HT on the smooth muscle cells.
3. 5-CT, 5-MeOT and 8-OH-DPAT mimicked the inhibitory response of 5-HT, and the rank order of the potency was 5-CT>5-HT>5-MeOT>8-OH-DPAT. On the other hand, oxymethazoline, α-methyl-5-HT, 2-methyl-5-HT, cisapride, BIMU-1, BIMU-8, ergotamine and dihydroergotamine had almost no effect on spontaneous contractions, even at 10–100 μM.
4. Inhibition by 5-HT was not decreased by either pindolol (1 μM), ketanserin (1 μM), tropisetron (10 μM), MDL72222 (1 μM) or {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808 (10 μM), but was antagonized by the following compounds in a competitive manner (with pA₂ values in parentheses): methiothepin (8.05), methysergide (7.92), metergoline (7.4), mianserin (7.08), clozapine (7.06) and spiperone (6.86).
5. Ro 20-1724 (20 μM) and rolipram (10 μM) significantly enhanced the inhibitory response of 5-HT, but neither zaprinast (10 μM) nor dipyridamole (10 μM) altered the response of 5-HT.
6. 5-HT (1 nM–1 μM) caused a concentration-dependent accumulation of intracellular cyclic AMP in the circular muscle.
7. From the present results, the 5-HT receptor, which is functionally correlated with the 5-HT₇ receptor, mediates the inhibitory effect of 5-HT on porcine myometrial contractility. This inhibitory response is probably due to an increase in intracellular cyclic AMP through the activation of adenylate cyclase that is positively coupled to 5-HT₇ receptors.

     

Both 5-hydroxytryptamine 5-HT2A and 5-HT1B receptors are involved in the vasoconstrictor response to 5-HT in the human isolated internal thoracic artery

1. The 5-hydroxytryptamine (5-HT, serotonin) receptor subtypes that mediate vasoconstriction in the human internal thoracic artery (ITA), which is frequently used as an arterial graft, remain unclear. The aim of the present study was to elucidate the 5-HT receptor subtypes responsible for 5-HT-induced contraction of the human ITA. 2. The contractile responses to 5-HT of endothelium-denuded human ITA obtained from patients undergoing coronary bypass surgery were examined. In addition, we investigated the effects of sarpogrelate and SB224289, antagonists of 5-HT(2A) and 5-HT(1B) receptors, respectively, on the 5-HT-induced vasoconstriction. Finally, 5-HT(2A) and 5-HT(1B) receptors in the human ITA were immunolabelled. 3. 5-Hydroxytryptamine (1 nmol/L-10 micromol/L) caused vasoconstriction in a concentration-dependent manner. Both sarpogrelate (1 micromol/L) and SB224289 (1 micromol/L) significantly, but not completely, inhibited 5-HT-induced vasoconstriction. 4. Conversely, simultaneous pretreatment with supramaximum concentrations (1 micromol/L for both) of sarpogrelate and SB224289 almost completely inhibited the 5-HT-induced vasoconstriction. 5. Immunopositive staining for 5-HT(2A) and 5-HT(1B) receptors was detected in smooth muscle cells of the human ITA. 6. These results demonstrate that, in human ITA, 5-HT-induced vasoconstriction is mediated by activation of both 5-HT(2A) and 5-HT(1B) receptors. Thus, when the human ITA is used as an arterial graft, a combination of 5-HT(2A) and 5-HT(1B) receptor antagonists would appear to be most useful to prevent 5-HT-induced vasospasm.