The 5-HT1-like receptors mediating inhibition of sympathetic vasopressor outflow in the pithed rat: operational correlation with the 5-HT1A, 5-HT1B and 5-HT1D subtypes

1. It has been suggested that the inhibition of sympathetically-induced vasopressor responses produced by 5-hydroxytryptamine (5-HT) in pithed rats is mediated by 5-HT₁-like receptors. The present study has re-analysed this suggestion with regard to the classification schemes recently proposed by the NC-IUPHAR subcommittee on 5-HT receptors.
2. Intravenous (i.v.) continuous infusions of 5-HT and the 5-HT₁ receptor agonists, 8-OH-DPAT (5-HT_1A), indorenate (5-HT_1A), CP 93,129 (5-HT_1B) and sumatriptan (5-HT_1B/1D), resulted in a dose-dependent inhibition of sympathetically-induced vasopressor responses.
3. The sympatho-inhibitory responses induced by 5-HT, 8-OH-DPAT, indorenate, CP 93,129 or sumatriptan were analysed before and after i.v. treatment with blocking doses of the putative 5-HT receptor antagonists, WAY 100635 (5-HT_1A), cyanopindolol (5-HT_1A/1B) or GR 127935 (5-HT_1B/1D). Thus, after WAY 100635, the responses to 5-HT and indorenate, but not to 8-OH-DPAT, CP 93,129 and sumatriptan, were blocked. After cyanopindolol, the responses to 5-HT, indorenate and CP 93,129 were abolished, whilst those to 8-OH-DPAT and sumatriptan (except at the lowest frequency of stimulation) remained unaltered. In contrast, after GR 127935, the responses to 5-HT, CP 93,129 and sumatriptan, but not to 8-OH-DPAT and indorenate, were abolished.
4. In additional experiments, the inhibition induced by 5-HT was not modified after 5-HT₇ receptor blocking doses of mesulergine.
5. The above results suggest that the 5-HT₁-like receptors, which inhibit the sympathetic vasopressor outflow in pithed rats, display the pharmacological profile of the 5-HT_1A, 5-HT_1B and 5-HT_1D, but not that of 5-HT₇, receptors.

     

Correlation between 5-HT7 receptor affinity and protection against sound-induced seizures in DBA/2J mice

Audiogenic seizures can be induced in DBA/2J mice following intense auditory stimulation. A number of neurotransmitters, including 5-hydroxytryptamine (5-HT), are believed to be involved in mediating this effect since it has been shown previously that depletion of 5-HT or blockade of 5-HT receptors protects DBA/2J mice from these audiogenic seizures. The present study was undertaken to determine whether antagonism of the newly identified 5-HT₇ receptor may protect DBA/2J mice from audiogenic seizures by attempting to correlate in vivo potency of compounds with their affinity at the 5-HT₇ receptor. All compounds used in the correlation were shown to be antagonists at the 5-HT₇ receptor and a statistically significant correlation was observed between 5-HT₇ affinity and doses for half-maximal response (ED₅₀) for protection of DBA/2J mice from sound-induced seizures (r = 0.80; P < 0.05). No significant correlation was observed between in vivo activity and affinity at either 5-HT_1A, 5-HT_2A or 5-HT_2C receptors. It is also unlikely that interactions between the 5-ht₅ receptor will protect DBA/2J mice from audiogenic seizures since metergoline and mesulergine which are both active in this in vivo model have no affinity for the 5-ht₅ receptor. There are similarities between the pharmacology of the 5-HT₇ receptor and that of the 5-HT_1A receptor, however the correlation between the in vivo potency in DBA/2J mice and 5-HT_1A affinity was not significant. Furthermore, the 5-HT_1A receptor antagonist WAY 100135 did not protect DBA/2J mice from audiogenic seizures at doses that antagonise 5-HT_1A receptor-mediated effects in mice. These data suggest that antagonism of 5-HT₇ receptors may protect against audiogenic seizures in DBA/2J mice although a definitive conclusion must await studies with selective 5-HT₇ antagonists. Received: 20 March 1997 / Accepted: 10 August 1997     

The effects of A 5-HT1A receptor agonist and antagonist on the 5-hydroxytryptamine release in the central nucleus of the amygdala: a microdialysis study with flesinoxan and WAY 100635

The modulation of extracellular 5-hydroxytryptamine (5-HT) in the central nucleus of the amygdala (CeA) by 5-HT_1A receptors was studied by intracerebral microdialysis in awake and freely moving rats. Local administration of 1 μM tetrodotoxin (TTX), 60 mM K⁺ and perfusion with Ca²⁺-free Ringer containing EGTA confirmed that the major part of dialysate 5-HT levels from the CeA is of neuronal origin. Administration of 300 nM of RU 24969, a 5-HT_1B receptor agonist, through the probe into the CeA decreased dialysate 5-HT levels to 67.2% of the baseline value. Systemic administration of the 5-HT_1A receptor agonists 8-OH-DPAT and flesinoxan dose-dependently decreased 5-HT levels in the CeA. The effect of 0.3 mg/kg of flesinoxan could be completely antagonized by systemic administration of 0.05 mg/kg WAY 100635, a 5-HT_1A receptor antagonist. WAY 100635 alone had only minimal effects at this dose. These data show that a major part of the extracellular 5-HT in the CeA stems from 5-HT neurons and that the amount of 5-HT released into this brain region can be modulated by 5-HT_1A receptors. Received: 11 September 1996 / Accepted: 25 November 1996     

Pharmacological profile of 5-hydroxytryptamine-induced inhibition on the pressor effect elicited by sympathetic stimulation in long-term diabetic pithed rats

We analysed the type and/or subtype of 5-hydroxytryptamine (5-HT) receptors involved in the inhibitory mechanisms of 5-HT on the pressor responses induced by stimulation of sympathetic vasopressor outflow in long-term diabetic pithed rats. Diabetes was induced in male Wistar rats by a single subcutaneous injection of alloxan. Eight weeks later, rats were anaesthetized, pre-treated with atropine, and pithed. The effect of 5-HT on the pressor responses elicited by stimulation of the sympathetic outflow was analysed in eight-week alloxan-induced diabetic pithed rats. 5-HT (20 μg/kg/min) reduced the pressor action obtained by electrical stimulation of the sympathetic outflow. However, there was no effect on exogenous noradrenaline-induced pressor responses. 5-CT (5 μg/kg/min), 8-OH-DPAT (5 μg/kg/min), and α-methyl-5-HT (5 μg/kg/min), selective 5-HT₁, 5-HT_1A and 5-HT₂ receptor agonists, respectively, reproduced the 5-HT inhibitory action. Nevertheless, infusion of 5 μg/kg/min of 1-phenylbiguanide, CGS-12066B, L-694,247, BW273C86 or MK212 (5-HT₃, 5-HT_1B, 5-HT_1D, 5-HT_2B and 5-HT_2C receptor agonists, respectively) had no effect on the pressor responses elicited by stimulation of the sympathetic outflow. Methiothepin (100 μg/kg) and a cocktail of WAY-100,635 (100 μg/kg) and spiperone (125 μg/kg) blocked the 5-HT inhibitory effect on the pressor action obtained by sympathetic stimulation. Moreover, WAY-100, 635 abolished the 8-OH-DPAT inhibitory effect and spiperone blocked α-methyl-5-HT action. In conclusion, this study revealed that long-term experimental diabetes induces changes in the receptor type/subtype involved in the 5-HT inhibitory action on the sympathetic pressor responses produced by electrical stimulation. This is mainly mediated by pre-junctional 5-HT_1A and 5-HT_2A receptors.     

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.     

Specific interaction of 5-HT-moduline with human 5-HT1b as well as 5-HT1d receptors expressed in transfected cultured cells

5-HT_1B receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters. 5-HT-moduline (Leu-Ser-Ala-Leu) has been shown to specifically interact with a very high apparent affinity and in a non-competitive manner with 5-HT_1B receptors (Massot et al. 1996; Rousselle et al. 1996). Using transfected cells expressing either 5-HT_1B or 5-HT_1D receptors, it was shown that 5-HT-moduline prevents the binding of [³H]5-HT to 5-HT_1B as well as to 5-HT_1D receptors with similar biochemical characteristics: the IC₅₀ of the peptide was 1.2×10^–12 M for 5-HT_1B and 9×10^–13 M for 5-HT_1D receptors. The observed effect corresponds to a marked decrease of the maximal binding for [³H]5-HT on 5-HT_1B (–51.2±1%) as well as 5-HT_1D binding (–47.2±7.7% of the control binding) whereas the affinity of 5-HT is increased by a factor close to 3. No effect is observed using the “scrambled” peptide (Ala-Leu-Leu-Ser). Parallel assays using transfected cells expressing 5-HT_1A or 5-ht₆ receptors did not show any significant change induced by the peptide under similar assay conditions. The interaction of the peptide was also studied on the functional activity related to the stimulation of the receptors as measured by the increase in [³⁵S]GTPγS binding reflecting the coupling of the receptor to the G-protein. 5-HT-moduline yields an antagonistic effect on the 5-HT induced coupling with a corresponding IC₅₀=1.2±0.7×10^–12 M for 5-HT_1B and 9.8±4.0×10^–12 M for 5-HT_1D receptors, respectively. The present results demonstrate that 5-HT-moduline interacts with 5-HT_1D as well as 5-HT_1B receptors and possesses a non-competitive antagonistic activity, likely corresponding to its role of endogenous allosteric modulator, specific for both 5-HT_1B and 5-HT_1D receptors. Received: 26 March 1998 / Accepted: 22 May 1998     

Interactions of GR127935, a 5-HT1B/D receptor ligand, with functional 5-HT receptors

GR127935 (N-[methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl) [1, 1-biphenyl]-4-carboxamide hydrochloride) has been recently introduced as an experimental tool to antagonize 5-HT_1B/D receptor-mediated functional responses. The compound indeed exhibits a very high affinity and selectivity for 5-HT_1B/D binding sites and it antagonizes a number of 5-HT_1B/D receptor-mediated responses. The present experiments were performed to investigate the selectivity of GR127935 against functional responses mediated by 5-HT₁-like, ‘orphan’ 5-HT₁-like (5-ht₇?), 5-HT₂, 5-HT₃ or 5-HT₄ receptors in several invivo preparations. Intravenous (i.v.) treatment with GR127935 (300μg?kg^-1) potently antagonized decreases in total carotid blood flow as well as hypotensive responses induced by the 5-HT₁-like receptor agonist sumatriptan in rabbits. I.v. bolus injections of GR127935 (up to 500 and/or 1500μg?kg^-1) did not significantly modify 5-HT-induced: (i) tachycardia in the pig (5-HT₄ receptor-mediated) and cat (‘orphan’ 5-HT₁-like or, perhaps, 5-ht₇ receptor-mediated); (ii) depressor effects in the rat and cat (‘orphan’ 5-HT₁-like or 5-ht₇ receptor-mediated); (iii) vonBezold-Jarisch reflex in the rat or the early phase of the urinary bladder contraction in the cat (both 5-HT₃ receptor-mediated). In contrast, high doses (500-1500μg?kg^-1) of GR127935 suppressed 5-HT-induced pressor responses in the rat and cat and urinary bladder contractions (secondary phase) in the cat as well as the DOI ((±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride)-induced pressor responses in the rat, which are all mediated by 5-HT_2A receptors. In conclusion, the present study demonstrates that GR127935 is a selective 5-HT_1B/D receptor antagonist devoid of interactions at ‘orphan’ 5-HT₁-like (5-ht₇?), 5-HT₃ and 5-HT₄ receptors. However, GR127935 possesses a moderate 5-HT_2A receptor blocking property, which is consistent with its binding profile (pK_i: 7.4). Lastly, in view of the potent antagonist action of GR127935, the sumatriptan-induced hypotension in rabbits seems to be mediated by 5-HT_1B/D 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.     

Further characterization of the 5-hydroxytryptamine1-like receptors mediating the increase in external carotid blood flow in the dog

It has recently been shown that the increase in external carotid blood flow (external CBF) produced by 5-hydroxytryptamine (5-HT) in the anaesthetized dog, being mimicked by 5-carboxamidotryptamine, potently blocked by methiothepin and resistant to blockade by ritanserin and MDL 72222, is mediated by 5-HT₁-like receptors. In the present investigation, we have further characterized these 5-HT₁-like receptors. Like 5-HT, 1 min intracarotid (i.c.) infusions of the 5-HT_1A receptor agonist, indorenate, produced an increase in external CBF without modifying mean arterial blood pressure or heart rate. Contrasting with indorenate, 1 min i.c. infusions of the 5-HT_1A receptor agonists, 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT), buspirone and ipsapirone, or the 5-HT_1A/5-HT_1B receptor agonist, 5-methoxy-3-[1,2,3,6-tetrahydro-4-pyridinyl]-1-H-indol succinate (RU 24969), resulted in dose-dependent decreases in external CBF; furthermore, both the 5-HT_1C/5-HT₂ receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl-)-aminopropane (DOI) and the 5-HT₃ receptor agonist, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT), were essentially inactive. Thus, only indorenate increased the external CBF in the dog; this effect of indorenate was not antagonized by intravenous (i.v.) administration of the 5-HT₁ and 5-HT₂ receptor antagonist, methiothepin, or completely abolished after sympathectomy. Unlike methiothepin, the 5-HT_1A and 5-HT_1B receptor antagonist, (±)-pindolol, did not block indorenate-induced external carotid vasodilatation. Together, the above results support the notion that indorenate is acting on the 5-HT₁-like receptors involved in the increase in external CBF in the dog. These receptors, which are probably located on carotid sympathetic nerve endings, do not seem to correspond to either the 5-HT_1A, 5-HT_1B, or 5-HT_1C binding sites. © 1993 Wiley-Liss, Inc.     

Analysis of the heart rate effects of 5-hydroxytryptamine in the cat; mediation of tachycardia by 5-HT1-like receptors

Summary The effects of 5-hydroxytryptamine (5-HT) on heart rate in anaesthetized cats were analysed both in intact animals and after spinal section plus vagotomy.The intact cat responded to 5-HT (3, 10 and 30 g·kg^–1, i.v.) with a brief, but intense, bradycardia and a longerlasting hypotension. Administration of MDL 72222, a selective antagonist of M-type 5-HT receptors, blocked bradycardia elicited by 5-HT without affecting that caused by stimulation of the vagus nerve.In spinal cats the same doses of 5-HT increased heart rate and blood pressure. These effects remained essentially unchanged after bilateral adrenalectomy, guanethidine, propranolol and burimamide, suggesting that 5-HT acted directly on the myocardium and blood vessels. The tachycardic responses to 5-HT in spinal cats were little affected by 0.5 mg·kg^–1 doses of MDL 72222 or of the 5-HT₂ receptor antagonists, ketanserin, ritanserin or cyproheptadine. In contrast, the non-selective 5-HT receptor antagonist, methysergide, which binds to both 5-HT₁ and 5-HT₂ recognition sites in rat brain membranes, potently antagonized the 5-HT-induced tachycardia in doses of 0.05 to 0.5 mg·kg^–1. However pizotifen and mianserin, two other 5-HT₂ antagonists which show poor affinity for 5-HT₁ recognition sites, were also effective against the tachycardic response to 5-HT in doses of 0.5–4.5 mg·kg^–1. The pressor responses to 5-HT in the spinal cat were markedly inhibited by all six 5-HT₂ antagonists at a dose of 0.5 mg·kg^–1.5-Carboxamido-tryptamine, which has a high and selective affinity for 5-HT₁ recognition sites, elicited marked tachycardia in doses of 0.1–10 g/kg^–1 in spinal cats treated with saline. These responses were not affected in animals treated with 4.5 mg·kg^–1 of ketanserin, which was able to shift the dose-response curve for 5-HT to the right. On the other hand, methysergide (0.5 mg·kg^–1) displaced the dose-response curves for both 5-carboxamidotryptamine and 5-HT to a similar extent.Unlike on the dog saphenous vein, methysergide showed no agonist effects on heart rate in the spinal cat.On the basis of the above results, we conclude that: (i) the reflexogenic bradycardic response elicited by 5-HT overshadows its direct tachycardic response on heart rate in the intact cat; (ii) M-type 5-HT receptors mediate the bradycardic response; (iii) the pressor response to 5-HT in the spinal cat involves 5-HT₂ receptors; (iv) tachycardia by 5-HT in the spinal cat is mediated mainly by 5-HT₁-like receptors, but an additional, though less important, non-5-HT₁ mechanism may also be involved; (v) the cardiac 5-HT₁ receptors are similar, but perhaps not identical, to those delineated in the dog saphenous vein or rat brain membrane preparations; and (vi) the tachycardic responses to 5-HT and, in particular the more selective, 5-carboxamidotryptamine may be conveniently utilized to characterize new chemical compounds designed for potential 5-HT₁ receptor antagonist activity.     

Effects of current and prospective antimigraine drugs on the porcine isolated meningeal artery

Vasoconstriction to agonists at serotonin (5-hydroxytryptamine; 5-HT) receptors and α-adrenoceptors, as well as vasodilatation induced by α-CGRP, have been well described in the porcine carotid circulation in vivo. The present study sets out to investigate the effects of current and prospective antimigraine drugs on porcine meningeal artery segments in vitro. Sumatriptan, ergotamine, dihydroergotamine, isometheptene and clonidine failed to contract the meningeal artery, but 5-HT, noradrenaline and phenylephrine induced concentration-dependent contractions. The contractions to 5-HT were competitively antagonized by the 5-HT_2A receptor antagonist ketanserin, whilst those to noradrenaline were antagonized by α₁-(prazosin), α₂-(rauwolscine and yohimbine) and α_2C/2B-(OPC-28326) adrenoceptor antagonists. Whilst dobutamine and salbutamol were ineffective, α-CGRP produced concentration-dependent relaxations that were antagonized by the CGRP₁ receptor antagonist olcegepant. In agreement with their lack of contractile effect, sumatriptan and ergotamine failed to influence forskolin-stimulated cyclic AMP accumulation in the porcine meningeal artery; in contrast, both compounds decreased forskolin-stimulated cyclic AMP accumulation in the human isolated saphenous vein, where they induced contractions. Finally, using RT-PCR, we could demonstrate the presence of mRNAs encoding for several 5-HT receptors (5-HT_1B, 5-HT_1D, 5-HT_1F, 5-HT_2A and 5-HT₇) and adrenoceptors (α_1A, α_1B, α_1D, α_2A, α_2B, α_2C, β₁ and β₂), as well as that for the calcitonin receptor like receptor, a component of the CGRP₁ receptor. These results suggest that: (i) the porcine meningeal artery may not be involved in the vasoconstriction of the carotid vascular bed elicited by antimigraine drugs in anaesthetized pigs, and (ii) the mismatch between the presence of receptor mRNA and the lack of response to sumatriptan, dobutamine and salbutamol implies that mRNAs for the 5-HT_1B receptor and β₁- and β₂-adrenoceptors are probably unstable, or that their density is too low for being translated as receptor protein in sufficient quantities.     

Further characterization of the 5-HT<Subscript>1</Subscript> receptors mediating cardiac sympatho-inhibition in pithed rats: pharmacological correlation with the 5-HT<Subscript>1B</Subscript> and 5-HT<Subscript>1D</Subscript> subtypes

Serotonin (5-hydroxytryptamine; 5-HT) is capable of inhibiting the tachycardic responses elicited by sympathetic stimulation, but not by exogenous noradrenaline, in pithed rats pre-treated with desipramine. More recently, it has been shown that this cardiac sympatho-inhibitory response to 5-HT, mediated by prejunctional 5-HT₁ receptors as well as putative 5-ht_5A/5B receptors, is mimicked dose-dependently by the agonists CP 93,129 (r5-HT_1B), sumatriptan (5-HT_1B/1D) and PNU-142633 (5-HT_1D). This study analysed further the pharmacological profile of the above 5-HT₁ receptors.Continuous i.v. infusions of CP 93,129, sumatriptan or PNU-142633 (30 µg kg^–1min^–1 each) failed to modify the tachycardic responses to exogenous noradrenaline but inhibited those elicited by preganglionic (C7–T1) stimulation of the cardiac sympathetic outflow. These sympatho-inhibitory responses were unaltered after i.v. administration of physiological saline (1 ml kg^–1) or the 5-HT_1A receptor antagonist WAY 100635 (10 µg kg^–1). In contrast, the antagonist GR 127935 (5-HT_1B/1D; 100 µg kg^–1, i.v.) abolished the responses to CP 93,129, sumatriptan and PNU-142633, whilst SB224289 (5-HT_1B; 300 µg kg^–1, i.v.) abolished the responses to CP 93,129 without affecting those to sumatriptan and PNU-142633. Interestingly, BRL15572 (5-HT_1D; 300 µg kg^–1, i.v.) abolished the responses to PNU-142633 and attenuated those to sumatriptan, but not those to CP 93,129.WAY 100635, GR 127935, SB224289 and BRL15572, given alone at the above doses, failed to modify the sympathetically induced tachycardic responses. The 5-HT₁ receptors producing cardiac sympatho-inhibition in pithed rats thus display the pharmacological profile of the 5-HT_1B and 5-HT_1D receptor subtypes.     

An inhibitory prejunctional 5-HT1-like receptor in the isolated perfused rat kidney

Summary The present study has identified a receptor for 5-hydroxytryptamine (5-HT) which functions to inhibit the stimulus-induced release of [³H] noradrenaline following sympathetic periarterial nerve stimulation to the isolated perfused rat kidney. In addition to 5-HT (IC₃₀=4.5×10^–8 mol/l), both 5-carboxamidotryptamine (IC₃₀=8×10^–9 mol/l) and 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl) indole (RU-24969, IC₃₀=2.5×10^–7 mol/l) acted as agonists whereas 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) was inactive. The inhibitory effect of 5-HT on the electrically-evoked release of tritium was antagonized in a concentration-dependent manner by methiothepin (IC₅₀=4×10^–9 mol/l), metergoline (IC₅₀=4×10^–8 mol/l) and methysergide (IC₅₀=1.3×10^–7 mol/l) but not by cyproheptadine, ketanserin, mesulergine, (–)-propranolol, (±)-pindolol, (±)-cyanopindolol, metoclopramide or phentolamine. It is concluded that the receptor to 5-HT conforms to general criteria defining 5-HT₁-like receptors but at the present time the receptor site cannot be fitted to the designated 5-HT_1A, 5-HT_1B or 5-HT_1C subtypes.Preliminary accounts of this work were presented to the British Pharmacological Society in London (December, 1984) and Southampton (July, 1985)     

Blockade of serotonin 5-HT1B and 5-HT2A receptors suppresses the induction of locomotor activity by 5-HT reuptake inhibitors,citalopram and fluvoxamine,in NMRI mice exposed to a novel environment: a comparison to other 5-HT receptor subtypes

Rationale Though 5-HT plays an important role in the modulation of motor function, which is perturbed in depressive states, little is known concerning the influence of serotonin reuptake inhibitors (SSRIs) on locomotor activity (LA). Recently, we demonstrated that SSRIs, such as citalopram, enhance LA in mice exposed to a novel environment. Objectives This study examined the role of multiple classes of 5-HT receptor in citalopram-induced LA. Methods The most selective antagonists currently available were used. Results Citalopram-induced LA was dose-dependently attenuated by the 5-HT_1B/1D receptor antagonists, S18127, GR125,743 and GR127,935, and by the selective 5-HT_1B antagonist, SB224,289, but unaffected by the selective 5-HT_1A antagonist, WAY100,635. The selective antagonists at 5-HT_2A receptors, MDL100,907 and SR46,349 also dose-dependently attenuated induction of locomotion by citalopram, whereas the 5-HT_2B antagonist, SB204,741, and the 5-HT_2B/2C antagonist, SB206,553 were ineffective. Further, the selective 5-HT_2C antagonist, SB242,084, potentiated the response to citalopram. Selective antagonists at 5-HT₃ (ondansetron), 5-HT₄ (GR125,487), 5-HT₆ (SB271,046) and 5-HT₇ (SB269,970) receptors did not significantly modify the action of citalopram. Underpinning these findings, SB224,289, GR125,743, MDL100,907 and SR46,349 likewise attenuated induction of locomotion by a further SSRI, fluvoxamine. Conclusions The locomotor response to SSRIs of mice exposed to a novel environment is mediated via 5-HT_1B and 5-HT_2A receptors. In view of the importance of motor function to the etiology and treatment of depression, the significance of these observations to the clinical actions of SSRIs will be of interest to elucidate.     

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

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

     

5-HT1-like and 5-HT2A receptors mediate 5-hydroxytryptamine-induced contraction of rabbit isolated mesenteric artery

The contractions induced by 5-hydroxytryptamine (5-HT) and the 5-HT₁-like receptor agonist, sumatriptan, were investigated in the open ring preparations of rabbit mesenteric artery in order to characterize the 5-HT receptors. 5-HT induced concentration-dependent contractions. Sumatriptan did not induce any contraction of unstimulated rings, whereas it elicited concentration-dependent contractions in preparations given a moderate tone by a threshold concentration of prostaglandin F₂ (PGF₂). Pargyline, cocaine or normetanephrine were without significant effect on the contractions induced by 5-HT and sumatripan. The 5-HT concentration-effect curve was clearly biphasic. Methiothepin (0.01 M) shifted the both phases of the concentration-effect curve to the right. Ketanserin (0.1 M) shifted the second, low affinity, phase and prazosin did not alter concentration-effect curve to 5-HT. The sumatriptan concentration-effect curve was shifted by methiothepin (0.01 M) to the right (pK_B = 9.19) but not by ketanserin (1 M). Concentration-effect curves to 5-HT and sumatriptan were not affected by the 5-HT₃ receptor antagonist tropisetron (1 M). These results suggest that 5-HT₁-like type receptors are responsible for the first phase of 5-HT-induced contraction and 5-HT_2A receptor for the second phase, in rabbit mesenteric artery. Sumatriptan-induced contractions appear to be mediated by 5-HT₁-like type receptors in this artery. These results also suggest that this kind of amplification may be a common feature of vascular 5-HT₁-like type receptor as has been shown in other vascular segments such as rabbit femoral, iliac and renal arteries, and guinea-pig iliac artery.     

5-Hydroxytryptamine receptors with a 5-HT6 receptor-like profile stimulating adenylyl cyclase activity in pig caudate membranes

This study deals with the characterization of 5-hydroxytryptamine (5-HT, serotonin) receptors positively linked to adenylyl cyclase in membranes from pig brain caudate. 5-HT and related agonists induced a concentration-dependent stimulation of adenylyl cyclase activity in pig caudate membranes, with the following rank order of potency (mean pEC₅₀ values): 5-HT (7.1) 5-methoxytryptamine (6.9) > 5-carboxamidotryptamine (5.6) > sumatriptan (<5). Maximal stimulation by 5-HT averaged 35 pmol cyclic AMP/min/mg protein over a basal activity of 159 pmol cyclic AMP/min/mg protein. 5-Methoxytryptamine and 5-carboxamidotryptamine had similar efficacies to that of 5-HT, whereas sumatriptan was about half efficacious. Other compounds known as agonists at some 5-HT receptors were weakly potent (mean pEC₅₀ values <5). They include the 5-HT_1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), the 5-HT₄ receptor agonist, renzapride and the 5-HT₂ receptor agonist, (1-(2,5-dimethoxy-4-iodophenyl)-2 aminopropane) (DOI). In antagonist studies, methiothepin (0.1 and 1 mol/l) shifted the 5-HT curve to the right with no depression of the E_max, yielding pK_B values of 7.4–8.0. Clozapine (1 mol/l) also produced surmountable antagonism of 5-HT-induced effects (pK_B 6.9). Ketanserin (10 mol/l) weakly antagonized 5-HT (pK_B 5.0). The 5-HT₄ receptor antagonists, tropisetron (ICS 205–930) and SDZ 205–557 (2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester), each at 1 mol/l, did not significantly alter the concentration-response curve of 5-HT. The present receptor shares some characteristics of the recently cloned 5-HT₆ receptor (Monsma et al. (1993) Mol Pharmacol 43:320–327): similar pharmacological profile, location (striatum) and ability to stimulate adenylyl cyclase. It may thus represent the functional 5-HT₆ receptor in its natural environment. Correspondence to: P. Schoeffter at the above address     

5-HT1B receptor-mediated contractions in human temporal artery: evidence from selective antagonists and 5-HT receptor mRNA expression

1. In the human temporal artery both 5-HT_1-like and 5-HT_2A receptors mediate the contractile effects of 5-hydroxytryptamine (5-HT) and we have suggested that the 5-HT_1-like receptors resemble more closely recombinant 5-HT_1B than 5-HT_1D receptors. To investigate further which subtype is involved, we investigated the blockade of 5-HT-induced contractions by the 5-HT_1B-selective antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2-methyl-4′[(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-yl] carbonyl} furo[2,3-f]indole-3-spiro-4′-piperidine oxalate) and the 5-HT_1D-selective antagonist BRL-15572 (1-phenyl-3[4-3-chlorophenyl piperazin-1-yl] phenylpropan-2-ol). We also used RT-PCR to search for the mRNA of 5-HT_1B, 5-HT_1D and other 5-HT receptors.
2. The contractile effects of 5-HT in temporal artery rings were partially antagonized by SB-224289 (20, 200 nM) (apparent K_B=1 nM) and ketanserin (1 μM) but not by BRL-15572 (500 nM).
3. Sumatriptan evoked contractions (EC₅₀, 170 nM) that were resistant to blockade by BRL-15572 (500 nM) but antagonized by SB-224289 (20, 200 nM).
4. The potency of 5-HT (EC₅₀) was estimated to be 94 nM for the ketanserin-sensitive receptor and 34 nM for the SB-224289-sensitive receptor. The fraction of maximal 5-HT response mediated through SB-224289-sensitive receptors was 0.20–0.67, the remainder being mediated through ketanserin-sensitive receptors.
5. We detected arterial receptor mRNA for the following receptors (incidence): 5-HT_1B (8/8), 5-HT_1D (2/8), 5-HT_1F (0/4), 5-HT_2A (0/8), 5-HT_2B (0/8), 5-HT_2C (0/8), 5-HT₄ (4/8) and 5-HT₇ (4/8).
6. We conclude that the ketanserin-resistant fraction of the 5-HT effects and the effects of sumatriptan are mediated by 5-HT_1B receptors. The lack of antagonism by BRL-15572 rules out 5-HT_1D receptors as mediators of the contractile effects of 5-HT and sumatriptan.

     

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.     

Vasoconstrictor and vasodilator responses to tryptamine of rat-isolated perfused mesentery: comparison with tyramine and β-phenylethylamine

BACKGROUND AND PURPOSE

Tryptamine increases blood pressure by vasoconstriction, but little is known about its actions on the mesentery, in particular the resistance arteries. Tryptamine interacts with trace amine-associated receptors (TAARs) and because of its structural similarity to 5-HT, it may also interact with 5-HT receptors. Our hypothesis is therefore that the rat mesenteric arterial bed will exhibit vasopressor and vasodepressor responses to tryptamine via both 5-HT and TAARs.

EXPERIMENTAL APPROACH

Tryptamine-evoked responses were assayed from pressure changes of the rat-isolated mesenteric vasculature perfused at constant flow rate in the absence and presence of adrenoceptor and 5-HT receptor antagonists.

KEY RESULTS

Tryptamine caused dose-dependent vasoconstriction of the mesenteric arterial bed as increases in perfusion pressure. These were unaffected by the α₁-adrenoceptor antagonist, prazosin, but were attenuated by the non-selective α-adrenoceptor antagonist, phentolamine. The 5-HT_2A receptor antagonists, ketanserin and ritanserin, abolished the tryptamine-induced pressure increases to reveal vasodilator responses in mesenteric beds preconstricted with phenylephrine. These tryptamine-induced vasodilator responses were unaffected by the 5-HT₇ receptor antagonist, SB269970, but were eliminated by the NOS inhibitor, N_ω-nitro-L-arginine methyl ester (L-NAME). Tyramine and β-phenylethylamine also caused vasodilatation in pre-constricted vasculature, which was also abolished by L-NAME.

CONCLUSIONS AND IMPLICATIONS

Tryptamine causes vasoconstriction of the mesenteric vasculature via 5-HT_2A receptors, which when inhibited exposed vasorelaxant effects in pre-constricted tissues. The vasodilatation was independent of 5-HT_2A and 5-HT₇ receptors but like that for tyramine and β-phenylethylamine was due to NO release. Potency orders suggest TAAR involvement in the vasodilatation by these trace amines.