Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

1. It has been suggested that the tachycardic response to 5-hydroxytryptamine (5-HT) in the spinal-transected cat is mediated by ‘5-HT₁-like'' receptors since this effect, being mimicked by 5-carboxamidotryptamine (5-CT), is not modified by ketanserin or MDL 72222, but it is blocked by methiothepin, methysergide or mesulergine. The present study was set out to reanalyse this suggestion in terms of the IUPHAR 5-HT receptor classification schemes proposed in 1994 and 1996.
2. Intravenous (i.v.) bolus injections of the tryptamine derivatives, 5-CT (0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μg kg⁻¹), 5-HT (3, 10 and 30 μg kg⁻¹) and 5-methoxytryptamine (3, 10 and 30 μg kg⁻¹) as well as the atypical antipsychotic drug, clozapine (1000 and 3000 μg kg⁻¹) resulted in dose-dependent increases in heart rate, with a rank order of agonist potency of 5-CT >> 5-HT > 5-methoxytryptamine >> clozapine.
3. The tachycardic effects of 5-HT and 5-methoxytryptamine were dose-dependently antagonized by i.v. administration of lisuride (30 and 100 μg kg⁻¹), ergotamine (100 and 300 μg kg⁻¹) or mesulergine (100, 300 and 1000 μg kg⁻¹); the highest doses of these antagonists used also blocked the tachycardic effects of 5-CT. Clozapine (1000 and 3000 μg kg⁻¹) did not affect the 5-HT-induced tachycardia, but attenuated, with its highest dose, the responses to 5-methoxytryptamine and 5-CT. However, these doses of clozapine as well as the high doses of ergotamine (300 μg kg⁻¹) and mesulergine (300 and 1000 μg kg⁻¹) also attenuated the tachycardic effects of isoprenaline. In contrast, 5-HT-, 5-methoxytryptamine- and 5-CT-induced tachycardia were not significantly modified after i.v. administration of physiological saline (0.1 and 0.3 ml kg⁻¹), the 5-HT_1B/1D receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (500 μg kg⁻¹) or the 5-HT_3/4 receptor antagonist, tropisetron (3000 μg kg⁻¹).
4. Intravenous injections of the 5-HT₁ receptor agonists, sumatriptan (30, 100 and 300 μg kg⁻¹) and indorenate (300 and 1000 μg kg⁻¹) or the 5-HT₄ receptor (partial) agonist cisapride (300 and 1000 μg kg⁻¹) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses.
5. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht₇ receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT₇ receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht₇ gene product, the 5-HT₇ receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT₇ receptors.

     

Mediation of 5-HT-induced external carotid vasodilatation in GR 127935-pretreated vagosympathectomized dogs by the putative 5-HT7 receptor

1. The vasodilator effects of 5-hydroxytryptamine (5-HT) in the external carotid bed of anaesthetized dogs with intact sympathetic tone are mediated by prejunctional sympatho-inhibitory 5-HT_1B/1D receptors and postjunctional 5-HT receptors. The prejunctional vasodilator mechanism is abolished after vagosympathectomy which results in the reversal of the vasodilator effect to vasoconstriction. The blockade of this vasoconstrictor effect of 5-HT with the 5-HT_1B/1D receptor antagonist, GR 127935, unmasks a dose-dependent vasodilator effect of 5-HT, but not of sumatriptan. Therefore, the present study set out to analyse the pharmacological profile of this postjunctional vasodilator 5-HT receptor in the external carotid bed of vagosympathectomized dogs pretreated with GR 127935 (20 μg kg⁻¹, i.v.).
2. One-minute intracarotid (i.c.) infusions of 5-HT (0.330 μg min⁻¹), 5-carboxamidotryptamine (5-CT; 0.010.3 μg min⁻¹), 5-methoxytryptamine (1100 μg min⁻¹) and lisuride (31000 μg min⁻¹) resulted in dose-dependent increases in external carotid blood flow (without changes in blood pressure or heart rate) with a rank order of agonist potency of 5-CT>>5-HT⩾5-methoxytryptamine>lisuride, whereas cisapride (1001000 μg min⁻¹, i.c.) was practically inactive. Interestingly, lisuride (mean dose of 85±7 μg kg⁻¹, i.c.), but not cisapride (mean dose of 67±7 μg kg⁻¹, i.c.), specifically abolished the responses induced by 5-HT, 5-CT and 5-methoxytryptamine, suggesting that a common site of action may be involved. In contrast, 1 min i.c. infusions of 8-OH-DPAT (33000 μg min⁻¹) produced dose-dependent decreases, not increases, in external carotid blood flow and failed to antagonize (mean dose of 200±33 μg kg⁻¹, i.c.) the agonist-induced vasodilator responses.
3. The external carotid vasodilator responses to 5-HT, 5-CT and 5-methoxytryptamine were not modified by intravenous (i.v.) pretreatment with either saline, (±)-pindolol (4 mg kg⁻¹) or ritanserin (100 μg kg⁻¹) plus granisetron (300 μg kg⁻¹), but were dose-dependently blocked by i.v. administration of methiothepin (10 and 30 μg kg⁻¹, given after ritanserin plus granisetron), mesulergine (10 and 30 μg kg⁻¹), metergoline (1 and 3 mg kg⁻¹), methysergide (1 and 3 mg kg⁻¹) or clozapine (0.3 and 1 mg kg⁻¹). Nevertheless, the blockade of the above responses, not significant after treatment with the lower of the two doses of metergoline and mesulergine, was nonspecific after administration of the higher of the two doses of methysergide and clozapine.
4. Based upon the above rank order of agonist potencies and the antagonism produced by a series of drugs showing high affinity for the cloned 5-ht₇ receptor, our results indicate that the 5-HT receptor mediating external carotid vasodilatation in GR 127935-pretreated vagosympathectomized dogs is operationally similar to the putative 5-HT₇ receptor mediating relaxation of vascular and non-vascular smooth muscles (e.g. rabbit femoral vein, canine coronary artery, rat systemic vasculature and guinea-pig ileum) as well as tachycardia in the cat.

     

Facilitation and inhibition of male rat ejaculatory behaviour by the respective 5-HT1A and 5-HT1B receptor agonists 8-OH-DPAT and anpirtoline,as evidenced by use of the corresponding new and selective receptor antagonists NAD-299 and NAS-181

1. Ejaculatory problems and anorgasmia are well-known side-effects of the SSRI antidepressants, and a pharmacologically induced increase in serotonergic neurotransmission inhibits ejaculatory behaviour in the rat. In the present study the role of 5-HT_1A and 5-HT_1B receptors in the mediation of male rat ejaculatory behaviour was examined by use of selective agonists and antagonists acting at these 5-HT receptor subtypes.
2. The 5-HT_1A receptor agonist 8-OH-DPAT (0.25–4.00 μmol kg⁻¹ s.c.) produced an expected facilitation of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the new selective 5-HT_1A receptor antagonist (R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R) tartrate monohydrate (NAD-299) (1.0 μmol kg⁻¹ s.c.). NAD-299 by itself (0.75–3.00 μmol kg⁻¹ s.c.) did not affect the male rat ejaculatory behaviour.
3. The 5-HT_1B receptor agonist anpirtoline (0.25–4.00 μmol kg⁻¹ s.c.) produced a dose-dependent inhibition of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the 5-HT_1B receptor antagonist isamoltane (16 μmol kg⁻¹ s.c.) as well as by the new and selective antagonist (R)-(+)-2-(3-morpholinomethyl-2H-chromene-8-yl)oxymethylmorpholino methansulphonate (NAS-181) (16 μmol kg⁻¹ s.c.). Isamoltane (1.0–16.0 μmol kg⁻¹ s.c.) and NAD-181 (1.0–16.0 μmol kg⁻¹ s.c.) had no, or weakly facilitatory effects on the male rat ejaculatory behaviour. The non-selective 5-HT₁ receptor antagonist (−)-pindolol (8 μmol kg⁻¹ s.c.), did not antagonize the inhibition produced by anpirtoline.
4. The present results demonstrate opposite effects, facilitation and inhibition, of male rat ejaculatory behaviour by stimulation of 5-HT_1A and 5-HT_1B receptors, respectively, suggesting that the SSRI-induced inhibition of male ejaculatory dysfunction is due to 5-HT_1B receptor stimulation.

     

Evidence for the involvement of 5-HT4 receptors in the 5-hydroxytryptamine-induced pattern of migrating myoelectric complex in sheep

1. The effects induced by 5-hydroxytryptamine (5-HT) on gastrointestinal myoelectric activity in conscious sheep were recorded through electrodes chronically implanted and analysed by computer. The 5-HT receptors and the cholinergic neuronal pathways involved in these actions were investigated.
2. The intravenous (i.v.) administration of 5-HT (2, 4 and 8 μg kg⁻¹ min⁻¹, 5 min) induced an antral inhibition concomitant with a duodenal activity front that migrated to the jejunum, followed by a period of intestinal inactivity. This myoelectric pattern closely resembled that observed in the phases III and I of the migrating myoelectric complex (MMC) in sheep. The 0.5 μg kg⁻¹ min⁻¹ dose evoked the same pattern in only two out of the six animals used. Likewise, the 1 μg kg⁻¹ min⁻¹ dose similarly affected four of the six animals. In addition, a transient stimulation was observed in the antrum and jejunum when the two highest doses were used.
3. The 5-HT₁ antagonist, methiothepin (0.1 mg kg⁻¹), the 5-HT₂ antagonists, ritanserin (0.1 mg  kg⁻¹) and ketanserin (0.3 mg  kg⁻¹), the 5-HT₃ antagonists, granisetron (0.2 mg kg⁻¹) and ondansetron (0.5 mg kg⁻¹), as well as the 5-HT₄ antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808 (0.2 mg kg⁻¹), did not modify the spontaneous gastrointestinal myoelectric activity. However, the cholinoceptor antagonists, atropine (0.2 mg kg⁻¹) and hexamethonium (2 mg kg⁻¹), inhibited gastrointestinal activity.
4. When these antagonists were injected i.v. 10 min before 5-HT (2 or 4 μg kg⁻¹ min⁻¹, 5 min), only {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808, atropine and hexamethonium were able to modify the 5-HT-induced actions, all of them being completely blocked by the three antagonists.
5. Our data show that 5-HT initiates a MMC-like pattern in the gastrointestinal area in sheep through 5-HT₄ receptors. Furthermore, these actions are mediated by cholinergic neural pathways involving muscarinic and nicotinic receptors. However, our results do not indicate a role for either 5-HT₁, 5-HT₂ or 5-HT₃ receptors in the 5-HT-induced effects.

     

Characterization of 5-HT receptors mediating constriction of porcine carotid arteriovenous anastomoses; involvement of 5-HT1B/1D and novel receptors

1. It was previously shown that porcine cranial arteriovenous anastomoses (AVAs) constrict to 5-hydroxytryptamine (5-HT), ergotamine, dihydroergotamine, as well as sumatriptan and that sumatriptan acts exclusively via 5-HT_1B/1D receptors. The present study was devoted to establish the contribution of 5-HT_1B/1D receptors in the constriction of AVAs elicited by 5-HT (in presence of 0.5 mg kg⁻¹ ketanserin), ergotamine and dihydroergotamine in anaesthetized pigs.
2. Intracarotid infusion of 5-HT (2 μg kg⁻¹ min⁻¹) and intravenous doses of ergotamine (2.5–20 μg kg⁻¹) and dihydroergotamine (3–100 μg kg⁻¹) reduced AVA and increased nutrient blood flows and vascular conductances. The vasodilator response to 5-HT, observed mainly in the skin and ear, was much more prominent than that of the ergot alkaloids.
3. Treatment with the 5-HT_1B/1D receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (0.5 mg kg⁻¹, i.v.) significantly attenuated both ergot-induced AVA constriction and arteriolar dilatation, whereas {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 only slightly affected the carotid vascular effects of 5-HT.
4. The results suggest that 5-HT constricts carotid AVAs primarily via receptors, which seem to differ from those (5-HT_1B/1D) stimulated by sumatriptan. The ergot alkaloids produce AVA constriction for a substantial part via 5-HT_1B/1D receptors, but also stimulate unidentified receptors. Both these non-5-HT_1B/1D receptors may be targets for the development of novel antimigraine drugs.
5. The moderate vasodilator response to the ergot derivatives seems to be mediated, at least in part, by 5-HT_1B/1D receptors, whereas the arteriolar dilatation caused by 5-HT may be mediated by other, possibly 5-HT₇ receptors.

     

Citalopram-induced hypophagia is enhanced by blockade of 5-HT1A receptors: role of 5-HT2C receptors

1. The selective 5-hydroxytryptamine reuptake inhibitor citalopram (10 and 20 mg kg⁻¹, i.p.) significantly reduced food intake in male rats (CD-COBS) habituated to eat their daily food during a 4-h period.
2. The 5-HT_1A receptor antagonist WAY100635 (0.3 mg kg⁻¹) administered systemically did not modify feeding but significantly potentiated the reduction in food intake caused by 10 mg kg⁻¹ i.p. citalopram. The dose of 5 mg kg⁻¹ i.p. citalopram was not active in animals pretreated with vehicle but significantly reduced feeding in animals pretreated with WAY100635.
3. WAY100635 (0.1 μg 0.5 μl⁻¹) injected into the dorsal raphe significantly potentiated the hypophagic effect of 10 mg kg⁻¹ citalopram.
4. WAY100635 (1.0 μg 0.5 μl⁻¹) injected into the median raphe did not modify feeding or the hypophagic effect of 10 mg kg⁻¹ citalopram.
5. The 5-HT_2B/2C receptor antagonist SB206553 (10 mg kg⁻¹, p.o.) slightly reduced feeding by itself but partially antagonized the effect of WAY100635 administered systemically (0.3 mg kg⁻¹, s.c.) or into the dorsal raphe (0.1 μg 0.5 μl⁻¹) in combination with 10 mg kg⁻¹ i.p. citalopram. The hypophagic effect of 10 mg kg⁻¹ i.p. citalopram alone was not significantly modified by SB206553.
6. Brain concentrations of citalopram and its metabolite desmethylcitalopram in rats pretreated with SB206553, WAY100635 and their combination were comparable to those of vehicle-pretreated rats, 90 min after citalopram injection.
7. The hypophagic effect of citalopram was potentiated by blocking 5-HT_1A receptors. Only the effect of the WAY100635/citalopram combination seemed to be partially mediated by central 5-HT_2C receptors.

     

Modulation of the firing activity of noradrenergic neurones in the rat locus coeruleus by the 5-hydroxtryptamine system

1. The aim of the present study was to investigate the putative modulation of locus coeruleus (LC) noradrenergic (NA) neurones by the 5-hydroxytryptaminergic (5-HT) system by use of in vivo extracellular unitary recordings and microiontophoresis in anaesthetized rats. To this end, the potent and selective 5-HT_1A receptor antagonist WAY 100635 (N-{2-[4(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide trihydroxychloride) was used.
2. In the dorsal hippocampus, both local (by microiontophoresis, 20 nA) and systemic (100 μg kg⁻¹, i.v.) administration of WAY 100635 antagonized the suppressant effect of microiontophorectically-applied 5-HT on the firing activity of CA₃ pyramidal neurones, indicating its antagonistic effect on postsynaptic 5-HT_1A receptors.
3. WAY 100635 and 5-HT failed to modify the spontaneous firing activity of LC NA neurones when applied by microiontophoresis. However, the intravenous injection of WAY 100635 (100 μg kg⁻¹) readily suppressed the spontaneous firing activity of LC NA neurones.
4. The lesion of 5-HT neurones with the neurotoxin 5,7-dihydroxytryptamine increased the spontaneous firing activity of LC NA neurones and abolished the suppressant effect of WAY 100635 on the firing activity of LC NA neurones.
5. In order to determine the nature of the 5-HT receptor subtypes mediating the suppressant effect of WAY 100635 on NA neurone firing activity, several 5-HT receptor antagonists were used. The selective 5-HT₃ receptor antagonist BRL 46470A (10 and 100 μg kg⁻¹, i.v.), the 5-HT_1D receptor antagonist GR 127935 (100 μg kg⁻¹, i.v.) and the 5-HT_1A/1B receptor antagonist (−)-pindolol (15 mg kg⁻¹, i.p.) did not prevent the suppressant effect of WAY 100635 on the firing activity of LC NA neurones. However, the suppressant effect of WAY 100635 was prevented by the non-selective 5-HT receptor antagonists spiperone (1 mg kg⁻¹, i.v.) and metergoline (1 mg kg⁻¹, i.v.), by the 5-HT₂ receptor antagonist ritanserin (500 μg kg⁻¹, i.v.). It was also prevented by the 5-HT_1A receptor/α_1D-adrenoceptor antagonist BMY 7378 (1 mg kg⁻¹, i.v.) and by the α₁-adrenoceptor antagonist prazosin (100 μg kg⁻¹, i.v.).
6. These data support the notion that the 5-HT system tonically modulates NA neurotransmission since the lesion of 5-HT neurones enhanced the LC NA neurones firing activity and the suppressant effect of WAY 100635 on the firing activity of NA neurones was abolished by this lesion. However, the location of the 5-HT_1A receptors involved in this complex circuitry remains to be elucidated. It is concluded that the suppressant effect of WAY 100635 on the firing activity of LC NA neurones is due to an enhancement of the function of 5-HT neurones via a presynaptic 5-HT_1A receptor. In contrast, the postsynaptic 5-HT receptor mediating this effect of WAY 100635 on NA neurones appears to be of the 5-HT_2A subtype.

     

Pharmacological analysis of the haemodynamic effects of 5-HT1B/D receptor agonists in the normotensive rat

1. The receptors involved in mediating the haemodynamic effects of three 5-HT_1B/D receptor agonists were investigated in pentobarbitone anaesthetized rats (n=6–17 per group).
2. Cumulative intravenous (i.v.) infusions of rizatriptan and sumatriptan (from 0.63 to 2500 μg kg⁻¹; each dose over 5 min) induced dose-dependent and marked hypotension (−42±6 and −34±4 mmHg at the highest dose, respectively; both P<0.05 vs vehicle: +5±3 mmHg) and bradycardia (−85±16 and −44±12 beats min⁻¹ at the highest dose, respectively; both P<0.05 vs vehicle: +16±6 beats min⁻¹). Zolmitriptan evoked only moderate hypotension at the highest dose (−19±9 mmHg; P<0.05 vs vehicle).
3. A high dose of the 5-HT_1B/D receptor antagonist, GR 127935 (0.63 mg kg⁻¹, i.v.), failed to antagonize the hypotension and bradycardia evoked by sumatriptan (−35±6 mmHg and −52±19 beats min⁻¹, respectively; both not significant vs sumatriptan in untreated rats), but moderately reduced the hypotension and bradycardia evoked by rizatriptan (−20±5 mmHg and −30±17 beats min⁻¹, respectively; both P<0.05 vs vehicle and vs rizatriptan in untreated rats).
4. The selective 5-HT_1A receptor antagonist, WAY 100635 (0.16 and 0.63 mg kg⁻¹, i.v.), dose-dependently attenuated the haemodynamic responses evoked by rizatriptan and sumatriptan, which were almost abolished by the higher dose of WAY 100635 (−4±3 mmHg and −15±8 beats min⁻¹; both not significant vs vehicle and P<0.05 vs rizatriptan in untreated rats). A slight but statistically significant reduction in mean arterial pressure (MAP) persisted at the highest dose of sumatriptan (−13±4 mmHg following the higher dose of WAY 100635; P<0.05 vs vehicle).
5. In pithed rats with MAP normalized by angiotensin II, rizatriptan failed to induce hypotension or bradycardia (+5±4 mmHg and −6±16 beats min⁻¹, respectively; both NS vs vehicle and P<0.05 vs rizatriptan in untreated rats). Similarly, sumatriptan failed to induce bradycardia in pithed rats (+5±6 beats min⁻¹; not significant vs vehicle and P<0.05 vs sumatriptan in untreated rats), whereas a slight but statistically significant reduction in MAP, compared to controls, occurred at the highest dose (−9±9 mmHg; P<0.05 vs both vehicle and sumatriptan in untreated rats).
6. In bilaterally vagotomized and atropine-treated (1 mg kg⁻¹, i.v.) rats, the reductions in MAP and heart rate evoked by rizatriptan (−31±4 mmHg and −64 ±9 beats min⁻¹, respectively; both P<0.05 vs vehicle and not significant vs rizatriptan in controls) and sumatriptan (−47±8 mmHg and −56±10 beats min⁻¹, respectively; both P<0.05 vs vehicle and not significant vs sumatriptan in controls) were not statistically significantly different from those observed in controls.
7. In conclusion, the 5-HT_1B/D receptor agonists, rizatriptan and sumatriptan, elicit hypotension and bradycardia in the normotensive anaesthetized rat predominantly via activation of central 5-HT_1A receptors, and a consequent reduction in sympathetic outflow.

     

Electrophysiological and neurochemical evidence that pindolol has agonist properties at the 5-HT1A autoreceptor in vivo

1. It has been hypothesized that 5-HT_1A autoreceptor antagonists may enhance the therapeutic efficacy of SSRIs and other antidepressants. Although early clinical trials with the β-adrenoceptor/5-HT₁ ligand, pindolol, were promising, the results of recent more extensive trials have been contradictory. Here we investigated the actions of pindolol at the 5-HT_1A autoreceptor by measuring its effect on 5-HT neuronal activity and release in the anaesthetized rat.
2. Pindolol inhibited the electrical activity of 5-HT neurones in the dorsal raphe nucleus (DRN). This effect was observed in the majority of neurones tested (10/16), was dose-related (0.2–1.0 mg kg⁻¹, i.v.), and was reversed by the 5-HT_1A receptor antagonist, WAY 100635 (0.1 mg kg⁻¹, i.v.), in 6/7 cases tested.
3. Pindolol also inhibited 5-HT neuronal activity when applied microiontophoretically into the DRN in 9/10 neurones tested. This effect of pindolol was current-dependent and blocked by co-application of WAY 100635 (3/3 neurones tested).
4. In microdialysis experiments, pindolol caused a dose-related (0.8 and 4 mg kg⁻¹, i.v.) fall in 5-HT levels in dialysates from the frontal cortex (under conditions where the perfusion medium contained 1 μM citalopram). In rats pretreated with WAY 100635 (0.1 mg kg⁻¹, i.v.), pindolol (4 mg kg⁻¹, i.v.) did not decrease, but rather increased 5-HT levels.
5. We conclude that, under the experimental conditions used in this study, pindolol displays agonist effects at the 5-HT_1A autoreceptor. These data are relevant to previous and ongoing clinical trials of pindolol in depression which are based on the rationale that the drug is an effective 5-HT_1A autoreceptor antagonist.

     

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.

     

Risperidone inhibits 5-hydroxytryptaminergic neuronal activity in the dorsal raphe nucleus by local release of 5-hydroxytryptamine

1. The effects of risperidone on brain 5-hydroxytryptamine (5-HT) neuronal functions were investigated and compared with other antipsychotic drugs and selective receptor antagonists by use of single cell recording and microdialysis in the dorsal raphe nucleus (DRN).
2. Administration of risperidone (25–400 μg kg⁻¹, i.v.) dose-dependently decreased 5-HT cell firing in the DRN, similar to the antipsychotic drug clozapine (0.25–4.0 mg kg⁻¹, i.v.), the putative antipsychotic drug amperozide (0.5–8.0 mg kg⁻¹, i.v.) and the selective α₁-adrenoceptor antagonist prazosin (50–400 μg kg⁻¹, i.v.).
3. The selective α₂-adrenoceptor antagonist idazoxan (10–80 μg kg⁻¹, i.v.), in contrast, increased the firing rate of 5-HT neurones in the DRN, whereas the D₂ and 5-HT_2A receptor antagonists raclopride (25–200 μg kg⁻¹, i.v.) and MDL 100,907 (50–400 μg kg⁻¹, i.v.), respectively, were without effect. Thus, the α₁-adrenoceptor antagonistic action of the antipsychotic drugs might, at least partly, cause the decrease in DRN 5-HT cell firing.
4. Pretreatment with the selective 5-HT_1A receptor antagonist WAY 100,635 (5.0 μg kg⁻¹, i.v.), a drug previously shown to antagonize effectively the inhibition of 5-HT cells induced by risperidone, failed to prevent the prazosin-induced decrease in 5-HT cell firing. This finding argues against the notion that α₁-adrenoceptor antagonism is the sole mechanism underlying the inhibitory effect of risperidone on the DRN cells.
5. The inhibitory effect of risperidone on 5-HT cell firing in the DRN was significantly attenuated in rats pretreated with the 5-HT depletor PCPA (p-chlorophenylalanine; 300 mg kg⁻¹, i.p., day⁻¹ for 3 consecutive days) in comparison with drug naive animals.
6. Administration of risperidone (2.0 mg kg⁻¹, s.c.) significantly enhanced 5-HT output in the DRN.
7. Consequently, the reduction in 5-HT cell firing by risperidone appears to be related to increased availability of 5-HT in the somatodendritic region of the neurones leading to an enhanced 5-HT_1A autoreceptor activation and, in turn, to inhibition of firing, and is probably only to a minor extent caused by its α₁-adrenoceptor antagonistic action.

     

Investigation of the mechanisms underlying the hypophagic effects of the 5-HT and noradrenaline reuptake inhibitor,sibutramine, in the rat

1. Sibutramine is a novel 5-hydroxytryptamine (5-HT) and noradrenaline reuptake inhibitor (serotonin- noradrenaline reuptake inhibitor, SNRI) which is currently being developed as a treatment for obesity. Sibutramine has been shown to decrease food intake in the rat. In this study we have used a variety of monoamine receptor antagonists to examine the pharmacological mechanisms underlying sibutramine-induced hypophagia.
2. Individually-housed male Sprague-Dawley rats were maintained on reversed phase lighting with free access to food and water. Drugs were administered at 09 h 00 min and food intake was monitored over the following 8 h dark period.
3. Sibutramine (10 mg kg⁻¹, p.o.) produced a significant decrease in food intake during the 8 h following drug administration. This hypophagic response was fully antagonized by the α₁-adrenoceptor antagonist, prazosin (0.3 and 1 mg kg⁻¹, i.p.), and partially antagonized by the β₁-adrenoceptor antagonist, metoprolol (3 and 10 mg kg⁻¹, i.p.) and the 5-HT receptor antagonists, metergoline (non-selective; 0.3 mg kg⁻¹, i.p.); ritanserin (5-HT_2A/2C; 0.1 and 0.5 mg kg⁻¹, i.p.) and SB200646 (5-HT_2B/2C; 20 and 40 mg kg⁻¹, p.o.).
4. By contrast, the α₂-adrenoceptor antagonist, RX821002 (0.3 and 1 mg kg⁻¹, i.p.) and the β₂-adrenoceptor antagonist, ICI 118,551 (3 and 10 mg kg⁻¹, i.p.) did not reduce the decrease in food intake induced by sibutramine.
5. These results demonstrate that β₁-adrenoceptors, 5-HT_2A/2C-receptors and particularly α₁-adrenoceptors, are involved in the effects of sibutramine on food intake and are consistent with the hypothesis that sibutramine-induced hypophagia is related to its ability to inhibit the reuptake of both noradrenaline and 5-HT, with the subsequent activation of a variety of noradrenaline and 5-HT receptor systems.

     

Antagonist properties of (?)-pindolol and WAY 100635 at somatodendritic and postsynaptic 5-HT1A receptors in the rat brain

1. The aim of the present work was to characterize the 5-hydroxytryptamine_1A (5-HT_1A) antagonistic actions of (−)-pindolol and WAY 100635 (N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide). Studies were performed on 5-HT_1A receptors located on 5-hydroxytryptaminergic neurones in the dorsal raphe nucleus (DRN) and on pyramidal cells in the CA1 and CA3 regions of the hippocampus in rat brain slices.
2. Intracellular electrophysiological recording of CA1 pyramidal cells and 5-hydroxytryptaminergic DRN neurones showed that the 5-HT_1A receptor agonist 5-carboxamidotryptamine (5-CT) evoked in both cell types a concentration-dependent cell membrane hyperpolarization and a decrease in cell input resistance. On its own, (−)-pindolol did not modify the cell membrane potential and resistance at concentrations up to 10 μM, but it antagonized the 5-CT effects in a concentration-dependent manner. Similar antagonism of 5-CT effects was observed in the CA3 hippocampal region. (−)-Pindolol also prevented the 5-HT_1A receptor-mediated hyperpolarization of CA1 pyramidal cells due to 5-HT (15 μM). In contrast, the 5-HT-induced depolarization mediated by presumed 5-HT₄ receptors persisted in the presence of 3 μM (−)-pindolol.
3. In the hippocampus, (−)-pindolol completely prevented the hyperpolarization of CA1 pyramidal cells by 100 nM 5-CT (IC₅₀=92 nM; apparent K_B=20.1 nM), and of CA3 neurones by 300 nM 5-CT (IC₅₀=522 nM; apparent K_B=115.1 nM). The block by (−)-pindolol was surmounted by increasing the concentration of 5-CT, indicating a reversible and competitive antagonistic action.
4. Extracellular recording of the firing rate of 5-hydroxytryptaminergic neurones in the DRN showed that (−)-pindolol blocked, in a concentration-dependent manner, the decrease in firing elicited by 100 nM 5-CT (IC₅₀=598 nM; apparent K_B=131.7 nM) or 100 nM ipsapirone (IC₅₀=132.5 nM; apparent K_B=124.9 nM). The effect of (−)-pindolol was surmountable by increasing the concentration of the agonist. Intracellular recording experiments showed that 10 μM (−)-pindolol were required to antagonize completely the hyperpolarizing effect of 100 nM 5-CT.
5. In vivo labelling of brain 5-HT_1A receptors by i.v. administration of [³H]-WAY 100635 ([O-methyl-³H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl-N-(2-pyridyl)cyclo-hexane-carboxamide) was used to assess their occupancy following in vivo treatment with (−)-pindolol. (−)-Pindolol (15 mg kg⁻¹) injected i.p. either subchronically (2 day-treatment before i.v. injection of [³H]-WAY 100635) or acutely (20 min before i.v. injection of [³H]-WAY 100635) markedly reduced [³H]-WAY 100635 accumulation in all 5-HT_1A receptor-containing brain areas. In particular, no differences were observed in the capacity of (−)-pindolol to prevent [³H]-WAY 100635 accumulation in the DRN and the CA1 and CA3 hippocampal areas.
6. Intracellular electrophysiological recording of 5-hydroxytryptaminergic DRN neurones showed that WAY 100635 prevented the hyperpolarizing effect of 100 nM 5-CT in a concentration-dependent manner (IC₅₀=4.9 nM, apparent K_B=0.25 nM). In CA1 pyramidal cells, hyperpolarization induced by 50 nM 5-CT was also antagonized by WAY 100635 (IC₅₀=0.80 nM, apparent K_B=0.28 nM).

     

Agonistic properties of alniditan,sumatriptan and dihydroergotamine on human 5-HT1B and 5-HT1D receptors expressed in various mammalian cell lines

1. Alniditan, a novel migraine abortive agent, is a potent 5-HT_1B/5-HT_1D receptor agonist of nM affinity. We compared the agonistic properties of alniditan, sumatriptan and dihydroergotamine on the cloned human 5-HT_1B receptor expressed at 200 fmol mg⁻¹ protein (B_max) in non-induced L929sA cells, at 740 fmol mg⁻¹ protein in HEK 293 and at 2300 fmol mg⁻¹ protein in mIFNβ-induced L929sA cells, and on the human cloned 5-HT_1D receptor expressed in C6 glioma cells (B_max 780 fmol mg⁻¹ protein).
2. Sodium butyrate treatment increased the expression level of human (h)5-HT_1B receptors in HEK 293 cells and h5-HT_1D receptors in C6 glioma cells approximately 3 fold, the binding affinities of [³H]-5-HT and [³H]-alniditan were unaffected.
3. Agonistic properties were evaluated based on inhibition of cyclic AMP accumulation in the cells after stimulation of adenylyl cyclase by forskolin or isoproterenol. Alniditan, sumatriptan and dihydroergotamine were full agonists at the h5-HT_1B receptor (IC₅₀ values were 1.7, 20 and 2 nM, respectively in HEK 293 cells) and h5-HT_1D receptors (IC₅₀ values of 1.3, 2.6 and 2.2 nM, respectively). At the h5-HT_1B receptor the agonist potency of the compounds slightly increased with higher receptor density. The opposite was seen for antagonists (ocaperidone, risperidone and ritanserin).
4. This comparative study demonstrated that alniditan was 10 times more potent than sumatriptan at the h5-HT_1B receptor, and twice as potent at the h5-HT_1D receptor. Dihydroergotamine was more potent an agonist at the h5-HT_1B receptor when expressed at high and low level in L929sA cells (but not in HEK 293 cells), and was less potent at the h5-HT_1D receptor.

     

Comparative desensitization of the human 5-HT2A and 5-HT2C receptors expressed in the human neuroblastoma cell line SH-SY5Y

1. We have used previously characterized clones of the human neuroblastoma cell line, SH-SY5Y, constitutively expressing either the human 5-HT_2A or 5-HT_2C receptor to compare their desensitization profiles after exposure to 5-HT.
2. 5-HT stimulated [³H]-inositol phosphate ([³H]-IP_x) production at both the 5-HT_2C (pEC₅₀=8.03±0.15) and 5-HT_2A receptors (pEC₅₀=7.15±0.08), with maximal responses occurring after exposure to 1 μM and 10 μM 5-HT, respectively.
3. Exposure of cells to maximally effective concentrations of 5-HT caused time- and concentration-dependent desensitization of [³H]-IP_x formation. The 5-HT_2A response desensitized slower (t_1/2=110 min) and with lower sensitivity than that of the 5-HT_2C receptor (t_1/2=12.5 min). In each case, desensitization was blocked by co-administration of a specific receptor antagonist. Following exposure to 10 μM 5-HT for 2 h, both receptors exhibited extensive desensitization, with subsequent responses to 5-HT reduced by more than 80%.
4. 5-HT stimulated Ins(1,4,5)P₃ production with a potency similar to that for [³H]-IP_x production at each receptor. In both cases Ins(1,4,5)P₃ levels peaked rapidly then returned to basal level within a short time. This peak consistently occurred earlier for the 5-HT_2C receptor (5 s) than for the 5-HT_2A receptor (20 s).
5. Prior exposure of SH-SY5Y/5-HT_2C cells to 5-HT (1 μM/15 min) caused a significant decrease in the 5-HT-stimulated peak in Ins(1,4,5)P₃ levels whereas no such change occurred in SH-SY5Y/5-HT_2A cells following exposure to 10 μM 5-HT for 15 min.
6. These results indicate that the human 5-HT_2A and 5-HT_2C receptors both exhibit desensitization at the level of inositol phosphate formation when expressed in the same cellular environment, with the 5-HT_2C receptor being more sensitive to 5-HT-mediated desensitization than the 5-HT_2A receptor.

     

Opposite effects of CCKB agonists in grooming behaviour in rats: further evidence for two CCKB subsites

1. The hypothesis of the existence of two CCK_B receptor subsites, CCK_B1 and CCK_B2 corresponding probably to different coupling states of CCK_B receptors, was studied by measuring grooming behaviour in rats.
2. The B1 receptor agonist, BC197 (300 μg kg⁻¹, i.p.) produced a 45–50% decrease in grooming activity, which was prevented by both the CCK_B receptor antagonists CI-988 (20 μg kg⁻¹ i.p.) and L-365,260 (200 μg kg⁻¹, i.p.).
3. In contrast, 3, 10 and 30 μg kg⁻¹, i.p., of the potent B2 receptor agonist, BC264, enhanced grooming (150–190%). This effect was prevented by previous injection of 75 μg kg⁻¹ of L-365,260 while higher doses (200 μg kg⁻¹, i.p.) produced only a partial antagonism. Moreover, CI-988 (20 μg kg⁻¹, i.p.), showed an opposite effect in potentiating the responses induced by BC264. However, 200 μg kg⁻¹ of CI-988 tended to suppress the increase of grooming induced by BC264.
4. The effects of BC264 were prevented by the D₁ receptor (SCH 23390) and D₂ receptor (sulpiride) antagonists, while those of BC197 were only antagonized by sulpiride, emphasizing the existence of a link between peptidergic (CCK) and dopaminergic systems.
5. This study brings additional evidence for the existence of the two CCK_B receptor subsites and suggests that particular attention should be focused on the selectivity of CCK_B receptor agonists, notably to explain the fact that some compounds such as Boc-CCK4 induce anxiogenic-like effects while others, including BC264, are devoid of these effects.

     

Effect of the selective 5-HT1A receptor antagonist WAY 100635 on the inhibition of e.p.s.ps produced by 5-HT in the CA1 region of rat hippocampal slices

1. The actions of N-(2-(-4(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), a novel and selective 5-hydroxytryptamine_1A (5-HT_1A) antagonist, on excitatory postsynaptic potentials (e.p.s.ps) were investigated by use of intracellular recordings in pyramidal cells of the CA1 region of rat hippocampal slices.
2. WAY 100635 (10 nM) did not affect any of the investigated parameters of cell excitability such as membrane potential, total input resistance (R_in), firing threshold, action potential amplitude, action potential frequency adaptation, and slow afterhyperpolarization (sAHP) which follows repetitive firing of action potentials. WAY 100635 did not have any effect on either the slope or the amplitude of e.p.s.ps evoked by stimulation of the CA1 stratum radiatum.
3. Bath application of either 5-hydroxytryptamine (5-HT, 10–30 μM) or 5-carboxamidotryptamine (5-CT, 300 nM) hyperpolarized the membrane potential (ΔV_m=−4.1±0.9 and −6.0±0.9 mV, respectively), and reduced R_in (−25±8% and −18±1%, respectively). 5-HT blocked the action potential frequency adaptation and significantly reduced the amplitude of the sAHP that follows repetitive firing of action potentials.
4. 5-HT significantly decreased the amplitude of evoked e.p.s.ps (−14±6%). This effect was greater in the presence of the GABA_A receptor antagonist bicuculline (10 μM, −45±12%) and was mimicked by 5-CT (−49±5%). Both AMPA and NMDA components of e.p.s.ps were significantly reduced in amplitude by 5–HT (−38±8%, n=6, and −29±12%, n=3, respectively; P<0.05).
5. WAY 100635 fully antagonized the hyperpolarization, the reduction of R_in, and the decrease in amplitude of e.p.s.ps elicited by 5-HT, while it did not affect the action of 5-HT on the action potential frequency adaptation. In the presence of WAY 100635, 5-HT elicited a depolarization which was blocked by 10–30 μM RS 23597-190, a selective 5-HT₄ receptor antagonist.
6. Our data demonstrate that WAY 100635 is devoid of direct effects on CA1 pyramidal cell excitability and on evoked e.p.s.ps, while it fully antagonizes the effects of 5-HT on excitatory synaptic transmission and on hyperpolarization, without affecting the 5-HT₄ receptor-mediated response. Since WAY 100635 selectively antagonizes 5-HT_1A receptor-mediated actions of 5-HT, our data also demonstrate that the inhibitory action of 5-HT on excitatory synaptic transmission in CA1 is mediated by 5-HT_1A receptors.

     

Investigation into the contractile response of melatonin in the guinea-pig isolated proximal colon: the role of 5-HT4 and melatonin receptors

1. The interaction of melatonin (N-acetyl-5-methoxytryptamine) with 5-hydroxytryptamine₄ (5-HT₄) receptors and/or with melatonin receptors (ML₁, ML₂ sites) has been assessed in isolated strips of the guinea-pig proximal colon. In the same preparation, the pharmacological profile of a series of melatonin agonists (2-iodomelatonin, 6-chloromelatonin, N-acetyl-5-hydroxytryptamine (N-acetyl-5-HT), 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT)) was investigated.
2. In the presence of 5-HT_1/2/3 receptor blockade with methysergide (1 μM) and ondansetron (10 μM), melatonin (0.1 nM–10 μM), 5-HT (1 nM–1 μM) and the 5-HT₄ receptor agonist, 5-methoxytryptamine (5-MeOT: 1 nM–1 μM) caused concentration-dependent contractile responses. 5-HT and 5-MeOT acted as full agonists with a potency (−log EC₅₀) of 7.8 and 8.0, respectively. The potency value for melatonin was 8.7, but its maximum effect was only 58% of that elicited by 5-HT.
3. Melatonin responses were resistant to atropine (0.1 μM), tetrodotoxin (0.3 μM), and to blockade of 5-HT₄ receptors by SDZ 205,557 (0.3 μM) and GR 125487 (3, 30 and 300 nM). The latter antagonist (3 nM) inhibited 5-HT-induced contractions with an apparent pA₂ value of 9.6. GR 125487 antagonism was associated with 30% reduction of the 5-HT response maximum. Contractions elicited by 5-HT were not modified when melatonin (1 and 10 nM) was used as an antagonist.
4. Like melatonin, the four melatonin analogues concentration-dependently contracted colonic strips. The rank order of agonist potency was: 2-iodomelatonin (10.8) >6-chloromelatonin (9.9) ⩾ N-acetyl-5-HT (9.8) ⩾5-MCA-NAT (9.6) >melatonin (8.7), an order typical for ML₂ sites. In comparison with the other agonists, 5-MCA-NAT had the highest intrinsic activity.
5. The melatonin ML_1B receptor antagonist luzindole (0.3, 1 and 3 μM) had no effect on the concentration-response curve to melatonin. Prazosin, an α-adrenoceptor antagonist possessing moderate/high affinity for melatonin ML₂ sites did not affect melatonin-induced contractions at 0.1 μM. Higher prazosin concentrations (0.3 and 1 μM) caused a non-concentration-dependent depression of the maximal response to melatonin without changing its potency. Prazosin (0.1 and 1 μM) showed a similar depressant behaviour towards the contractile responses to 5-MCA-NAT.
6. In the guinea-pig proximal colon, melatonin despite some structural similarity with the 5-HT₄ receptor agonist 5-MeOT, does not interact with 5-HT₄ receptors (or with 5-HT_1/2/3 receptors). As indicated by the rank order of agonist potencies and by the inefficacy of luzindole, the most likely sites of action of melatonin are postjunctional ML₂ receptors. However, this assumption could not be corroborated with the use of prazosin as this ‘ML₂ receptor antagonist'' showed only a non-concentration-dependent depression of the maximal contractile response to both melatonin and 5-MCA-NAT. Further investigation with the use of truly selective antagonists at melatonin ML₂ receptors is required to clarify this issue.

     

Mechanism of actions of sumatriptan on coronary flow before and after endothelial dysfunction in guinea-pig isolated heart

1. The mechanism of action of sumatriptan on coronary flow was examined before and after two different forms of endothelial ablation in guinea-pig isolated hearts. The mechanism was assessed in terms of the influence of the integrity of the coronary endothelium, the role of release of nitric oxide (NO) from the endothelium, and the receptor subtypes mediating the effects.
2. Continuous perfusion with sumatriptan reduced coronary flow, but the concentration-response curve was v-shaped. Sumatriptan (0.001–0.1 μM) caused a concentration-dependent decrease in coronary flow with the maximum effect achieved at 0.23±0.04 μM. The pEC₅₀ was 8.49±0.07. At higher concentrations (0.1–10 μM) there was a concentration-dependent diminution of the vasoconstrictor effect. Endothelial ablation by saponin removed the diminution in the vasoconstrictor effect. In contrast, pretreatment with N^G-nitro L-arginine methyl ester (L-NAME) (100 μM, 45 min perfusion) did not affect it. This was despite both saponin and L-NAME being effective in reducing basal release of NO into the coronary effluent (measured by chemiluminescence) to the same extent (71±3 and 73±2%, respectively).
3. {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935, a selective 5-hydroxytryptamine_1D (5-HT_1D) receptor antagonist (3 and 10 nM), which by itself had no effect on coronary flow or NO release, antagonized the vasoconstrictor response to sumatriptan and unmasked a sumatriptan-induced concentration-dependent increase in coronary flow and NO release. These increases in coronary flow and NO release were abolished by pretreatment with either saponin or L-NAME.
4. Mesulergine, a 5-HT₂ receptor antagonist which had no effect by itself on basal coronary flow or NO release, inhibited the vasodilator response to sumatriptan that occurred in the presence of {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935, and actually enhanced the vasoconstrictor response, increasing the maximum fall in coronary flow from −3.9±0.4 to −5.2±0.4 ml min⁻¹ g⁻¹ (P<0.05). The diminution of vasoconstrictor effect of sumatriptan was abolished by mesulergine and by pretreatment with saponin, but not by L-NAME.
5. In conclusion, guinea-pig coronary arteries constrict to low concentrations of sumatriptan, causing a reduction in coronary flow. This effect appears to be caused by 5-HT_1D agonism with the receptors located on the coronary vascular smooth muscle. With higher concentrations of sumatriptan this is partially offset by a weaker vasodilator effect, which is caused by low affinity 5-HT₂ agonism. Although this effect is endothelium-dependent, it is not caused by the release of NO. Interestingly, when the vasoconstrictor effect of sumatriptan was inhibited by the 5-HT_1D antagonist {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935, a high affinity vasodilator effect of sumatriptan was unmasked. This is 5-HT₂ receptor mediated and is caused by release of NO from the coronary endothelium.
6. In man, sumatriptan and 5-HT may both be capable of causing pathogenic coronary vasoconstriction. The implications of the present data are that the scope for this may depend greatly on (i) the extent of underlying endothelial dysfunction, (ii) the extent of endothelial 5-HT₂ receptor-mediated release of vasodilator autacoids (which include NO) and (iii) the extent of smooth muscle 5-HT_1D receptor-mediated vasoconstriction.

     

Effect of chronic m-CPP on locomotion,hypophagia, plasma corticosterone and 5-HT2C receptor levels in the rat

1. The present study examined 5-HT_2C receptor agonist-induced behavioural tolerance and 5-HT_2C receptor down-regulation in adult rat brain. The effect of chronic subcutaneous infusion of the 5-HT_2C receptor agonist, m-chlorophenylpiperazine (m-CPP, 10 mg kg⁻¹, day⁻¹), for 14 days was examined on daily food intake, the ability of acute m-CPP (2.5 mg kg⁻¹, i.p.) to induce hypolocomotion in a novel arena and elevate plasma corticosterone levels and on ex vivo cortical [³H]-mesulergine binding and hippocampal 5-HT_2C receptor protein levels.
2. Before chronic infusion, m-CPP (2.5 mg kg⁻¹, i.p.) attenuated the number of turns and rears made in a novel open field arena. In contrast, while m-CPP still elicited this hypolocomotion following 14 days, saline infusion, no such hypolocomotion occurred in rats given chronic m-CPP (10 mg kg⁻¹ day⁻¹), indicating that almost complete tachyphylaxis of this behaviour occurred with chronic 5-HT_2C receptor agonist injection.
3. During chronic infusion of m-CPP, rats consumed less food per day than saline-treated controls. Acute challenge with m-CPP following two weeks, treatment still attenuated food intake over the next four hours (by 43% and 30%, respectively from that on the previous day) in saline and m-CPP infusion groups, showing that only partial tolerance to 5-HT_2C receptor agonist-induced hypophagia occurred.
4. In naive home cage rats, plasma corticosterone was elevated in a dose-dependent manner 35 min after m-CPP injection (0.5, 1 and 3 mg kg⁻¹, i.p.) but levels were comparable to control values 16 h after m-CPP (2, 5 and 10 mg kg⁻¹, i.p.). Sixteen hours after a single m-CPP injection (2.5 mg kg⁻¹, i.p.), plasma corticosterone levels were comparable in a group of rats which had received 14 days infusion of m-CPP or saline. However, following a similar acute m-CPP injection (2.5 mg kg⁻¹, i.p., −16 h) in rats previously infused for 14 days with m-CPP, plasma corticosterone levels were lower than those in a separate group which received no chronic infusions (but only acute m-CPP injection), even though the plasma m-CPP levels were comparable in both groups. The data are consistent with the proposal that chronic m-CPP induced some down-regulation of hypothalamic 5-HT_2C receptors which contribute, in a tonic manner, to plasma corticosterone secretion under the conditions investigated.
5. Chronic m-CPP infusion reduced the amount of [³H]-mesulergine binding (by 27%, without altering the K_D) in membranes prepared from parietal/occipital/temporal cortex (under conditions to exclude binding to 5-HT_2A receptors) and 5-HT_2C receptor protein-like immunoreactive levels measured by radioimmunoassay in the hippocampus by 38%, confirming that 5-HT_2C receptor down-regulation had occurred.
6. Even after 14 days m-CPP infusion only partial behavioural tolerance and 5-HT_2C receptor down-regulation were observed, which may vary in different brain regions of the rat. Thus the hypophagia produced by m-CPP may involve activation of 5-HT_2C receptors in the hypothalamus, where there is a greater receptor reserve or which are more resistant to agonist-induced down-regulation than 5-HT_2C receptors in limbic areas (striatum and nucleus accumbens) mediating m-CPP-induced hypolocomotion.