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.

     

Characterization of prejunctional 5-HT1 receptors that mediate the inhibition of pressor effects elicited by sympathetic stimulation in the pithed rat

1. A study was made of the effects of 5-carboxamidotryptamine (5-CT) on pressor responses induced in vivo by electrical stimulation of the sympathetic outflow from the spinal cord of pithed rats. All animals had been pretreated with atropine. Sympathetic stimulation (0.1, 0.5, 1 and 5 Hz) resulted in frequency-dependent increases in blood pressure. Intravenous infusion of 5-CT at doses of 0.01, 0.1 and 1 μg kg⁻¹ min⁻¹ reduced the pressor effects obtained by electrical stimulation. The inhibitory effect of 5-CT was significantly more pronounced at lower frequencies of stimulation. In the present study we characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-CT.
2. The inhibition induced by 0.01 μg kg⁻¹ min⁻¹ of 5-CT on sympathetically-induced pressor responses was partially blocked after i.v. treatment with methiothepin (10  μg kg⁻¹), WAY-100,635 (100 μg kg⁻¹) or GR127935T (250 μg kg⁻¹), but was not affected by cyanopindolol (100 μg kg⁻¹).
3. The selective 5-HT_1A receptor agonist 8-OH-DPAT and the selective 5-HT_1B/1D receptor agonists sumatriptan and L-694,247 inhibited the pressor response, whereas the 5-HT_1B receptor agonists CGS-12066B and CP-93,129 and the 5-HT_2C receptor agonist m-CPP did not modify the pressor symapthetic responses.
4. The selective 5-HT_1A receptor antagonist WAY-100,635 (100 μg kg⁻¹) blocked the inhibition induced by 8-OH-DPAT and the selective 5-HT_1B/1D receptor antagonist GR127935T (250 μg kg⁻¹) abolished the inhibition induced either by L-694,247 or sumatriptan.
5. None of the 5-HT receptor agonists used in our experiments modified the pressor responses induced by exogenous noradrenaline (NA).
6. These results suggest that the presynaptic inhibitory action of 5-CT on the electrically-induced pressor response is mediated by both r-5-HT_1D and 5-HT_1A receptors.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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.

     

Role of α2-adrenoceptors in the regulation of intestinal water transport

1. The influence of the sympathetic nervous system on intestinal fluid transport by the jejunum and ileum of the anaesthetized rat was investigated under basal conditions and during active secretion induced by intra-arterial infusion of vasoactive intestinal peptide (VIP).
2. Intra-arterial infusion of noradrenaline (3, 10, 30 nmol min⁻¹, i.a.) and i.v. injection of the selective α₂-adrenoceptor agonist UK 14,304 (1 μmol kg⁻¹, i.v.) increased the rate of basal fluid absorption. The effect of UK 14,304 was blocked by yohimbine (10 μmol kg⁻¹, i.v). However, the selective α₁-adrenoceptor agonist phenylephrine (5 μmol kg⁻¹, i.v.) did not alter either the jejunal or ileal absorption rate.
3. The α₂-adrenoceptor antagonists yohimbine (0.3, 1.0, 3 and 10 μmol kg⁻¹, i.v.) and rauwolscine (10 μmol kg⁻¹, i.v.) decreased the basal absorption rate, while the α₁-adrenoceptor antagonist prazosin (3 μmol kg⁻¹, i.v.) was without effect. Intracerebroventricular injection of yohimbine (3 μmol kg⁻¹) caused a significant antiabsorptive effect in the jejunum but not ileum.
4. Peripheral chemical sympathectomy induced by pretreating animals with 6-hydroxydopamine (150 mg kg⁻¹, i.p., total dose) induced a trend towards impaired absorption in the jejunum and ileum.
5. The findings provide evidence that the sympathetic nervous system exerts tonic control on intestinal fluid transport and that the effect is mainly through peripheral α₂-adrenoceptors.
6. The subtype determination of α₂-adrenoceptors in modulating intestinal fluid transport was assessed by determining the effects of α₂-adrenoceptor agents on intestinal fluid secretion induced by i.a. infusion of VIP (0.8 μg min⁻¹).
7. Intravenous administration of UK 14,304 caused a dose-dependent reversal of the secretory phase of the VIP-induced response, but failed to restore fluid transport to the control level of net absorption. EC₅₀ values were 0.17 μmol kg⁻¹ in the jejunum and 0.22 μmol kg⁻¹ in the ileum.
8. The effect of UK 14,304 was blocked by the selective α_2A/D antagonist BRL 44408 and the non-selective α₂ antagonist yohimbine (each 10 μmol kg⁻¹). The selective α_2B/C antagonist ARC 239 (10 μmol kg⁻¹) did not affect the antisecretory action of UK 14,304. It is suggested that the α₂-adrenoceptors in the rat intestinal epithelium are the α_2D or α_2A-like subtype.

     

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.

     

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.

     

The role of A3 adenosine receptors in central regulation of arterial blood pressure

1. Pharmacological studies have suggested that A₃ receptors are present on central neurons. Recently this adenosine receptor subtype has been identified in the rat and its presence in the central nervous system has been confirmed.
2. In this study we investigated the effects of acute intracerebroventricular (i.c.v.) injections of N⁶-2-(4-aminophenyl)-ethyladenosine (APNEA), a non-selective A₃ adenosine receptor agonist, on arterial blood pressure (ABP) and heart rate (HR), after treatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective antagonist of A₁ adenosine receptors.
3. Anaesthetized rats, after DPCPX (12 μg⁻¹ kg i.c.v.), were treated with APNEA (0.4–4 μg kg⁻¹ i.c.v.) resulting in a transitory and dose-dependent decrease in arterial blood pressure without a change in heart rate. APNEA also induced hypotensive responses after i.c.v. pretreatment with aminophylline, at a dose of 20 μg kg⁻¹. In contrast, pretreatment 48 h before, with 4 μg kg⁻¹ i.c.v. of pertussis toxin reduced the hypotensive effect induced by APNEA. Administration of APNEA at a higher dose (20 μg kg⁻¹ i.c.v.), after DPCPX, induced a decrease in ABP of −66±5.4 mmHg and after 3 min a decrease in heart rate of −62±6.0 beats min⁻¹. Transection of the spinal cord abolished this significant fall in ABP, but not the decrease of HR.
4. These results suggest that a population of A₃-receptors is present in the CNS, whose activation induces a decrease in blood pressure with no change of heart rate.

     

Receptor subtypes Y1 and Y5 are involved in the renal effects of neuropeptide Y

1. Systemic infusion of neuropeptide Y (NPY) reduces renal blood flow and can concomitantly increase diuresis, natriuresis and calciuresis in anaesthetized rats. The present study was designed to investigate whether the apparently contradictory NPY effects on renal blood flow and urine formation and composition are mediated by distinct NPY receptor subtypes.
2. NPY and its analogues, peptide YY (PYY), [Leu³¹, Pro³⁴]NPY and NPY₁₃₃₆, were infused in incremental doses of 0.3, 1 and 3 μg kg⁻¹ min⁻¹ for 45 min each and the results compared to those obtained in vehicle-infused rats. Renal blood flow was monitored in 15 min intervals, while urine excretion and composition were determined in 15 min collection periods. Plasma renin activity and aldosterone concentrations were measured at the end of the final infusion period.
3. Relative to vehicle NPY, PYY and [Leu³¹, Pro³⁴]NPY dose-dependently reduced renal blood flow and increased diuresis, natriuresis and calciuresis with roughly similar potency; NPY₁₃₃₆ slightly but significantly increased renal blood flow but had no effect on diuresis, natriuresis and calciuresis. None of the peptides significantly affected endogenous creatinine clearance or kaliuresis.
4. Plasma renin activity was significantly reduced by PYY. Quantitatively similar reductions were observed with NPY and [Leu³¹, Pro³⁴]NPY but failed to reach statistical significance with the given number of experiments. NPY₁₃₃₆ did not reduce plasma renin activity. None of the peptides significantly affected plasma aldosterone concentrations.
5. In another series of experiments infusion of PYY₃₃₆ (2 μg kg⁻¹ min⁻¹ for 120 min) did not reduce renal blood flow but significantly enhancd diuresis and natriuresis to a similar extent as the NPY 2 μg kg⁻¹ min⁻¹.
6. In a final series of experiments the Y₁-selective antagonist, BIBP 3226 (1 or 10 μg kg⁻¹ min⁻¹) dose-dependently antagonized reductions of renal blood flow elicited by bolus injections of NPY (0.130 μg kg⁻¹). BIBP 3226 (10 μg kg⁻¹ min⁻¹) also inhibited the effects of a 120 min infusion of NPY (2 μg kg⁻¹ min⁻¹) on renal blood flow but had only minor inhibitory effects on enhancements of diuresis and did not significantly affect enhancements of natriuresis.
7. We conclude that NPY reduces renal blood via a classical Y₁ subtype of NPY receptor. In contrast enhancements of diuresis, natriuresis and calciuresis occur via a distinct subtype which resembles the receptor that mediates NPY-induced enhancement of food intake.

     

The role of α2-adrenoceptor antagonism in the anti-cataleptic properties of the atypical neuroleptic agent,clozapine, in the rat

1. The mechanism underlying the anticataleptic properties of the atypical neuroleptic agent, clozapine, has been investigated in the rat.
2. The close structural analogues of clozapine, loxapine (0.1 mg kg⁻¹ s.c.) and iso-clozapine (1 and 3 mg kg⁻¹ s.c.) induced catalepsy in rats. In contrast, clozapine and the regio-isomer of loxapine, iso-loxapine (up to 10 mg kg⁻¹ s.c.) did not produce catalepsy, but at a dose of 1 mg kg⁻¹ significantly inhibited catalepsy induced by loxapine (0.3 mg kg⁻¹ s.c.).
3. Radioligand binding assays showed that cataleptogenic potential was most clearly predicted by the D₂/5-HT_1A, D₂/5-HT_1B/1D and D₂/α₂-receptor affinity (K_D) ratios: i.e. 30–100-fold higher ratios were calculated for loxapine and iso-clozapine, whereas the ratios were less than 1 for clozapine and iso-loxapine. The ratios of affinities for D₂ to 5-HT_2A, 5-HT_2C or D₁ did not reflect the grouping of cataleptic and non-cataleptic compounds.
4. Co-treatment with the α₂-adrenoceptor antagonists, yohimbine (1–10 mg kg⁻¹ s.c.), RX 821002 (1–10 mg kg⁻¹ s.c.) and MK-912 (0.3 and 1 mg kg⁻¹ s.c.) dose-dependently inhibited the cataleptic response to loxapine (0.3 mg kg⁻¹). Yohimbine (1–10 mg kg⁻¹ s.c.) also dose-dependently inhibited the cateleptic response to haloperidol (0.3 mg kg⁻¹ s.c.). The α₂-adrenoceptor antagonists had no effect per se.
5. Neither yohimbine (10 mg kg⁻¹) nor RX821002 (3 mg kg⁻¹) altered the cataleptic response to the D₁ receptor antagonist, SCH 23390 (1 mg kg⁻¹ s.c.), while, like clozapine, both compounds abolished the response to the 5-HT_2A receptor antagonist, MDL 100,151 (3 mg kg⁻¹ s.c.).
6. The present data strongly implicate α₂-adrenoceptor blockade in the anticataleptic properties of clozapine and suggest that its lack of extrapyramidal side effects in the clinic may also be a consequence of this property.

     

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.

     

Vasodilator effects of adrenomedullin on small pulmonary arteries and veins in anaesthetized cats

1. This study was conducted to determine adrenomedullin (AM) action sites in the pulmonary vascular bed and the relation between its vasodilator effects and vascular tone. Moreover, an examination was made into whether calcitonin gene-related peptide (CGRP) receptors mediate pulmonary vasodilatations induced by AM. To this end, we directly measured internal diameter (i.d.) changes in small pulmonary arteries and veins (100–1100 μm i.d.) by use of an X-ray televison system on the in vivo cat lung.
2. Under control (resting vascular tone) conditions, AM injections into the left main pulmonary artery caused dose-related i.d. increases in both small arteries and veins. The mean i.d. increase of the 100–1100 μm arteries (4±1, 11±2, and 17±2% with 0.01, 0.1, and 1 nmol kg⁻¹ AM, respectively) was significantly larger than that for the veins (1±1, 5±2, and 7±2% with 0.01, 0.1 and 1 nmol kg⁻¹ AM, respectively) whatever the injected dose of AM.
3. When unilobar hypoxia (5% O₂) had decreased the i.d. of the 100–1100 μm arteries and veins by 16±3 and 6±3%, respectively, AM (0.1 nmol kg⁻¹) was able to induce significantly larger i.d. increases in the arteries (28±3%) and veins (11±3%) than those under control conditions.
4. The AM-induced i.d. response pattern in the serially connected pulmonary arteries was quite different from that induced by CGRP; AM caused a greater increase in smaller vessels (100–500 μm) than in larger vessels (500–1100 μm). In the case of CGRP, a greater increase was observed in the larger vessels.
5. CGRP_8–37 (100 nmol kg⁻¹, i.v., followed by a continuous infusion of 0.2 nmol kg⁻¹ min⁻¹) had no significant effect on the i.d. increase induced by AM (0.1 nmol kg⁻¹) in any serial segments of the arteries and veins.
6. The results indicate that, in the cat, AM induces greater vasodilatation in small pulmonary arteries and lesser vasodilatation in small veins, the maximum dilatation being in the more peripheral arterial segment (100–500 μm). The vasodilator effect of AM was enhanced when vascular tone was elevated. The data suggest that the AM-induced pulmonary vasodilatation is not mediated by CGRP receptors but by its own specific receptor.

     

The effect of the selective 5-HT1A agonists alnespirone (S-20499) and 8-OH-DPAT on extracellular 5-hydroxytryptamine in different regions of rat brain

1. We have examined the effects of the systemic administration of the selective 5-HT_1A agonist alnespirone (S-20499) on in vivo 5-hydroxytryptamine (5-HT) release in the dorsal raphe nucleus, the median raphe nucleus and four forebrain areas innervated differentially by both (dorsal striatum, frontal cortex, ventral hippocampus and dorsal hippocampus).
2. Alnespirone (0.1–3 mg kg⁻¹, s.c.) dose-dependently reduced extracellular 5-HT in the six areas examined. In forebrain, the maximal reductions occurred in striatum and frontal cortex (maximal reduction to 23 and 29% of baseline, respectively). Those in dorsal and ventral hippocampus were more moderate (to ca 65% of baseline). In contrast, the decrease in 5-HT elicited in the median raphe nucleus was more marked than that in the dorsal raphe nucleus (to ca 30 and 60% of baseline, respectively). The selective 5-HT_1A antagonist WAY-100635 (0.5 mg kg⁻¹, s.c.) prevented the decrease in 5-HT induced by alnespirone (0.3 mg kg⁻¹, s.c.) in frontal cortex.
3. 8-OH-DPAT (0.025, 0.1 and 0.3 mg kg⁻¹, s.c.) also reduced extracellular 5-HT in a regionally-selective manner (e.g., to 32% of baseline in striatum and to 69% in dorsal hippocampus at 0.1 mg kg⁻¹, s.c.). In midbrain, 8-OH-DPAT reduced the dialysate 5-HT slightly more in the median than in the dorsal raphe nucleus at all doses examined.
4. Doses of both compounds close to their respective ED₅₀ values (0.3 mg kg⁻¹ alnespirone, 0.025 mg kg⁻¹ 8-OH-DPAT) reduced 5-HT to a comparable extent in all regions examined. However, the reductions attained at higher doses were more pronounced for 8-OH-DPAT.
5. These data show that the reduction of 5-HT release elicited by alnespirone and 8-OH-DPAT is more important in forebrain areas innervated by 5-hydroxytryptaminergic neurones of the dorsal raphe nucleus. This regional selectivity seems unlikely to be accounted for by differences in the sensitivity of 5-HT_1A autoreceptors controlling 5-HT release, given the dissimilar effects of these two 5-HT_1A agonists in regions rich in cell bodies and nerve terminals. This suggests the presence of complex mechanisms of control of 5-HT release by 5-HT_1A receptors.

     

RPR 106541, a novel,airways-selective glucocorticoid: effects against antigen-induced CD4+T lymphocyte accumulation and cytokine gene expression in the Brown Norway rat lung

1. The effects of a novel 17-thiosteroid, RPR 106541, were investigated in a rat model of allergic airway inflammation.
2. In sensitized Brown Norway rats, challenge with inhaled antigen (ovalbumin) caused an influx of eosinophils and neutrophils into the lung tissue and airway lumen. In the lung tissue there was also an accumulation of CD4⁺ T lymphocytes and increased expression of mRNA for interleukin-4 (IL-4) and IL-5, but not interferon-γ (IFN-γ). These findings are consistent with an eosinophilia orchestrated by activated Th2-type cells.
3. RPR 106541 (10–300 μg kg⁻¹), administered by intratracheal instillation into the airways 24 h and 1 h before antigen challenge, dose-dependently inhibited cell influx into the airway lumen. RPR 106541 (100 μg kg⁻¹) caused a significant (P<0.01) (98%) inhibition of eosinophil influx and a significant (P<0.01) (100%) inhibition of neutrophil influx. RPR 106541 was approximately 7 times and 4 times more potent than budesonide and fluticasone propionate, respectively.
4. When tested at a single dose (300 μg kg⁻¹), RPR 106541 and fluticasone each caused a significant (P<0.01) (100%) inhibition of CD4⁺ T cell accumulation in lung tissue. Budesonide (300 μg kg⁻¹) had no significant effect. RPR 106541 and fluticasone (300 μg kg⁻¹), but not budesonide (300 μg kg⁻¹), significantly (P<0.05) inhibited the expression within lung tissue of mRNA for IL-4. RPR 106541 (300 μg kg⁻¹) also significantly (P<0.05) inhibited expression of mRNA for IL-5.
5. The high topical potency of RPR 106541 in this model, which mimics important aspects of airway inflammation in human allergic asthmatics, suggests that this glucocorticoid may be useful in the treatment of bronchial asthma.

     

Regulation of bradykinin B2-receptor expression by oestrogen

1. Tissue kallikrein is overexpressed in the kidney of female rats, this sexual dimorphism being associated with a greater effect of early blockade of bradykinin B₂-receptors on female blood pressure phenotype. We evaluated the effect of ovariectomy and oestradiol benzoate (50 μg kg⁻¹ every two days for two weeks) on the vasodepressor response to intra-arterial injection of bradykinin (150–900 ng kg⁻¹) and on the expression of bradykinin B₂-receptors.
2. Ovariectomy reduced the magnitude of the vasodepressor response to bradykinin and unmasked a secondary vasopressor effect. Oestrogen replacement restored the vasodepressor response to bradykinin in ovariectomized rats.
3. The vasodepressor responses to sodium nitroprusside (3–18 μg kg⁻¹), acetylcholine (30–600 ng kg⁻¹), desArg⁹-bradykinin (150–900 ng kg⁻¹) or prostaglandin E₂ (30–600 ng kg⁻¹) were significantly reduced by ovariectomy. Oestrogen restored to normal the responses to desArg⁹-bradykinin, acetylcholine and prostaglandin E₂, but not that to sodium nitroprusside.
4. B₂-receptor mRNA levels were decreased by ovariectomy in the aorta and kidney and they were restored to normal levels by oestrogen. Neither ovariectomy nor oestradiol affected receptor expression in the heart and uterus.
5. These results indicate that oestrogen regulates B₂-receptor gene expression and function. Since kinins exert a cardiovascular protective action, reduction in their vasodilator activity after menopause might contribute to the increased risk of pathological cardiovascular events. Conversely, the cardioprotective effects of oestrogen replacement might be, at least in part, mediated by activation of the kallikrein-kinin system.