Differential autoreceptor control of extracellular 5-HT in guinea pig and rat: species and regional differences

Rationale Central 5-hydroxytryptamine (5-HT) release is regulated by inhibitory 5-HT autoreceptors, including 5-HT_1A and 5-HT_1B receptors.Objectives The purpose of this study was to use combinations of selective autoreceptor antagonists to elucidate the role of these receptors in controlling extracellular 5-HT in terminal areas.Methods Microdialysis was carried out in awake rats and guinea pigs to measure extracellular 5-HT in the frontal cortex and dentate gyrus. Using the selective 5-HT_1A receptor antagonist, WAY-100635, and the selective 5-HT_1B receptor antagonist, SB-224289, we have compared the roles of 5-HT_1A and 5-HT_1B autoreceptors in controlling extracellular 5-HT.Results SB-224289 (4 mg/kg i.p.) alone produced a significant 50% increase in extracellular 5-HT in the dentate gyrus of guinea pigs, but not in the frontal cortex of the same animals. Co-administration of WAY-100635 (0.3 mg/kg s.c.), did not change the SB-224289-induced increase in dentate gyrus 5-HT but did produce a significant augmentation (60% increase) of guinea pig frontal cortex 5-HT. In contrast, neither autoreceptor antagonist, alone or in combination, affected extracellular 5-HT in the frontal cortex or dentate gyrus of rats.Conclusions These data indicate that there is a species difference in the autoreceptor control of 5-HT release. Furthermore, in the guinea pig there is a divergence between dorsal and median raphe innervated brain regions. On the basis that antagonism of 5-HT_1A and 5-HT_1B receptors produced an immediate increase in extracellular 5-HT in multiple brain regions in the guinea pig, it is suggested that this might be a novel mechanism for achieving antidepressant efficacy.     

Characterization of 4-(2-hydroxyphenyl)-1-[2'-[N-(2'-pyridinyl)-p-fluorobenzamido]ethyl]piperazine (p-DMPPF) as a new potent 5-HT1A antagonist

BACKGROUND AND PURPOSE: The identification of potent and selective radioligands for the mapping of 5-HT receptors is interesting both for clinical and experimental research. The aim of this study was to compare the potency of a new putative 5-HT(1A) receptor antagonist, p-DMPPF, (4-(2-hydroxyphenyl)-1-[2'-[N-(2'-pyridinyl)-p-fluorobenzamido]ethyl]piperazine) with that of the well-known 5-HT(1A) antagonists, WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide) and its fluorobenzoyl analogue, p-MPPF (4-(2-methoxyphenyl)-1-[2'-[N-(2'-pyridinyl)-p-fluorobenzamido]ethyl]piperazine). EXPERIMENTAL APPROACH: Single cell extracellular recordings of dorsal raphe (DR) neurones were performed in rat brain slices. The potency of each compound at antagonizing the effect of the 5-HT(1A) agonist, 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)-tetraline], was quantified using the Schild equation. The pharmacological profile of p-DMPPF was defined using competition binding assays. KEY RESULTS: Consistently with a 5-HT(1A) receptor antagonist profile, incubation of slices with an equimolar (10 nM) concentration of each compound markedly reduced the inhibitory effect of 8-OH-DPAT on the firing rate of DR neurones, causing a significant rightward shift in its concentration-response curve. The rank order of potency of the antagonists was WAY-100635>p-DMPPF>or=p-MPPF. The sensitivity of DR neurones to the inhibitory effect of 8-OH-DPAT was found to be heterogeneous. The binding experiments demonstrated that p-DMPPF is highly selective for 5-HT(1A) receptors, with a K(i) value of 7 nM on these receptors. CONCLUSIONS AND IMPLICATIONS: The potency of the new compound, p-DMPPF, as a 5-HT(1A) antagonist is similar to that of p-MPPF in our electrophysiological assay. Its selectivity towards 5-HT(1A) receptors makes it a good candidate for clinical development.     

Characterisation of the selective 5-HT1B receptor antagonist SB-616234-A (1-[6-(cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride): in vivo neurochemical and behavioural evidence of anxiolytic/antidepressant activity

The 5-HT_1B receptor has attracted significant interest as a potential target for the development of therapeutics for the treatment of affective disorders such as anxiety and depression. Here we present the in vivo characterisation of a novel, selective and orally bioavailable 5-HT_1B receptor antagonist, SB-616234-A (1-[6-(cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride). SB-616234-A reversed the 5-HT_1/7 receptor agonist, SKF-99101H-induced hypothermia in guinea pigs in a dose related manner with an ED₅₀ of 2.4 mg/kg p.o. Using in vivo microdialysis in freely moving guinea pigs, SB-616234-A (3–30 mg/kg p.o.) caused a dose-related increase in extracellular 5-HT in the dentate gyrus. Evaluation of antidepressant- and anxiolytic-like effects of this 5-HT_1B receptor antagonist was performed in a variety of models and species. SB-616234-A produced a decrease in immobility time in the mouse forced swim test; an effect suggestive of antidepressant activity. Furthermore, SB-616234-A produced dose-related anxiolytic effects in both rat and guinea pig maternal separation-induced vocalisation models with an ED₅₀ of 1.0 and 3.3 mg/kg i.p., respectively (vs fluoxetine treatment ED₅₀ = 2.2 mg/kg i.p. in both species). Also a significant reduction in posturing behaviours was observed in the human threat test in marmosets; an effect indicative of anxiolytic activity. In summary, SB-616234-A is a novel, potent and orally bioavailable 5-HT_1B receptor antagonist which exhibits a neurochemical and behavioural profile that is consistent with both anxiolytic- and antidepressant-like activity in a variety of species. Taken together these data suggest that SB-616234-A may have therapeutic efficacy in the treatment of affective disorders.     

Cloning and pharmacological characterisation of the guinea pig 5-ht5A receptor

The guinea pig 5-hydroxytryptamine(5A) (gp5-ht(5A)) receptor was cloned from guinea pig brain using degenerate polymerase chain reaction (PCR) and shows 88%, 85% and 84% amino acid sequence identity versus the human, rat and mouse 5-ht(5A) receptors, respectively. The receptor was transiently expressed in human embryonic kidney (HEK) 293 cells. [(3)H]-Lysergic acid diethylamide (LSD) bound saturably to gp5-ht(5A)/HEK293 membranes with a K(d) of 2.3+/-0.1 nM and B(max) of 15.7+/-3.4 pmol/mg protein. The receptor binding profile, determined by competition with [(3)H]LSD, correlated well with that for the human 5-ht(5A) receptor. 5-HT stimulated [(35)S]GTPgammaS binding to gp5-ht(5A)/HEK293 membranes (pEC(50) 8.1+/-0.2), and the response was surmountably antagonised by methiothepin and ritanserin, giving apparent pK(B) values of 8.0 and 7.2, respectively. The 5-HT response was absent using membranes prepared from gp5-ht(5A)/HEK293 cells pretreated with pertussis toxin (PTX). These data suggest that the gp5-ht(5A) receptor couples to G(i)-proteins in this expression system and shows a similar pharmacological profile to that for the human 5-ht(5A) receptor.     

SB-616234-A (1-[6-(cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2'methyl-4'-(5-methyl-1,2,3-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride): a novel, potent and selective 5-HT1B receptor antagonist

SB-616234-A possesses high affinity for human 5-HT_1B receptors stably expressed in Chinese hamster ovary (CHO) cells (pK_i 8.3 ± 0.2), and is over 100-fold selective for a range of molecular targets except h5-HT_1D receptors (pK_i 6.6 ± 0.1). Similarly, affinity (pK_i) for rat and guinea pig striatal 5-HT_1B receptors is 9.2 ± 0.1. In [³⁵S]-GTPγS binding studies in the human recombinant cell line, SB-616234-A acted as a high affinity antagonist with a pA₂ value of 8.6 ± 0.2 whilst providing no evidence of agonist activity in this system. In [³⁵S]-GTPγS binding studies in rat striatal membranes, SB-616234-A acted as a high affinity antagonist with an apparent pK_B of 8.4 ± 0.5, again whilst providing no evidence of agonist activity in this system. SB-616234-A (1 μM) potentiated electrically stimulated [³H]-5-HT release from guinea pig and rat cortical slices (S₂/S₁ ratios of 1.8 and 1.6, respectively). SB-616234-A (0.3–30 mg kg⁻¹ p.o.) caused a dose-dependent inhibition of ex vivo [³H]-GR125743 binding to rat striatal 5-HT_1B receptors with an ED₅₀ of 2.83 ± 0.39 mg kg⁻¹ p.o. Taken together these data suggest that SB-616234-A is a potent and selective 5-HT_1B autoreceptor antagonist that occupies central 5-HT_1B receptors in vivo following oral administration.     

Effect of 5-HT5A antagonists in animal models of schizophrenia, anxiety and depression

The few available data on the pharmacological effect of 5-HT5A receptors suggest that antagonists may have anxiolytic, antidepressant and antipsychotic activity. The aim of our study was to verify these suggestions in relevant animal models. Two 5-HT5A antagonist ligands, SB-699551-A (N-[2-(dimethylamino)ethyl]-N-[[4'-[[(2-phenylethyl)amino]methyl][1,1'-biphenyl]-4-yl]methyl]cyclopentanepropanamide dihydrochloride) (3-60 mg/kg, intraperitoneally) and A-843277 (N-(2,6-dimethoxybenzyl)-N'[4-(4-fluorophenyl)thiazol-2-yl]guanidine) (3-30 mg/kg, intraperitoneally), were examined in the open-field test, in a foot-shock-induced ultrasonic vocalization test, in the forced swim test (FST) and in the amphetamine-induced and phencyclidine-induced hyperlocomotion tests to examine their effect on general behavioural patterns, and their anxiolytic-like, antidepressant-like and antipsychotic-like properties, respectively. In the open-field test, SB-699551-A induced sedation and A-843277 induced writhing. In the ultrasonic vocalization test, SB-699551-A reduced vocalizations, whereas A-843277 was ineffective. In the FST, SB-699551-A was ineffective and A-843277 reduced immobility, but only at the highest dose. In the amphetamine-induced and phencyclidine-induced hyperlocomotion test, both compounds were ineffective. SB-699551-A showed an anxiolytic-like property in the ultrasonic vocalization test; however, this compound has a sedative effect. A-843277 showed an antidepressant-like property in the FST, but its immobility-reducing effect may also be a consequence of abdominal irritation. Consequently, further investigations are required to define the therapeutic potential of 5-HT5A receptor ligands in anxiety, depression and schizophrenia models.     

Involvement of 5-hydroxytryptamine(1A) receptors in nicotine-induced tail tremor in rats

Involvement of the serotonergic system in tail tremor induced by repeated administration of nicotine was investigated in rats. Tail tremor induced by nicotine (0.5 mg/kg, s.c.) was suppressed by a 5-HT(1A) receptor antagonist, N-?2-[4-(2-methoxyphenyl)-1-piperazinyl-]ethyl?-N-(2-pyridinyl)cycloh exanecarboxamide trihydrochloride (WAY-100635; 0.3-3 mg/kg, i.p.), but not by a 5-HT(2) receptor antagonist, ketanserin (0.1-0.3 mg/kg, i.p). The 5-HT(1A) receptor agonists, buspirone (1-20 mg/kg, i.p.), gepirone (1-10 mg/kg, i.p.), tandospirone (1-10 mg/kg, i.p.) and (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.01-0.1 mg/kg, s.c.), enhanced the tail tremor. The enhancement of tail tremor by buspirone (10 mg/kg, i.p.) was blocked by WAY-100635 (0.3-3 mg/kg, i.p.). These findings suggest that nicotine-induced tail tremor is mediated by 5-HT(1A) receptors and that 5-HT(1A) receptor antagonists are effective in the treatment of tremor.     

WAY-100635 has high selectivity for serotonin 5-HT(1A) versus dopamine D(4) receptors

The serotonin 5-HT(1A) receptor antagonist WAY-100635 was recently reported to have potent agonist properties at dopamine D(4) receptors (Chemel et al., 2006, Psychopharmacology 188, 244-251.). Herein WAY-100635 (pK(i) at human (h) serotonin 5-HT(1A) receptors=9.51; pK(i) at dopamine hD(4.4) receptors=7.42) stimulated [(35)S]GTPgammaS incorporation in membranes of Chinese Hamster Ovary cells expressing dopamine hD(4.4) receptors with only moderate potency and modest efficacy (pEC(50)=6.63; E(max)=19% of dopamine). Moreover, in antagonism experiments, WAY-100635 had a much lower potency at dopamine hD(4.4) receptors (pK(B)=7.09), than at serotonin h5-HT(1A) receptors (pK(B)=9.47). These data demonstrate that WAY-100635 has high selectivity for serotonin h5-HT(1A)versus dopamine hD(4.4) receptors.     

Chronic fluoxetine-induced desensitization of 5-HT1A and 5-HT1B autoreceptors: regional differences and effects of WAY-100635

Desensitization of 5-HT(1A) and 5-HT(1B) autoreceptors is thought to be the mechanism underlying the therapeutic effects of fluoxetine and other selective serotonin reuptake inhibitors when these are administered chronically. The blockade of 5-HT(1A) autoreceptors occurring on administration of a selective serotonin reuptake inhibitor together with a 5-HT(1A) autoreceptor antagonist is responsible for the acute increase in 5-hydroxytryptamine (serotonin, 5-HT) levels observed under these circumstances. The effects of repeated administration of selective serotonin reuptake inhibitors together with 5-HT(1A) receptor antagonists have not been widely studied. In this work, we found that the effects of fluoxetine (5 mg/kg, i.p., daily for 12 days) to desensitize 5-HT(1B) autoreceptors in the frontal cortex, as measured by the effect of the locally administered 5-HT(1B) receptor agonist, 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP 93129), and to desensitize 5-HT(1A) autoreceptors as measured by the action of the 5-HT(1A) receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 50 microg/kg, s.c.) to reduce 5-HT levels in cortex, were prevented by concomitant administration of the 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide (WAY-100635; 0.3 mg/kg, s.c.). 5-HT(1B) receptor activity in the hypothalamus, as measured by the effects of locally administered CP 93129, and 5-HT(1A) autoreceptor activity, as determined by the effects of subcutaneous 8-OH-DPAT to reduce 5-HT levels in hypothalamus, were not altered either by fluoxetine alone or by fluoxetine in the presence of WAY-100635. The data suggest that the regulation of extracellular levels of 5-HT in the cortex and hypothalamus is subject to different autoregulatory mechanisms.     

Desensitization of 5-HT(1A) autoreceptors by a low chronic fluoxetine dose effect of the concurrent administration of WAY-100635.

Using microdialysis, receptor autoradiography and in situ hybridization, we examined the effects of fluoxetine alone or with WAY-100635 on: (a) extracellular 5-HT in frontal cortex; and (b) density and sensitivity of 5-HT(1A) autoreceptors in rat brain. WAY-100635 (0.3 mg/kg, s.c.) doubled the increase in extracellular 5-HT produced by fluoxetine (3 mg/kg, i.p.) in frontal cortex. Two-week minipump treatments with these daily doses significantly raised extracellular 5-HT to 275 +/- 33% (fluoxetine) and 245 +/- 10% (fluoxetine plus WAY-100635) of controls. Fluoxetine 3 mg/kg.day desensitized dorsal raphe 5-HT(1A) autoreceptors, an effect prevented by the concurrent WAY-100635 administration. However, WAY-100635 (alone or with fluoxetine) did not change 5-HT(1A) autoreceptor sensitivity. The density of 5-HT(1A) receptors and its encoding mRNA, was unaffected by these treatments. These results suggest that prolonged blockade of 5-HT(1A) receptors in vivo prevents the autoreceptor desensitization induced by fluoxetine but does not result in receptor sensitization.     

Endogenous 5-HT tonically inhibits spontaneous firing activity of dorsal hippocampus CA1 pyramidal neurons through stimulation of 5-HT(1A) receptors in quiet awake rats: in vivo electrophysiological evidence.

The present study was performed to examine an overall effect of endogenous serotonin (5-HT) on the spontaneous firing activity of the dorsal hippocampus CA1 pyramidal neurons in quiet awake rats. A selective 5-HT(1A) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohe xanecarboxamide (WAY-100635: 0.03-0.2 mg/kg, s.c.) significantly increased the firing activity. A depletion of 5-HT with parachlorophenylalanine (PCPA: 500 mg/kg/day x 3 days) completely abolished this increasing effect of WAY-100635. The baseline spike frequency of the PCPA-treated rats (3.90 +/- 0.39 Hz) was significantly higher than that of the vehicle-treated rats (2.09 +/- 0.19 Hz). A 5-HT(2A) antagonist ritanserin (1 mg/kg, i.p.) and a 5-HT(3/4) antagonist 2-methoxy-4-amino-5-chloro benzoic acid 2-(diethylamino) ethyl ester (SDZ-205557: 3 mg/kg, s.c.) did not modify the firing activity and the increasing effect of WAY-100635. These results suggest that, in quiet awake rats, endogenous 5-HT would tonically inhibit the spontaneous firing activity of the CA1 pyramidal neurons mainly through stimulating 5-HT(1A) receptors.     

Evidence that hypophagia induced by d-fenfluramine and d-norfenfluramine in the rat is mediated by 5-HT2C receptors

The present series of studies is the first to investigate the pharmacological mechanisms underlying d-fenfluramine- and d-norfenfluramine-induced hypophagia in the rat using highly selective serotonin 5-HT2 receptor antagonists. Administration of d-fenfluramine, and its major metabolite d-norfenfluramine, suppresses food intake in animals. Both compounds stimulate the release of serotonin and are potent inhibitors of the re-uptake of 5-HT into nerve terminals. In addition, d-norfenfluramine also acts as a direct 5-HT(2B/2C) receptor agonist. Pre-treatment with the selective 5-HT2C receptor antagonist, SB-242084 (0.3-3 mg/kg), dose-dependently inhibited both d-fenfluramine- (3 mg/kg) and d-norfenfluramine-induced (2 mg/kg) hypophagia. In contrast, the hypophagic effect of d-fenfluramine and d-norfenfluramine was unaffected by prior treatment with the highly selective 5-HT2B receptor antagonists, SB-215505 (0.3-3 mg/kg) and RS-127445 (1-3 mg/kg) or the 5-HT2A receptor antagonists MDL 100,907 (0.003-0.03 mg/kg) and ketanserin (0.2, 0.5 mg/kg). In addition, the 5-HT1A receptor antagonist WAY-100635 (0.3, 1 mg/kg) and the 5-HT1B receptor antagonists GR-127935 (1, 2 mg/kg) and SB-224289 (2-10 mg/kg) did not affect d-fenfluramine-induced hypophagia. These data provide unequivocal evidence for an important role of the 5-HT2C receptor in the mediation of d-fenfluramine and d-norfenfluramine-induced hypophagia in the rat and do not support the involvement of 5-HT1A/1B/2A/2B receptors.     

Characterization of SB-269970-A, a selective 5-HT(7) receptor antagonist

The novel 5-HT(7) receptor antagonist, SB-269970-A, potently displaced [(3)H]-5-CT from human 5-HT(7(a)) (pK(i) 8.9+/-0.1) and 5-HT(7) receptors in guinea-pig cortex (pK(i) 8.3+/-0.2). 5-CT stimulated adenylyl cyclase activity in 5-HT(7(a))/HEK293 membranes (pEC(50) 7.5+/-0.1) and SB-269970-A (0.03 - 1 microM) inhibited the 5-CT concentration-response with no significant alteration in the maximal response. The pA(2) (8.5+/-0.2) for SB-269970-A agreed well with the pK(i) determined from [(3)H]-5-CT binding studies. 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes (pEC(50) of 8.4+/-0.2) was inhibited by SB-269970-A (0.3 microM) with a pK(B) (8.3+/-0.1) in good agreement with its antagonist potency at the human cloned 5-HT(7(a)) receptor and its binding affinity at guinea-pig cortical membranes. 5-HT(7) receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. SB-269970-A was CNS penetrant (steady-state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min(-1) kg(-1)). Following a single dose (3 mg kg(-1)) SB-269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea-pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested. 5-CT (0.3 mg kg(-1) i.p.) induced hypothermia in guinea-pigs was blocked by SB-269970-A (ED(50) 2.96 mg kg(-1) i.p.) and the non-selective 5-HT(7) receptor antagonist metergoline (0.3 - 3 mg kg(-1) s.c.), suggesting a role for 5-HT(7) receptor stimulation in 5-CT induced hypothermia in guinea-pigs. SB-269970-A (30 mg kg(-1)) administered at the start of the sleep period, significantly reduced time spent in Paradoxical Sleep (PS) during the first 3 h of EEG recording in conscious rats.     

WAY-100635 and GR127935: Effects on 5-hydroxytryptamine-containing neurones

N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY-100635) is a potent and selective 5-HT_1A receptor antagonist in a slice preparation of the guinea pig dorsal raphe nucleus: the inhibitory actions on 5-HT neuronal firing of 5-hydroxytryptamine (5-HT, serotonin), 5-carboxamidotryptamine (5-CT) and 8-hydroxy-2-(di-n- propylamino)tetralin (8-OH-DPAT), but not those of baclofen, were abolished by 30 nM WAY-100635. The selective 5-HT_1D receptor antagonist N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1-biphenyl]-4-carboxamide (GR127935, 300 nM) did not attenuate the 5-HT induced inhibition, indicating that 5-HT_1D receptors do not contribute to the inhibitory action of exogenous 5-HT on 5-HT neurones.     

The role of alpha(1)-adrenoceptors and 5-HT(1A) receptors in the control of the micturition reflex in male anaesthetized rats

1. The effects of the alpha(1)-adrenoceptor antagonists doxazosin (0.1 -- 2 mg kg(-1)), RS-100329 (alpha(1A); 0.01 -- 1 mg kg(-1)), RS-513815 (Ro 151-3815, alpha(1B); 0.3 -- 3 mg kg(-1)) and BMY 7378 (alpha(1D); 0.1 -- 1 mg kg(-1)), the 5-HT(1A) receptor agonist, 8-OH-DPAT (0.03 -- 0.3 mg kg(-1)) and antagonist WAY-100635 (0.03 -- 0.3 mg kg(-1)) were investigated (i.v.) on the 'micturition reflex' in the urethane anaesthetized male rat. 2. Reflex-evoked urethra contractions were most sensitive to the inhibitory action of RS-100329, followed by doxazosin, BMY 7378 and WAY-100635 and then RS-513815. The maximum inhibition was 66, 63, 54, 46 and 22% at doses of 0.3, 0.5, 0.3, 0.3 and 3 mg kg(-1) respectively. 3. BMY 7378 and 8-OH-DPAT decreased, while WAY-100635 increased, the pressure threshold to induce bladder contraction. WAY-100635 (0.01 mg kg(-1)) blocked the effects of BMY 7378 (1 mg kg(-1)) on bladder pressure and volume threshold. 4. Doxazosin, RS-100329 and BMY 7378 had a similar potency in inducing a fall in arterial blood pressure while WAY-100635 only caused a fall at the highest dose. 5. Therefore, reflex-evoked urethral contraction involves the activation of alpha(1A/1D)-adrenoceptors, as BMY 7378 and RS-100329 are similarly potent in attenuating this effect. The ability of WAY-100635 to attenuate this contraction may suggest that 5-HT(1A) receptors are also involved. However, as this inhibition occurred at the highest dose of WAY-100635, which also caused a fall in arterial blood pressure; this effect is considered to be due to blockade of alpha(1)-adrenoceptors not 5-HT(1A) receptors. Nevertheless the initiation of the 'micturition reflex' involves the activation of 5-HT(1A) receptors.     

The relaxant 5-HT receptor in the dog coronary artery smooth muscle: pharmacological resemblance to the cloned 5-ht7 receptor subtype.

1. The relaxant effect of 5-hydroxytryptamine (5-HT) in the dog isolated coronary artery deprived of endothelium is mediated by a receptor unrelated to the 5-HT1, 5-HT2, 5-HT3 or 5-HT4 types. Based upon the pharmacological characteristics of this relaxant 5-HT receptor and those reported for the new members of the 5-HT receptor family, the present study explored the possibility that the relaxant 5-HT receptor referred to above, corresponds to the cloned 5-ht7 subtype. Thus, the relaxing and/or blocking effects of several 5-HT receptor drugs as well as some typical and atypical antipsychotic drugs with high affinity for the cloned 5-ht7 receptor in precontracted ring segments were analyzed. 2. 5-HT, 5-carboxamidotryptamine (5-CT) and 5-methoxytryptamine, but not 8-OH-DPAT or sumatriptan, produced concentration-dependent relaxations in endothelium-denuded canine coronary artery rings precontracted with prostaglandin F2a (2 microM). Clozapine (1 microM) produced in some cases a small relaxing effect and antagonized 5-HT- and 5-CT-induced relaxation suggesting a partial agonist effect. In the presence of the 5-HT1D receptor antagonist, GR127935 (100 nM), the rank order of agonist potency was 5-CT > 5-HT > clozapine > or = 5-methoxytryptamine. 8-OH-DPAT and sumatriptan remained inactive as agonists. 3. In GR127935-treated preparations, methiothepin (3 nM) and mianserin (1 microM), as well as the antipsychotics, clozapine (1 microM), pimozide (300 nM), risperidone (3 nM) and spiperone (1 microM), failed to induce a significant relaxation in prostaglandin F2x-precontracted vessels, but produced significant rightward displacements of the concentration-response curves to 5-HT and 5-CT without significantly reducing the Emax. In a final set of experiments with 5-CT, metergoline (100 nM) and mesulergine (300 nM) behaved as competitive antagonists. In contrast, lisuride (3 nM) noncompetitively antagonized 5-CT-induced relaxation. The estimated affinity (apparent pKa values) of the above antagonist drugs for the relaxant 5-HT receptor significantly correlated with their reported affinity at the cloned 5-ht7 receptor. 4. Taken together, the above pharmacological data may suggest that the relaxant 5-HT receptor in the smooth muscle of the canine coronary artery is similar to the cloned 5-ht7 receptor subtype.     

SB-656104-A,a novel selective 5-HT7 receptor antagonist,modulates REM sleep in rats

1 (6-((R)-2-[2-[4-(4-Chloro-phenoxy)-piperidin-1-yl]-ethyl]-pyrrolidine-1-sulphonyl)-1H-indole hydrochloride) (SB-656104-A), a novel 5-hydroxytryptamine (5-HT(7)) receptor antagonist, potently inhibited [(3)H]-SB-269970 binding to the human cloned 5-HT(7(a)) (pK(i) 8.7+/-0.1) and 5-HT(7(b)) (pK(i) 8.5+/-0.2) receptor variants and the rat native receptor (pK(i) 8.8+/-0.2). The compound displayed at least 30-fold selectivity for the human 5-HT(7(a)) receptor versus other human cloned 5-HT receptors apart from the 5-HT(1D) receptor ( approximately 10-fold selective). 2 SB-656104-A antagonised competitively the 5-carboxamidotryptamine (5-CT)-induced accumulation of cyclic AMP in h5-HT(7(a))/HEK293 cells with a pA(2) of 8.5. 3 Following a constant rate iv infusion to steady state in rats, SB-656104 had a blood clearance (CL(b)) of 58+/-6 ml min(-1) kg(-1) and was CNS penetrant with a steady-state brain : blood ratio of 0.9 : 1. Following i.p. administration to rats (10 mg kg(-1)), the compound displayed a t(1/2) of 1.4 h with mean brain and blood concentrations (at 1 h after dosing) of 0.80 and 1.0 micro M, respectively. 4 SB-656104-A produced a significant reversal of the 5-CT-induced hypothermic effect in guinea pigs, a pharmacodynamic model of 5-HT(7) receptor interaction in vivo (ED(50) 2 mg kg(-1)). 5 SB-656104-A, administered to rats at the beginning of the sleep period (CT 0), significantly increased the latency to onset of rapid eye movement (REM) sleep at 30 mg kg(-1) i.p. (+93%) and reduced the total amount of REM sleep at 10 and 30 mg kg(-1) i.p. with no significant effect on the latency to, or amount of, non-REM sleep. SB-269970-A produced qualitatively similar effects in the same study. 6 In summary, SB-656104-A is a novel 5-HT(7) receptor antagonist which has been utilised in the present study to provide further evidence for a role for 5-HT(7) receptors in the modulation of REM sleep.     

Activation of 5-HT(1A) autoreceptors enhances the inhibitory effect of galanin on hippocampal 5-HT release in vivo

The microdialysis technique was used to examine interactions between 5-HT(1A) and galanin receptors in the dorsal raphe nucleus (DRN), by measuring the extracellular levels of 5-HT in the ventral hippocampus of awake rats. The rats were pretreated with the 5-HT(1A) receptor agonist (R,S)-8-OH-DPAT (0.3 mg/kg, s.c.) or saline. 8-OH-DPAT caused a time-dependent reduction of basal 5-HT levels down to 43-48% at 40 min while at 140 min, the hippocampal 5-HT had returned to control values. At that time point, the rats received a second injection of 8-OH-DPAT or galanin (0.15, 0.5 and 1.5 nmol/0.5 microl) infused into the lateral ventricle. The second injection of 8-OH-DPAT caused a significantly smaller reduction of hippocampal 5-HT levels. In contrast, galanin at all three doses in the 8-OH-DPAT-pretreated groups, was significantly more potent in reducing 5-HT levels (maximal reduction to 74%, 52% and 49%, respectively) than it was in saline-pretreated rats (maximal reduction to 96%, 85% and 69%, respectively). The inhibitory effect of galanin (1.5 nmol) on extracellular 5-HT levels in the rat hippocampus was significantly attenuated by co-administration of the 5-HT(1A) receptor antagonists WAY-100635 (0.3 and 0.6 mg/kg s.c.) and, to a lesser extent, with pindolol (20 mg/kg s.c.). These data provide direct in vivo evidence of agonistic 5-HT(1A)-galanin receptor interaction at the presynaptic level. Furthermore, the findings indicate that a down-regulation of the somato-dendritic 5-HT(1A) autoreceptors, following their stimulation with 8-OH-DPAT and possibly also indirectly with 5-HT reuptake inhibitors, may be compensated by a subsequent 'sensitization' of the inhibitory galanin receptors in the DRN. Thus, the enhanced galanin receptor-mediated inhibition of 5-HT neurotransmission may contribute to the pathophysiology of depression or to the reduced and delayed efficacy of antidepressant therapies.     

A 5-HT(2C) receptor-mediated interaction between 2,5-dimethoxy-4-methylamphetamine and citalopram in the rat

Previous studies conducted in our laboratory have shown that acute administration of the selective serotonin re-uptake inhibitor (SSRI), citalopram, potentiates the stimulus effects of the phenethylamine hallucinogen [-]-2,5-dimethoxy-4-methylamphetamine (DOM) in the rat while neither substituting for the DOM stimulus when administered alone nor altering brain levels of DOM. The present investigation was designed to determine the mechanism by which citalopram acts on DOM-induced stimulus control. To that end, we tested the following hypotheses: (a) citalopram blocks the transport of DOM by the serotonin transporter, (b) citalopram acts via the 5-HT(1A) receptor, and (c) citalopram acts via the 5-HT(2C) receptor. Hypothesis (a) was rejected on the basis of equilibrium saturation studies of [3H]citalopram binding, which revealed no significant affinity of DOM for the 5-HT transporter of rat brain membranes. Hypotheses (b) and (c) were tested in a group of 20 rats in which stimulus control was established with DOM (0.6 mg/kg; 75 min pretreatment time). A two-lever, fixed ratio 10 (FR10), positively reinforced task with saline controls was employed. Hypothesis (b), a role for the 5-HT(1A) receptor, was rejected on the basis of an absence of antagonism of the effects of citalopram on DOM by the selective 5-HT(1A) receptor antagonist, WAY-100635. In contrast, Hypothesis (c), a role for the 5-HT(2C) receptor, gained support from the observation of significant antagonism of the effects of citalopram on DOM by the selective 5-HT(2C) receptor antagonist, SB-242084.     

Activation and blocking of 5-HT1A receptors influence on the immune response in CBA mice

5-HT1A type serotonin receptors influence the immunomodulating action of the selective preparations 8-OH-DPAT (5-HT1A receptor agonist) and WAY-100635 (5-HT1A receptor antagonist) in CBA mice. The activation of 5HT1A receptors with 8-OH-DPAT (1 mg/kg) decreased, while their blocking with WAY-100635 (1 mg/kg) increased the reaction intensity at the peak of response to immunization with ram erythrocytes. Preliminary blocking of the 5-HT1A receptors with WAY-100635 prevented the inhibiting action of 8-OH-DPAT.