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Background and Purpose: Lurasidone is an atypical mood-stabilizing antipsychotic with a unique receptor-binding profile, including 5-HT 7 receptor antagonism; however, the detailed effects of 5-HT 7 receptor antagonism on various transmitter systems relevant to schizophrenia, particularly the thalamo-insular glutamatergic system and the underlying mechanisms, are yet to be clarified., Experimental Approach: We examined the mechanisms underlying the clinical effects of lurasidone by measuring the release of l-glutamate, GABA, dopamine, and noradrenaline in the reticular thalamic nucleus (RTN), mediodorsal thalamic nucleus (MDTN) and insula of freely moving rats in response to systemic injection or local infusion of lurasidone or MK-801 using multiprobe microdialysis with ultra-HPLC., Key Results: Systemic MK-801 (0.5 mg·kg -1 ) administration increased insular release of l-glutamate, dopamine, and noradrenaline but decreased GABA release. Systemic lurasidone (1 mg·kg -1 ) administration also increased insular release of l-glutamate, dopamine, and noradrenaline but without affecting GABA. Local lurasidone administration into the insula (3 μM) did not affect MK-801-induced insular release of l-glutamate or catecholamine, whereas local lurasidone administration into the MDTN (1 μM) inhibited MK-801-induced insular release of l-glutamate and catecholamine, similar to the 5-HT 7 receptor antagonist SB269970., Conclusions and Implications: The present results indicate that MK-801-induced insular l-glutamate release is generated by activation of thalamo-insular glutamatergic transmission via MDTN GABAergic disinhibition resulting from NMDA receptor inhibition in the MDTN and RTN. Lurasidone inhibited this MK-801-evoked insular l-glutamate release through inhibition of excitatory 5-HT 7 receptor in the MDTN. These effects on thalamo-insular glutamatergic transmission may contribute to the antipsychotic and mood-stabilizing actions of lurasidone., (© 2019 The British Pharmacological Society.)

Background and Purpose: Although the antinociceptive efficacies of imidazoline I 2 receptor agonists have been established, the exact post-receptor mechanisms remain unknown. This study tested the hypothesis that monoaminergic transmission is critical for I 2 receptor agonist-induced antinociception., Experimental Approach: von Frey filaments were used to assess antinociceptive effects of two I 2 receptor agonists, 2-BFI and CR4056 on chronic constriction injury (CCI)-induced neuropathic pain or complete Freund's adjuvant (CFA)-induced inflammatory pain in rats. Rectal temperature was measured to assess hypothermic effects of 2-BFI. A two-lever drug discrimination paradigm in which rats were trained to discriminate 5.6 mg·kg -1 2-BFI (i.p.) from its vehicle was used to examine the discriminative stimulus effects of 2-BFI. In each experiment, pharmacological mechanisms were investigated by combining 2-BFI or CR4056 with various pharmacological manipulations of the monoaminergic system including selective reuptake inhibition, monoamine depletion and monoamine receptor antagonism., Key Results: In the CCI model, selective reuptake inhibitors of 5-HT (fluoxetine) or noradrenaline (desipramine), but not dopamine (GBR12909), enhanced 2-BFI-induced antinociception. Selective depletion of 5-HT or noradrenaline almost abolished 2-BFI-induced antinociception. 5-HT 1A , 5-HT 2A and α 1 -adrenoceptor antagonists, but not other monoaminergic antagonists, attenuated 2-BFI and CR4056-induced antinociception in CCI and/or CFA models. However, none of these monoamine receptor antagonists significantly altered 2-BFI-induced hypothermia or discriminative stimulus effects., Conclusions and Implications: Antinociception induced by I 2 receptor agonists was mediated by serotonergic and noradrenergic mechanisms with 5-HT 1A , 5-HT 2A and α 1 -adrenoceptor being particularly important. In contrast, the hypothermic and discriminative stimulus effects of I 2 receptor agonists were mediated by distinct, independent mechanisms., (© 2018 The British Pharmacological Society.)

Background and Purpose: The bioactive monoamine 5-HT, implicated in the pathogenesis of functional gastrointestinal disorders, is abundantly synthesized and stored in rat proximal colonic mucosa and released to the gut lumen and subepithelial space. Despite much data regarding its expression and function, the effects of luminal 5-HT on colonic anion secretion have not been fully investigated., Experimental Approach: We measured short-circuit current (Isc ) as an indicator of ion transport in mucosa-submucosa or mucosa-only preparations of rat proximal colon. Total CO2 output was measured in vitro and in vivo. Immunohistochemistry was performed to investigate the localization of 5-HT4 , NOS1 and NOS2., Key Results: Luminal 5-HT gradually increased the amplitude and sustained the elevation of Isc . Luminal 5-HT-evoked ΔIsc was acetazolamide sensitive and HCO3 (-) dependent, consistent with cytosolic carbonic anhydrase-dependent electrogenic HCO3 (-) secretion, while not affected by tetrodotoxin (TTX), atropine or indomethacin. Pretreatment with the selective 5-HT4 antagonist GR113808, but not antagonists for 5-HT3 , 5-HT6 or 5-HT7 , inhibited luminal 5-HT-evoked ΔIsc . Furthermore, luminal cisapride and tegaserod increased Isc to the same extent as did 5-HT in the presence of indomethacin and TTX. Removal of the submucosa or pretreatment with NOS inhibitors enhanced luminal 5-HT-evoked ΔIsc , suggesting that NO synthesized in the submucosa suppresses mucosal anion secretion. NOS1 and NOS2 were immunostained in the submucosal neurons and glial cells respectively. Luminal 5-HT-evoked HCO3 (-) secretion was confirmed in vivo, inhibited by co-perfusion of GR113808, but not by ondansetron., Conclusions and Implications: A novel apical 5-HT4 -mediated HCO3 (-) secretory pathway and an NO-dependent inhibitory mechanism are present in the proximal colon. Luminal 5-HT-evoked HCO3 (-) secretion may be important for the maintenance of mucosal integrity by regulating luminal pH., (© 2015 The British Pharmacological Society.)

Background and Purpose: The 5-HT3 receptor is a member of the pentameric ligand-gated ion channel family and is pharmacologically targeted to treat irritable bowel syndrome and nausea/emesis. Furthermore, many antidepressants elevate extracellular concentrations of 5-HT. This study investigates the functional consequences of exposure of recombinant 5-HT3 A receptors to agonists and antagonists., Experimental Approach: We used HEK cells stably expressing recombinant 5-HT3 A receptors and the ND7/23 (mouse neuroblastoma/dorsal root ganglion hybrid) cell line, which expresses endogenous 5-HT3 receptors. Surface expression of recombinant 5-HT3 A receptors, modified to contain the bungarotoxin (BTX) binding sequence, was quantified using fluorescence microscopy to image BTX-conjugated fluorophores. Whole cell voltage-clamp electrophysiology was used to measure the density of current mediated by 5-HT3 A receptors., Key Results: 5-HT3 A receptors were up-regulated by the prolonged presence of agonists (5-HT and m-chlorophenylbiguanide) and antagonists (MDL-72222 and morphine). The up-regulation of 5-HT3 A receptors by 5-HT and MDL-72222 was time- and concentration-dependent but was independent of newly translated receptors. The phenomenon was observed for recombinant rodent and human 5-HT3 A receptors and for endogenous 5-HT3 receptors in neuronal ND7/23 cells., Conclusions and Implications: Up-regulation of 5-HT3 A receptors, following exposure to either agonists or antagonists suggests that this phenomenon may occur in response to different therapeutic agents. Medications that elevate 5-HT levels, such as the antidepressant inhibitors of 5-HT reuptake and antiemetic inhibitors of 5-HT3 receptor function, may both raise receptor expression. However, this will require further investigation in vivo., (© 2015 The British Pharmacological Society.)

Background and Purpose: Classically, ligands of GPCRs have been classified primarily upon their affinity and efficacy to activate a signal transduction pathway. Recent reports indicate that the efficacy of a particular ligand can vary depending on the receptor-mediated response measured (e.g. activating G proteins, other downstream responses, internalization). Previously, we reported that inverse agonists induce both homo- and heterologous desensitization, similar to agonist stimulation, at the Gs -coupled 5-HT7 receptor. The primary objective of this study was to determine whether different inverse agonists at the 5-HT7 receptor also induce internalization and/or degradation of 5-HT7 receptors., Experimental Approach: HEK293 cells expressing 5-HT7(a, b or d) receptors were pre-incubated with 5-HT, clozapine, olanzapine, mesulergine or SB269970 and their effects upon receptor density, AC activity, internalization, recruitment of β-arrestins and lysosomal trafficking were measured., Key Results: The agonist 5-HT and three out of four inverse agonists tested increased internalization independently of β-arrestin recruitment. Among these, only the atypical antipsychotics clozapine and olanzapine promoted lysosomal sorting and reduced 5-HT7 receptor density (∼60% reduction within 24 h). Inhibition of lysosomal degradation with chloroquine blocked the clozapine- and olanzapine-induced down-regulation of 5-HT7 receptors. Incubation with SB269970 decreased both 5-HT7(b) constitutive internalization and receptor density but increased 5-HT7(d) receptor density, indicating differential ligand regulation among the 5-HT7 splice variants., Conclusions and Implications: Taken together, we found that various ligands differentially activate regulatory processes governing receptor internalization and degradation in addition to signal transduction. Thus, these data extend our understanding of functional selectivity at the 5-HT7 receptor., (© 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

Background and Purpose: Pattern separation, that is, the formation of distinct representations from similar inputs, is an important hippocampal process implicated in cognitive domains like episodic memory. A deficit in pattern separation could lead to memory impairments in several psychiatric and neurological disorders. Hence, mechanisms by which pattern separation can be increased are of potential therapeutic interest., Experimental Approach: 5-HT1A receptors are involved in spatial memory. Herein we tested the 'biased' 5-HT1A receptor agonists F15599, which preferentially activates post-synaptic heteroreceptors, and F13714, which preferentially activates raphe-located autoreceptors, in rats in a novel spatial task assessing pattern separation, the object pattern separation (OPS) task., Key Results: The acetylcholinesterase inhibitor donepezil, which served as a positive control, significantly improved spatial pattern separation at a dose of 1 mg·kg(-1) , p.o. F15599 increased pattern separation at 0.04 mg·kg(-1) , i.p., while F13714 decreased pattern separation at 0.0025 mg·kg(-1) , i.p. The selective 5-HT1A receptor antagonist WAY-100635 (0.63 mg·kg(-1) , s.c.) counteracted the effects of both agonists. These data suggest that acute preferential activation of post-synaptic 5-HT1A heteroreceptors improves spatial pattern separation, whereas acute preferential activation of raphe-located 5-HT1A autoreceptors impairs performance., Conclusions and Implications: We successfully established and validated a novel, simple and robust OPS task and observed a diverging profile of response with 'biased' 5-HT1A receptor agonists based on their targeting of receptors in distinct brain regions. Our data suggest that the post-synaptic 5-HT1A receptor consists of a potential novel molecular target to improve pattern separation performance., (© 2015 The British Pharmacological Society.)

Background and Purpose: Although substance P (SP) and neurokinin NK1 receptors have been reported to be involved in cisplatin-induced acute and delayed emesis, their precise roles remain unclear. Pica, the consumption of non-nutrient materials such as kaolin in rats, can be used as a model of nausea in humans. We investigated the time-dependent changes in cisplatin-induced pica and the involvement of SP and NK1 receptors in this behaviour., Experimental Approach: Rats were administered cisplatin with or without a daily injection of a 5-HT3 receptor antagonist (granisetron) or an NK1 receptor antagonist (aprepitant), and kaolin intake was then monitored for 5 days. The effects of granisetron on the cisplatin-induced expression of preprotachykinin-A (PPT-A) mRNA, which encodes mainly for SP, and on SP release in the medulla, measured by in vivo brain microdialysis, were also investigated., Key Results: Cisplatin induced pica within 8 h of its administration that continued for 5 days. Granisetron inhibited the acute phase (day 1), but not the delayed phase (days 2-5), of pica, whereas aprepitant abolished both phases. Within 24 h of the injection of cisplatin, PPT-A mRNA expression and SP release in the medulla were significantly increased; these findings lasted during the observation period and were inhibited by granisetron for up to 24 h., Conclusions and Implications: The profiles of cisplatin-induced pica in rats are similar to clinical findings for cisplatin-induced emesis in humans, and we showed that SP production in the medulla and activation of NK1 receptors are involved in this cisplatin-induced pica., (© 2014 The British Pharmacological Society.)

Background and Purpose: Many dementia patients exhibit behavioural and psychological symptoms (BPSD) that include psychosis, aggressivity, depression and anxiety. Antipsychotic drugs are frequently prescribed but fail to significantly attenuate mood deficits, may interfere with cognitive function and are associated with motor and cardiac side effects, which are problematic in elderly patients. A need therefore exists for drugs that are better suited for the treatment of BPSD., Experimental Approach: We used in vitro cellular and in vivo behavioural tests to characterize ADN-1184, a novel arylsulfonamide ligand with potential utility for treatment of BPSD., Key Results: ADN-1184 exhibits substantial 5-HT6 /5-HT7 /5-HT2A /D2 receptor affinity and antagonist properties in vitro. In tests of antipsychotic-like activity, it reversed MK-801-induced hyperactivity and stereotypies and inhibited conditioned avoidance response (MED = 3 mg·kg(-1) i.p.). Remarkably, ADN-1184 also reduced immobility time in the forced swim test at low doses (0.3 and 1 mg·kg(-1) i.p.; higher doses were not significantly active). Notably, up to 30 mg·kg(-1) ADN-1184 did not impair memory performance in the passive avoidance test or elicit significant catalepsy and only modestly inhibited spontaneous locomotor activity (MED = 30 mg·kg(-1) i.p.)., Conclusions and Implications: ADN-1184 combines antipsychotic-like with antidepressant-like properties without interfering with memory function or locomotion. This profile is better than that of commonly used atypical antipsychotics tested under the same conditions and suggests that it is feasible to identify drugs that improve BPSD, without exacerbating cognitive deficit or movement impairment, which are of particular concern in patients with dementia., (© 2013 The British Pharmacological Society.)

Background and Purpose: Here, we have compared the neurochemical profile of three new cathinones, butylone, mephedrone and methylone, in terms of their potential to inhibit plasmalemmal and vesicular monoamine transporters. Their interaction with 5-HT and dopamine receptors and their psychostimulant effect was also studied., Experimental Approach: Locomotor activity was recorded in mice following different doses of cathinones. Monoamine uptake assays were performed in purified rat synaptosomes. Radioligand-binding assays were carried out to assess the affinity of these compounds for monoamine transporters or receptors., Key Results: Butylone, mephedrone and methylone (5-25 mg·kg(-1) ) caused hyperlocomotion, which was prevented with ketanserin or haloperidol. Methylone was the most potent compound inhibiting both [(3) H]5-HT and [(3) H]dopamine uptake with IC(50) values that correlate with its affinity for dopamine and 5-HT transporter. Mephedrone was found to be the cathinone derivative with highest affinity for vesicular monoamine transporter-2 causing the inhibition of dopamine uptake. The affinity of cathinones for 5-HT(2A) receptors was similar to that of MDMA., Conclusions and Implications: Butylone and methylone induced hyperlocomotion through activating 5-HT(2A) receptors and increasing extra-cellular dopamine. They inhibited 5-HT and dopamine uptake by competing with substrate. Methylone was the most potent 5-HT and dopamine uptake inhibitor and its effect partly persisted after withdrawal. Mephedrone-induced hyperlocomotion was dependent on endogenous 5-HT. Vesicular content played a key role in the effect of mephedrone, especially for 5-HT uptake inhibition. The potency of mephedrone in inhibiting noradrenaline uptake suggests a sympathetic effect of this cathinone., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background and Purpose: 5-HT(6) receptors are abundant in the hippocampus, nucleus accumbens and striatum, supporting their role in learning and memory. Selective 5-HT(6) receptor antagonists produce pro-cognitive effects in several learning and memory paradigms while 5-HT(6) receptor agonists have been found to enhance and impair memory., Experimental Approach: The conditioned emotion response (CER) paradigm was validated in rats. Then we examined the effect of the 5-HT(6) receptor antagonist, EMD 386088 (10 mg·kg(-1) , i.p.), and agonists, E-6801 (2.5 mg·kg(-1) , i.p.) and EMD 386088 (5 mg·kg(-1) , i.p.) on CER-induced behaviour either alone or after induction of memory impairment by the muscarinic receptor antagonist, scopolamine (0.3 mg·kg(-1) , i.p) or the NMDA receptor antagonist, MK-801 (0.1 mg·kg(-1) , i.p)., Key Results: Pairing unavoidable foot shocks with a light and tone cue during CER training induced a robust freezing response, providing a quantitative index of contextual memory when the rat was returned to the shock chamber 24 h later. Pretreatment (-20 min pre-training) with scopolamine or MK-801 reduced contextual freezing 24 h after CER training, showing production of memory impairment. Immediate post-training administration of 5-HT(6) receptor antagonist, SB-270146, and agonists, EMD 386088 and E-6801, had little effect on CER freezing when given alone, but all significantly reversed scopolamine- and MK-801-induced reduction in freezing. CONCLUSION AND IMPLICATIONS Both the 5-HT(6) receptor agonists and antagonist reversed cholinergic- and glutamatergic-induced deficits in associative learning. These findings support the therapeutic potential of 5-HT(6) receptor compounds in the treatment of cognitive dysfunction, such as seen in Alzheimer's disease and schizophrenia., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background and Purpose: 5-HT is taken up by and stored in adrenergic nerves and periarterial nerve stimulation (PNS) releases 5-HT to cause vasoconstriction in rat mesenteric arteries. The present study investigated whether PNS-released 5-HT stored in adrenergic nerves affects the function of perivascular calcitonin gene-related peptide-containing (CGRPergic) nerves., Experimental Approach: Rat mesenteric vascular beds without endothelium and with active tone were perfused with Krebs solution. Changes in perfusion pressure in response to PNS and CGRP injection were measured before (control) and after perfusion of Krebs solution containing 5-HT (10 µM) for 20 min. Distributions of 5-HT- and TH-immunopositive fibres in mesenteric arteries were studied using immunohistochemical methods., Key Results: PNS (1-4 Hz) frequency dependently caused adrenergic nerve-mediated vasoconstriction followed by CGRPergic nerve-mediated vasodilatation. 5-HT treatment inhibited PNS-induced vasodilatation without affecting exogenous CGRP-induced vasodilatation, while it augmented PNS-induced vasoconstriction. Guanethidine (adrenergic neuron blocker), methysergide (non-selective 5-HT receptor antagonist) and BRL15572 (selective 5-HT1D receptor antagonist) abolished inhibition of PNS-induced vasodilatation in 5-HT-treated preparations. Combined treatment with 5-HT and desipramine (catecholamine transporter inhibitor), but not fluoxetine (selective 5-HT reuptake inhibitor), did not inhibit PNS-induced vasodilatation. Exogenous 5-HT inhibited PNS-induced vasodilatation, which was antagonized by methysergide. In immunohistochemical experiments, 5-HT-immunopositive nerves, colocalized with adrenergic TH-immunopositive nerves, were observed only in 5-HT-treated mesenteric arteries, but not in control preparations or arteries co-treated with desipramine., Conclusions and Implications: These results suggest that 5-HT can be taken up by and released from adrenergic nerves in vitro by PNS to inhibit CGRPergic nerve transmission in rat mesenteric arteries., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background and Purpose: The vasomodulating actions of 5-HT in vein grafts, and the underlying mechanisms, remain to be fully clarified. Here, we characterized the actions by which 5-HT affects electrical and mechanical activities in rabbit autologous jugular vein grafts., Experimental Approach: Smooth muscle cell (SMC) membrane potential and isometric tension were measured in vein grafts 4 weeks after implantation into carotid arteries. Changes in the expression of 5-HT receptor subtypes and in myosin heavy chain isoforms (SM1, SM2 and SMemb) were examined by immunohistochemistry and Western blot analysis., Key Results: The walls of grafted veins displayed massive increases in the number of SM1- and SM2-positive SMCs. 5-HT induced a large depolarization and contraction that were each reduced by both 5-HT(2A) - and 5-HT(1B/1D) -receptor antagonists. The 5-HT-induced contraction was not modified by a 5-HT₇ -receptor antagonist. The 5-HT₇ -receptor-selective agonist AS 19 did not induce relaxation during the contraction to prostaglandin F(2α) . Immunohistochemical and Western blot analyses revealed that immunoreactive responses against 5-HT(2A) and 5-HT(1B/1D) receptors were increased in the vein graft., Conclusions and Implications: 5-HT is able to induce a large contraction in rabbit autologous jugular vein grafts through (i) an increased number of differentiated contractile SMCs; (ii) an increased number of SMCs expressing contractile 5-HT(2A) - and 5-HT(1B/1D) receptors; and (iii) a down-regulation of the function of the relaxant SMC 5-HT₇ receptors. These changes in the vein graft may help it to resist the higher pressure present on the arterial side of the circulation., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background and Purpose: Biochemical identification of 5-HT has revealed similar projection patterns across vertebrates. In CNS, 5-HT regulates major physiological functions but its peripheral functions are still emerging. The pharmacology of 5-HT is mediated by a diverse range of receptors that trigger different responses. Interestingly, 5-HT receptors have been detected in pigment cells indicating their role in skin pigmentation. Hence, we investigated the role of this monoaminergic system in amphibian pigment cells, melanophores, to further our understanding of its role in pigmentation biology together with its evolutionary significance., Experimental Approach: Pharmacological profiling of 5-HT receptors was achieved using potent/selective agonists and antagonists. In vitro responses of melanophores were examined by Mean Melanophores Size Index assay. The melanophores of lower vertebrates are highly sensitive to external stimuli. The immediate cellular responses to drugs were defined in terms of pigment translocation within the cells., Key Results: 5-HT exerted strong concentration-dependent pigment dispersion at threshold dose of 1 × 10(-6) g·mL(-1). Specific 5-HT(1) and 5-HT(2) receptor agonists, sumatriptan and myristicin. also induced dose-dependent dispersion. Yohimbine and metergoline synergistically antagonized sumatriptan-mediated dispersion, whereas trazodone partially blocked myristicin-induced dispersion. Conversely, 5-HT(3) and 5-HT(4) receptor agonists, 1 (3 chlorophenyl) biguanide (1,3 CPB) and 5-methoxytryptamine (5-MT), caused a dose-dependent pigment aggregation. The aggregatory effect of 1,3 CPB was completely blocked by ondansetron, whereas L-lysine partially blocked the effect of 5-MT., Conclusions and Implications: The results suggest that 5-HT-induced physiological effects are mediated via distinct classes of receptors, which possibly participate in the modulation of pigmentary responses in amphibian., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: The human 5-hydroxytryptamine(7) (h5-HT(7)) receptor is G(s) -coupled and stimulates the production of the intracellular signalling molecule cAMP. Previously, we reported a novel property of the h5-HT(7) receptor: pseudo-irreversible antagonists irreversibly inhibit forskolin-stimulated (non-receptor-mediated) cAMP production. Herein, we sought to determine if competitive antagonists also affect forskolin-stimulated activity and if this effect is common among other G(s) -coupled receptors., Experimental Approach: Recombinant cell lines expressing h5-HT(7) receptors or other receptors of interest were briefly exposed to antagonists; cAMP production was then stimulated by forskolin and quantified by an immunocompetitive assay., Key Results: In human embryonic kidney 293 cells stably expressing h5-HT(7) receptors, all competitive antagonists inhibited nearly 100% of forskolin-stimulated cAMP production. This effect was insensitive to pertussis toxin, that is, not G(i/o) -mediated. Potency to inhibit forskolin-stimulated activity strongly correlated with h5-HT(7) binding affinity (r(2) = 0.91), indicating that the antagonists acted through h5-HT(7) receptors to inhibit forskolin. Potency and maximal effects of clozapine, a prototypical competitive h5-HT(7) antagonist, were unaffected by varying forskolin concentration. Antagonist interaction with h5-HT(6), human β(1), β(2), and β(3) adrenoceptors did not inhibit forskolin's activity., Conclusions and Implications: The inhibition of adenylate cyclase, as measured by forskolin's activity, is an underlying property of antagonist interaction with h5-HT(7) receptors; however, this is not a common property of other G(s) -coupled receptors. This phenomenon may be involved in the roles played by h5-HT(7) receptors in human physiology. Development of h5-HT(7) antagonists that do not elicit this effect would aid in the elucidation of its mechanisms and shed light on its possible physiological relevance., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: β-Adrenoceptor (β-AR)-mediated inotropic effects are attenuated and G(i) proteins are up-regulated in heart failure (HF). Muscarinic receptors constitutively inhibit cAMP formation in normal rat cardiomyocytes. We determined whether constitutive activity of muscarinic receptors to inhibit adenylyl cyclase (AC) increases in HF and if so, whether it modifies the reduced β-AR- or emergent 5-HT₄-mediated cAMP-dependent inotropic effects., Experimental Approach: Contractility and AC activity were measured and related to each other in rat ventricle with post-infarction HF and sham-operated (Sham) controls with or without blockade of muscarinic receptors by atropine and inactivation of G(i) protein by pertussis toxin (PTX)., Key Results: Isoprenaline-mediated inotropic effects were attenuated and basal, isoprenaline- and forskolin-stimulated AC activity was reduced in HF compared with Sham. Atropine or PTX pretreatment increased forskolin-stimulated AC activity in HF hearts. β-AR-stimulated AC and maximal inotropic response were unaffected by atropine in Sham and HF. In HF, the potency of serotonin (5-HT) to evoke an inotropic response was increased in the presence of atropine with no change in the maximal inotropic response. Interestingly, PTX pretreatment reduced the potency of 5-HT to evoke inotropic responses while increasing the maximal inotropic response., Conclusions and Implications: Although muscarinic constitutive inhibition of AC is increased in HF, it does not contribute to the reduced β-AR-mediated inotropic effects in rat ventricle in HF. The data support the hypothesis that there are differences in the functional compartmentation of 5-HT₄ and β-AR AC signalling in myocardium during HF., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Glioblastoma treatment as now constituted offers increased survival measured in months over untreated patients. Because glioblastomas are active in synthesizing a bewildering variety of growth factors, a systematic approach to inhibiting these is being undertaken as treatment adjunct. The serotonin 7 receptor is commonly overexpressed in glioblastoma. Research documentation showing agonists at serotonin receptor 7 cause increased extracellular regulated kinase 1/2 activation, increased interleukin-6 synthesis, increased signal transducer and activator of transcription-3 activation, increased resistance to apoptosis and other growth enhancing changes in glioblastoma is reviewed in this paper. Because three drugs in wide use to treat thought disorders - paliperidone, pimozide and risperidone - are also potent and well-tolerated inhibitors at serotonin receptor 7, these drugs should be studied for growth factor deprivation in an adjunctive role in glioblastoma treatment.

Background and Purpose: 5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT(1) receptor of an insect model for neurobiology, physiology and pharmacology., Experimental Approach: A cDNA encoding for the Periplaneta americana 5-HT(1) receptor was amplified from brain cDNA. The receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal antiserum., Key Results: The P. americana 5-HT(1) receptor (Pea5-HT(1)) shares pronounced sequence and functional similarity with mammalian 5-HT(1) receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT(1) was expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands., Conclusions and Implications: This study marks the first pharmacological identification of an inverse agonist and a neutral antagonist at an insect 5-HT(1) receptor. The results presented here should facilitate further analyses of 5-HT(1) receptors in mediating central and peripheral effects of 5-HT in insects.

Background and Purpose: 5-Hydroxytryptamine (5-HT) is a key regulator of the gastrointestinal system and we have shown that submucosal neuronal 5-HT(3) receptors exerted a novel inhibitory effect on colonic ion transport. The aim of the present study was to investigate the precise mechanism(s) underlying this inhibitory effect., Experimental Approach: Mucosa/submucosa or mucosa-only preparations from rat distal colon were mounted in Ussing chambers for measurement of short-circuit current (I(sc)) as an indicator of ion secretion. Somatostatin release was determined with radioimmunoassay. Intracellular cAMP content was measured with enzyme-linked immunoadsorbent assay (elisa). Immunohistochemical techniques were used to study the expression of 5-HT(3) receptors, somatostatin and somatostatin receptors in colonic tissue., Key Results: In rat distal colonic mucosa/submucosa preparations, pretreatment with 5-HT(3) receptor antagonists enhanced 5-HT-induced increases in I(sc). However, in mucosa-only preparations without retained neural elements, pretreatment with 5-HT(3) receptor antagonists inhibited 5-HT-induced DeltaI(sc). Pretreatment with a somatostatin-2 (sst(2)) receptor antagonist in mucosa/submucosa preparations augmented 5-HT-induced DeltaI(sc). Combination of sst(2) and 5-HT(3) receptor antagonists did not cause further enhancement of 5-HT-induced DeltaI(sc). Moreover, both sst(2) and 5-HT(3) receptor antagonists enhanced 5-HT-induced increase in intracellular cAMP concentration in the mucosa/submucosa preparations. 5-HT released somatostatin from rat colonic mucosa/submucosa preparations, an effect prevented by pretreatment with 5-HT(3) receptor antagonists. Immunohistochemical staining demonstrated the presence of 5-HT(3) receptors on submucosal somatostatin neurons and of sst(2) receptors on colonic mucosa., Conclusion and Implications: Activation of neuronal 5-HT(3) receptors in the submucosal plexus of rat colon suppressed 5-HT-induced ion secretion by releasing somatostatin from submucosal neurons.

Background and Purpose: Human and rat 5-HT(7) receptors were studied with a particular emphasis on the molecular interactions involved in ligand binding, searching for an explanation to the interspecies selectivity observed for a set of compounds. We performed affinity studies, molecular modelling and site-directed mutagenesis, with special focus on residue Phe(7.38) of the human 5-HT(7) receptor [Cys(7.38) in rat]., Experimental Approach: Competition binding studies were performed for seven 5-HT(7) receptor ligands at three different 5-HT(7) receptors. The functional behaviour was evaluated by measuring 5-carboxytryptamine-stimulated cAMP production. Computational simulations were carried out to explore the structural bases in ligand binding observed for these compounds., Key Results: Competition experiments showed a remarkable selectivity for the human receptor when compared with the rat receptor. These results indicate that mutating Cys to Phe at position 7.38 profoundly affects the binding affinities at the 5-HT(7) receptor. Computational simulations provide a structural interpretation for this key finding. Pharmacological characterization of compounds mr25020, mr25040 and mr25053 revealed a competitive antagonistic behaviour. Compounds mr22423, mr22433, mr23284 and mr25052 behaved as partial agonists., Conclusions and Implications: We propose that the interspecies difference in binding affinities observed for the compounds at human and rat 5-HT(7) receptors is due to the nature of the residue at position 7.38. Our molecular modelling simulations suggest that Phe(7.38) in the human receptor is integrated in the hydrophobic pocket in the central part of the binding site [Phe(6.51)-Phe(6.52)] and allows a tighter binding of the ligands when compared with the rat receptor.

Background and Purpose: Despite decreased presynaptic 5-HT(1A) and altered 5-HT(2A) receptor function in genetically-deficient serotonin (5-HT) transporter (SERT) mice, the 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate salt (WAY 100635) still induced head twitches in these mice, a well-established 5-HT(2A) receptor-mediated response., Experimental Approach: Interactions between 5-HT(1A) and 5-HT(2A) receptors were assessed using the head-twitch response following 5-HT(1A) and 5-HT(2A) receptor agonists and antagonists in SERT wild-type (+/+), heterozygous (+/-), and knockout (-/-) mice. The role of brain 5-HT availability in WAY 100635 induced head twitches was also examined., Key Results: WAY 100635 induced head twitches in a SERT gene-dose dependent manner, inducing 5-fold more head twitches in SERT -/- versus SERT +/+ mice. In SERT -/- mice, inhibition of 5-HT synthesis with p-chlorophenylalanine (PCPA) markedly depleted tissue 5-HT in all five brain areas examined and abolished WAY 100635 induced head twitches. Further, the selective 5-HT reuptake inhibitor fluvoxamine increased WAY 100635 induced head twitches in SERT +/+ and +/- mice. Head twitches following the 5-HT(2A) receptor agonist (+/-)-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI) were robust in SERT +/+ and +/- mice but much reduced in SERT -/- mice. DOI-induced head twitches were decreased by the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) in SERT +/+ and +/- mice. All drug-induced head twitches were blocked by the 5-HT(2A) receptor antagonist a-Phenyl-1-(2-phenylethyl)-4-piperidinemethanol (MDL 11,939)., Conclusions and Implications: These data show that indirect activation of 5-HT(2A) receptors via blockade of presynaptic 5-HT(1A) receptors potentiated head-twitch responses, suggesting functional interactions between these receptors, interactions affected by altered 5-HT availability. Our findings strongly support the correlation of WAY 100635 induced head twitches with increased 5-HT availability, induced genetically or pharmacologically.

Background and Purpose: Cannabis is the source of at least seventy phytocannabinoids. The pharmacology of most of these has been little investigated, three notable exceptions being Delta(9)-tetrahydrocannabinol, cannabidiol and Delta(9)-tetrahydrocannabivarin. This investigation addressed the question of whether the little-studied phytocannabinoid, cannabigerol, can activate or block any G protein-coupled receptor., Experimental Approach: The [(35)S]GTPgammaS binding assay, performed with mouse brain membranes, was used to test the ability of cannabigerol to produce G protein-coupled receptor activation or blockade. Its ability to displace [(3)H]CP55940 from mouse CB(1) and human CB(2) cannabinoid receptors and to inhibit electrically evoked contractions of the mouse isolated vas deferens was also investigated., Key Results: In the brain membrane experiments, cannabigerol behaved as a potent alpha(2)-adrenoceptor agonist (EC(50)= 0.2 nM) and antagonized the 5-HT(1A) receptor agonist, R-(+)-8-hydroxy-2-(di-n-propylamino)tetralin (apparent K(B)= 51.9 nM). At 10 microM, it also behaved as a CB(1) receptor competitive antagonist. Additionally, cannabigerol inhibited evoked contractions of the vas deferens in a manner that appeared to be alpha(2)-adrenoceptor-mediated (EC(50)= 72.8 nM) and displayed significant affinity for mouse CB(1) and human CB(2) receptors., Conclusions and Implications: This investigation has provided the first evidence that cannabigerol can activate alpha(2)-adrenoceptors, bind to cannabinoid CB(1) and CB(2) receptors and block CB(1) and 5-HT(1A) receptors. It will now be important to investigate why cannabigerol produced signs of agonism more potently in the [(35)S]GTPgammaS binding assay than in the vas deferens and also whether it can inhibit noradrenaline uptake in this isolated tissue and in the brain.

Background and Purpose: Central 5-hydroxytryptamine (5-HT)-containing pathways utilizing 5-HT(7) receptors are known to be critical for the mediation of cardiovascular reflexes. The nucleus tractus solitarius (NTS) is a site involved in the integration of cardiovascular afferent information. The present experiments examined the involvement of the 5-HT(7) receptor in the processing of cardiovascular reflexes in the NTS., Experimental Approach: In anaesthetized rats extracellular recordings were made from 104 NTS neurones that were excited by electrical stimulation of the vagus nerve and/or activation of cardiopulmonary afferents. Drugs were applied ionophoretically in the vicinity of these neurones., Key Results: The non-selective 5-HT(7) receptor agonist 5-carboxamidotryptamine maleate (5-CT) applied to 78 neurones increased the firing rate in 18 by 59% and decreased it in 38 neurones by 47%. Similarly, the 5-HT(1A) agonist 8-OH-DPAT applied to 20 neurones had an excitatory (8), inhibitory (7) or no effect (5) on the 20 neurones tested. In the presence of the 5-HT(7) antagonist SB 258719 the 5-CT excitation was attenuated. Furthermore, the excitatory response of NTS neurones evoked by electrical stimulation of the vagus nerve or activation of cardiopulmonary afferents with intra atrial phenylbiguanide was attenuated by SB 258719. The inhibitory action of 5-CT was unaffected by SB 258719 and the 5-HT(1A) antagonist WAY-100635. WAY-100635 failed to have any effect on 5-CT and vagal afferent-evoked excitations., Conclusions and Implications: Vagal afferent-evoked excitation of NTS neurones can be blocked by SB 258719, a selective 5-HT(7) antagonist. This observation further supports the involvement of 5-HT neurotransmission in NTS afferent processing.

Background and Purpose: As a combination of 5-HT selective reuptake inhibitor (SSRI) with 5-HT(1A) receptor antagonism may yield a rapidly acting antidepressant, WAY-211612, a compound with both SSRI and 5-HT(1A) receptor antagonist activities, was evaluated in preclinical models., Experimental Approach: Occupancy studies confirmed the mechanism of action of WAY-211612, while its in vivo profile was characterized in microdialysis and behavioural models., Key Results: WAY-211612 inhibited 5-HT reuptake (K(i) = 1.5 nmol.L(-1); K(B) = 17.7 nmol.L(-1)) and exhibited full 5-HT(1A) receptor antagonist activity (K(i) = 1.2 nmol.L(-1); K(B) = 6.3 nmol.L(-1); I(max) 100% in adenyl cyclase assays; K(B) = 19.8 nmol.L(-1); I(max) 100% in GTPgammaS). WAY-211612 (3 and 30 mg.kg(-1), po) occupied 5-HT reuptake sites in rat prefrontal cortex (56.6% and 73.6% respectively) and hippocampus (52.2% and 78.5%), and 5-HT(1A) receptors in the prefrontal cortex (6.7% and 44.7%), hippocampus (8.3% and 48.6%) and dorsal raphe (15% and 83%). Acute or chronic treatment with WAY-211612 (3-30 mg.kg(-1), po) raised levels of cortical 5-HT approximately twofold, as also observed with a combination of an SSRI (fluoxetine; 30 mg.kg(-1), s.c.) and a 5-HT(1A) antagonist (WAY-100635; 0.3 mg.kg(-1), s.c). WAY-211612 (3.3-30 mg.kg(-1), s.c.) decreased aggressive behaviour in the resident-intruder model, while increasing the number of punished crossings (3-30 mg.kg(-1), i.p. and 10-56 mg.kg(-1), po) in the mouse four-plate model and decreased adjunctive drinking behaviour (56 mg.kg(-1), i.p.) in the rat scheduled-induced polydipsia model., Conclusions and Implications: These findings suggest that WAY-211612 may represent a novel antidepressant.

Background and Purpose: Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa which induces anxiolytic- and antipsychotic-like effects in rodents. These effects could be mediated by facilitation of the endocannabinoid system or by the activation of 5-HT(1A) receptors. As either of these mechanisms could promote adaptation to inescapable stress, the aim of the present work was to test the hypothesis that CBD would attenuate the autonomic and behavioural consequences of restraint stress (RS). We also investigated if the responses to CBD depended on activation of 5-HT(1A) receptors., Experimental Approach: Male Wistar rats received i.p. injections of vehicle or CBD (1, 10 or 20 mg kg(-1)) and 30 min later were submitted to 60 min of restraint where their cardiovascular responses were recorded. The protocol of the second experiment was similar to the first one except that animals received i.p. injections of the 5-HT(1A) receptor antagonist WAY100635 (0.1 mg kg(-1)) before CBD treatment and exposure to restraint. 24 h later they were also tested in the elevated plus-maze (EPM), an animal model of anxiety., Key Results: Exposure to RS increased blood pressure and heart rate and induced an anxiogenic response in the EPM 24 h later. These effects were attenuated by CBD. WAY100635 by itself did not change the cardiovascular and anxiogenic response to RS, but blocked the effects of CBD., Conclusion and Implications: The results suggest that CBD can attenuate acute autonomic responses to stress and its delayed emotional consequences by facilitating 5-HT(1A) receptor-mediated neurotransmission.

Background and Purpose: Micturition is controlled by central 5-HT-containing pathways. 5-HT2 receptors have been implicated in this system especially in control of the urethra, which is a drug target for treating urinary incontinence. This study investigates the role of each of the three subtypes of this receptor with emphasis on sphincter regulation., Experimental Approach: Recordings of urethral and bladder pressure, external urethral sphincter (EUS) EMG, as well as the micturition reflex induced by bladder distension along with blood pressure and heart rate were made in anaesthetized rats. The effects of agonists and antagonists for 5-HT2 receptor subtypes were studied on these variables., Key Results: The 5-HT2C agonists Ro 60-0175, WAY 161503 and mCPP, i.v., activated the EUS, increased urethral pressure and inhibited the micturition reflex. The effects of Ro 60-0175 on the EUS were blocked by the 5-HT2C antagonist SB 242084 and the 5-HT2A antagonists, ketanserin and MDL 100907. SB 242084 also blocked the inhibitory action on the reflex, while the 5-HT2B antagonist RS 127445 only blocked the increase in urethral pressure. The 5-HT2A receptor agonist DOI given i.v. or i.t. but not i.c.v. activated the EUS., Conclusions and Implications: 5-HT2A/2C receptors located in the sacral spinal cord activate the EUS, while central 5-HT2C receptors inhibit the micturition reflex and 5-HT2B receptors, probably at the level of the urethra, increase urethral smooth muscle tone. Furthermore, 5-HT2B and 5-HT2C receptors do not seem to play an important role in the physiological regulation of micturition.

Background and Purpose: The beneficial effect of 5-HT6 receptor antagonism in cognition remains controversial. This study has been undertaken to reassess the cognition enhancing properties of acute vs subchronic treatment with the selective 5-HT6 receptor antagonist SB-271046 in unimpaired rats, as well as against scopolamine (cholinergic-) or MK-801 (glutamatergic-mediated) deficits., Experimental Approach: The Morris water maze was used, measuring behaviour acquisition and retention, and swim speed. Other behavioural measures included yawning and motor activity. SB-271046 was given acutely before each trial or subchronically for 7 days before the trials. The AChE inhibitor galanthamine was also used alone or in combination with SB-271046., Key Results: Subchronic treatment with SB-271046 improved acquisition in the Morris water maze, while the acute treatment only improved retention. Neither acute nor subchronic SB-271046 treatment reversed scopolamine-induced learning deficits. MK-801 induced learning impairment associated with a behavioural syndrome, reversed by acute, but not subchronic, SB-271046 treatment. Interestingly, combined treatment with galanthamine and SB-271046 reversed the scopolamine- or MK-801-induced learning impairments. Subchronic treatment with SB-271046 did not modify motor activity or the increased number of yawns, a cholinergic-mediated behaviour, induced by single administration of SB-271046., Conclusions and Implications: These data suggest a potential therapeutic role of 5-HT6 receptor antagonists such as SB-271046, alone or in combination with galanthamine, in the treatment of cognitive dysfunction, such as those seen in Alzheimer's disease and schizophrenia.

Background and Purpose: Ketanserin, a selective 5-HT receptor antagonist, prolongs the QT interval of ECG in patients. The purpose of the present study was to determine whether ketanserin would block human cardiac ether-à-go-go-related gene (hERG) potassium channels., Experimental Approach: Whole-cell patch voltage-clamp technique was used to record membrane currents in HEK 293 cells expressing wild type or mutant hERG channel genes., Key Results: Ketanserin blocked hERG current (I(hERG)) in a concentration-dependent manner (IC50=0.11 microM). The drug showed an open channel blocking property, the block increasing significantly at depolarizing voltages between +10 to +60 mV. Voltage-dependence for inactivation of hERG channels was negatively shifted by 0.3 microM ketanserin. A 2.8 fold attenuation of inhibition by elevation of external K+ concentration (from 5.0 to 20 mM) was observed, whereas the inactivation-deficient mutants S620T and S631A had the IC50s of 0.84 +/- 0.2 and 1.7 +/-0.4 microM (7.6 and 15.4 fold attenuation of block). In addition, the hERG mutants in pore helix and S6 also significantly reduced the channel block (2-59 fold) by ketanserin., Conclusions and Implications: These results suggest that ketanserin binds to and blocks the open hERG channels in the pore helix and the S6 domain; channel inactivation is also involved in the blockade of hERG channels. Blockade of hERG channels most likely contributes to the prolongation of QT intervals in ECG observed clinically at therapeutic concentrations of ketanserin.

Background and Purpose: 5-HT (serotonin) is known to be involved in neuroinflammation and immunoregulation. The human immunodeficiency virus (HIV) targets cells such as monocytes/macrophages, which colocalize with 5-HT-releasing cell types, mostly platelets. In this study, we investigated the effects of 5-HT on HIV-1-infected macrophages in vitro., Experimental Approach: Human macrophages cultured in serum-free medium were treated over 7 days with 5-HT at three concentrations (0.01, 1 and 100 microM) with or without agonists and antagonists of 5-HT(1A) and 5-HT(2) receptors. After 7 days of treatment, macrophages were infected with HIV-1/Ba-L and virus replication was monitored over 16 days and expression of proviral HIV DNA was investigated by PCR after 24 h of infection. Cell surface expression of HIV-1/Ba-L receptor (CD4) and coreceptor (CCR5) was investigated by flow cytometry. The CCR5 ligand, macrophage inflammatory protein-1alpha (MIP-1alpha), was quantified by ELISA in cell culture supernatants and MIP-1alpha mRNA expression was assessed by reverse transcriptase-PCR., Key Results: In vitro, 5-HT downregulated the membranous expression of CCR5 and led to a decrease of HIV-1 infection, probably through its action on 5-HT(1A) receptors. 5-HT (100 microM) was also able to induce overexpression of MIP-1alpha mRNA leading to an increase of MIP-1alpha secretion by human macrophages., Conclusions and Implications: The effects of 5-HT on HIV infection could be a consequence of the increase in MIP-1alpha concentrations and/or CCR5 receptor downregulation. These results suggest that 5-HT can inhibit the replication of HIV-1 in primary culture of human macrophages through its action on 5-HT(1A) receptors.

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.

Background and Purpose: Nocistatin (NST) is a neuropeptide generated from cleavage of the nociceptin/orphanin FQ (N/OFQ) precursor. Evidence has been presented that NST acts as a functional antagonist of N/OFQ, although NST receptor and transduction pathways have not yet been identified. We previously showed that N/OFQ inhibited [(3)H]5-hydroxytryptamine ([(3)H]5-HT) release from mouse cortical synaptosomes via activation of NOP receptors. We now investigate whether NST regulates [(3)H]5-HT release in the same preparation., Experimental Approach: Mouse and rat cerebrocortical synaptosomes in superfusion, preloaded with [(3)H]5-HT and stimulated with 1 min pulses of 10 mM KCl, were used., Key Results: Bovine NST (b-NST) inhibited the K(+)-induced [(3)H]5-HT release, displaying similar efficacy but lower potency than N/OFQ. b-NST action underwent concentration-dependent and time-dependent desensitization, and was not prevented either by the NOP receptor antagonist [Nphe(1) Arg(14),Lys(15)]N/OFQ(1-13)-NH(2) (UFP-101) or by the non-selective opioid receptor antagonist, naloxone. Contrary to N/OFQ, b-NST reduced [(3)H]5-HT release from synaptosomes obtained from NOP receptor knockout mice. However, both N/OFQ and NST were ineffective in synaptosomes pre-treated with the G(i/o) protein inhibitor, Pertussis toxin. NST-N/OFQ interactions were also investigated. Co-application of maximal concentrations of both peptides did not result in additive effects, whereas pre-application of maximal b-NST concentrations partially attenuated N/OFQ inhibition., Conclusions and Implications: We conclude that b-NST inhibits [(3)H]5-HT release via activation of G(i/o) protein linked pathways, not involving classical opioid receptors and the NOP receptor. The present data strengthen the view that b-NST is, per se, a biologically active peptide endowed with agonist activity.

Background and Purpose: The study investigated whether eugenosedin-A, a 5-hydroxytryptamine and alpha/beta adrenoceptor antagonist, enhanced delayed-rectifier potassium (K(DR))- or large-conductance Ca(2+)-activated potassium (BK(Ca))-channel activity in basilar artery myocytes through cyclic AMP/GMP-dependent and -independent protein kinases., Experimental Approach: Cerebral smooth muscle cells (SMCs) were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K(+)- and Ca(2+)-channel activities., Key Results: Eugenosedin-A (1 microM) did not affect the K(DR) current but dramatically augmented BK(Ca) channel activity in a concentration-dependent manner. Increased BK(Ca) current was abolished by charybdotoxin (ChTX, 0.1 microM) or iberiotoxin (IbTX, 0.1 microM), but not affected by a small-conductance K(Ca) blocker (apamin, 100 microM). BK(Ca) current activation by eugenosedin-A was significantly inhibited by an adenylate cyclase inhibitor (SQ 22536, 10 microM), a soluble guanylate cyclase inhibitor (ODQ, 10 microM), competitive antagonists of cAMP and cGMP (Rp-cAMP, 100 microM and Rp-cGMP, 100 microM), and cAMP- and cGMP-dependent protein kinase inhibitors (KT5720, 0.3 microM and KT5823, 0.3 microM). Eugenosedin-A reversed the inhibition of BK(Ca) current induced by the protein kinase C activator, phorbol myristyl acetate (PMA, 0.1 microM). Eugenosedin-A also prevented BK(Ca) current inhibition induced by adding PMA, KT5720 and KT5823. Moreover, eugenosedin-A reduced the amplitude of voltage-dependent L-type Ca(2+) current (I(Ca,L)), but without modifying the voltage-dependence of the current., Conclusions and Implications: Eugenosedin-A enhanced BK(Ca) currents by stimulating the activity of cyclic nucleotide-dependent protein kinases. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and thereby relax cerebral SMCs.

Background and Purpose: F15063 is a high affinity D(2)/D(3) antagonist, D(4) partial agonist, and high efficacy 5-HT(1A) agonist, with little affinity (40-fold lower than for D(2) receptors) at other central targets. Here, the profile of F15063 was evaluated in models of positive symptoms of schizophrenia and motor side-effects., Experimental Approach: Rodent behavioural tests were based on reversal of hyperactivity induced by psychostimulants and on measures of induction of catalepsy and 'serotonin syndrome'., Key Results: F15063 potently (ED(50)s: 0.23 to 1.10 mg kg(-1) i.p.) reversed methylphenidate-induced stereotyped behaviors, blocked d-amphetamine and ketamine hyperlocomotion, attenuated apomorphine-induced prepulse inhibition (PPI) deficits, and was active in the conditioned avoidance test. In mice, it reversed apomorphine-induced climbing (ED(50)=0.30 mg kg(-1) i.p.). F15063, owing to its 5-HT(1A) agonism, did not produce (ED(50)>40 mg kg(-1) i.p.) catalepsy in rats and mice, a behavior predictive of occurrence of extra-pyramidal syndrome (EPS) in man. This absence of cataleptogenic activity was maintained upon sub-chronic treatment of rats for 5 days at 40 mg kg(-1) p.o. Furthermore, F15063 did not induce the 'serotonin syndrome' in rats (flat body posture and forepaw treading: ED(50) >32 mg kg(-1) i.p.)., Conclusions and Implications: F15063 conformed to the profile of an atypical antipsychotic, with potent actions in models of hyperdopaminergic activity but without inducing catalepsy. These data suggest that F15063 may display potent antipsychotic actions with low EPS liability. This profile is complemented by a favourable profile in rodent models of negative symptoms and cognitive deficits of schizophrenia (companion paper).

Background and Purpose: Central 5-HT-containing pathways are known to be important in cardiovascular regulation and a crucial area for this regulation is the nucleus tractus solitarius (NTS), which contains many of the known 5-HT receptor subtypes. In this study the role of 5-HT(1B) and 5-HT(1D) receptors, targets for the antimigraine drugs known collectively as triptans, was examined in the NTS. EXPERIMENT APPROACH: Extracellular recordings were made, in anaesthetized rats, from 109 NTS neurones that were excited by electrical stimulation of the vagus and drugs were applied ionophoretically to these neurones., Key Results: The 5-HT(1B/1D) receptor agonist sumatriptan applied to 64 neurones produced a 64% reduction in the firing rate of 54 of these neurones. Ketanserin, a 5-HT(1D/2A) receptor antagonist, alone had little effect, but co-applied with sumatriptan significantly attenuated this inhibition, whilst co-application of the 5-HT(1B) receptor antagonist GR55562 resulted in potentiation of this inhibition. Sumatriptan also caused a 25% reduction in vagal afferent evoked activity as well as that caused by stimulation of cardiopulmonary afferents. In another 41 neurones the 5-HT(1B) receptor agonist CP-93 129 produced a doubling of the background firing rate in 31 of these neurones and a significant increase in both vagal afferent evoked activity and that evoked by cardiopulmonary afferent activation., Conclusions and Implications: Activation of 5-HT(1B) and 5-HT(1D) receptors have opposing actions on NTS neurones of excitation and inhibition, respectively. As both receptors are negatively coupled to adenylate cyclase this would indicate that they have different anatomical locations within NTS.

Background and Purpose: A strategy to treat Alzheimer's disease (AD) is to increase the soluble form of amyloid precursor protein (sAPPalpha), a promnesic protein, in the brain. Because strong evidence supports beneficial effects of 5-hydroxytryptamine 5-HT(4) receptor agonists in memory and learning, we investigated the role of 5-HT(4) receptors on APP processing in 8 weeks-old male C57BL/6j mice., Experimental Approach: Mice were given, subcutaneously, prucalopride or ML 10302 (s.c.), two highly selective 5-HT(4) receptor agonists and, up to 240 min later, the hippocampus and cortex were analysed by Western blot for sAPPalpha determination., Key Results: Prucalopride (5 or 10 mg kg(-1)) significantly increased sAPPalpha levels in the hippocampus and cortex, but did not modify the expression level of APP mRNA as detected by quantitative RT-PCR. A selective 5-HT(4) receptor antagonist, GR125487 (1 mg kg(-1), s.c.) inhibited prucalopride induced- increase in sAPPalpha levels. In addition, levels of sAPPalpha were increased by ML10302 only at 20 mg kg(-1) and was limited to the cortex. Also, prucalopride increased sAPPalpha levels in the cortex of a transgenic mouse model of AD, expressing the London mutation of APP. Furthermore, the combined injection of a selective acetylcholinesterase inhibitor, donepezil and prucalopride induced a synergic increase in sAPPalpha levels in the cortex and hippocampus., Conclusions and Implications: Our results demonstrate that the 5-HT(4) receptor plays a key role in the non-amyloidogenic pathway of APP metabolism in vivo and give support to the beneficial use of 5-HT(4) agonists for AD treatment.

Background and Purpose: The serotonergic system within the spinal cord have been proposed to play an important role in the analgesic effects of systemic morphine. Currently, seven groups of 5-HT receptors (5-HT1-7) have been characterized. One of the most recently identified subtypes of 5 HT receptor is the 5-HT7 receptor. We aimed to examine the role of spinal 5-HT7 receptors in the antinociceptive effects of systemic morphine., Experimental Approach: The involvement of spinal 5-HT7 receptor in systemic morphine antinociception was compared to that of the 5-HT1A and 5-HT2 receptors by using the selective 5-HT7 receptor antagonist, SB-269970, the selective 5-HT1A receptor antagonist, WAY 100635, the selective 5-HT2 antagonist ketanserin as well as the non-selective 5-HT1,2,7 receptor antagonist, metergoline. Nociception was evaluated by the radiant heat tail-flick test., Key Results: I.t. administration of SB-269970 (10 microg) and metergoline (20 microg) completely blocked the s.c. administered morphine-induced (1, 3, 5 and 10 mg kg(-1)) antinociception in a time-dependent manner. Additionally, i.t. administration of SB-269970 (1, 3, 10 and 20 microg) and metergoline (1, 5, 10 and 20 microg) dose dependently inhibited the antinociceptive effects of a maximal dose of morphine (10 mg kg(-1), s.c.). I.t. administration of WAY 100635 (20 microg) or ketanserine (20 microg) did not alter morphine-induced (1, 3, 5 and 10 mg kg(-1), s.c.) antinociception., Conclusion and Implications: These findings indicate that the involvement of spinal 5-HT7, but not of 5-HT1A or of 5-HT2 receptors in the antinociceptive effects of systemic morphine.

This study provides a comprehensive evaluation of 5-HT(3) receptor functional distribution in both the rat and mouse intestinal tract. 5-HT(3A-S) receptor splice variant mRNA was expressed throughout the intestine of the rat and mouse; the 5-HT(3A-L) variant being more common in the rat.5-HT, m-CPB, 1-PBG and 2-methyl-5-hydroxytryptamine (2m5-HT) induced contraction in the jejunum, ileum, proximal colon and distal colon of the rat (pEC(50) range: 2m5-HT, 5.86+/-0.40 to m-CPB, 7.47+/-0.27) and mouse (pEC(50) range: 1-PBG, 5.34+/-0.06 to m-CPB, 6.49+/-0.14) in the presence of nontarget 5-HT receptor antagonists, methysergide (1 muM) and GR125487 (0.1 microM). The rank orders of potency in the four regions of the rat and mouse intestine were concordant with the accepted order and the responses to 5-HT were inhibited by ondansetron (0.1 microM).5-HT(3)-induced contractions to 5-HT were reduced by tetrodotoxin (1 microM). Pargyline (10 muM) and fluoxetine (1 microM) potentiated responses in the rat jejunum. Atropine (0.1 microM) potentiated 5-HT(3)-induced responses in the rat jejunum (E(max) 49-65%), but attenuated responses in most other regions of the rat and mouse (e.g. mouse ileum: E(max) 57-26%). In the rat jejunum, L-NAME (100 microM) mimicked the effect of atropine, hexamethonium (100 microM) suppressed 5-HT(3)-induced responses, but tachykinin receptor antagonists were without effect. It is concluded that functional 5-HT(3) receptors are present in nerves along the length of the rat and mouse intestinal tract. The mouse proximal colon was found to discriminate 5-HT(3) receptor agonist profiles better than any other region in the rat or mouse. The rat jejunum shows evidence of 5-HT uptake and inactivation processes as well as inhibitory nitrergic and nontachykinin excitatory pathways associated with the 5-HT(3)-induced response.

1. We previously demonstrated that p-chloroamphetamine (PCA) intravenously (i.v.) evokes a specific patterned bursting response in the vas deferens nerve (VDN) of anaesthetised male rats that is associated with contraction of the vas deferens, and ejaculation and contraction of the bulbospongiosus muscles. The present study used selective 5-HT agonists to induce similar rhythmic bursting responses in the VDN in order to reveal the 5-HT receptor subtypes involved. 2. The 5-HT(2C) receptor agonist (1.0 mg kg(-1) Ro600175 i.v.) evoked the characteristic bursting pattern responses in the VDN. The 5-HT(1A) receptor agonist (1.0 mg kg(-1) 8-OH-DPAT i.v.) failed to elicit any responses. However, 8-OH-DPAT coadministered in combination with Ro600175 induced a potentiation of the responses. 3. Responses were also evoked in rats with a mid-thoracic spinalisation, with a more predictable response being observed following the combination of agonists. This suggests an action of both agonists in the lumbosacral spinal cord. 4. Responses were blocked by 0.5 mg kg(-1) SB206553 i.v. (5-HT(2B/C) receptor antagonist) or 0.5 mg kg(-1) WAY100635 i.v. (5-HT(1A) receptor antagonist), but not 0.1 or 1.0 mg kg(-1) SB269970 i.v. (5-HT(7) receptor antagonist). 5. We suggest that activation of 5-HT(2C) and 5-HT(1A) receptor subtypes synergistically elicits contraction of the vas deferens through the activation of sympathetic preganglionic neurones in the spinal cord. 6. These data support the idea of a proejaculatory action of 5-HT(2C) receptors in the lumbosacral spinal cord, suggesting a descending 5-HT excitatory pathway in addition to a 5-HT inhibitory pathway. An excitatory action of 8-OH-DPAT at lumbosacral sites is also evident.

1. We investigated the effect of alloxan-induced diabetes on the inhibitory mechanisms of 5-hydroxytryptamine (5-HT) in the pressor responses induced by stimulation of sympathetic vasopressor outflow in pithed rats, and analysed the type and/or subtype of 5-HT receptors involved. 2. Diabetes was induced in male Wistar rats by a single s.c. injection of alloxan, then 4 weeks later, they were anaesthetized, pretreated with atropine and pithed. Electrical stimulation of the sympathetic outflow from the spinal cord (0.1, 0.5, 1 and 5 Hz) resulted in frequency-dependent increases in blood pressure. 3. Intravenous infusions of 5-HT (1-80 microg kg(-1) min(-1)) reduced the pressor effects obtained by electrical stimulation. The 5-HT(1) receptor agonist 5-carboxamidotryptamine, 5-CT (5 microg kg(-1) min(-1)), caused an inhibition of the pressor response, whereas the selective 5-HT(2) receptor agonist, alpha-methyl-5-HT (5 microg kg(-1) min(-1)) and the selective 5-HT(3) receptor agonist, 1-phenylbiguanide (40 microg kg(-1) min(-1)), did not modify the sympathetic pressor responses. 5-HT had no effect on exogenous noradrenaline (NA)-induced pressor responses. 4. The inhibition of electrically induced pressor responses by 5-HT (10 microg kg(-1) min(-1)) was unable to be elicited after i.v. treatment with methiothepin (100 microg kg(-1)) because of the marked inhibition produced by methiothepin alone. The 5-HT-induced inhibition was blocked after i.v. administration of WAY-100,635 (100 microg kg(-1)) and not affected by ritanserin (1 mg kg(-1)), MDL 72222 (2 mg kg(-1)). 5. The selective 5-HT(1A) receptor agonist, 8-hydroxydipropylaminotretalin hydrobromide (8-OH-DPAT) (5-20 microg kg(-1) min(-1)) but neither the rodent 5-HT(1B) receptor agonist, CGS-12066B (5 microg kg(-1) min(-1)), nor the selective nonrodent 5-HT(1B) and 5-HT(1D) receptor agonist, L-694,247 (5 and 40 microg kg(-1) min(-1)), inhibited the electrically induced pressor response. The selective 5-HT(1A) receptor antagonist, WAY-100,635 (100 microg kg(-1)), blocked the inhibition induced by 8-OH-DPAT (10 microg kg(-1) min(-1)). 8-OH-DPAT had no effect on exogenous NA-induced pressor responses. 6. Experimental diabetes produces changes in the inhibitory effect induced by 5-HT on electrically induced sympathetic pressor responses, such that the inhibitory action induced by 5-HT in diabetic pithed rats is mediated by prejunctional 5-HT(1A) receptors.

1. Although an important regulatory role for serotonin (5-HT) in seizure activation and propagation is well established, relatively little is known of the function of specific 5-HT receptor subtypes on seizure modulation. 2. The aim of the present study was to investigate the role of 5-HT(1A, 1B and 1D) receptors in modulating generalised seizures in the rat maximal electroshock seizure threshold (MEST) test. 3. The mixed 5-HT receptor agonists SKF 99101 (5-20 mg kg(-1) i.p.) and RU 24969 (1-5 mg kg(-1) i.p.), 0.5 h pretest, both produced marked dose-related increases in seizure threshold. These agents share high affinity for 5-HT(1A, 1B and 1D) receptors. 4. Antiseizure effects induced by submaximal doses of these agonists were maintained following p-chlorophenylalanine (150 mg kg(-1) i.p. x 3 days)-induced 5-HT depletion. 5. The anticonvulsant action of both SKF 99101 (15 mg kg(-1) i.p.) and RU 24969 (2.5 mg kg(-1) i.p.) was dose-dependently abolished by the selective 5-HT1B receptor antagonist SB-224289 (0.1-3 mg kg(-1) p.o., 3 h pretest) but was unaffected by the selective 5-HT1A receptor antagonist WAY 100635 (0.01-0.3 mg kg(-1) s.c., 1 h pretest). This indicates that 5-HT1B receptors are primarily involved in mediating the anticonvulsant properties of these agents. 6. In addition, the ability of the 5-HT(1B/1D) receptor antagonist GR 127935 (0.3-3 mg kg(-1) s.c., 60 min pretest) to dose-dependently inhibit SKF 99101-induced elevation of seizure threshold also suggests possible downstream involvement of 5-HT1D receptors in the action of this agonist, although confirmation awaits the identification of a selective 5-HT1D receptor antagonist. 7. Overall, these data demonstrate that stimulation of postsynaptic 5-HT1B receptors inhibits electroshock-induced seizure spread in rats.

1 Tegaserod (Zelnorm) is a potent 5-hydroxytryptamine4 (5-HT4) receptor agonist with clinical efficacy in disorders associated with reduced gastrointestinal motility and transit. The present study investigated the interaction of tegaserod with 5-HT2 receptors, and compared its potency in this respect to its 5-HT4 receptor agonist activity. 2 Tegaserod had significant binding affinity for human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors (pKi=7.5, 8.4 and 7.0, respectively). The 5-HT2B receptor-binding affinity of tegaserod was identical to that at human recombinant 5-HT4(c) receptors (mean pKi=8.4) in human embryonic kidney-293 (HEK-293) cells stably transfected with the human 5-HT4(c) receptor. 3 Tegaserod (0.1-3 microm) inhibited 5-HT-mediated contraction of the rat isolated stomach fundus potently (pA2=8.3), consistent with 5-HT(2B) receptor antagonist activity. Tegaserod produced, with similar potency, an elevation of adenosine 3',5' cyclic monophosphate in HEK-293 cells stably transfected with the human 5-HT4(c) receptor (mean pEC50=8.6), as well as 5-HT4) receptor-mediated relaxation of the rat isolated oesophagus (mean pEC50=8.2) and contraction of the guinea-pig isolated colon (mean pEC50=8.3). 4 Following subcutaneous administration, tegaserod (0.3 or 1 mg kg(-1)) inhibited contractions of the stomach fundus in anaesthetized rats in response to intravenous dosing of alpha-methyl 5-HT (0.03 mg kg(-1)) and BW 723C86 (0.3 mg kg(-1)), selective 5-HT2B receptor agonists. At similar doses, tegaserod (1 and 3 mg kg(-1) subcutaneously) evoked a 5-HT4 receptor-mediated increase in colonic transit in conscious guinea-pigs. 5 The data from this study indicate that tegaserod antagonizes 5-HT2B receptors at concentrations similar to those that activate 5-HT4 receptors. It remains to be determined whether this 5-HT2B receptor antagonist activity of tegaserod contributes to its clinical profile.

In the presence of NMDA receptor open-channel blockers [Mg(2+); (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801); 1-amino-3,5-dimethyladamantane (memantine)] and TTX, high concentrations (30-100 microm) of either 5-hydroxytryptamine (5-HT) or alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT) significantly potentiated NMDA-induced depolarizations of frog spinal cord motoneurones. Potentiation was blocked by LY-53,857 (10-30 microm), SB 206553 (10 microm), and SB 204741 (30 microm), but not by spiroxatrine (10 microm), WAY 100,635 (1-30 microm), ketanserin (10 microm), RS 102221 (10 microm), or RS 39604 (10-20 microm). Therefore, alpha-Me-5-HT's facilitatory effects appear to involve 5-HT(2B) receptors. These effects were G-protein dependent as they were prevented by prior treatment with guanylyl-5'-imidodiphosphate (GMP-PNP, 100 microm) and H-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH(2) (GP antagonist 2A, 3-6 microm), but not by pertussis toxin (PTX, 3-6 ng ml(-1), 48 h preincubation). This potentiation was not reduced by protein kinase C inhibition with staurosporine (2.0 microm), U73122 (10 microm) or N-(2-aminoethyl)-5-isoquinolinesulfonamide HCl (H9) (77 microm) or by intracellular Ca(2+) depletion with thapsigargin (0.1 microm) (which inhibits Ca(2+)/ATPase). Exposure of the spinal cord to the L-type Ca(2+) channel blockers nifedipine (10 microm), KN-62 (5 microm) or gallopamil (100 microm) eliminated alpha-Me-5-HT's effects. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7) (100 microm) diminished the potentiation. However, the calcium/calmodulin-dependent protein kinase II (CaM Kinase II) blocker KN-93 (10 microm) did not block the 5-HT enhancement of the NMDA responses. In summary, activation of 5-HT(2B) receptors by alpha-Me-5-HT facilitates NMDA-depolarizations of frog motoneurones via a G-protein, a rise in [Ca(2+)](i) from the entry of extracellular Ca(2+) through L-type Ca(2+) channels, the binding of Ca(2+) to calmodulin and a lessening of the Mg(2+) -produced open-channel block of the NMDA receptor.

This study aimed to determine, quantify and explain regional differences in the relaxant response to the selective 5-HT(1) and 5-HT(7) receptor agonist 5-carboxamidotryptamine (5-CT) throughout the canine stomach. Longitudinal muscle strips from eight gastric corpus regions and six antrum regions were mounted for isotonic measurement. The 5-CT-induced relaxation was examined on a prostaglandin F(2alpha)-induced submaximal response, expressed as percentage of this response and fitted to the operational model of agonism (OMOA). 5-HT(7) receptor messenger RNA (mRNA) expression was compared by means of quantitative PCR. 5-CT inhibited PGF(2alpha)-induced tonic contraction (corpus) and increase of phasic contraction amplitude (antrum). The consistent antagonism produced by the selective 5-HT(7) receptor antagonist SB-269970 (10 nm, pA(2) estimates 8.2-8.9) confirmed that in every region, the inhibition by 5-CT was 5-HT(7) receptor mediated. However, variation in the maximum effect (61-108%) and pEC(50) (6.4-8.6) was observed throughout the different regions. The OMOA explained these differences as differences in the efficacy parameter tau (ratio of receptor density and coupling efficiency; log tau estimates ranging from 0.1 to 2.1). The log tau gradient decreases going from the lesser to the greater curvature. A proportional difference (68%) in the relative expression of 5-HT(7) receptor mRNA between the lesser and the greater curvature indicates that differences in receptor density contribute to the observed functional differences. This study illustrates that 5-HT(7) receptors are present throughout the ventral wall of the canine stomach, but the efficacy (expressed as log tau) is clearly greater close to the lesser curvature. Differences in 5-HT(7) receptor expression at least partially explain the functional differences.

Serotonin-2 receptor antagonists, like ritanserin, greatly enhance deep slow wave sleep (SWS-2) and low-frequency EEG power in humans and rodents. 5-HT(2A) and 5-HT(2C) receptors may be involved in these effects, but the role of the 5-HT(2B) receptor is still unclear. To investigate the role of the 5-HT(2B) receptor in regulation of the sleep-wake cycle, the subtype-selective antagonist SB-215505 (0.1, 0.3 and 1.0 mg kg(-1) i.p.) was administered to Sprague-Dawley rats at light onset (beginning of passive phase). EEG, EMG and motor activity were recorded during the subsequent 8 h. SB-215505 dose-dependently increased wakefulness (W) at the expense of the intermediate stage of sleep, paradoxical sleep (PS) and SWS-2 in the first hour. Parallel to increased W, significantly increased motor activity was found. Spectral analysis of the EEG in W showed a dose-dependent decrease in power density in the 3-8 Hz frequency range (maximum effect at 6 Hz). In light slow wave sleep and SWS-2, the drug reduced low-frequency (<8 Hz) EEG power, suggesting decreased sleep intensity after SB-215505 treatment. In PS, the drug dose-dependently decreased EEG power solely in the theta (6-9 Hz) band, primarily affecting the peak power value (7 Hz). The well-known SWS-2 enhancing effect of 5-HT(2) receptor antagonists is mediated by 5-HT(2A) and/or 5-HT(2C) receptors. In contrast, blockade of 5-HT(2B) receptors increases motor activity and W along with decreased theta activity during W and PS. Activation of 5-HT(2B) receptors may contribute to initiation of sleep and to theta generation during W and PS under physiological conditions.

1 The influence of sodium ion substitutes on the 5-hydroxytryptamine (5-HT)-induced flux of the organic cation [14C]guanidinium through the ion channel of the mouse 5-HT3 receptor and on the competition of 5-HT with the selective 5-HT3 receptor antagonist [3H]GR 65630 was studied, unless stated otherwise, in mouse neuroblastoma N1E-115 cells. 2 Under physiological conditions (135 mm sodium), 5-HT induced a concentration-dependent [14C]guanidinium influx with an EC50 (1.3 microm) similar to that in electrophysiological studies. 3 The stepwise replacement of sodium by increasing concentrations of the organic cation hydroxyethyl trimethylammonium (choline) concentration dependently caused both a rightward shift of the 5-HT concentration-response curve and an increase in the maximum effect of 5-HT. Complete replacement of sodium resulted in a 34-fold lower potency of 5-HT and an almost two times higher maximal response. A low potency of 5-HT in choline buffer was also observed in other 5-HT3 receptor-expressing rodent cell lines (NG 108-15 or NCB 20). 4 Replacement of Na+ by Li+ left the potency and maximal effects of 5-HT almost unchanged. Replacement by tris (hydroxymethyl) methylamine (Tris), tetramethylammonium (TMA) or N-methyl-d-glucamine (NMDG) caused an increase in maximal response to 5-HT similar to that caused by choline. The potency of 5-HT was only slightly reduced by Tris, to a high degree decreased by TMA (comparable to the decrease by choline), but not influenced by NMDG. 5 The potency of 5-HT in inhibiting [3H]GR65630 binding to intact cells was 35-fold lower when sodium was completely replaced by choline, but remained unchanged after replacement by NMDG. 6 The results are compatible with the suggestion that choline competes with 5-HT for the 5-HT3 receptor; the increase in maximal response may be partly due to a choline-mediated delay of the 5-HT-induced desensitization. For studies of 5-HT-evoked [14C]guanidinium flux through 5-HT3 receptor channels, NMDG appears to be an 'ideal' sodium substituent since it increases the signal-to-noise ratio without interfering with 5-HT binding.

1 Microdialysis was used to study the acute and chronic effects of escitalopram (S-citalopram; ESCIT) and chronic citalopram (CIT), together with the 5-HT1A receptor antagonist WAY100,635 (N-[2-[methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide trihydrochloride) and the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on extracellular 5-hydroxytryptamine (5-HT) levels in the rat prefrontal cortex. 2 Extracellular 5-HT rose to 234 and 298% of basal values after subcutaneous (s.c.) acute doses of 0.15 and 0.63 mg kg(-1) ESCIT. No further increase was observed at 2.5 mg kg(-1) ESCIT (290%). 3 The effect of 13-day s.c. infusion of 10 mg kg(-1) day(-1) ESCIT on extracellular 5-HT (422% of baseline) was greater than after 2 days (257% of baseline), whereas exposure to ESCIT was similar. In contrast, the increase in extracellular 5-HT induced by the infusion of CIT for 2 (306%) and 13 days (302%) was similar. However, brain and plasma levels of S-citalopram in rats infused with CIT for 13 days were lower than after 2 days. 4 Acute treatment with 2.5 mg kg(-1) ESCIT or 5 mg kg(-1) CIT raised extracellular 5-HT by 243 and 276%, respectively, in rats given chronic vehicle but had no effect in rats given ESCIT (10 mg kg(-1) day(-1)) or CIT (20 mg kg(-1) day(-1)) for 2 or 13 days, suggesting that the infused doses had maximally increased extracellular 5-HT. WAY100,635 (0.1 mg kg(-1) s.c.) increased extracellular 5-HT levels by 168, 174 and 169% of prechallenge values in rats infused with vehicle or ESCIT for 2 or 13 days, respectively. WAY100,635 enhanced extracellular 5-HT levels to 226, 153 and 164% of prechallenge values in rats infused with vehicle or CIT for 2 and 13 days, respectively. 5 8-OH-DPAT (0.025 mg kg(-1)) reduced extracellular 5-HT by 54% in control rats, but had no effect in those given ESCIT and CIT for 13 days. 6 This series of experiments led to the conclusion that chronic treatment with ESCIT desensitizes the 5-HT1A receptors, regulating the release of 5-HT in the prefrontal cortex and enhances the effect of the drug on extracellular 5-HT. They also indicate that chronic treatment with ESCIT and CIT did not prevent WAY100,635 from raising extracellular 5-HT.

1. Binding of the novel radioligand (3)H-2-(2-dimethylaminomethyl-phenylsulphanyl)-5-methyl-phenylamine ((3)H-MADAM) to the serotonin transporter (SERT) was used to characterise a range of selective serotonin re-uptake inhibitors (SSRIs) in vitro and in vivo. 2. (3)H-MADAM bound with high affinity in a saturable manner to both human SERT expressed in CHO cells (K(d)=0.20 nm (pK(d)=9.74+/-0.12), B(max)=35+/-4 fmol mg(-1) protein) and mouse cerebral cortex membranes (K(d)=0.21 nm (pK(d)=9.66+/-0.10), B(max)=50+/-24 fmol mg(-1) protein). 3. Binding of (3)H-MADAM was highly selective for SERT in vitro as demonstrated by the in vitro profile of MADAM tested at 75 different receptors, ion channels and transporters. This was further substantiated by the pharmacological profile of the binding. Hence, the binding of (3)H-MADAM was potently inhibited by SSRIs but not by selective inhibitors of noradrenaline transport and dopamine transport. Likewise, a 5-HT(2A/2C) receptor antagonist did not inhibit (3)H-MADAM binding. 4. (3)H-MADAM binding in vivo was inhibited only by compounds which also inhibited the binding of (3)H-MADAM in vitro (the SSRIs, mixed SERT/noradrenaline transport inhibitors and clomipramine), confirming the selectivity of (3)H-MADAM for SERT also in vivo. Moreover, compounds effective in inhibiting (3)H-MADAM binding were the only ones found to be active in the mouse 5-HTP potentiation test confirming the model as a behavioural correlate to in vivo 5-HT uptake. 5. Finally, it was found that a SERT occupancy of 85-95% was necessary to produce 50% of the maximum behavioural response (ED(50)).

(1) The effects of the selective 5-HT7 receptor antagonists SB-269970 (3-300 microg kg-1; n=5-6) and SB-656104 (30 microg kg-1; n=5) administered centrally (i.c.v.) were investigated on the 'micturition reflex' in the urethane anaesthetized female rat. (2) In cystometric recordings, SB-269970 caused significant increases in volume of 58+/-15 and 138+/-33% and pressure of 140+/-46 and 149+/-60% thresholds at 10 and 30 microg kg-1. These changes were associated with significant decreases in distension-induced bladder contraction of 62+/-14 and 60+/-11%, respectively. However, there was no change in residual volume. At the higher doses, SB-269970 blocked the micturition reflex. SB-656104 had similar effects to SB-269970 but in addition significantly increased the residual volume. (3) SB-269970 (10 microg kg-1; n=5) given i.v. had no effect on the micturition reflex. (4) SB-269970 (30 microg kg-1; n=4) given intrathecally (i.t.) had no effect on micturition reflex, although the selective 5-HT1A receptor antagonist WAY-100635 given i.t. after SB-269970 caused a significant increase in the volume threshold. (5) Using an isovolumetric method in which urethral changes were measured, SB-269970 (30 microg kg-1; n=4; i.c.v.) failed to have any effect on these urethral-evoked changes although they significantly reduced the amplitude of the bladder contraction. (6) These data demonstrate that 5-HT7 receptors located supraspinally in the rat are involved in the control of micturition.

1. The venom of Phoneutria nigriventer spider (PNV) causes intense pain and inflammation following an attack. We have investigated the involvement of capsaicin-sensitive nerve fibres by utilizing an in vitro nerve preparation. Extracellular DC potential recordings were made from the rat-isolated vagus nerve, a preparation that is rich in capsaicin-sensitive, that is, nociceptive, C-fibres. 2. PNV (1-10 microg ml(-1)), capsaicin (0.03-0.3 microM) or 5-hydroxytriptamine (5-HT) (0.3-3 microM) induced dose-dependent depolarizations of vagus nerve fibres. Depolarizing responses to capsaicin were blocked by ruthenium red (RR, 10 microM), but responses to PNV were not. Depolarizing responses to PNV or veratridine (50 microM) were inhibited by tetrodotoxin (TTX, 10 microM), but those to capsaicin were not. This suggests that capsaicin and PNV depolarize the nerve fibres by distinct mechanisms. 3. Depolarization in response to 5-HT (3 microM) was reduced by the 5-HT(3) receptor antagonists Y25130 (0.5 micro M) and tropisetron (10 nM) or, to a lesser extent, by the 5-HT(4) receptor antagonist RS39604 (1 or 10 microM). Depolarizing responses to PNV were not affected significantly by Y25130 or tropisetron, but were blocked by RS39604. 4. These data show that 5-HT(4) receptors play a significant role in the activation of nociceptive sensory nerve fibres by PNV and suggest that this is of importance in the development of the pain and inflammation associated with bites from the P. nigriventer spider.

1 Constriction measurements and intracellular microelectrode recordings were performed in vitro on lymphatic vessels isolated from the guinea-pig mesentery to investigate whether 5-hydroxytryptamine (5-HT) affected lymphatic pumping and smooth muscle membrane potential. 2 5-HT decreased in a concentration-dependent manner the frequency of constrictions induced by intraluminal vessel perfusion. In nonperfused vessels, 5-HT hyperpolarized the lymphatic smooth muscle membrane potential and decreased the frequency and amplitude of spontaneous transient depolarizations (STDs). 3 The actions of 5-HT were significantly reversed by the 5-HT(7) receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine (SB269970, 0.5 micro M) and by the 5-HT(1/2/5/7) receptor antagonists methysergide (0.5 micro M), and were mimicked by the 5-HT(1/7)-receptor agonist, 5-CT. 4 The 5-HT(4)-receptor antagonists 1-methyl-1H-indole-3-carboxylic acid [1-2-[(methyl sulfonyl) amino] ethyl-4-piperidinyl] methyl ester (GR113808, 1 micro M) and (1-piperidinyl) ethyl 1H-indole 3-carboxylate (SB203186, 1 micro M) did not significantly affect the 5-HT-induced responses. The 5-HT(4)-receptor agonist 1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-(2-methylsulfonylamino) ethyl-4-piperidinyl]-1-propanone hydrochloride (RS67506) decreased the constriction frequency, albeit only at 50 micro M and without affecting the smooth muscle membrane potential. 5 Responses to 5-HT were attenuated by the nitric oxide synthase inhibitor N(G)-nitro L-arginine (100 micro M), whereas indomethacin (10 micro M) and tetrodotoxin (1 micro M) were without effects. 6 5-HT-induced responses were inhibited by the ATP-sensitive K(+) channel blocker, glibenclamide (10 micro M) and the cAMP-dependent protein kinase inhibitor N-[2-(p-bromociannamylamino)-ethyl]-5-isoquinolinesulfonamide-dichloride (H89, 10 micro M) blocked the hyperpolarization. 7 These results suggest that 5-HT modulates the rate of lymphatic vessel pumping by eliciting K(ATP) channel-mediated smooth muscle hyperpolarization and decrease in STD activity, which appear to be mediated by activation of 5-HT(7) receptors coupled to cAMP production.

(1) 5-HT moduline (5-HTm) is tetrapeptide (Leu-Ser-Ala-Leu) previously shown to act as a specific endogenous antagonist to central 5-HT(1B/1D) receptors. Its effects were investigated in rat and rabbit pulmonary arteries (PAs). (2) In rabbit PAs, contractile responses to the 5-HT(1B/1D) receptor agonist 5-carboxamidotryptamine (5-CT) were inhibited by 1 and 10 micro M 5-HTm in a non-competitive fashion with the maximum contractile response (E(max), per cent of response to 50 mM KCl) being reduced from 65.6+/-7% (n=6) to 39.7+/-6.5% (n=6) and 25.2+/-7.9 (n=4), respectively. The ability of 5-HTm to inhibit responses to 5-CT was increased by the aminopeptidase inhibitor bestatin (10 micro M). (3) In the rabbit PAs, the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methylester (L-NAME) potentiated responses to 5-CT (E(max): 106+/-22.5 (n=4)) and this response was also inhibited by 10 micro M 5-HTm (E(max): 38+/-13% (n=8)). (4) 5-HTm (10 micro M) inhibited responses to 5-CT in rat PAs, the E(max) being reduced from 24.8+/-4.1% (n=7) to 15.5+/-3.7% (n=9). 5-HTm induced relaxation of 5-CT-pre-constricted rat PAs with a pIC(50) of 9.0+/-0.6 (n=9). (5) In PAs from chronic hypoxic, pulmonary hypertensive rats, the maximum response to 5-CT was increased to 80+/-8.5% (n=11). 5-HTm reduced this response to 34.4+/-6.3% (n=12). L-NAME markedly inhibited the ability of 5-HTm to inhibit responses to 5-CT (E(max) before 5-HTm: 100.5+/-16% (n=5), E(max) after 5-HTm: 107+/-11.3% (n=4)). (6) In conclusion we show here for the first time that 5-HTm is a non-competitive inhibitor of 5-HT(1B/1D) receptor-mediated constriction in PAs. In rat PAs, L-NAME can inhibit this effect of 5-HTm.