Influence of the 5-HT(2C) receptor antagonist SB242,084 on behaviour produced by the 5-HT(2) agonist Ro60-0175 and the indirect 5-HT agonist dexfenfluramine.
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Ro60-0175 has been described as a selective agonist at the 5-HT(2C) receptor, yet it has only 10- fold higher affinity at the 5-HT(2C) compared to the 5-HT(2A) subtype, and equivalent affinity for the 5-HT(2B) receptor. The selective 5-HT(2C) receptor antagonist SB242,084 (0.5 mg kg(-1) i.p.), blocked the hypoactivity and penile grooming induced by Ro60-0175 (1 mg kg(-1) s.c.). The combination of SB242,084 (0.5 mg kg(-1) i.p.) and Ro60-0175 (3 - 10 mg kg(-1)) produced a completely different pattern of behaviours including wet-dog shakes, hyperactivity and back muscle contractions. These latter effects were blocked by the selective 5-HT(2A) receptor antagonist MDL100,907 (0.5 mg kg(-1) i.p.), but not the 5-HT(2B) receptor antagonist SB215,505 (3 mg kg(-1) p.o.). The indirect 5-HT releaser/reuptake inhibitor dexfenfluramine (1 - 10 mg kg(-1) i.p.) produced a mild increase in locomotor activity, penile grooming, and occasional back muscle contractions and wet-dog shakes. Pre-treatment with SB242,084 (0.5 mg kg(-1)), blocked the incidence of penile grooming, and markedly potentiated both the dexfenfluramine-induced hyperactivity, the incidence of back muscle contractions, and to a lesser extent wet-dog shakes. Some toxicity was also evident in animals treated with dexfenfluramine (10 mg kg(-1))/SB242,084 (0.5 mg kg(-1)), but not in any other treatment groups. The hyperactivity and toxicity produced by the dexfenfluramine (10 mg kg(-1))/SB242,084 (0.5 mg kg(-1)) combination was replicated in a further study, and hyperthermia was also recorded. Both hyperthermia and toxicity were blocked by MDL100,907 (0.5 mg kg(-1)) but not SB215,505 (3 mg kg(-1)). An attenuation of the hyperlocomotor response was also observed following MDL100,907. These findings suggest that 5-HT(2C) receptor activation can inhibit the expression of behaviours mediated through other 5-HT receptor subtypes. (+info)
RNA editing of the 5-HT(2C) receptor is reduced in schizophrenia.
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5-HT(2C) receptor (5HT(2C)R, serotonin-2C) RNA undergoes editing to produce several receptor variants, some with pharmacological differences. This investigation comprised two parts: the characterisation of 5-HT(2C)R RNA editing in a larger human control sample than previously examined, and a comparative study in subjects with schizophrenia. Secondary structure analysis of the putative edited region of the human 5-HT(2C)R gene predicted the existence of a double stranded (ds) RNA loop, essential for RNA editing in this receptor. RNA was then extracted from frontal cortex of five controls and five subjects with schizophrenia. RT-PCR products of the edited region were cloned and sequenced (n = 100). Reduced RNA editing, increased expression of the unedited 5-HT(2C-INI) isoform in schizophrenia (P = 0.001) and decreased expression of the 5-HT(2C-VSV) and 5-HT(2C-VNV) isoforms were detected in the schizophrenia group. In addition, two novel mRNA edited variants were identified: 5-HT(2C-MNI) and 5-HT(2C-VDI). Screening of the 5-HT(2C)R gene did not reveal any mutations likely to disrupt the dsRNA loop, suggesting that the reduced RNA editing in schizophrenia may instead be caused by altered activity of the editing enzyme(s). Since the unedited 5-HT(2C-INI) is more efficiently coupled to G proteins than the other isoforms, its increased expression in schizophrenia may lead to enhanced 5-HT(2C)R-mediated effects. The results also illustrate that potentially important receptor alterations may occur in schizophrenia which are not detectable merely in terms of receptor abundance. (+info)
Differences in conformational properties of the second intracellular loop (IL2) in 5HT(2C) receptors modified by RNA editing can account for G protein coupling efficiency.
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Adenosine-to-inosine RNA editing events that have been demonstrated for 5HT (2C) receptors resulted in alterations of the amino acid sequence at positions 156, 158 and 160 in the intracellular loop 2 (IL2) region. The edited receptor isoforms were shown to have reduced basal activity, but similar maximum responses to agonist binding. To identify the molecular mechanism of these pharmacological effects of editing we explored the conformational properties of the edited IL2 in comparison with the wild type. The results from conformational studies of the IL2 isoforms, using biased Monte Carlo simulations with an implicit solvent model based on a screened Coulomb potential, show that the compared loops differ in their preferred spatial orientations as a result of differences in the conformational space that is accessible to them by energy criteria. For the IL2 of the unedited (5HT (2C-INI) ) receptor, the preference for structures oriented towards the 7TM bundle is larger than for the 5HT (2C-VGV) edited receptor. This difference in preferred orientation can affect the association of IL2 with other intracellular loop domains involved in G protein coupling and hence the coupling efficiency. The results illustrate the high sensitivity of the system to small changes in the interaction surface presented to other intracellular loops, and/or the G protein. (+info)
Variability of 5-HT2C receptor cys23ser polymorphism among European populations and vulnerability to affective disorder.
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Substantial evidence supports a role for dysfunction of brain serotonergic (5-HT) systems in the pathogenesis of major affective disorder, both unipolar (recurrent major depression) and bipolar.(1) Modification of serotonergic neurotransmission is pivotally implicated in the mechanism of action of antidepressant drugs(2) and also in the action of mood stabilizing agents, particularly lithium carbonate.(3) Accordingly, genes that code for the multiple subtypes of serotonin receptors that have been cloned and are expressed in brain,(4) are strong candidates for a role in the genetic etiology of affective illness. We examined a structural variant of the serotonin 2C (5-HT2C) receptor gene (HTR2C) that gives rise to a cysteine to serine substitution in the N terminal extracellular domain of the receptor protein (cys23ser),(5) in 513 patients with recurrent major depression (MDD-R), 649 patients with bipolar (BP) affective disorder and 901 normal controls. The subjects were drawn from nine European countries participating in the European Collaborative Project on Affective Disorders. There was significant variation in the frequency of the HT2CR ser23 allele among the 10 population groups included in the sample (from 24.6% in Greek control subjects to 9.2% in Scots, chi(2) = 20.9, df 9, P = 0.01). Logistic regression analysis demonstrated that over and above this inter-population variability, there was a significant excess of HT2CR ser23 allele carriers in patients compared to normal controls that was demonstrable for both the MDD (chi(2) = 7.34, df 1, P = 0.006) and BP (chi(2) = 5.45, df 1, P = 0.02) patients. These findings support a possible role for genetically based structural variation in 5-HT2C receptors in the pathogenesis of major affective disorder. (+info)
Inverse agonist actions of typical and atypical antipsychotic drugs at the human 5-hydroxytryptamine(2C) receptor.
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Atypical antipsychotic drugs, which are distinguished from typical antipsychotic drugs by a lower incidence of extra-pyramidal side effects and less propensity to elevate serum prolactin levels (e.g., clozapine, olanzapine, risperidone, quetiapine, ziprasidone), have become the most widely used treatments for schizophrenia, although their precise mechanism of action remains controversial. It has been suggested that this group of atypical antipsychotic drugs is characterized by preferentially high affinities for 5-hydroxytryptamine (5-HT)2A serotonin receptors and relatively low affinities for D2-dopamine receptors. It has recently been proposed that these atypical antipsychotic drugs may also be distinguished from typical antipsychotic drugs (e.g., haloperidol, fluphenazine, chlorpromazine, and so on) by inverse agonist actions at the 5-HT2C-INI RNA edited isoform of the human 5-HT2C receptor transiently expressed in COS-7 cells. We have examined the relationship among 5-HT2C inverse agonist potency, efficacy, and atypical antipsychotic drug status in HEK-293 cells of a large number of typical and atypical antipsychotic drugs using human embryonic kidney (HEK)-293 cells stably transfected with the h5-HT2C-INI receptor. Inverse agonist actions at h5-HT2C-INI receptors were measured for both typical and atypical antipsychotic drugs. Thus, some typical antipsychotic drugs (chlorpromazine, mesoridazine, fluphenazine, and loxapine) were efficient inverse agonists, whereas several clinically effective atypical antipsychotic drugs (remoxapride, quetiapine, sulpiride, melperone, amperozide) were not. Additionally, several drugs without significant antipsychotic actions (M100907, ketanserin, mianserin, ritanserin, and amitriptyline) were potent inverse agonists at the 5-HT2C-INI isoform expressed in HEK-293 cells. Taken together, these results demonstrate that both typical and atypical antipsychotic drugs may exhibit inverse agonist effects at the 5-HT2C-INI isoform of the human 5-HT2C receptor and that no relationship exists between inverse agonist actions and atypicality. (+info)
RNA-editing of the 5-HT(2C) receptor alters agonist-receptor-effector coupling specificity.
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1. The serotonin(2C) (5-HT(2C)) receptor couples to both phospholipase C (PLC)-inositol phosphate (IP) and phospholipase A(2) (PLA(2))-arachidonic acid (AA) signalling cascades. Agonists can differentially activate these effectors (i.e. agonist-directed trafficking of receptor stimulus) perhaps due to agonist-specific receptor conformations which differentially couple to/activate transducer molecules (e.g. G proteins). Since editing of RNA transcripts of the human 5-HT(2C) receptor leads to substitution of amino acids at positions 156, 158 and 160 of the putative second intracellular loop, a region important for G protein coupling, we examined the capacity of agonists to activate both the PLC-IP and PLA(2)-AA pathways in CHO cells stably expressing two major, fully RNA-edited isoforms (5-HT(2C-VSV), 5-HT(2C-VGV)) of the h5-HT(2C) receptor. 2. 5-HT increased AA release and IP accumulation in both 5-HT(2C-VSV) and 5-HT(2C-VGV) expressing cells. As expected, the potency of 5-HT for both RNA-edited isoforms for both responses was 10 fold lower relative to that of the non-edited receptor (5-HT(2C-INI)) when receptors were expressed at similar levels. 3. Consistent with our previous report, the efficacy order of two 5-HT receptor agonists (TFMPP and bufotenin) was reversed for AA release and IP accumulation at the non-edited receptor thus demonstrating agonist trafficking of receptor stimulus. However, with the RNA-edited receptor isoforms there was no difference in the relative efficacies of TFMPP or bufotenin for AA release and IP accumulation suggesting that the capacity for 5-HT(2C) agonists to traffic receptor stimulus is lost as a result of RNA editing. 4. These results suggest an important role for the second intracellular loop in transmitting agonist-specific information to signalling molecules. (+info)
Differential regulation of the mesoaccumbens circuit by serotonin 5-hydroxytryptamine (5-HT)2A and 5-HT2C receptors.
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Serotonin [5-hydroxytryptamine (5-HT)] 5-HT(2A) and 5-HT(2C) receptors (5-HT(2A)Rs and 5-HT(2C)Rs), which innervate the dopamine mesoaccumbens pathway, may play an important role in the behavioral effects of cocaine. To test this hypothesis, the present study measured cocaine-evoked locomotor activity after bilateral microinjection of selective 5-HT(2A)R and 5-HT(2C)R antagonists into the ventral tegmental area (VTA) or the nucleus accumbens (NAc) shell. Locomotor activity was measured after intracranial microinjection of saline (0.2 microl/side), the selective 5-HT(2A)R antagonist R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine methanol (M100907) (0.1 or 0.3 microg. 0.2 microl(-1). side(-1)), or the selective 5-HT(2C)R antagonist 8-[5-(2,4-dimethoxy-5-(4-trifluoromethylphenylsulfon-amido)phenyl-5-oxopentyl)]-1 ,3,8-triazaspiro[4.5]decane-2,4-dione hydrochloride (RS 102221) (0.05-0.5 microg. 0.2 microl(-1). side(-1)) followed by an injection of saline (1 ml/kg, i.p.) or cocaine (10 mg/kg, i.p.). Microinjection of M100907 (0.1-0.3 microg/side) into the VTA or RS 102221 (0.15-0.5 microg/side) into the NAc shell attenuated cocaine-induced hyperactivity in a dose-related manner. However, hyperactivity evoked by cocaine was not altered by microinjection of RS 102221 into the VTA or M100907 into the NAc shell. No changes in basal activity were observed after microinjection of M100907 or RS 102221 into either brain region. These findings are the first to demonstrate that the behavioral effects of cocaine are generated in part by activation of 5-HT(2A)Rs in the VTA and by activation of 5-HT(2C)Rs in the NAc shell. The selective regulation of the mesoaccumbens circuit by 5-HT(2A)Rs and 5-HT(2C)Rs implicates these 5-HT receptors as important in the behavioral outcomes of systemic cocaine administration. (+info)
RNA editing of the human serotonin 5-HT2C receptor alters receptor-mediated activation of G13 protein.
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The recent completion of the human genome predicted the presence of only 30,000 genes, stressing the importance of mechanisms that increase molecular diversity at the post-transcriptional level. One such post-transcriptional event is RNA editing, which generates multiple protein isoforms from a single gene, often with profound functional consequences. The human serotonin 5-HT(2C) receptor undergoes RNA editing that creates multiple receptor isoforms. One consequence of RNA editing of cell surface receptors may be to alter the pattern of activation of heterotrimeric G-proteins and thereby shift preferred intracellular signaling pathways. We examined the ability of the nonedited 5-HT(2C) receptor isoform (INI) and two extensively edited isoforms, VSV and VGV, to interact with various G-protein alpha subunits. Two functional assays were utilized: the cell-based functional assay, Receptor Selection/Amplification Technology(TM), in which the pharmacological consequences of co-expression of 5HT(2C) receptor isoforms with G-protein alpha subunits in fibroblasts were studied, and 5HT(2C) receptor-mediated rearrangements of the actin cytoskeleton in stable cell lines. These studies revealed that the nonedited 5-HT(2C) receptor functionally couples to G(q) and G(13). In contrast, coupling to G(13) was not detected for the extensively edited 5-HT(2C) receptors. Thus, RNA editing represents a novel mechanism for regulating the pattern of activation of heterotrimeric G-proteins, molecular switches that control an enormous variety of biological processes. (+info)