Yuji Odagaki

Trazodone and its active metabolite m-chlorophenylpiperazine as partial agonists at 5-HT1A receptors assessed by [35S]GTPγS binding

Trazodone is an effective antidepressant drug with a broad therapeutic spectrum, including anxiolytic efficacy. Although trazodone is usually referred to as a serotonin (5-HT) reuptake inhibitor, this pharmacological effect appears to be too weak to fully account for its clinical effectiveness. The present study aimed to elucidate the agonist properties of trazodone and its active metabolite, m-chlorophenylpiperazine (m-CPP), at 5-HT1A receptors by means of the guanosine-5′-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding assay. In membranes prepared from Chinese hamster ovary cells expressing human 5-HT1A receptors (CHO/h5-HT1A), trazodone behaved as an almost full agonist and m-CPP was also a highly efficacious partial agonist at 5-HT1A receptors. The intrinsic activities of both compounds were higher than those of tandospirone and buspirone, which are clinically effective anxiolytics with well-known 5-HT1A partial agonist properties. These effects were replicated in the 5-HT1A receptor-mediated [35S]GTPγS binding assay in native rat brain membranes (at least in hippocampal membranes), although the intrinsic activities of the compounds were low and differently ranked compared to those in CHO/h5-HT1A cell membranes. When considering the implications of 5-HT1A receptors in anxiety and/or depression, as well as the clinical effectiveness of azapirone anxiolytics with partial 5-HT1A receptor agonist properties such as buspirone, it is possible that the agonist effects on 5-HT1A receptors of trazodone and its active metabolite m-CPP presented in this study contribute, at least in part, to the clinical efficacy of the atypical antidepressant trazodone.

5-HT1A RECEPTOR AGONIST PROPERTIES OF ANTIPSYCHOTICS DETERMINED BY [35S]GTPγS BINDING IN RAT HIPPOCAMPAL MEMBRANES

1 5‐Hydroxytryptamine 1A (5‐HT1A) receptors have attracted increasing attention as a promising target for antipsychotic therapy. Although many atypical antipsychotic drugs, including the prototype clozapine, have been reported to be partial agonists at 5‐HT1A receptors, these results are often fragmental and derived mainly from experiments that used cultured cells. 2 In the present study, [35S]guanosine 5′‐O‐(3‐thiotriphosphate) ([35S]GTPγS) binding assay in rat hippocampal membranes was applied to a series of antipsychotic drugs, especially atypical antipsychotics. 3 Most, but not all, of atypical antipsychotic drugs and the classical antipsychotic drug nemonapride behaved as partial agonists at 5‐HT1A receptors with varied potencies and relative efficacies. The most potent compound was perospirone with a mean EC50 of 27 nmol/L, followed by aripiprazole (45 nmol/L) > ziprasidone (480 nmol/L) > nemonapride (790 nmol/L) > clozapine (3900 nmol/L) > quetiapine (26 000 nmol/L). The maximal percentage increases over the basal binding (%Emax) for these antipsychotic drugs were 30–50%, with the exception of perospirone (∼ 15%), whereas 5‐HT stimulated the binding to a mean %Emax of 105%. 4 Increasing concentrations of the selective and neutral 5‐HT1A antagonist WAY100635 shifted the concentration–response curve of nemonapride‐stimulated [35S]GTPγS binding to the right and in parallel. 5 The relative efficacy or intrinsic activity of a compound was affected differently by the differing concentrations of guanosine diphosphate (GDP) in the assay buffer, which should be taken into consideration when determining the relative efficacies of these antipsychotics as 5‐HT1A receptor agonists. 6 These results provide important information concerning the relevance of 5‐HT1A receptor partial agonist properties in the treatment for schizophrenic patients with most, if not all, of atypical antipsychotic drugs.

Pharmacological characterization of M1 muscarinic acetylcholine receptor-mediated Gq activation in rat cerebral cortical and hippocampal membranes

This study aimed to pharmacologically characterize the response derived from functional activation of Gq proteins coupled with native muscarinic acetylcholine receptors in rat cerebral cortex and hippocampus. Rat cerebral cortical and hippocampal membranes were prepared, and the effects of a range of mAChR agonists and antagonists, allosteric modulators, and muscarinic toxins were determined by an antibody-capture scintillation proximity assay combined with [35S]GTPγS binding, using the anti-Gαq antibody sc-393. Increased specific [35S]GTPγS binding, elicited by carbachol (CCh), was selectively inhibited by the muscarinic toxin MT7, and was resistant to membrane pretreatment with N-ethylmaleimide, indicating that the response derived exclusively from Gαq, selectively coupled with the M1 mAChR. In addition to CCh, many mAChR agonists, including oxotremorine, arecholine, and methacholine, stimulated binding in a concentration-dependent manner with varied potencies and efficacies. The intrinsic activities of partial M1 mAChR agonists in the present study were generally lower than previously reported in M1-expressing cells. Xanomeline and N-desmethylclozapine had negligible or minimal agonist properties. CCh-stimulated [35S]GTPγS binding to Gαq was inhibited by mAChR antagonists, including scopolamine, ipratropium, atropine, 4-DAMP, pirenzepine, and AF-DX 116, with a rank order of potency consistent with previous studies of M1-expressing cells. There was a highly significant correlation between the potencies of 13 agonists and 19 antagonists in the cerebral cortex and hippocampus. The effects of several allosteric mAChR modulators were also investigated. These data provide a comprehensive pharmacological profile of the Gq-coupled M1 mAChR subtype natively expressed at physiological levels in rat cerebral cortex and hippocampus.

Disturbances in the FGFR1-5-HT1A heteroreceptor complexes in the raphe-hippocampal 5-HT system develop in a genetic rat model of depression

The FGFR1-5-HT1A heteroreceptor complexes are involved in neuroplasticity in the rat hippocampus and in the mesencephalic raphe 5-HT nerve cells. There exists a 5-HT1A protomer enhancement of FGFR1 protomer signaling. Acute and 10 day treatment with intracerebroventricular (i.c.v.) FGF-2 and the 5-HT1A agonist 8-OH-DPAT produced enhanced antidepressant effects in the forced swim test (FST). We studied in the current work the disturbances in the FGFR1-5-HT1A heterocomplexes in a genetic rat model of depression, the Flinders sensitive line (FSL) rats of Sprague-Dawley (SD) origin, by means of neurochemical, neurophysiological and behavioral techniques. In control SD rats, the FGFR1 agonist SUN11602 and FGF2 produced a significant reduction of G protein-coupled inwardly rectifying K+ channel (GIRK) currents induced by 8-OH-DPAT in the CA1 area of the hippocampus. In FSL rats, only i.c.v. 8-OH-DPAT alone treatment produced a significant reduction in the immobility time. The combined i.c.v. treatment (FGF2 + 8-OH-DPAT) in FSL rats did not cause a significant decrease in immobility time in the FST. However, in the SD rats this combined treatment produced a significant reduction. Furthermore, in the FSL rat a significant increase in the density of FGFR1-5-HT1A proximity ligation assay (PLA) positive clusters was only found after i.c.v. 8-OH-DPAT treatment alone in the CA2 and CA3 areas. In the SD rat a significant increase in the density of specific PLA clusters was only observed in the CA2 area of the i.c.v. combined treatment (FGF2 + 8-OH-DPAT) group. No treatment led to significant changes in the PLA clusters of the dorsal raphe in the FSL rat. However, significant changes in the density of specific PLA clusters were only found in the dorsal raphe of SD rats after combined treatment and treatment with 8-OH-DPAT alone. The results indicate that in FSL rats compared with SD rats alterations may develop in the ability of 8-OH-DPAT and combined FGFR1 and 5-HT1A agonist treatment to increase the density of FGFR1-5-HT1A heteroreceptor complexes of the dorsal raphe. It is proposed that such deficits in FSL rats may possibly reflect a failure of the combined agonist treatment to uncouple the 5-HT1A autoreceptors from the GIRK channels. This may contribute to the failure of producing antidepressant-like effects in the FSL rat by combined agonist treatment as seen in the SD rat. The antidepressant-like effects seen with the 5-HT1A agonist alone treatment in FSL but not in SD rats may instead involve significant increases in the FGFR1-5-HT1A complexes of the CA2 and CA3 areas of the hippocampus.

Activation of Gq Proteins Coupled with 5-HT2 Receptors in Rat Cerebral Cortical Membranes Assessed by Antibody-Capture Scintillation Proximity Assay/[35S]GTPγS Binding

Background/Aims: Functional activation of Gq coupled with 5-HT2 receptors was investigated in rat cerebral cortical membranes. Methods: Antibody-capture scintillation proximity assay (SPA)/[35S]GTPγS binding with anti-Gαq antibody was performed. Results: The specific [35S]GTPγS binding to Gαq was increased by 5-hydroxytryptamine (5-HT) in a concentration-dependent but unsaturable manner. The increase elicited by micromolar concentrations of 5-HT was inhibited completely by ketanserin, whereas it inhibited the response by submillimolar to millimolar concentrations of 5-HT only partially. Analysis of the concentration-dependent increases by 5-HT in the absence and presence of ketanserin, methiothepin, WAY100635, and pirenzepine clearly indicates that there are two distinct components of 5-HT-stimulated [35S]GTPγS binding, one of which is a pharmacologically relevant increase elicited by lower concentrations (-30 μmol/l) of 5-HT mediated through 5-HT2 receptors and the other is pharmacologically undefined stimulation by higher concentrations of 5-HT. When 5-HT and carbachol were added simultaneously, there was apparently lack of additivity. Conclusion: It is concluded that by means of antibody-capture SPA/[35S]GTPγS binding it is possible to detect two distinct components of 5-HT-elicited activation of Gq shared by M1 muscarinic receptors, one of which is mediated through 5-HT2 receptors and the other is derived from unknown origin in rat cerebral cortical membranes.

Lack of G protein-coupled sigma receptors in rat brain membranes: receptor-mediated high-affinity GTPase activity and [35S]GTPγS binding studies

Summary.Although sigma (σ) receptors have been identified as an independent receptor family distinct from opioid and phencyclidine receptors, the physiological roles of these receptors are largely unknown. It is controversial whether there exist metabotropic σ receptors that are coupled with heterotrimeric G proteins. In the present study, the stimulatory effects of σ ligands on high-affinity GTPase activity and [35S]GTPγS binding were determined in the membranes prepared from rat cerebral cortex, hippocampus, and striatum. In either G protein activation assay, none of the σ ligands examined had stimulatory effect in any brain regions, except for unambiguous concentration-dependent increase in [35S]GTPγS binding by (+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine [(+)-3-PPP] in striatal membranes. However, the competition study clearly showed this response was mediated through dopamine D2-like receptors, but not σ receptors. It is concluded that σ receptors are not coupled to heterotrimeric G proteins, at least those of Gi/o type.

Comparative analysis of pharmacological properties of xanomeline and N-desmethylclozapine in rat brain membranes

Background: 3(3-Hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) and N-desmethylclozapine are of special interest as promising antipsychotics with better efficacy, especially for negative symptoms and/or cognitive/affective impairment. Methods: The guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding experiments were performed using (1) conventional filtration technique, (2) antibody-capture scintillation proximity assay, and (3) immunoprecipitation method, in brain membranes prepared from rat cerebral cortex, hippocampus, and striatum. Results: Xanomeline had agonistic activity at the M1 muscarinic acetylcholine receptor (mAChR) in all brain regions, as well as at the 5-HT1A receptor in the cerebral cortex and hippocampus. On the other hand, N-desmethylclozapine exhibited slight agonistic effects on the M1 mAChR, and agonistic properties at the 5-HT1A receptor in the cerebral cortex and hippocampus. This compound also behaved as an agonist at the δ-opioid receptor in the cerebral cortex and striatum. In addition, the stimulatory effects of N-desmethylclozapine on [35S]GTPγS binding to Gαi/o were partially mediated through mAChRs (most likely M4 mAChR subtype), at least in striatum. Conclusions: The agonistic effects on the mAChRs (particularly M1 subtype, and also probably M4 subtype), the 5-HT1A receptor and the δ-opioid receptor expressed in native brain tissues, some of which are common to both compounds and others specific to either, likely shape the unique beneficial effectiveness of both compounds in the treatment for schizophrenic patients. These characteristics provide us with a clue to develop newer antipsychotics, beyond the framework of dopamine D2 receptor antagonism, that are effective not only on positive symptoms but also on negative symptoms and/or cognitive/affective impairment.

Functional coupling between adenosine A 1 receptors and G-proteins in rat and postmortem human brain membranes determined with conventional guanosine-5′- O -(3-[ 35 S]thio)triphosphate ([ 35 S]GTPγS) binding or [ 35 S]GTPγS/immunoprecipitation assay

Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A1 receptor and G-protein was assessed by means of two [35S]GTPγS binding assays, i.e., conventional filtration method and [35S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A1 receptor-mediated Gαi-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [35S]GTPγS binding determined with conventional assay derives from functional activation of Gαi/o proteins (not restricted only to Gαi-3) coupled to adenosine A1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [35S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %Emax values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A1 receptor/G-protein interaction in postmortem human brain tissue.