Ryoichi Toyoshima

Association of structural polymorphisms in the human period3 gene with delayed sleep phase syndrome

Recent progress in biological clock research has facilitated genetic analysis of circadian rhythm sleep disorders, such as delayed sleep phase syndrome (DSPS) and non‐24‐h sleep–wake syndrome (N‐24). We analyzed the human period3 (hPer3) gene, one of the human homologs of the Drosophila clock‐gene period (Per), as a possible candidate for rhythm disorder susceptibility. All of the coding exons in the hPer3 gene were screened for polymorphisms by a PCR‐based strategy using genomic DNA samples from sleep disorder patients and control subjects. We identified six sequence variations with amino acid changes, of which five were common and predicted four haplotypes of the hPer3 gene. One of the haplotypes was significantly associated with DSPS (Bonferronis corrected P = 0.037; odds ratio = 7.79; 95% CI 1.59–38.3) in our study population. Our results suggest that structural polymorphisms in the hPer3 gene may be implicated in the pathogenesis of DSPS.

A Missense Variation in Human Casein Kinase I Epsilon Gene that Induces Functional Alteration and Shows an Inverse Association with Circadian Rhythm Sleep Disorders

Recent studies have shown that functional variations in clock genes, which generate circadian rhythms through interactive positive/negative feedback loops, contribute to the development of circadian rhythm sleep disorders in humans. Another potential candidate for rhythm disorder susceptibility is casein kinase I epsilon (CKIɛ), which phosphorylates clock proteins and plays a pivotal role in the circadian clock. To determine whether variations in CKIɛ induce vulnerability to human circadian rhythm sleep disorders, such as delayed sleep phase syndrome (DSPS) and non-24-h sleep–wake syndrome (N-24), we analyzed all of the coding exons of the human CKIɛ gene. One of the variants identified encoded an amino-acid substitution S408N, eliminating one of the putative autophosphorylation sites in the carboxyl-terminal extension of CKIɛ. The N408 allele was less common in both DSPS (p=0.028) and N-24 patients (p=0.035) compared to controls. When DSPS and N-24 subjects were combined, based on an a priori prediction of a common mechanism underlying both DSPS and N-24, the inverse association between the N408 allele and rhythm disorders was highly significant (p=0.0067, odds ratio=0.42, 95% confidence interval: 0.22–0.79). In vitro kinase assay revealed that CKIɛ with the S408N variation was ∼1.8-fold more active than wild-type CKIɛ. These results indicate that the N408 allele in CKIɛ plays a protective role in the development of DSPS and N-24 through alteration of the enzyme activity.

Psychiatric problems of heart transplant candidates with left ventricular assist devices

Most heart transplant candidates are equipped with left ventricular assist devices (LVADs). LVAD therapy is associated with characteristic psychiatric and psychosocial problems. To investigate the mental states of heart transplant candidates, psychiatric diagnosis, psychological or behavioral problems, and the need for treatment were evaluated around the time of registration to the waiting list and during follow-up. Saitama Medical University Hospital has been designated a hospital for heart transplantation since October 2002. The subjects were 14 heart transplant candidates (9 male candidates and 5 female candidates, mean age 29 years) at the hospital from September 1997 to October 2005. These 14 candidates were equipped with LVADs. The waiting periods on LVAD support were from 119 days to 1028 days, and the average waiting period was 313 days. Six candidates among the 14 had more than one DSM-IV diagnosis. Seven candidates were diagnosed with adjustment disorder, which was the most frequent diagnosis. Three candidates had depressive disorder, one had psychotic disorder, and one had dissociative disorder. Three candidates had acute cognitive dysfunction (delirium) due to their general medical condition. All three had other disorders with mainly psychological elements. Nine candidates (64%) were diagnosed with disorders with mainly psychological elements. Antipsychotics were used for the candidates in psychotic states and with delirium, and there was a need for crisis intervention. Antidepressants and antianxiety drugs were used for the candidates with depressive disorder; they needed intensive observation. Four candidates (28%) needed some attention and some antianxiety drugs or hypnotics. Psychiatric interventions were not necessary in five candidates (36%).

Genetic polymorphisms of human melatonin 1b receptor gene in circadian rhythm sleep disorders and controls

Recent studies suggest that melatonin 1b (Mel1b) receptor, as well as melatonin 1a (Mel1a) receptor, is involved in the modulation of circadian rhythms in mammals. Mutational analysis was performed in the entire coding region of the human Mel1b receptor gene using genomic DNA from sleep disorder subjects. We have identified two missense mutations, G24E and L66F. However, neither is likely to be associated with sleep disorders in our study population. One of the subjects with non-24-h sleep-wake syndrome carries missense mutations in both the Mel1a and Mel1b receptor genes.

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.

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.