Yuji Odagaki

Aim: Postmortem brain studies offer enormous opportunities to study molecular mechanisms associated with suicide. In the present study, conventional [ ³⁵ S]GTPγS binding assay and its version‐up method ([ ³⁵ S]GTPγS binding/immunoprecipitation assay) were applied to postmortem human hippocampal membranes prepared from suicide victims and control subjects. Methods: By using conventional [ ³⁵ S]GTPγS binding assay, functional activations of G _i/o proteins coupled with multiple GPCRs (5‐HT _1A receptor, α _2A ‐adrenoceptor, M ₂ /M ₄ mAChRs, adenosine A ₁ receptor, histamine H ₃ receptor, group II mGlu, GABA _B receptor, μ‐opioid receptor, δ‐opioid receptor, and NOP receptor) were detected by using 15 different agonists. Furthermore, 5‐HT _2A receptor‐ and M ₁ mAChR‐mediated Gα _q/11 activation and adenosine A ₁ receptor‐mediated Gα _i‐3 activation were detectable by means of [ ³⁵ S]GTPγS binding/immunoprecipitation assay. Results: No significant differences in pharmacological parameters of all concentration‐response curves investigated were found between suicide victims and control subjects. Significant correlations were obtained for the maximal percent increases between some distinct signaling pathways. Conclusion: Although only preliminary and auxiliary results were obtained as to the potential differences between suicide victims and control subjects because of the limited number of subjects as well as unmatched age and postmortem delay, adenosine A ₁ receptor‐mediated Gα _i/o activation and 5‐HT _2A receptor‐mediated Gα _q/11 activation appear worth focusing on in the future investigations. This study also indicates the possibility that some distinct signaling pathways are interrelated with each other, for example, functional activations of G _i/o proteins coupled to M ₂ /M ₄ mAChR and 5‐HT _1A receptor, NOP receptor, and GABA _B receptor, and NOP receptor and δ‐opioid receptor.

Background Many psychoactive compounds have been developed to have more beneficial clinical efficacy than conventional drugs by adding agonistic action at 5-HT_1A receptors. The aim of the present study was to evaluate several psychotropic drugs that had been reported to behave as an agonist at 5-HT_1A receptor (aripiprazole, brexpiprazole, asenapine, lurasidone, and vortioxetine) in both rat and postmortem human brain membranes. Methods The [³⁵S]GTPγS binding assay for G_i/o proteins coupled with 5-HT_1A receptors was performed in rat brain membranes and postmortem human brain membranes. Results The specific binding was stimulated by brexpiprazole in rat hippocampus, human hippocampus, and human prefrontal cortex. Aripiprazole also behaved as an agonist in the same brain regions. Interestingly, its potency was much higher in rat hippocampal membranes than in human brain membranes, indicating the possibility of species differences. Although vortioxetine was an efficacious stimulator at high concentrations, its potency was undeterminable because of a lack of saturability. In addition to 5-HT_1A receptor agonism, involvement of other components, e.g., 5-HT_1B receptor agonism, was speculated by the biphasic inhibitory effects of the selective 5-HT_1A receptor neutral antagonist. Negligible stimulatory effects were obtained as to lurasidone and asenapine. Conclusions Our previous studies have raised the concept of a psychoactive drug group with a common pharmacological mechanism of action, i.e., 5-HT_1A receptor agonism, consisting of perospirone, aripiprazole, ziprasidone, clozapine, quetiapine, nemonapride, and trazodone. The present study demonstrates the data indicating that brexpiprazole and probably vortioxetine are included in this drug group. Lurasidone and asenapine are excluded from this group.

G-protein-coupled receptors (GPCRs) have an enormous biochemical importance as they bind to diverse extracellular ligands and regulate a variety of physiological and pathological responses. G-protein activation measures the functional consequence of receptor occupancy at one of the earliest receptor-mediated events. Receptor coupling to G-proteins promotes the GDP/GTP exchange on Gα subunits. Thus, modulation of the binding of the poorly hydrolysable GTP analog [³⁵S]GTPγS to the Gα-protein subunit can be used as a functional approach to quantify GPCR interaction with agonist, antagonist or inverse agonist drugs. In order to determine receptor-mediated selective activation of the different Gα-proteins, [³⁵S]GTPγS binding assays combined with immunodetection by specific antibodies have been developed and applied to physiological and pathological brain conditions. Currently, immunoprecipitation with magnetic beads and scintillation proximity assays are the most habitual techniques for this purpose. The present review summarizes the different procedures, advantages and limitations of the [³⁵S]GTPγS binding assays combined with selective Gα-protein sequestration methods. Experience of functional coupling of several GPCRs to different Gα-proteins and recommendations for optimal performance in brain membranes are described. One of the biggest opportunities opened by these techniques is that they enable evaluation of biased agonism in the native tissue, which results in high interest in drug discovery. The available results derived from application of these functional methodologies to study GPCR dysfunctions in neuro-psychiatric disorders are also described. In conclusion, [³⁵S]GTPγS binding combined with antibody-mediated immunodetection represents an useful method to separately evaluate the functional activity of drugs acting on GPCRs over each Gα-protein subtype.

Background Chronic exposure to opiates causes the development of tolerance and physical dependence as well as persistent brain neuroplasticity. Despite a wealth of postmortem human studies for opiate addicts, little direct information regarding the functional status of serotonergic and cholinergic receptor-mediated signaling pathways in the human brain of opiate addicts is yet available. Methods Functional activation of Gα_q/11 proteins coupled to 5-HT_2A and M₁ type muscarinic acetylcholine receptor (mAChR) was assessed by using the method named [³⁵S]GTPγS binding/immunoprecipitation in frontal cortical membrane preparations from postmortem human brains obtained from opiate addicts and matched controls. Results Concentration–response curves for 5-HT and carbachol in individual subjects were analyzed according to a nonlinear regression model, which generated the values of maximum percent increase (%E_max), negative logarithm of the half-maximal effect (pEC₅₀) and slope factor. As for 5-HT_2A receptor-mediated Gα_q/11 activation, the %E_max values were reduced significantly and the pEC₅₀ values were decreased significantly in opiate addicts as compared to the control group. Regarding carbachol-induced Gα_q/11 activation, no significant difference in %E_max or pEC₅₀ values was detected between the both groups, whereas the slope factor was increased significantly in opiate addicts as compared to the control group. Conclusion Our data demonstrate that the signaling pathways mediated by Gα_q/11 proteins coupled with 5-HT_2A receptors and M₁ mAChRs in prefrontal cortex are functionally altered in opiate addicts in comparison with control subjects. These alterations may underpin some aspects of addictive behavior to opiate as well as neuropsychological consequences or comorbid mental disorders associated with opioid use.

Serotonin-2A (5-HT2A) receptors play an important role in the regulation of many brain functions that are disturbed in patients with such psychiatric diseases as mood disorders and schizophrenia. T...

Accumulating studies have implicated intracellular signaling through muscarinic acetylcholine receptors (mAChRs) in psychiatric illness. In the present study, carbamylcholine chloride (carbachol)-induced Gα_i/o and Gα_q/11 activation was identified in postmortem human prefrontal cortical membranes. The following two sample cohorts were used: subjects [1], consisting of 40 controls without neuropsychiatric disorders, and subjects [2], consisting of 20 with bipolar disorder (BP), 20 major depressive disorder (MDD), 20 schizophrenia, and 20 controls, strictly sex- and age-matched. Carbachol-stimulated [³⁵S]GTPγS binding to human brain membranes was assessed by the two methods, i.e., conventional method using filtration techniques (Gα_i/o activation coupled to M₂/M₄ mAChRs) applied to subjects [1], and [³⁵S]GTPγS binding/immuno precipitation assay (Gα_q/11 activation coupled to M₁ mAChR) applied to subjects [1] and [2]. The concentration eliciting the half-maximal effect (EC₅₀), maximum percent increase (%E_max), and slope factor were obtained from concentration–response curve of carbachol-induced Gα_i/o and Gα_q/11 activation. The pEC₅₀ values of both carbachol-induced Gα_i/o and Gα_q/11 activations in subjects [1] were significantly correlated, though its implications or underlying molecular processes are unclear. The results of M₁ mAChR-mediated Gα_q/11 activation in subjects [2] indicated no significant disorder-specific alterations. However, the distribution patterns of the pEC₅₀ values showed unequal variances among the groups. There was a significant inverse correlation between the %E_max values and the pEC₅₀ values in subjects with schizophrenia, but not in those with BP or MDD, or controls. These data support the notion that schizophrenia patients consist of biologically heterogeneous subgroups with respect to M₁ mAChR-mediated signaling pathways.