P.1.g.032 35 Receptor-mediated activation of Gαq and Gαi-3 assessed by [ S]GTPγS binding/immunoprecipitation assay in postmortem human brain membranes ○Yuji Odagaki1), Masakazu Kinoshita1), Toshio Ota1), J. Javier Meana2), Luis F. Callado2), and Jesús A. García-Sevilla3) 1) Department of Psychiatry, Faculty of Medicine, Saitama Medical University, Japan 2) Department of Pharmacology, University of the Basque Country (UPV/EHU), Spain 3) Laboratory of Neuropharmacology, Institut Universitarid’ Investigacióen Ciències de la Salut (IUNICS), University of the Balearic Islands, Spain 【Backgrounds and Objective】 Abnormalities in signal transduction mediated through G-protein-coupled receptors are implicated in the pathogenesis of many neuropsychiatric disorders such as schizophrenia, mood disorders, and Alzheimer’s disease. Studies using postmortem human brain tissues have potential to address this issue directly. In the present 35 study, we performed [ S]GTPγS binding/immunoprecipitation assay using anti-Gαq and anti-Gαi-3 antibodies in postmortem human prefrontal cortex obtained from control subjects, who had not suffered from any neuropsychiatric disorder. The targeted receptors are muscarinic acetylcholine receptor (mAChR) M1 subtype and 5-HT2A receptor coupled with Gαq, and adenosine A1 receptor coupled with Gαi-3. 【Materials and Methods】 Dorsolateral prefrontal cortical tissues excised from 40 control subjects (26 male/14 female, age range from 16 to 80 y, postmortem delay from 4 to 64 h) were used. 35 Functional activation of Gαq by carbachol and 5-HT was determined by [ S]GTPγS binding/immunoprecipitation assay [1], with minor modifications. Adenosine A1 35 receptor-mediated [ S]GTPγS binding to Gαi-3 was assessed by the method described in [2]. Receptor-mediated [35S]GTPγS binding was expressed as percent increase over the basal unstimulated binding, and analyzed by using non-linear regression method to determine the concentration eliciting the half-maximal effect (EC50) and the maximal increase (%Emax). 【Results】 1) Comparison of the response between gray and white matter When we started to prepare membranes from brain tissue, it was noticed that some brain blocks contained substantial volume of white matter contaminated. To investigate the possible influence of contamination of white matter, brain pieces were divided into the two portions, gray matter (which consisted of as much gray matter and as little white matter as possible) and white matter (which consisted of as little gray matter and as much white matter as possible), and the responses induced by 5-HT and carbachol were compared between these two membranes. As shown in Fig. 1, gray matter-enriched membranes had much higher %Emax values than those determined in white matter-enriched membranes, for 5-HT- stimulated (Fig. 1A) as well as carbachol-stimulated (Fig. 1B) responses. Based on these results, white matter portions were excised from brain pieces as much as possible when preparing membranes in subsequent experiments. 2) 5-HT-stimulated Gαq activation is mediated through 5-HT2A receptor Concentration-response curve for 5-HT was shifted rightward in parallel in the presence of increasing concentrations of ketanserin (Fig. 2A), with a KB value of around 0.7 nM (Fig. 2B), indicative of involvement of 5-HT2A receptor. 3) Carbachol-stimulated Gαq activation is mediated through M1 mAChR Concentration-response curve for carbachol was shifted rightward in parallel in the presence of increasing concentrations of telenzepine (Fig. 3A), with a KB value of around 2 nM (Fig. 3B), indicative of involvement of M1 mAChR. 4) 5-HT2A receptor-mediated Gαq activation The concentration-dependent curve for 5-HT determined from 40 control samples

is depicted in Fig. 4. The mean EC50 was 130 nM (pEC50 = 6.88 ± 0.07), while the %Emax was 155 ± 66 % over the basal binding. 5) M1 mACh-mediated Gαq activation The concentration-dependent curve for carbacol determined from 40 control

samples is depicted in Fig. 5. The mean EC50 was 16 µM (pEC50 = 4.82 ± 0.02), while the %Emax was 470 ± 23 % over the basal binding. 6) Adenosine A1 receptor-mediated Gαi-3 activation The concentration-dependent curve for adenosine determined from 40 control

samples is depicted in Fig. 6. The mean EC50 was 820 nM (pEC50 = 6.09 ± 0.06), while the %Emax was 160 ± 9 % over the basal binding. 7) Interaction among the three measures As shown in Fig. 7, the %Emax values for M1 mAChR/Gαq coupling were plotted as a function of those for 5-HT2A/Gαq coupling. There was a tendency of positive correlation between them (p=0.0827), and this correlation became significant (p=0.0018) when one outlier (indicated by an arrow) was omitted. No significant correlation was obtained as for other comparisons (e.g., %Emax values for 5- HT2A/Gαq coupling vs. those for adenosine A1 receptor/Gαi-3 coupling, and %Emax values for M1 mAChR/Gαq coupling vs those for adenosine A1 receptor/Gαi-3 coupling; Fig. 8). 8) Effects of demographic factors There were no statistically significant differences in pEC50 values, %Emax, and slope factors for all three measures, when the subjects were divided by sex or by the presence/absence of any drug(s) detected in blood. There were no significant correlations between the values and postmortem delay, between the values and storage duration, between the values and pH, and the values and age, except for the negative correlation between the %Emax values for M1 mAChR/Gαq coupling and age (Fig. 9). 【Conclusion】 The functional activation of Gαq coupled to 5-HT2A receptor and M1 mAChR, and the functional activation of Gαi-3 coupled to adenosine A1 receptor, are detectable in postmortem human prefrontal cortex membranes. In future studies, special caution is required as to the negative correlation of %Emax values for M1 mAChR/Gαq coupling with age, likely associated with cognitive deterioration by aging process. 【References】 [1] Odagaki Y, Kinoshita M and Toyoshima R (2014) Functional activation of Gαq via serotonin2A (5-HT2A) and muscarinic acetylcholine M1 receptors assessed by guanosine-5’-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation in rat brain membranes. Eur J Pharmacol 726: 109- 115. [2] Odagaki Y, Kinoshita M, Ota T, Meana JJ, Callado LF, García-Sevilla JA (2015) Adenosine A1 receptors are selectively coupled to Gαi-3 in postmortem human brain cortex: Guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation study. Eur J Pharmacol 764: 592-598. 【Disclosure】 This poster is supported by the Saitama Medical University Internal Grant 26-B-1-12.