title: Adenosine A1( )receptors are selectively coupled to Gα(i-3) in postmortem human brain cortex: Guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding/immunoprecipitation study.
abstract: By means of guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding assay combined with immunoprecipitation using anti-Gα subunit antibody, we recently reported 5-HT2A receptor- and M1 muscarinic acetylcholine receptor-mediated Gαq activation in rat cerebral cortical membranes (Odagaki et al., 2014). In the present study, this method has been applied to postmortem human brains, with focusing on adenosine receptor-mediated G-protein activation. In the exploratory experiments using a series of agonists and the antibodies specific to each Gα subtypes in the presence of low (10 nM) or high (50 μM) concentration of GDP, the most prominent increases in specific [(35)S]GTPγS binding in the membranes prepared from human prefrontal cortex were obtained for the combinations of adenosine (1mM)/anti-Gαi-3 in the presence of 50 μM GDP as well as 5-HT (100 μM)/anti-Gαq and carbachol (1mM)/anti-Gαq in the presence of 10nM GDP. Adenosine-induced activation of Gαi-3 emerged only when GDP concentrations were increased higher than 10 μM, and the following experiments were performed in the presence of 300 μM GDP. Adenosine increased specific [(35)S]GTPγS binding to Gαi-3 in a concentration-dependent manner to 251.4% of the basal unstimulated binding, with an EC50 of 1.77 μM. The involvement of adenosine A1 receptor was verified by the experiments using selective agonists and antagonists at adenosine A1 or A3 receptor. Among the α subunits of Gi/o class (Gαi-1, Gαi-2, Gαi-3, and Gαo.), only Gαi-3 was activated by 1mM adenosine, indicating that human brain adenosine A1 receptor is coupled preferentially, if not exclusively, to Gαi-3.
authors.affiliation: Department of Psychiatry, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Iruma-gun, Moroyama-machi, Saitama 350-0495, Japan. Electronic address: odagaki@saitama-med.ac.jp.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Odagaki
authors.affiliation: Department of Psychiatry, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Iruma-gun, Moroyama-machi, Saitama 350-0495, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Kinoshita
authors.affiliation: Department of Psychiatry, Faculty of Medicine, Saitama Medical University, 38 Morohongo, Iruma-gun, Moroyama-machi, Saitama 350-0495, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Ota
authors.affiliation: Department of Pharmacology, University of the Basque Country, UPV/EHU, E-48940 Leioa, Bizkaia, and Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Javier Meana
authors.affiliation: Department of Pharmacology, University of the Basque Country, UPV/EHU, E-48940 Leioa, Bizkaia, and Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM, Spain.
authors.forename: Luis F
authors.initials: LF
authors.lastname: Callado
authors.affiliation: Laboratory of Neuropharmacology, IUNICS/IdISPa, University of the Balearic Islands (UIB), and Redes Temáticas de Investigación Cooperativa en Salud-Red de Trastornos Adictivos (RETICS-RTA), Spain.
authors.forename: Jesús A
authors.initials: JA
authors.lastname: García-Sevilla
mesh_terms: D000241:Adenosine / Q000378:metabolism; D058907:Adenosine A1 Receptor Agonists / Q000378:metabolism / Q000494:pharmacology; D058916:Adenosine A1 Receptor Antagonists / Q000378:metabolism / Q000494:pharmacology; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001344:Autopsy; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D008875:Middle Aged; D017397:Prefrontal Cortex / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding; D043682:Receptor, Adenosine A1 / Q000187:drug effects / Q000378:metabolism*; D055815:Young Adult
chemical_list: D058907:Adenosine A1 Receptor Agonists; D058916:Adenosine A1 Receptor Antagonists; D043682:Receptor, Adenosine A1; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C498819:GNAI3 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000241:Adenosine
keywords: Adenosine A(1) receptor; G-protein; Immunoprecipitation; Postmortem human brain; [(35)S]GTPγS binding