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

By means of guanosine-5'-O-(3-[S-35]thio)triphosphate ([S-35]GTP gamma S) binding assay combined with immunoprecipitation using anti-G alpha subunit antibody, we recently reported 5-HT2A receptor- and M-1 muscarinic acetylcholine receptor-mediated G alpha(q) 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 Ga subtypes in the presence of low (10 nM) or high (50 mu M) concentration of GDP, the most prominent increases in specific [S-35]GTP gamma S binding in the membranes prepared from human prefrontal cortex were obtained for the combinations of adenosine (1mM)/anti-Gai-3 in the presence of 50 mu M GDP as well as 5-HT (100 mu M)/anti-Gaq and carbachol (1 mM)/anti-G alpha(q) in the presence of 10 nM GDP. Adenosine-induced activation of G alpha(i-3) emerged only when GDP concentrations were increased higher than 10 mu M, and the following experiments were performed in the presence of 300 mu M GDP. Adenosine increased specific [S-35]GTP gamma S binding to G alpha(i-3) in a concentration-dependent manner to 251. 4% of the basal unstimulated binding, with an EC50 of 1. 77 mu M. The involvement of adenosine A(1) receptor was verified by the experiments using selective agonists and antagonists at adenosine A(1) or A(3) receptor. Among the a subunits of Gi/o class (G alpha(i-1), G alpha(i-2), G alpha(i-3), and G alpha(o).), only G alpha(i-3) was activated by 1 mM adenosine, indicating that human brain adenosine A(1) receptor is coupled preferentially, if not exclusively, to G alpha(i-3). (C) 2015 Elsevier B.V. All rights reserved.