Functional coupling between adenosine A1 receptors and G-proteins in rat and postmortem human brain membranes determined with conventional guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding or [35S]GTPγS/immunoprecipitation assay.

Abstract

Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A1 receptor and G-protein was assessed by means of two [35S]GTPγS binding assays, i.e., conventional filtration method and [35S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A1 receptor-mediated Gαi-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [35S]GTPγS binding determined with conventional assay derives from functional activation of Gαi/o proteins (not restricted only to Gαi-3) coupled to adenosine A1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [35S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %Emax values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A1 receptor/G-protein interaction in postmortem human brain tissue.