Adenosine A1-A2A Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects

Guanosine, a guanine-based purine nucleoside, has been described as a neuromodulator that exerts neuroprotective effects in animal and cellular ischemia models. However, guanosine's exact mechanism of action and molecular targets have not yet been identified. Here, we aimed to elucidate a role of adenosine receptors (ARs) in mediating guanosine effects. We investigated the neuroprotective effects of guanosine in hippocampal slices from A(2A)R-deficient mice (A(2A)R(-/-)) subjected to oxygen/glucose deprivation (OGD). Next, we assessed guanosine binding at ARs taking advantage of a fluorescent-selective A(2A)R antagonist (MRS7396) which could engage in a bioluminescence resonance energy transfer (BRET) process with NanoLuc-tagged A(2A)R. Next, we evaluated functional AR activation by determining cAMP and calcium accumulation. Finally, we assessed the impact of A(1)R and A(2A)R co-expression in guanosine-mediated impedance responses in living cells. Guanosine prevented the reduction of cellular viability and increased reactive oxygen species generation induced by OGD in hippocampal slices from wild-type, but not from A(2A)R(-/-) mice. Notably, while guanosine was not able to modify MRS7396 binding to A(2A)R-expressing cells, a partial blockade was observed in cells co-expressing A(1)R and A(2A)R. The relevance of the A(1)R and A(2A)R interaction in guanosine effects was further substantiated by means of functional assays (i.e., cAMP and calcium determinations), since guanosine only blocked A(2A)R agonist-mediated effects in doubly expressing A(1)R and A(2A)R cells. Interestingly, while guanosine did not affect A(1)R/A(2A)R heteromer formation, it reduced A(2A)R agonist-mediated cell impedance responses. Our results indicate that guanosine-induced effects may require both A(1)R and A(2A)R co-expression, thus identifying a molecular substrate that may allow fine tuning of guanosine-mediated responses.