Abstract
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 A2AR-deficient mice (A2AR−/−) subjected to oxygen/glucose deprivation (OGD). Next, we assessed guanosine binding at ARs taking advantage of a fluorescent-selective A2AR antagonist (MRS7396) which could engage in a bioluminescence resonance energy transfer (BRET) process with NanoLuc-tagged A2AR. Next, we evaluated functional AR activation by determining cAMP and calcium accumulation. Finally, we assessed the impact of A1R and A2AR 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 A2AR−/− mice. Notably, while guanosine was not able to modify MRS7396 binding to A2AR-expressing cells, a partial blockade was observed in cells co-expressing A1R and A2AR. The relevance of the A1R and A2AR interaction in guanosine effects was further substantiated by means of functional assays (i.e., cAMP and calcium determinations), since guanosine only blocked A2AR agonist-mediated effects in doubly expressing A1R and A2AR cells. Interestingly, while guanosine did not affect A1R/A2AR heteromer formation, it reduced A2AR agonist-mediated cell impedance responses. Our results indicate that guanosine-induced effects may require both A1R and A2AR co-expression, thus identifying a molecular substrate that may allow fine tuning of guanosine-mediated responses.
Highlights
Guanosine is a guanine-based purine nucleoside that has been shown to exert neuroprotective and neurotrophic effects in both in vitro and in vivo studies
We have demonstrated that guanosine prevents reactive oxygen species (ROS) generation and cell death in hippocampal slices subjected to the oxygen/glucose deprivation (OGD) [11]
Production (p = 0.0279) (Figure 1A,B), losing its neuroprotective effect. These results suggested that A2A receptor (A2A R) expression was necessary for guanosine-mediated neuroprotection
Summary
Guanosine is a guanine-based purine nucleoside that has been shown to exert neuroprotective and neurotrophic effects in both in vitro and in vivo studies (for review, see [1]). It has been postulated as a good candidate for the management of several central nervous system (CNS) disorders, including neurodegenerative diseases (i.e., Parkinson’s, Alzheimer’s) or ischemia [1,2]. Brain ischemia is one of the major health disability conditions worldwide [3] It occurs after a blood supply collapse that leads to a reduced level of oxygen and glucose within the affected brain area. We have demonstrated that guanosine prevents reactive oxygen species (ROS) generation and cell death in hippocampal slices subjected to the oxygen/glucose deprivation (OGD) [11]
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