The adenosine metabolite inosine is a functional agonist of the adenosine A2A receptor with a unique signaling bias

Inosine is an endogenous purine nucleoside that is produced by catabolism of adenosine. Adenosine has a short half-life (approximately 10 s) and is rapidly deaminated to inosine, a stable metabolite with a half-life of approximately 15 h. Resembling adenosine, inosine acting through adenosine receptors (ARs) exerts a wide range of anti-inflammatory and immunomodulatory effects in vivo. The immunomodulatory effects of inosine in vivo, at least in part, are mediated via the adenosine A(2A), receptor (A(2A)R), an observation that cannot be explained fully by in vitro pharmacological characterization of inosine at the A(2A)R. It is unclear whether the in vivo effects of inosine are due to inosine or a metabolite of inosine engaging the A(2A)R. Here, utilizing a combination of label free, cell-based, and membrane-based functional assays in conjunction with an equilibrium agonist-binding assay we provide evidence for inosine engagement at the A(2A)R and subsequent activation of downstream signaling events. Inosine-mediated A(2A)R activation leads to CAMP production with an EC50 of 300.7 mu M and to extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation with an EC50 of 89.38 mu M. Our data demonstrate that inosine produces ERK1/2-biased signaling whereas adenosine produces cAMP-biased signaling at the A(2A)R, highlighting pharmacological differences between these two agonists. Given the in vivo stability of inosine, our data suggest an additional, previously unrecognized, mechanism that utilizes inosine to functionally amplify and prolong A(2A)R activation in vivo. (C) 2016 Elsevier Inc. All rights reserved.