The nucleoside adenosine inhibits intracellular microvascular α2C-adrenoceptor surface trafficking

Abstract

Vascular smooth muscle (VSM) G protein-coupled α2C-adrenoceptors (α2C-ARs) regulate the vasoconstriction of small blood vessels, particularly arterioles. The VSM α2C-ARs are intracellular receptors and in response to cellular stress, translocate to the cell surface, eliciting a biological response of vasoconstriction. This translocation is mediated by the cyclic-AMP dependent EPAC-RAP1A signaling pathway, and the protein-protein interaction of α2C-ARs with the actin-binding filamin-2 protein plays a crucial role in this process.This study utilized an in-silico (3D structure-based virtual screening) approach to identify small candidate molecule(s) that could disrupt α2C-AR-filamin-2 protein-protein interactions and in-vitro studies to test the activity of candidate molecule(s) on receptor trafficking. The nucleoside adenosine was identified as a potent inhibitor of α2C-AR-filamin-2 interactions. To experimentally validate adenosine activity, primary VSM cells were explanted from the tail artery of a C57BL/6 mouse, which expresses endogenous α2C-ARs. The cytotoxic activity of adenosine (1mM-0.1µM) was assessed using MTT assay. The localization and function of α2C-ARs in the presence or absence of adenosine were assessed using immunocytochemistry and cAMP assays. Adenosine at ≤ 5µM concentration inhibited receptor trafficking from intracellular compartments to the cell surface and inhibited the function of α2C-ARs.This study identifies a hitherto unknown α2C-AR mediated vasoconstriction inhibitory effect of adenosine on the microcirculation.