Evidence for the involvement of NADPH oxidase in adenosine receptor-mediated control of coronary flow using A1and A3knockout mice

Abstract

The NADPH oxidase (Nox) subunits 1, 2 (gp91 phox), and 4 are the major sources for reactive oxygen species (ROS) in cardiovascular system. In conditions such as ischemia–reperfusion injury, and hypoxia, both ROS and adenosine are released suggesting a possible interaction. We hypothesized that ROS generated through Nox is involved in adenosine-induced coronary flow (CF) responses. Adenosine (10−8–10−5.5 mol/L) increased CF in isolated hearts from wild-type (WT; C57BL/6), A1 adenosine receptor (AR) knockout (A1KO), A3AR KO (A3KO) and A1 and A3AR double KO (A1/A3DKO) mice. The Nox inhibitors apocynin (10−5 mol/L) and gp91 ds-tat (10−6 mol/L) or the superoxide dismutase (SOD) and catalase-mimicking agent EUK134 (50 μmol/L) decreased the adenosine-enhanced CF in the WT and all the KOs. Additionally, adenosine increased phosphorylation of p47-phox subunit and extracellular signal-regulated kinase (ERK) 1/2 without changing protein expression of Nox isoforms in WT. Moreover, intracellular superoxide production was increased by adenosine and CGS-21680 (a selective A2A agonist), but not BAY 60-6583 (a selective A2B agonist), in mouse coronary artery smooth muscle cells (CASMCs) and endothelial cells (CAECs). This superoxide increase was inhibited by the gp91 ds-tat and ERK 1/2 inhibitor (PD98059). In conclusion, adenosine-induced increase in CF in isolated heart involves Nox2-generated superoxide, possibly through ERK 1/2 phosphorylation with subsequent p47-phox subunit phosphorylation. This adenosine/Nox/ROS interaction occurs in both CASMCs and CAECs, and involves neither A1 nor A3 ARs, but possibly A2A ARs in mouse.