Evidence for the involvement of NADPH oxidase in adenosine receptor‐mediated control of coronary flow using A 1 and A 3 knockout mice

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 ; C57 BL /6), A 1 adenosine receptor ( AR ) knockout (A 1 KO ), A 3 AR KO (A 3 KO ) and A 1 and A 3 AR double KO (A 1 /A 3 DKO ) 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 EUK 134 (50 μmol/L) decreased the adenosine‐enhanced CF in the WT and all the KO s. 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 A 2A agonist), but not BAY 60‐6583 (a selective A 2B agonist), in mouse coronary artery smooth muscle cells ( CASMC s) and endothelial cells ( CAEC s). This superoxide increase was inhibited by the gp91 ds‐tat and ERK 1/2 inhibitor ( PD 98059). 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 CASMC s and CAEC s, and involves neither A 1 nor A 3 AR s, but possibly A 2A AR s in mouse.