Src is an upstream regulator of NADPH oxidase in human vascular smooth muscle cells

Ang II is a potent inducer of NADPH oxidase-derived reactive oxygen species (ROS) in vascular smooth muscle cells (VSMC). Mechanisms whereby Ang II regulates NADPH oxidase are unclear. We investigated whether c-Src plays a role in the activation of NADPH oxidase and if this non-receptor tyrosine kinase influences generation of ROS in human VSMCs. VSMC were derived from small arteries from gluteal subcutaneous biopsies of healthy volunteers. Expression of p22phox, gp91phox, p40phox, p47phox and p67phox was assessed by Western blots. Activation of NADPH oxidase and O2− were measured by lucigenin chemiluminescence. The fluoroprobe CM-H2DCFDA was used to detect H2O2. Ang II increased expression of the NADPH oxidase subunits and significantly augmented activity of NADPH oxidase (5±1 vs 31±3 nmol O2−/min/mg protein). Generation of O2− and H2O2 was increased 4-5 fold by Ang II. PP2 selective Src inhibitor, decreased expression of p47phox and p67phox and reduced activity of NADPH oxidase (9±2 nmol O2−/min/mg protein). Ang II-induced production of ROS was attenuated by PP2. To further examine the role of Src in the regulation of NADPH oxidase-derived ROS, VSMCs from c-Src-/- mice were examined. Ang II-stimulated activity of NADPH oxidase was significantly lower in c-Src-/- mice compared with wild-type counterparts (15±3 vs 24±5 nmol O2−/min/mg protein). Our data demonstrate that c-Src influences NADPH oxidase by stimulating synthesis of NADPH oxidase subunits, as well as by influencing activity of the enzyme. These findings suggest that Ang II regulates NADPH oxidase in human VSMCs via c-Src-dependent mechanisms.