c-Src induces phosphorylation and translocation of p47phox - Role in superoxide generation by angiotensin II in human vascular smooth muscle cells

Objectives-The aim of this study was to determine molecular mechanisms whereby c-Src regulates angiotensin II (Ang II)-mediated NAD(P)H oxidase-derived .O-2(-) in human vascular smooth muscle cells (VSMCs). Methods and Results-VSMCs from human small arteries were studied. Ang II increased NAD(P) H oxidase-mediated generation of .O-2(-) and H2O2 (P < 0.01). PP2, c-Src inhibitor, attenuated these effects by 70% to 80%. Immunoprecipitation of p47phox, followed by immunoblotting with antiphosphoserine antibody, demonstrated a rapid increase (1.5- to 2-fold) in p47phox phosphorylation in Ang II-stimulated cells. This was associated with p47phox translocation from cytosol to membrane, as assessed by immunoblotting and immunofluorescence. PP2 abrogated these effects. Long-term Ang II stimulation (6 to 24 hours) increased NAD(P) H oxidase subunit expression. c-Src inhibition decreased abundance of gp91phox, p22phox, and p47phox. Confirmation of c-Src-dependent regulation of NAD(P) H oxidase was tested in VSMCs from c-Src(-/-) mice. Ang II-induced center dot O-2(-) generation was lower in c-Src(-/-) than c-Src(+/+) counterparts. This was associated with decreased p47phox phosphorylation, blunted Ang II-stimulated NAD(P)H oxidase activation, and failure of Ang II to increase subunit expression. Conclusions-c-Src regulates NAD(P)H oxidase-derived .O-2(-) generation acutely by stimulating p47phox phosphorylation and translocation and chronically by increasing protein content of gp91phox, p22phox, and p47phox in Ang II-stimulated cells. These novel findings identify NAD(P)H oxidase subunits, particularly p47phox, as downstream targets of c-Src.