c-Src induces phosphorylation and translocation of p47phox: role in superoxide generation by angiotensin II in human vascular smooth muscle cells.
OBJECTIVE
The aim of this study was to determine molecular mechanisms whereby c-Src regulates angiotensin II (Ang II)-mediated NAD(P)H oxidase-derived *O2- in human vascular smooth muscle cells (VSMCs).


RESULTS
VSMCs from human small arteries were studied. Ang II increased NAD(P)H oxidase-mediated generation of *O2- 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 *O2- 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 *O2- 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.
Touyz
R M
RM
Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Montreal, Canada. touyzr@ircm.qc.ca
Yao
G
G
Schiffrin
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EL
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D051356:Cortactin; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019255:NADPH Oxidases; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D021122:Protein Subunits; D021381:Protein Transport; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000502:physiology*; D011743:Pyrimidines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D044140:Receptor, Angiotensin, Type 1 / Q000096:biosynthesis / Q000235:genetics; D013481:Superoxides / Q000378:metabolism*
C412373:AG 1879; D051356:Cortactin; D004791:Enzyme Inhibitors; D008840:Microfilament Proteins; D010750:Phosphoproteins; D021122:Protein Subunits; D011743:Pyrimidines; D017382:Reactive Oxygen Species; D044140:Receptor, Angiotensin, Type 1; D013481:Superoxides; D000804:Angiotensin II; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D016392:Proto-Oncogene Proteins pp60(c-src)