We demonstrated that in vascular smooth muscle cells (VSMC) Ang II induces phosphorylation of p47phox, which together with p67phox translocates to cytochrome b558 to assemble active NAD(P)H oxidase. Mechanisms underlying Ang II-stimulated p47phox translocation in VSMC remain unclear. We tested the hypothesis that p47phox associates with cytoskeletal proteins enabling site-directed activation of membrane-associated NAD(P)H oxidase. VSMCs derived from human resistance and coronary arteries were studied. The importance of p47 phox in Ang II-mediated generation of •O₂^– was demonstrated in p47phox antibody-electroporated VSMCs. Whereas Ang II increased •O₂^– production 2-3-fold in IgG-electroporated cells, in p47phox antibody-electroporated cells responses were significantly reduced (p<0.01). Apocynin, which blocks p47phox interaction with membrane-associated subunits, inhibited Ang II-induced NAD(P)H oxidase activation, as assessed by lucigenin (5 mol/L) chemiluminescence. Immunfluorescence confocal microscopy demonstrated a perinuclear distribution of p47phox in basal conditions. Following Ang II stimulation (10^–7 mol/l), p47phox rearranged in a linear fashion co-localizing with filamentous actin and migrating to the cell membrane. Co-immunoprecipitation studies confirmed an association between p47phox and actin in stimulated cells. Cytoskeletal disruption with cytochalasin B (10^–6 mol/L) prevented p47phox:actin interaction and significantly attenuated (p<0.01) Ang II-mediated production of H₂O₂ by >60%. Our data indicate that upon cell stimulation p47phox migrates from the perinuclear region to the cell membrane through actin-binding processes, which is critically involved in VSMC •O₂^– generation. These findings suggest that p47phox:actin interaction plays an important role in the temporal and spatial organization of p47phox enabling efficient translocation and site-directed assembly of functionally active NAD(P)H oxidase in Ang II-stimulated human VSMCs.

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