Mechanism of endothelial cell NADPH oxidase activation by angiotensin II -: Role of the p47Phox subunit

Endothelial cells express a constitutively active phagocyte-type NADPH oxidase whose activity is augmented by agonists such as angiotensin II. We recently reported (Li, J.-M., and Shah, A. M. (2002) J. Biol. Chem. 277, 19952-19960) that in contrast to neutrophils a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as preassembled intracellular complexes. Here, we investigate the mechanism of angiotensin II-induced endothelial NADPH oxidase activation. Angiotensin II (100 nmol/liter)-induced reactive oxygen species production (as measured by dichloro-hydrofluorescein fluorescence or lucigenin chemiluminescence) was completely absent in coronary microvascular endothelial cells isolated from p47(phox) knockout mice. Transfection of p47(phox) cDNA into p47(phox-/-) cells restored the angiotensin II response, whereas transfection of antisense p47(phox) cDNA into wild-type cells depleted p47(phox) and inhibited the angiotensin II response. In unstimulated human microvascular endothelial cells, there was significant p47(phox)-p22(phox) complex formation but minimal detectable p47(phox) phosphorylation. Angiotensin II induced rapid serine phosphorylation of P47(phox) (within 1 min, peaking at similar to15 min), a 1.9 +/- 0.1-fold increase in p47(phox)-p22(phox) complex formation and a 1.6 +/- 0.2-fold increase in NADPH-dependent O-2(radical anion) production (p < 0.05). p47(phox) was redistributed to "nuclear" and membrane-enriched cell fractions. These data indicate that angiotensin II-stimulated endothelial NADPH oxidase activity is regulated through serine phosphorylation of p47(phox) and its enhanced binding to p22(phox).