Mechanism of Endothelial Cell NADPH Oxidase Activation by Angiotensin II

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 dichlorohydrofluorescein fluorescence or lucigenin chemiluminescence) was completely absent in coronary microvascular endothelial cells isolated from p47 knockout mice. Transfection of p47 cDNA into p47 / cells restored the angiotensin II response, whereas transfection of antisense p47 cDNA into wild-type cells depleted p47 and inhibited the angiotensin II response. In unstimulated human microvascular endothelial cells, there was significant p47-p22 complex formation but minimal detectable p47 phosphorylation. Angiotensin II induced rapid serine phosphorylation of p47 (within 1 min, peaking at 15 min), a 1.9 0.1fold increase in p47-p22 complex formation and a 1.6 0.2-fold increase in NADPH-dependent O2 . production (p < 0.05). p47 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 and its enhanced binding to p22.