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 dichlorohydrofluorescein 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 approximately 15 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)-* 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).
Li
Jian-Mei
JM
Department of Cardiology, Guy's, King's, and St. Thomas's School of Medicine, King's College London, Bessemer Road, London SE5 9PJ, United Kingdom.
Shah
Ajay M
AM
D000804:Angiotensin II / Q000494:pharmacology*; D057911:Angiotensin Receptor Antagonists; D000818:Animals; D003331:Coronary Vessels / Q000166:cytology; D003573:Cytochrome b Group / Q000378:metabolism; D016373:DNA, Antisense; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D026901:Membrane Transport Proteins; D051379:Mice; D008815:Mice, Inbred Strains; D008817:Mice, Mutant Strains; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
D057911:Angiotensin Receptor Antagonists; D003573:Cytochrome b Group; D016373:DNA, Antisense; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; C036888:cytochrome b558; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase