NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II

Recently, it has been shown that the exogenous addition of hydrogen peroxide (H₂O₂) increases endothelial nitric oxide (NO.) production. The current study is designed to determine whether endogenous levels of H₂O₂ are ever sufficient to stimulate NO. production in intact endothelial cells. NO production was detected by a NO.. specific microelectrode or by an electron spin resonance spectroscopy using Fe²⁺-(DETC)₂ as a NO^.·specific spin trap. The addition of H₂O₂ to bovine aortic endothelial cells caused a potent and dose-dependent increase in NO. release. Incubation with angiotensin II (10^-7 mol) elevated intracellular H₂O₂ levels, which were attenuated with PEG-catalase. Angiotensin II increased NO. production by 2-fold, and this was prevented by Losartan and by PEG-catalase, suggesting a critical role of AT1 receptor and H₂O₂ in this response In contrast, NO. production evoked by either hradykinin or calcium ionophore A23187 was unaffected by PEG-catalase. As in bovine aortic endothelial cells, angiotensin II doubled NO. production in aortic endothelial cells from C57BL/6 mice but had no effect on NO. production in endothelial cells from p47^phox-/- mice. In contrast, A23187 stimulated NO. production to a similar extent in endothelial cells from wild-type and p47^phox-/ mice. In summary, the present study provides direct evidence that endogenous H₂O₂ derived from the NAD(P)H oxidase, mediates endothelial NO. production in response to angiotensin II. Under disease conditions associated with elevated levels of angiotensin II, this response may represent a compensatory mechanism. Because angiotensin II also stimulates O₂-production from the NAD(P)H oxidase, the H₂O₂ stimulation of NO. may facilitate peroxynitrite formation in response to this octapeptide.