Abstract

Hydrogen peroxide mediates vasodilation, but the mechanisms responsible for this process remain undefined. We examined the effect of H₂O₂ on nitric oxide (NO^⋅) production and the signaling events involved. NO^⋅ release from bovine aortic endothelial cells was detected with an NO^⋅-specific microelectrode. The addition of H₂O₂ caused a potent dose-dependent increase in NO^⋅ production. This was partially Ca²⁺-dependent because BAPTA/AM reduced NO^⋅ production at low (<50 μM) but not high (>100 μM) concentrations of H₂O₂. Phosphatidylinositol (PI) 3-kinase inhibition [with wortmannin or 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], infection with a dominant-negative mutant of Akt, or mitogen-activated protein kinase kinase/extracellular signal-regulated kinase ½ (MEK/ERK1/2) inhibition (with PD98059 or U0126) partially attenuated, whereas inhibition of both PI 3-kinase and MEK1/2 abolished H₂O₂-dependent NO^⋅ production. ERK1/2 seemed necessary for NO^⋅ production early (<5 min) after H₂O₂ addition, whereas PI 3-kinase/Akt was more important at later time points. Phosphorylation of endothelial nitric-oxide synthase (eNOS) at serine 1179 was observed >10 min after the addition of H₂O₂, and this was prevented by wortmannin but not by PD98059. c-Src family tyrosine kinase(s) was found to be upstream of H₂O₂-dependent Akt and eNOS serine 1179 phosphorylation and subsequent NO^⋅ production. In summary, H₂O₂ causes endothelial NO^⋅release mediated by cooperative effects between PI 3-kinase/Akt-dependent eNOS serine 1179 phosphorylation and activation of MEK/ERK1/2. This may represent an acute cellular adaptation to an increase in oxidant stress.