Akt-Dependent Phosphorylation of Serine 1179 and Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase 1/2 Cooperatively Mediate Activation of the Endothelial Nitric-Oxide Synthase by Hydrogen Peroxide

Hydrogen peroxide mediates vasodilation, but the mechanisms responsible for this process remain undefined. We examined the effect of H 2 O 2 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 H 2 O 2 caused a potent dose-dependent increase in NO ⋠production. This was partially Ca 2+ -dependent because BAPTA/AM reduced NO ⋠production at low (100 μM) concentrations of H 2 O 2 . 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 H 2 O 2 -dependent NO ⋠production. ERK1/2 seemed necessary for NO ⋠production early (10 min after the addition of H 2 O 2 , and this was prevented by wortmannin but not by PD98059. c-Src family tyrosine kinase(s) was found to be upstream of H 2 O 2 -dependent Akt and eNOS serine 1179 phosphorylation and subsequent NO ⋠production. In summary, H 2 O 2 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.