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

Hua Cai, Zongming Li, Michael E. Davis, William Kanner, David G. Harrison, Samuel C. Dudley

Research output: Contribution to journalArticlepeer-review

158 Scopus citations

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. Phosphatidyl-inositol (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 1/2 (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.

Original languageEnglish (US)
Pages (from-to)325-331
Number of pages7
JournalMolecular Pharmacology
Volume63
Issue number2
DOIs
StatePublished - Feb 1 2003

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