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

Abstract

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 ⅐ ) production and the signaling events involved. NO ⅐ release from bovine aortic endothelial cells was detected with an NO ⅐ -specific microelectrode. The addition of H 2 O 2 caused a potent dose-dependent increase in NO ⅐ production. This was partially Ca 2ϩ -dependent because BAPTA/AM reduced NO ⅐ production at low (Ͻ50 M) but not high (Ͼ100 M) 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 mitogenactivated 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 H 2 O 2 -dependent NO ⅐ production. ERK1/2 seemed necessary for NO ⅐ production early (Ͻ5 min) after H 2 O 2 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 Ͼ10 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 ⅐ production. In summary, H 2 O 2 causes endothelial NO ⅐ 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.

Mechanisms of H2O2-induced oxidative stress in endothelial cells

“…(Massaro et al 1989). In the absence of (Drummond et al 2000;Thomas et al 2002;Cai et al 2003b;Kuzkaya et al 2003;Landmesser et al 2003 This was reduced to 24.9 ± 8.1% in the presence of N G -nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS activity (n=6, p<0.001, (Pritchard et al 1995;Munzel et al 2000;Pritchard et al 2001)). These findings suggest that treatment with H 2 O 2 resulted in increased NOS activity in PAEC.…”

“…When eNOS-transfected PAEC were exposed to H 2 O 2 , 57.7 ± 6.0% of nuclei exhibited fluorescence compared with 47.9 ± 5.0% of vehicletreated, H 2 O 2 -exposed PAEC (Figure 4.3;n=24,p>0.25 Kuzkaya et al 2003;Landmesser et al 2003). Oxidation prevents NOS from transferring electrons to its physiologic substrate L-arginine (termed "uncoupling" of NOS) in static culture (Thomas et al 2002;Cai et al 2003b;Kuzkaya et al 2003;Landmesser et al 2003 (Coyle et al 2004). Therefore, we probed the effects of the combined contribution of NOS and NADPH oxidase to H 2 O 2 -induced increases in O 2 .-in PAEC using L-NAME and apocynin under shear conditions.…”

“…H 2 O 2 is initially formed from O 2 ·-produced by NADPH oxidase during inflammation, possibly through angiotensin II activation of NADPH oxidase (Pueyo et al 2000;Cai et al 2003b;Granger et al 2004). Angiotensin II can also stimulate NF-kB translocation to the nucleus resulting in VCAM-1 expression (Pueyo et al 2000) and inflammation.…”

“…Recent studies have documented various effects of H 2 O 2 in the vascular milieu (Griendling et al 2000a;Li et al 2001;Landmesser et al 2002;Cai et al 2003b), including smooth muscle cell and endothelial cell migration, and activation of inflammatory signaling mechanisms such as NF-κB (Wolin et al 2002;Cai et al 2003b;Landmesser et al 2003;Davis et al 2004). H 2 O 2 has also been found to stimulate increased eNOS expression, eNOS activity, and nitric oxide production in endothelial cells (Drummond et al 2000;Thomas et al 2002;Cai et al 2003b).…”

“…H 2 O 2 has also been found to stimulate increased eNOS expression, eNOS activity, and nitric oxide production in endothelial cells (Drummond et al 2000;Thomas et al 2002;Cai et al 2003b). Its ability to induce both nitric oxide (NO) and O 2 .-production makes H 2 O 2 a potent oxidative stimulus as it provides the components necessary for peroxynitrite formation (NO and O 2 .-interaction, (Beckman et al 1990;Pryor et al 1995).…”