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.
Footnotes
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This study was supported by National Institutes of Health grants HL39006 (to D.G.H.), HL64828 (to S.C.D.), and HL59248 (to D.G.H.), National Institutes of Health Program Project grant 58000 (to D.G.H.), a Department of Veterans Affairs merit grant (to D.G.H.), a Procter & Gamble University Exploratory Research grant (to S.C.D.), a Scientist Development Award from the American Heart Association (to S.C.D.), and a Postdoctoral Fellowship Award from the American Heart Association (to H.C.).
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This article was presented in part at the 74th Scientific Sessions of American Heart Association, Anaheim, CA, November 11–14, 2001.
- Abbreviations:
- eNOS
- endothelial nitric-oxide synthase
- MEK
- mitogen-activated protein kinase kinase
- ERK1/2
- extracellular signal-regulated kinase 1/2
- PI
- phosphatidylinositol
- CaM kinase II
- calcium/calmodulin-dependent protein kinase II
- ERK5
- extracellular signal-regulated kinase 5
- AMPK
- AMP-dependent protein kinase
- BAPTA/AM
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester
- LY294002
- 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride
- PD98059
- 2′-amino-3′-methoxyflavone
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene
- MOI
- multiplicity of infection
- PP1
- protein phosphatase 1
- Received July 16, 2002.
- Accepted October 28, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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