Bidirectional Actions of Hydrogen Peroxide on Endothelial Nitric-oxide Synthase Phosphorylation and Function: CO-COMMITMENT AND INTERPLAY OF Akt AND AMPK

Zhuangli Hu,Juan Chen,Qin Wei,Yong Xia

doi:10.1074/jbc.m802455200

Zhuangli Hu, Juan Chen + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m802455200

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Abstract

Endothelial NO synthase (eNOS) is critically modulated by kinases via the phosphorylation of its Ser(1179) (bovine) or Ser(1177) (human) residue. Reactive oxygen species such as H(2)O(2) was reported to activate Akt, leading to increased eNOS Ser(1179) phosphorylation and activity. But reactive oxygen species are also known to attenuate eNOS function in cardiovascular diseases. Prior studies showing H(2)O(2)-stimulated eNOS phosphorylation were performed on serum-starved cells, and only the short term effect of H(2)O(2) was examined. Here we found that the effects of H(2)O(2) on eNOS Ser(1179) phosphorylation and function were bidirectional. With endothelial cells cultured with serum, H(2)O(2) initially raised eNOS Ser(1179) phosphorylation and activity. However, after the peak increase at 30 min, eNOS Ser(1179) phosphorylation dramatically declined. Parallel to the alterations of eNOS Ser(1179) phosphorylation, Akt was transiently activated by H(2)O(2) and subsequently became dormant. In contrast, AMP-activated protein kinase (AMPK) was progressively activated in H(2)O(2)-treated cells. Blocking Akt activation abolished the initial rise of eNOS Ser(1179) phosphorylation after H(2)O(2) treatment. In long term H(2)O(2)-treated cells where Akt was deactivated, significant amounts of Ser(1179)-phosphorylated eNOS remained. AMPK inhibition eradicated the remaining eNOS Ser(1179) phosphorylation. Taken together, these studies revealed that Akt and AMPK orchestrated a bidirectional action on eNOS Ser(1179) phosphorylation in H(2)O(2)-treated cells. Long term H(2)O(2) exposure decreased eNOS Ser(1179) phosphorylation, and this might account for the loss of eNOS function in cardiovascular diseases where chronic oxidative injury occurs.

Highlights

Nitric oxide generated from endothelial cells plays a key role in maintaining cardiovascular homeostasis [1,2,3]
We found that the effects of H2O2 on Endothelial NO synthase (eNOS) Ser1179 phosphorylation and function were bidirectional in cells cultured with serum
Serum starvation facilitates detecting the changes of protein phosphorylation in cells, the effect of H2O2 on eNOS phosphorylation still needs to be characterized in cells under normal serum present conditions

Summary

EXPERIMENTAL PROCEDURES

Materials—Cell culture materials were obtained from Invitrogen. Bovine aortic endothelial cells (BAECs) and growth media were purchased from Cell Systems (Kirkland, WA). 2Ј, 5Ј-ADP-Sepharose 4B was the product of Amersham Biosciences. Western Blotting—The cells were harvested and lysed on ice for 30 min in the modified radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 50 mM NaF, 1 mM Na3VO4, 5 mM sodium pyrophosphate, and protease inhibitor tablet). The cell lysates (45 ␮g of protein) were added to the reaction mixture containing 50 mM Tris-HCl, pH 7.4, 0.5 mM NADPH, 10 nM CaCl2, 10 ␮g/ml calmodulin, 10 ␮M tetrahydrobiopterin, 5 ␮M L-[14C]arginine, and 45 ␮M L-arginine. Pull-down Assay—The cells were harvested and lysed on ice for 30 min in lyses buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM Na3VO4, 5 mM sodium pyrophosphate, and protease inhibitor tablet). Differences were considered statistically significant at p Ͻ 0.05

RESULTS

Previous studies reported that Akt can negatively regulate

OA at this concentration has been shown to only inhibit

DISCUSSION