Bidirectional actions of hydrogen peroxide on endothelial nitric-oxide synthase phosphorylation and function - Co-commitment and interplay of Akt and AMPK

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 H2O2 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 H2O2-stimulated eNOS phosphorylation were performed on serum-starved cells, and only the short term effect of H2O2 was examined. Here we found that the effects of H2O2 on eNOS Ser(1179) phosphorylation and function were bidirectional. With endothelial cells cultured with serum, H2O2 initially raised eNOSSer(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 H2O2 and subsequently became dormant. In contrast, AMP-activated protein kinase (AMPK) was progressively activated in H2O2-treated cells. Blocking Akt activation abolished the initial rise of eNOS Ser(1179) phosphorylation after H2O2 treatment. In long term H2O2-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 H2O2-treated cells. Long term H2O2 exposure decreased eNOS Ser(1179) phosphorylation, and this might account for the loss of eNOS function in cardiovascular diseases where chronic oxidative injury occurs.