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 Ser¹¹⁷⁹ (bovine) or Ser¹¹⁷⁷ (human) residue. Reactive oxygen species such as H₂O₂ was reported to activate Akt, leading to increased eNOS Ser¹¹⁷⁹ 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 Ser¹¹⁷⁹ phosphorylation and function were bidirectional. With endothelial cells cultured with serum, H₂O₂ initially raised eNOS Ser¹¹⁷⁹ phosphorylation and activity. However, after the peak increase at 30 min, eNOS Ser¹¹⁷⁹ phosphorylation dramatically declined. Parallel to the alterations of eNOS Ser¹¹⁷⁹ 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 Ser¹¹⁷⁹ phosphorylation after H₂O₂ treatment. In long term H₂O₂-treated cells where Akt was deactivated, significant amounts of Ser¹¹⁷⁹-phosphorylated eNOS remained. AMPK inhibition eradicated the remaining eNOS Ser¹¹⁷⁹ phosphorylation. Taken together, these studies revealed that Akt and AMPK orchestrated a bidirectional action on eNOS Ser¹¹⁷⁹ phosphorylation in H₂O₂-treated cells. Long term H₂O₂ exposure decreased eNOS Ser¹¹⁷⁹ phosphorylation, and this might account for the loss of eNOS function in cardiovascular diseases where chronic oxidative injury occurs.