Abstract 153: Co-commitment and Interplay between Akt and AMPK in the Regulation of Endothelial NO Synthase Phosphorylation by Reactive Oxygen Species
Reactive oxygen species critically modulates eNOS function via affecting eNOS phosphoryla-tion. With serum-starved cells, hydrogen peroxide (H2O2) was reported to induce eNOS Ser 1179/1177 (bovine/human) phosphorylation via PI3K-Akt pathway. H2O2 can also activate AMP-activated protein kinase (AMPK). It remains unclear whether AMPK participates in the effects of H2O2 on eNOS phosphorylation. Moreover, the interrelationship between these two pathways in the actions of H2O2 on eNOS phosphorylation is also unknown. To address these issues, we treated bovine aortic endothelial cells or bovine eNOS stably transfected HEK 293 cells with H2O2 and monitored the alterations of phospho-eNOS, Akt, and AMPK. H2O2 (1–500 μM) exhibited a bidirectional effect on eNOS Ser 1179 phosphorylation in cells grown in normal media with serum. At the early phase (30 min), H2O2 triggered eNOS Ser 1179 phosphorylation. But prolonged H2O2 incubation markedly reduced eNOS Ser 1179 phosphorylation. After 8-hr H2O2 treatment, eNOS Ser 1179 phosphorylation levels were decreased more than 3 folds (P<0.01, n=5). By monitoring the phosphorylation of Akt (Thr308/Ser473) and AMPK (Thr172), we found that the time course of Akt activation was dramatically different from that of AMPK. Akt was transiently activated during the first 30 min of H2O2 treatment and quickly became dormant. Conversely, AMPK activation was progressively enhanced along the time of H2O2 incubation. Blocking Akt activation by PI3K inhibition (LY294002, 10 μM) largely abolished the initial rise of eNOS Ser 1179 phosphorylation in H2O2-treated cells. However, in long-term H2O2-treated cells where Akt was completely deactivated, significant amounts of Ser 1179-phosphorylated eNOS remained (33±5.1% of control, P<0.01, n=8). Addition of AMPK inhibitor (compound C, 10 μM) abolished the remained amounts of Ser 1179-phosphrylated eNOS. Thus, these findings demonstrated that the effects of H2O2 on eNOS phosphorylation involved in both Akt and AMPK. It is the integrated actions of these two pathways that dictate the dynamic changes of eNOS phosphorylation in H2O2-treated cells. Coordination of these two pathways may represent a crucial mechanism by which endothelial cells motivate eNOS to cope with oxidant stress.