title: Bidirectional actions of hydrogen peroxide on endothelial nitric-oxide synthase phosphorylation and function: co-commitment and interplay of Akt and AMPK.
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.
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Molecular and Cellular Biochemistry, The Ohio State University Medical Center, Columbus, Ohio 43210.
authors.forename: Zhuangli
authors.initials: Z
authors.lastname: Hu
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Molecular and Cellular Biochemistry, The Ohio State University Medical Center, Columbus, Ohio 43210.
authors.forename: Juan
authors.initials: J
authors.lastname: Chen
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Molecular and Cellular Biochemistry, The Ohio State University Medical Center, Columbus, Ohio 43210.
authors.forename: Qin
authors.initials: Q
authors.lastname: Wei
authors.affiliation: Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Molecular and Cellular Biochemistry, The Ohio State University Medical Center, Columbus, Ohio 43210. Electronic address: yong.xia@osumc.edu.
authors.forename: Yong
authors.initials: Y
authors.lastname: Xia
mesh_terms: D055372:AMP-Activated Protein Kinases; D000818:Animals; D001011:Aorta / Q000166:cytology; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D013997:Time Factors
chemical_list: D009097:Multienzyme Complexes; D016877:Oxidants; D012694:Serine; D006861:Hydrogen Peroxide; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases