title: Endothelial nitric oxide synthase phosphorylation at Threonine 495 and mitochondrial reactive oxygen species formation in response to a high H₂O₂ concentration.
abstract: BACKGROUND
  Hydrogen peroxide (H₂O₂) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr(495)) in human umbilical vein endothelial cells (HUVECs) exposed to H₂O₂.
  
  
  METHODS
  HUVECs were exposed to 400 μM H₂O₂ for 30 min. Phosphorylation at Thr(495) was assessed by Western blotting and reactive oxygen species (ROS) monitored by flow cytometry. Protein kinase C (PKC) pathways were investigated by pretreatment with PKC-β inhibitor ruboxistaurin or pan-PKC inhibitor GF109203X. In addition, we investigated ROCK and ERK pathways by MEKK1/2 inhibitor U0126 and ROCK inhibitor Y27632.
  
  
  RESULTS
  H₂O₂ increased eNOS phosphorylation at Thr(495) (to 176% vs. control (100%), p < 0.001) along with increased mitochondrial ROS formation (from 19.7 to 45.3%, p < 0.01). This rise in phosphorylation could be prevented by U0126 and Y27632 in a dose-dependent manner, but did not result in lowered mitochondrial ROS formation. Conversely, addition of the antioxidant N-acetyl-L-cysteine only prevented mitochondrial ROS formation but did not prevent phosphorylation of eNOS Thr(495).
  
  
  CONCLUSIONS
  H₂O₂-mediated phosphorylation of eNOS Thr(495) is mediated by ROCK and ERK activity, but not by PKC, and is uncoupled from mitochondrial ROS signaling. Furthermore, ERK inhibition increased mitochondrial ROS formation.
authors.affiliation: The Danish National Research Foundation Center for Cardiac Arrhythmia, University of Copenhagen, Copenhagen, Denmark.
authors.forename: T J
authors.initials: TJ
authors.lastname: Guterbaum
authors.forename: T H
authors.initials: TH
authors.lastname: Braunstein
authors.forename: A
authors.initials: A
authors.lastname: Fossum
authors.forename: N-H
authors.initials: NH
authors.lastname: Holstein-Rathlou
authors.forename: C T
authors.initials: CT
authors.lastname: Torp-Pedersen
authors.forename: H
authors.initials: H
authors.lastname: Domínguez
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism; D002070:Butadienes / Q000494:pharmacology; D002478:Cells, Cultured; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D008301:Maleimides / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000502:physiology
chemical_list: D002070:Butadienes; D007211:Indoles; D008301:Maleimides; D009570:Nitriles; D017382:Reactive Oxygen Species; C113580:U 0126; D013912:Threonine; C099154:ruboxistaurin; D006861:Hydrogen Peroxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D020930:MAP Kinase Kinase Kinases; C070515:bisindolylmaleimide I; D000111:Acetylcysteine