title: Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelial cells via eNOS phosphorylation.
abstract: OBJECTIVE
  Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC).
  
  
  METHODS
  BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO⁻), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured.
  
  
  RESULTS
  Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser¹¹⁷⁹ via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O₂⁻) with SIN-1 treatment, and a producer of NO in the presence of DMPO.
  
  
  CONCLUSIONS
  Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases.
authors.affiliation: Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, OH, USA.
authors.forename: Amlan
authors.initials: A
authors.lastname: Das
authors.forename: Bhavani
authors.initials: B
authors.lastname: Gopalakrishnan
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Druhan
authors.forename: Tse-Yao
authors.initials: TY
authors.lastname: Wang
authors.forename: Francesco
authors.initials: F
authors.lastname: De Pascali
authors.forename: Antal
authors.initials: A
authors.lastname: Rockenbauer
authors.forename: Ira
authors.initials: I
authors.lastname: Racoma
authors.forename: Saradhadevi
authors.initials: S
authors.lastname: Varadharaj
authors.forename: Jay L
authors.initials: JL
authors.lastname: Zweier
authors.forename: Arturo J
authors.initials: AJ
authors.lastname: Cardounel
authors.forename: Frederick A
authors.initials: FA
authors.lastname: Villamena
mesh_terms: D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000201:enzymology*; D002417:Cattle; D002478:Cells, Cultured; D003497:Cyclic N-Oxides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D057809:HEK293 Cells; D006801:Humans; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000633:toxicity; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D013113:Spin Labels
chemical_list: D003497:Cyclic N-Oxides; D017382:Reactive Oxygen Species; D013113:Spin Labels; C002385:linsidomine; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D008981:Molsidomine; D052250:Nitric Oxide Synthase Type III
keywords: DMPO; cardiovascular diseases; eNOS; endothelial cells; endothelial dysfunction; nitric oxide; oxidative stress; peroxynitrite; reactive oxygen species; spin trapping