title: Peroxynitrite is not a major mediator of endothelial cell injury by activated neutrophils in vitro.
abstract: OBJECTIVE
  Human polymorphonuclear leukocytes (PMN) produce nitric oxide (NO), superoxide (O2.-) and peroxynitrite (ONOO-) upon stimulation. We investigated the role of ONOO- in PMN-induced injury to cultured bovine aortic endothelial cells (BAEC).
  
  
  METHODS
  BAEC were cocultured with phorbol 12-myristate 13-acetate (PMA)-activated human PMN (effector-to-target ratio, 10:1) and injury to BAEC was evaluated at intervals by 51Cr release assay. The levels of NO, O2.-, ONOO- and nitrotyrosine, a reaction product of ONOO-, were also measured, and the influence of NO synthase inhibitors, O2.- and hydroxyl radical scavengers and other effectors was examined.
  
  
  RESULTS
  In BAEC cocultured with PMA-activated PMN, 51Cr release was significantly increased [14.6 +/- 2.2% at 2 h (p < 0.05) and 42.6 +/- 2.7% at 4 h (p < 0.01); control (nonactivated PMN), < 4%]. Superoxide dismutase (100 U/ml) reduced 51Cr release to 4.6 +/- 2.2% at 2 h (p < 0.05). N-Iminoethyl-L-ornithine (L-NIO, 0.1 mM) potentiated 51Cr release (30.6 +/- 3.8% at 2 h, p < 0.01), and the potentiation was eliminated by anti-CD18 monoclonal antibody. The 51Cr release was completely prevented by dimethyl sulfoxide or by deferoxamine. Treatment of PMN with L-NIO inhibited NO generation and increased O2.- production. The nitrotyrosine level did not increase in BAEC cocultured with PMA-activated PMN.
  
  
  CONCLUSIONS
  NO-derived ONOO- is not a major cytotoxic mediator in BAEC injury by activated PMN. NO may have a cytoprotective effect by inhibiting PMN adherence to endothelial cells.
authors.affiliation: Department of Physiology, Tokai University School of Medicine, Kanagawa, Japan.
authors.forename: Z
authors.initials: Z
authors.lastname: Su
authors.forename: H
authors.initials: H
authors.lastname: Ishida
authors.forename: N
authors.initials: N
authors.lastname: Fukuyama
authors.forename: R
authors.initials: R
authors.lastname: Todorov
authors.forename: C
authors.initials: C
authors.lastname: Genka
authors.forename: H
authors.initials: H
authors.lastname: Nakazawa
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D018821:CD18 Antigens / Q000276:immunology; D002417:Cattle; D002478:Cells, Cultured; D018920:Coculture Techniques; D003676:Deferoxamine / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D018375:Neutrophil Activation; D009504:Neutrophils / Q000378:metabolism*; D009566:Nitrates / Q000032:analysis / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009573:Nitrites / Q000032:analysis; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D018821:CD18 Antigens; D009566:Nitrates; D009573:Nitrites; C110881:S-nitro-N-acetylpenicillamine; C039642:peroxynitric acid; C002744:3-nitrotyrosine; C065027:N(G)-iminoethylornithine; D014443:Tyrosine; D009952:Ornithine; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; D010396:Penicillamine; D003676:Deferoxamine; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide