Depletion of iNOS-derived nitric oxide by prostaglandin H synthase-2 in inflammation-activated J774.2 macrophages through lipohydroperoxidase turnover.
PGHS-2 (prostaglandin H synthase-2) is induced in mammalian cells by pro-inflammatory cytokines in tandem with iNOS [high-output ('inducible') nitric oxide synthase], and is co-localized with iNOS and nitrotyrosine in human atheroma macrophages. Herein, murine J774.2 macrophages incubated with lipopolysaccharide and interferon gamma showed induction of PGHS-2 and generated NO using iNOS that could be completely depleted by 12(S)-HPETE [12(S)-hydroperoxyeicosatetraenoic acid; 2.4 muM] or hydrogen peroxide (500 microM) (0.42+/-0.084 and 0.38+/-0.02 nmol x min(-1) x 10(6) cells(-1) for HPETE and H2O2 respectively). COS-7 cells transiently transfected with human PGHS-2 also showed HPETE- or H2O2-dependent NO decay (0.44+/-0.016 and 0.20+/-0.04 nmol x min(-1) x 10(6) cells(-1) for 2.4 microM HPETE and 500 microM H2O2 respectively). Finally, purified PGHS-2 consumed NO in the presence of HPETE or H2O2 (168 and 140 microM x min(-1) x microM enzyme(-1) for HPETE and H2O2 respectively), in a haem-dependent manner, with 20 nM enzyme consuming up to 4 microM NO. K(m) (app) values for NO and 15(S)-HPETE were 1.7+/-0.2 and 0.45+/-0.16 microM respectively. These data indicate that PGHS-2 catalytically consumes NO during peroxidase turnover and that pro-inflammatory cytokines simultaneously upregulate NO synthesis and degradation pathways in murine macrophages. Catalytic NO consumption by PGHS-2 represents a novel interaction between NO and PGHS-2 that may impact on the biological effects of NO in vascular signalling and inflammation.
Clark
Stephen R
SR
Department of Medical Biochemistry and Immunology, School of Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.
Anning
Peter B
PB
Coffey
Marcus J
MJ
Roberts
Andrew G
AG
Marnett
Lawrence J
LJ
O'Donnell
Valerie B
VB
D000818:Animals; D019556:COS Cells; D002384:Catalysis; D051546:Cyclooxygenase 2; D004566:Electrodes; D004790:Enzyme Induction; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007249:Inflammation / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D015289:Leukotrienes / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D009569:Nitric Oxide / Q000172:deficiency* / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009195:Peroxidase / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D006893:Hydroxyeicosatetraenoic Acids; D015289:Leukotrienes; D008054:Lipid Peroxides; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D009569:Nitric Oxide; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D007371:Interferon-gamma; D006861:Hydrogen Peroxide; D009195:Peroxidase; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases