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.
Department of Medical Biochemistry and Immunology, School of Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.
Stephen R
SR
Clark
Peter B
PB
Anning
Marcus J
MJ
Coffey
Andrew G
AG
Roberts
Lawrence J
LJ
Marnett
Valerie B
VB
O'Donnell
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