nitroarginine and cobaltous-chloride

Since it is known that hypoxia increases inducible nitric oxide synthase (iNOS) gene expression through the hypoxia responsive element, it was hypothesized that nitric oxide could be a mediator of hypoxia to inhibit Cyp1a1 promoter activity. In order to test this hypothesis, we have undertaken a study to examine the effects of hypoxia and nitric oxide on Cyp1a1 promoter activity in Hepa I cells. Mouse Cyp1a1 5' flanking DNA, 1.6kb, was cloned into pGL3 expression vector in order to construct pmCyp1a1-Luc. Hepa I cells were transfected with pmCyp1a1-Luc and were treated with 10(-9)M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of various hypoxic agents such as 10(-6)-10(-4) cobalt chloride or 10(-6)-10(-4)M picolinic acid or 10(-6)-10(-4) M desferrioxamine. The luciferase activity of the reporter gene was measured from pmCyp1a1-Luc transfected Hepa I cell lysate which contains 2 microg total protein using luciferin as a substrate. Hypoxic agents such as cobalt chloride, picolinic acid, and desferrioxamine showed inhibition of luciferase activity that was induced by 10(-9) M TCDD treatment in a dose dependent manner. Concomitant treatment of 1 mM N(G)-nitro-l-arginine with 10(-6)-10(-4) M cobalt chloride or 10(-6)-10(-4) M desferrioxamine or 10(-6)-10(-4) M picolinic acid or 10(-6)-10(-4) M sodium nitroprusside recovered luciferase activity from the TCDD induced luciferase activity that was inhibited by hypoxic agents. These data demonstrated that nitric oxide might be a mediator of iron chelating agents and hypoxic agents to inhibit dioxin induced Cyp1a1 promoter activity in Hepa I cells.

Topics: Animals; Arginine; Cells, Cultured; Cobalt; Cytochrome P-450 CYP1A1; Deferoxamine; Dioxins; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Genes, Reporter; Lipopolysaccharides; Luciferases; Mice; Nitric Oxide; Nitroarginine; Nitroprusside; Picolinic Acids; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Teratogens; Transfection

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced by hypoxia in endothelial cells (EC). To define the mechanisms by which GAPDH is regulated by hypoxia, EC were exposed to cobalt, other transition metals, carbon monoxide (CO), deferoxamine, or cycloheximide in the presence or absence of hypoxia for 24 h, and GAPDH protein and mRNA levels were measured. GAPDH was induced in cells by the transition metals cobalt, nickel, and manganese and by deferoxamine, and GAPDH mRNA induction by hypoxia was blocked by cycloheximide. GAPDH induction by hypoxia, unlike that of other hypoxia-regulated genes, was not inhibited by CO or by 4,6-dioxoheptanoic acid, an inhibitor of heme synthesis. GAPDH induction was not altered by mediators of protein phosphorylation, a calcium channel blocker, a calcium ionophore, or alterations in redox state. GAPDH induction by hypoxia or transitional metals was partially blocked by sodium nitroprusside but was not altered by the inhibitor of nitric oxide synthase N omega-nitro-L-arginine. These findings suggest that GAPDH induction by hypoxia in EC occurs via mechanisms other than those involved in other hypoxia-responsive systems.

Topics: Animals; Antidotes; Calcium; Cattle; Cell Hypoxia; Cells, Cultured; Chlorides; Cobalt; Deferoxamine; Endothelium, Vascular; Enzyme Induction; Enzyme Inhibitors; Ferrous Compounds; Gene Expression Regulation, Enzymologic; Glyceraldehyde-3-Phosphate Dehydrogenases; Heptanoates; Manganese Compounds; Nickel; Nitric Oxide; Nitroarginine; Nitroprusside; Oxidation-Reduction; RNA, Messenger; Up-Regulation; Zinc Compounds