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KMID : 0617320000090010186
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Journal of Pharmacetical Sceiences Ewha Womans University
2000 Volume.9 No. 1 p.186 ~ p.191
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Nitric Oxide Inhibits Dioxin Action for the Stimulation of Cyplal Promoter Activity
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Kim Ji-E.
SHEEN Yhun-Y.
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Abstract
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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 Cyplal promoter activity. In order to test this hypothesis, we have undertaken a study to examine the effects of hypoxia and nitric oxide on Cyplal promoter activity in Hep* 1 cells. Mouse Cyplal 5´ flanking DNA, 1.6kb, was cloned into pGL3 expression vector in order to construct pmCyplal-Luc. Hepa I cells were transfected with pmCyplal-Luc and were treated with 10^-9M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of various hypoxic agents such as 10^-6-10^-4M cobalt chloride or 10^-6-10^-4M picolinic acid or 10^-6-10^-4M desferrioxamine. The luciferase activity of the reporter gene was measured from pmCyplal-Loc trans-fee ted Hepa I cell lysate which contains 2 ㎍, 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^-9M TCDD treatment in a dose dependent manner. Concomitant treatment of 1 mM N^G-nitro-l-arginine with 10^-6-10^-4M cobalt chloride or 10^-6-10^-4M desferrioxamine or 10^-6-10^-4M picolinic acid or 10^-6-10^-4M 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 Cyplal promoter activity in Hepa I cells.
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KEYWORD
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