Histochemical detection of quinone reductase activity in situ using LY 83583 reduction and oxidation.
The application of enzymatic staining techniques, using tetrazolium dyes, to aldehyde-treated brain sections has revealed the presence of NADPH-diaphorase activity attributed to nitric oxide synthase. When evaluating the specificity of the putative guanylyl cyclase inhibitor LY 83583, a robust and novel staining pattern was noted in epithelial, endothelial, and astrocytic cells when LY 83583 was included in the NADPH-diaphorase histochemical reaction. This LY 83583-dependent staining could be blocked by the NAD(P)H:quinone oxidoreductase inhibitor dicumarol. Based on its quinone structure, we hypothesized that LY 83583 was a substrate for the enzyme NAD(P)H:quinone oxidoreductase. Transfection of human embryonic kidney 293 cells with the rat liver isoform of NAD(P)H:quinone oxidoreductase resulted in robust NADPH- and LY 83583-dependent staining that was completely blocked by dicumarol and was not observed in untransfected cells. Analysis of transfected cell extracts and brain homogenates indicated that LY 83583 was a substrate for NAD(P) H:quinone oxidoreductase, with a Km similar to the well-characterized substrate menadione. Sensitivity of the nitroblue tetrazolium reduction to superoxide dismutase indicated that the reduction of LY 83583 by NAD(P)H:quinone oxidoreductase leads to superoxide generation. The localization of NAD(P)H:quinone oxidoreductase activity to astrocytic cells suggests a role for glia in combating oxidative insults to brain and in activating quinone-like drugs such as LY 83583.
Murphy
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TH
Departments of Psychiatry and Physiology, University of British Columbia, Vancouver, Canada.
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Vincent
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D000634:Aminoquinolines / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006651:Histocytochemistry; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000502:physiology; D051381:Rats; D013194:Staining and Labeling
D000634:Aminoquinolines; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D009580:Nitroblue Tetrazolium; C041715:6-anilino-5,8-quinolinedione; D019255:NADPH Oxidases; C041831:NADH dehydrogenase (quinone); D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases