• LY 83583;
  • Quinone reductase;
  • Redox activation


  1. Top of page
  2. Abstract

Abstract: 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.

Abbreviations used: BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; BSA, bovine serum albumin fraction V; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GC, guanylyl cyclase; HEK, human embryonic kidney; LY 83583, 6-anilino-5,8-quinolinedione; MEM, minimum essential medium; NBT, nitro blue tetrazolium; NO, nitric oxide; NOS, nitric oxide synthase; PBS, phosphate-buffered saline; QR, NAD(P)H:quinone oxidoreductase; ROI, reactive oxygen intermediate; SOD, superoxide dismutase; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactoside.