title: Histochemical characterization of neuronal NADPH-diaphorase.
abstract: We examined the properties of neuronal NADPH-diaphorase in sections of rat striatum, using histochemical procedures. NADPH-diaphorase histochemistry stained discrete populations of central neurons and provided a Golgi-like image of the neurons exhibiting this activity. The NADPH-diaphorase reaction appeared to be enzyme catalyzed, since it was abolished by pre-treatment with proteases, heat, and acid or alkaline denaturation. Under anaerobic conditions, any tetrazolium salt with a redox potential more positive than NADPH could be reduced by the enzyme. NADPH-diaphorase activity was sensitive to inhibition by sulfhydryl reagents but was unaffected by metal chelators, superoxide dismutase, and catalase. Therefore, the enzyme is unlikely to be a metalloenzyme or to reduce tetrazoliums by producing superoxide anions or hydrogen peroxide. Various analogues of beta-NADPH could be used by the enzyme; however, beta-NADH, which can be used by DT-diaphorase, was ineffective. The enzyme was also resistant to dicumarol, an inhibitor of DT-diaphorase activity. Electron microscopy indicated that the NADPH-diaphorase reaction resulted in staining of various membranous organelles. We conclude that neuronal NADPH-diaphorase is a membrane-bound enzyme distinct from DT-diaphorase and other known enzymes with diaphorase activity. The histochemical characteristics presented here should now enable meaningful biochemical studies of neuronal NADPH-diaphorase to be undertaken.
authors.affiliation: Department of Psychiatry, University of British Columbia, Vancouver, Canada.
authors.forename: B T
authors.initials: BT
authors.lastname: Hope
authors.forename: S R
authors.initials: SR
authors.lastname: Vincent
mesh_terms: D000818:Animals; D006651:Histocytochemistry / Q000379:methods; D008297:Male; D008854:Microscopy, Electron; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology* / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D009247:NADH, NADPH Oxidoreductases; D009252:NADPH Dehydrogenase