ALPHA-NADPH APPEARS TO BE PRIMARILY OXIDIZED BY THE NADPH-DIAPHORASE ACTIVITY OF NITRIC-OXIDE SYNTHASE (NOS)

Biochemical studies have shown that the NADPH-diaphorase (NADPH-d) act ivity of nitric oxide synthase (NOS) represents only a part of the tot al cellular diaphorase pool. Histochemically, NADPH-d activity can be demonstrated in cells expressing no constitutive NOS. Therefore, attem pts aimed to improve the specificity of the NADPH-d reaction are curre ntly being undertaken. In this study, the effect of replacing the natu ral and common diaphorase substrate beta-NADPH with the artificial ste reoisomer alpha-NADPH on the extent of NADPH-d staining was examined. When beta-NADPH served as the substrate, discrete populations of centr al and peripheral neurons as well as numerous non-neural cells in many organs of common laboratory rodents (mouse, rat, gerbil, hamster, gui nea pig) and marmosets were found to generate formazan. Substitution o f alpha-NADPH for beta-NADPH resulted in reduced staining intensity of nerve cells and muscle fibers. Furthermore, alpha-NADPH-d staining of macula densa cells, enterocytes and granulocytes varied according to the species examined. No reaction was observed in most other cells whi ch stained positively for beta-NADPH-d activity. Examination of adjace nt sections, incubated for the demonstration of NOS-immunoreactivity, revealed that alpha-NADPH-d activity and NOS immunostaining are strict ly colocalized in neurons, striated muscle fibers and, species-depende ntly, in macula densa cells. It can thus be concluded that, with the e xception of gut granulocytes, alpha-NADPH is primarily metabolized by the reductase activity of NOS.