Neuronal NADPH diaphorase is a nitric oxide synthase.

Abstract

NADPH diaphorase histochemistry selectively labels a number of discrete populations of neurons throughout the nervous system. This simple and robust technique has been used in a great many experimental and neuropathological studies; however, the function of this enzyme has remained a matter of speculation. We, therefore, undertook to characterize this enzyme biochemically. With biochemical and immunochemical assays, NADPH diaphorase was purified to apparent homogeneity from rat brain by affnity chromatography and anion-exchange HPLC. Western (immunoblot) transfer and immunostaining with an antibody specific for NADPH diaphorase labeled a single protein of 150 kDa. Nitric oxide synthase was recently shown to be a 150-kDa, NADPHdependent enzyme in brain. It is responsible for the calcium/calmodulin-dependent synthesis of the guanylyl cyclase activator nitric oxide from L-arginine. We have found that nitric oxide synthase activity and NADPH diaphorase copurify to homogeneity and that both activities could be immunoprecipitated with an antibody recognizing neuronal NADPH diaphorase. Furthermore, nitric oxide synthase was competitively inhibited by the NADPH diaphorase substrate, nitro blue tetrazolium. Thus, neuronal NADPH diaphorase is a nitric oxide synthase, and NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide.The NADPH diaphorase histochemical technique is based on the presence in certain neurons of an enzyme that can catalyze the NADPH-dependent conversion of a soluble tetrazolium salt to an insoluble, visible formazan (1,2). This method has proven useful for the examination of select populations of neurons in both experimental studies and in human neuropathology. In particular, NADPH diaphorase has been shown to be a selective marker for forebrain neurons containing both somatostatin and neuropeptide Y (3) and for the ascending cholinergic reticular system in the mesopontine tegmentum (4). This method has, therefore, been used to examine these neurons in Huntington disease (5), Alzheimer disease (6, 7), progressive supranuclear palsy (8), and ischemia (9, 10). NADPH diaphorase-containing neurons appear relatively resistant to anoxia and excitotoxic damage (11-13), and those in the striatum are selectively spared in Huntington disease (5).Although the NADPH diaphorase activity has been well defined histochemically (14), the function of this enzyme has remained a mystery. Previous attempts to characterize the enzyme biochemically have been hampered by lack of a specific assay (15, 16) because several proteins can exhibit NADPH-dependent diaphorase activity in brain homogenate (17). Therefore, we have used both a biochemical assay and an antibody that specifically recognizes neuronal NADPH diaphorase (18,19) to monitor purification of this enzyme from rat brain. Because NADPH diaphorase is an NADPHdependent enzyme, a purification protocol similar to that recently used for the NADPH-dependent brain enzyme, nitric oxide synthase, was att...

Permanently compromised NADPH-diaphorase activity within the osmotically activated supraoptic nucleus after in utero but not adult exposure to Aroclor 1254

“…In agreement with others, we found that hyperosmotic stimulation of early and late adulthood rats triggered an activitydependent increase in NADPH-d staining density in the SON (Soinila et al, 1999;Yun et al, 2005). The increased NADPH-d staining is likely representative of neuronal NOS (nNOS) since NADPH-d has been shown to colocalize with nNOS (Dawson et al, 1991;Hope et al, 1991) but less reliably with endothelial NOS (eNOS) (Dinerman et al, 1994) and inducible NOS (iNOS) (Tracey et al, 1993). In support of this we show that stimulated NADPH-d staining is suppressed by in vitro treatment with the general NOS inhibitor, L-NNA, with highest affinity for nNOS (K i = 25 nM) and whose binding sites in the rat CNS correspond well with anti-NOS immunohistochemical staining (Kidd et al, 1995;Reif and McCreedy, 1995).…”

“…NADPH is a required cofactor for NOS enzyme activity (Dawson et al, 1991) and NOS has NADPH-d activity (Hope et al, 1991). An enhancement of NADPH-d staining in fixed sections coincides with a respective rise in the amount of NOS protein (Kishimoto et al, 1996) and NOS gene expression (Kadowaki et al, 1994); therefore, NAPDH-d staining can be used as an indicator of NOS activity in paraformaldehyde-fixed tissue (Matsumoto et al, 1993;Young et al, 1997).…”

Evidence that nitric oxide regulates cell‐cycle progression in the developing chick neuroepithelium

“…This enzyme survives fixation, whereas other similar enzymes are inactivated (Hope et al, 1991;Dawson et al, 1991;Hiramatsu et al, 1999). Applying this method to whole embryos, we found that NOS is present in the developing NT and adjacent tissues at 8-to 12-somite stages (see Fig.…”

Nitric oxide synthase and background adaptation in Xenopus laevis

“…The distribution of NADPH-d + neurons corroborates the findings of Brü ning and Mayer (1996) in Xenopus lae6is and is also in line with recent studies in other amphibian species Muñ oz et al, 1996) and in some reptiles (Brü ning et al, 1994;Smeets et al, 1997) (reviewed in Allaerts et al, 1997). In the rat, it was shown that NOS catalytic activity is responsible for NADPH-d staining (Hope et al, 1991;Dawson et al, 1991), although some tissues like the adrenal cortex and the liver display NADPH-d activity in the absence of NOS (Dawson et al, 1991). The faint NADPH-d reactivity in the PI of PLP-fixed Xenopus (except for the reactivity in some endothelial cells) may eventually be explained by assuming some cross-reactivity with mitochondrial cytochrome P450 reductase , as was also suggested by Smeets et al (1997) for some regions in the brain of Gekko gecko.…”