Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues.

Abstract

NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferasecontaining cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons ofthe intestine, and ganglion cells ofthe adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.Nitric oxide, NO, is a prominent vascular and neuronal messenger molecule first identified as the chemical responsible for endothelium-derived relaxing factor activity (1)(2)(3). NO is also formed in macrophages and other peripheral blood cells (4, 5), though NO synthase (EC 1.14.23.-) activity of macrophages involves a distinct enzyme protein with different cofactors than the brain/endothelial enzyme (6, 7). NO synthase of brain tissue has been purified to homogeneity and shown to be a monomer of 150 kDa with an absolute requirement for calmodulin, calcium, and NADPH for enzyme activity (8). Utilizing selective antisera, we have localized the brain/endothelial enzyme by immunohistochemistry (9); besides endothelial cells, the only other localization throughout the body is in neurons and nerve processes. In the brain, NO synthase is selectively localized to discrete populations of medium-to-large aspiny neurons of the cerebral cortex and corpus striatum, basket and granule cells of the cerebellum, and other selected sites (9). In the periphery NO synthase is highly concentrated in neurons of the myenteric plexus of the small intestine, ganglion cells in the adrenal medulla, and in nerve fibers of the posterior pituitary derived from NO synthase-containing cells in the supraoptic and paraventricular hypothalamic nuclei (9).The unique pattern of NO synthase localization throughout the brain does not match precisely with any known neurotransmitters. Identifying some property that is uniquely characteristic of NO synthase-containing neurons might shed light on the biological role of NO. In the present study we show that NO synthase-containing neurons are identical with populations of neurons selectively stained for NADPH diaphorase, an oxidative enzyme localized t...

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.…”

Potentiation of excitotoxicity in HIV-1-associated Dementia and the significance of glutaminase

“…Improper regulation of these factors can lead to impaired cellular functioning, modified neurotransmitter action, amplified inflammation and neuronal damage. Various potential neurotoxins are generated and released during the inflammatory process including reactive oxygen species, tumor necrosis factor-α (TNF-α), arachidonic acid, platelet-activating factor (PAF), nitric oxide, quinolinic acid, and glutamate (20,30,(46)(47)(48)(49)(50)(51)(52).…”