Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.1 (NADPH-diaphorase)
 3,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An NADPH-dependent membrane-bound flavoprotein dehydrogenase, assayed as a catalyst of electron transfer from NADPH to cytochrome c, was extracted from membranes of rabbit peritoneal neutrophils with Triton X-100 and sodium deoxycholate in the presence of diisopropylfluorophosphate as antiprotease, and purified to electrophoretic homogeneity. The purified enzyme in detergent was able to enhance the rate of formation of the superoxide anion O2- in a cell-free system, consisting of membrane and cytosolic fractions from resting neutrophils complemented with arachidonic acid, guanosine 5'-[gamma- thio]triphosphate and Mg2+. This suggested that the NADPH dehydrogenase was a component of the rabbit neutrophil oxidase complex. The purification factor of the enzyme with respect to the membrane fraction was close to 1000 and the recovery of activity was 33%. FMN and FAD were associated with the enzyme in a molar ratio close to 1. On SDS/PAGE, the enzyme migrated with a molecular mass of 77 kDa. A similar mass was determined by filtration on a molecular sieve. The isoelectric point of this enzyme was 4.7 +/- 0.1. Its activity was maximal between pH 7.5 and pH 8.5, and depended on the ionic strength of the medium, with a maximum at an ionic strength of 0.5. Reduction of cytochrome c by NADPH obeyed Michaelis-Menten kinetics with a KM value of 15 microM for cytochrome c. When NADPH was the variable substrate, a KM value of 1.9 microM for NADPH was found, but a significant deviation from Michaelis-Menten kinetics was observed at high concentrations of NADPH. Mersalyl strongly inhibited the reductase activity when added to the enzyme prior to NADPH; preincubation of the enzyme with NADPH considerably reduced the inhibitory efficiency of mersalyl. A partially proteolyzed water-soluble, active, form of enzyme with a molecular mass of 67 kDa was prepared. The proteolyzed enzyme exhibited the same specificity, and kinetic behavior with respect to NADPH, and the same dependency on the ionic strength, as the native enzyme.
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PMID:NADPH-cytochrome c reductase from rabbit peritoneal neutrophils. Purification, properties and function in the respiratory burst. 184 86

Superoxide (.O2-) production by the NADPH oxidase of a membrane fraction derived from rabbit peritoneal neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate (PMA) was studied at 25 degrees C under different conditions, and measured by the superoxide dismutase inhibitable reduction of cytochrome c. Whereas PMA-activated rabbit neutrophils incubated in a glucose-supplemented medium exhibited a substantial rate of production of .O2-, the membranes prepared by sonication of the activated neutrophils were virtually unable to generate .O2- in the presence of NADPH. Instead, they exhibited an NADPH-dependent diaphorase activity, measured by the superoxide-dismutase-insensitive reduction of cytochrome c. Upon addition of arachidonic acid, which is known to elicit oxidase activation, the NADPH diaphorase activity of the rabbit neutrophil membranes vanished and was stoichiometrically replaced by an NADPH oxidase activity. The emerging oxidase activity was fully sensitive to iodonium biphenyl, a potent inhibitor of the respiratory burst, whereas the diaphorase activity was not affected. Addition of 0.1% Triton X-100 or an excess of arachidonic acid, acting as detergent, resulted in the reappearance of the diaphorase activity at the expense of the oxidase activity. These results indicate that the diaphorase-oxidase transition is reversible. When the rabbit neutrophil membranes were supplemented with rabbit neutrophil cytosol, guanosine 5'-[gamma-thio]triphosphate and Mg2+, in addition to arachidonic acid, not only the NADPH diaphorase activity disappeared, but the emerging NADPH oxidase activity was markedly enhanced (about 10 times compared to that of membranes treated with arachidonic acid alone). The diaphorase-oxidase transition was accompanied by a 10-fold increase in the Km for NADPH, suggesting a change of conformation propagated to the NADPH-binding site during the transition. The treatment of PMA-activated rabbit neutrophils with cross-linking reagents, like glutaraldehyde or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, prevented the loss of the PMA-elicited oxidase activity upon disruption of the cells by sonication, suggesting that the interactions between the components of the oxidase complex are stabilized by cross-linking.
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PMID:Respiratory burst of rabbit peritoneal neutrophils. Transition from an NADPH diaphorase activity to an .O2(-)-generating oxidase activity. 217 79

This study examined the distribution of NADPH-diaphorase (NADPH-d) activity in cat pancreatic ganglia and the electrophysiological effects of nitric oxide (NO) donors, NO and the effect of endogenously released NO. The majority (64%) of pancreatic ganglion neurons stained positive for NADPH-d. Large nerve trunks contained numerous non-varicose NADPH-d positive fibers. NADPH-d positive nerve fibers within individual ganglia were varicosed. L-Arginine, sodium nitroprusside and NO, applied in the vicinity of the impaled neuron, evoked a hyperpolarizing response and initiated fast excitatory postsynaptic potentials (EPSPs) in the majority of neurons tested. The hyperpolarizing response was not affected by low Ca2+ (0.1 mM), high Mg2+ (15 mM). Pretreatment with nitro-L-arginine increased the amplitude of slow EPSPs in about 50% of neurons tested. These results support the hypothesis that NO plays a role in ganglionic transmission in the cat pancreas.
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PMID:Nitric oxide is a neuromodulator in cat pancreatic ganglia: histochemical and electrophysiological study. 767 25

NADPH diaphorase is a histochemical activity which, in formaldehyde-fixed tissue, is rather specific for nitric oxide synthase. Recently, it was shown that NADPH diaphorase activity is inhibited by ethylenediaminetetraacetic acid (EDTA) in neurons but not in the choroid plexus epithelium. The present study, while confirming these results, demonstrates that the apparent sensitivity of NADPH diaphorase for EDTA reflects only the dependence of malic enzyme, which is used as the source of reduced cofactor, on Mg2+ or Mn2+ ions. Furthermore, evidence is provided that the apparent EDTA-insensitive NADPH diaphorase activity in the choroid plexus reflects the activity of alkaline phosphatase in conjunction with NADH diaphorase. Apart from these pitfalls, the use of the indirect, malic enzyme based method for NADPH diaphorase was found to cause much higher background staining compared to the direct method using NADPH, and is therefore proposed to be abandoned.
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PMID:NADPH diaphorase is not inhibited by ethylenediaminetetraacetic acid and is not specific for nitric oxide synthase in the choroid plexus of rat and mouse. 773 45

Inhibition of brainstem cholinergic neurons by noradrenergic neurons of the locus ceruleus has long been suggested as a key mechanism of behavioral state control. In particular, the commonly held view is that noradrenaline (NA) plays a permissive role in rapid eye movement (REM) sleep generation by disinhibiting brainstem cholinergic neurons. While this notion has been supported by numerous investigations, the inhibition of cholinergic neurons by NA has never been directly demonstrated. The purpose of this study was to investigate the effects of NA upon identified cholinergic neurons in the rat mesopontine tegmentum. Using whole-cell patch-clamp recordings in slices, 175 cells were studied during bath application of 50 microM NA. Cholinergic neurons were positively identified by intracellular labeling with biocytin and subsequent staining with NADPH-diaphorase, a reliable marker for brainstem cholinergic neurons (Vincent et al., 1983). Successful intracellular labeling was obtained in 96 cells. Ninety-two percent (36 of 39) of cholinergic neurons hyperpolarized in response to NA, while noncholinergic cells (n = 57) exhibited mixed responses. Application of NA in a low-Ca2+, high-Mg2+ solution elicited the same hyperpolarizing effect as in normal solution, which indicated that the effect of NA on cholinergic neurons was direct. The noradrenergic hyperpolarization was mimicked by the alpha 2-adrenoceptor agonist UK-14,304, and was blocked by the alpha 2-adrenoceptor antagonist idazoxan, which suggested an alpha 2-mediated response. Finally, voltage-clamp experiments revealed that NA activates the inwardly rectifying potassium current, IKG.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Noradrenaline hyperpolarizes identified rat mesopontine cholinergic neurons in vitro. 810 53

Intermittent hypobaric hypoxia induces functional and morphological changes of the brain in 25-day-old rats. Administration of magnesium has partial pro-convulsion effect in hypoxia not exposed rats and it practically does not influence the excitability of cortical neurones in rats exposed to intermittent hypoxia. Magnesium administration decreases the number of NADPH-diaphorase neurones in rats exposed to hypoxia in all studied areas of the hippocampus and dentate gyrus. In control rats this effect was only in CA1, CA3 and in the ventral blade of the dentate gyrus. Increased concentration of magnesium in cells of the hypoxia exposed rats after the repeated magnesium administration was found.
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PMID:Functional and morphological changes of the brain in rats exposed to intermittent hypobaric hypoxia after the repetitive magnesium administration. 1600 10

Influence of magnesium pre-treatment during repetitive hypoxia was studied in the hippocampus of rats by histochemical analysis (NADPH-diaphorase staining). NADPH-diaphorase occurs concurrently with NO-synthase that is responsible for NO synthesis. Rat pups were kept together with their mother for 8 hours a day in a hypobaric chamber at a simulated altitude of 7,000 m since the day of birth till the 17th day. The first group of animals was exposed to the repeated hypoxia; the second group under the same conditions was pre-treated by magnesium before the exposition to the hypoxia. Both groups were compared with intact control animals and intact animals treated with magnesium. The experimental and control animals were the transaortically perfused with 4% buffered neutral formaldehyde under thiopental anaesthesia at the age of 35 days. Brains were processed for NADPH-d staining. We estimated the density of NADPH-d positive neurons in CA1 and CA3 areas of the hippocampus and in the dentate gyrus. Intermittent hypoxia brings about higher numbers of NADPH-diaphorase positive neurons of CA1 and CA3 of the hippocampus and of the dorsal blade of dentate gyrus, in the comparison with either group of control animals. In the hilus and ventral blade of the dentate gyrus, on the contrary, the number of NADPH-d positive neurons was smaller. Magnesium pre-treatment during hypoxia decreased number of nitrergic neurons in all areas of the hippocampus except CA1 area, where the effect of magnesium was not significant. These results demonstrate that magnesium can probably have a neuroprotective effect.
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PMID:Effect of magnesium pre-treatment on the hippocampal NOS activity during long-lasting intermittent hypoxia. 1675 10

We used NADPH-diaphorase staining to study effects of magnesium pre-treatment during long-lasting hypoxia on the brain structure of rats. NADPH-diaphorase is an enzyme co-localized in neurons with NO-synthase that is responsible for NO synthesis. NO participates in hypoxic-ischaemic injury of the brain. Hypoxia was induced in consecutive days from the 2nd till the 11th day of postnatal life in a hypobaric chamber (for 8 hours per day). Magnesium was administered before each hypoxia exposition. At the age of 12 days, the animals were transcardially perfused with 4% buffered neutral paraformaldehyde under the deep thiopental anaesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in five hippocampal regions. In comparison to the control animals, intermittent hypoxia brought about higher density of NADPH-diaphorase positive neurons in all studied areas of the hippocampal structure: in CA1 and CA3 areas of the hippocampus and in hilus, in the dorsal and ventral blades of the dentate gyrus. Magnesium pre-treatment during hypoxia reduced number of NADPH-diaphorase positive neurons in all studied areas.
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PMID:Magnesium and posthypoxic changes of nitrergic population in rat hippocampus. 2035 38