Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Nitric oxide is an important intercellular messenger in the central nervous system. NADPH-diaphorase, reported to be identical to nitric oxide synthase, is present in specific groups of cells in several neural tissues, including the retina. We determined NADPH-diaphorase activity in homogenates of the chick embryo retina. The enzyme activity was measured spectrophotometrically at 585 nm after incubating retinal total homogenates (100-150 micrograms protein) with 1 mM NADPH and 0.5 mM nitroblue tetrazolium in 50 mM Tris buffer, pH 8.1, at 37 degrees C. NADPH-diaphorase was detected in 14-day old retinas and 53-65% of the enzyme activity was inhibited by 3 mM NG-nitro-L-arginine (NARG), the arginine analog. One mM L-N5-(1-iminoethyl)ornithine (NIO) was the most potent inhibitor (63% inhibition) while 3 mM NG-nitro-L-arginine methyl ester (NAME) (33% inhibition) and 1 mM NG-monomethyl-L-arginine acetate (NMMA) (14% inhibition) were less effective. Enzyme activity was increased by 48% by 2 mM calcium chloride, an effect reversed by 1 mM EGTA or EDTA. Basal enzyme levels were also partially inhibited by the chelators, indicating the presence of calcium-dependent and -independent isoforms of nitric oxide synthase in the retina. The results show that the NADPH-diaphorase assay is simple and sensitive and that the different isoforms of nitric oxide synthase expressed in chick retinal cells during development can be demonstrated.
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PMID:Biochemical characterization of NADPH-diaphorase in the chick embryo retina: stimulation by calcium ions and inhibition by arginine analogs. 758 Oct 50

The effects of tumor necrosis factor-alpha (TNF-alpha) on the production of the vasoactive substances nitric oxide (NO) and endothelin-1 (ET-1) were investigated in cerebrovascular cells in culture. Bovine cerebral endothelial cells (BCEC) stained positively for NADPH-diaphorase/NO synthase activity and spontaneously produced nitrite, a stable NO oxidation product, which accumulated in the culture medium in a linear way for 48 h. Low concentrations of TNF-alpha (0.5-2 ng/ml) significantly enhanced nitrite production after a 24-h incubation. Higher concentrations or longer exposure times resulted in a cytotoxic effect that altered cell morphology, released lactate dehydrogenase (LDH) to the culture medium, and reduced the protein content. Dexamethasone, but not the NO synthase inhibitor N-iminoethyl-L-ornithine (L-NIO), prevented the cytotoxic effect of TNF-alpha in BCEC. TNF-alpha also significantly enhanced nitrite production in bovine cerebral smooth muscle cells (BCSMC). The enhancement was detected at all times between 8 and 72 h and at all concentrations tested (2-100 ng/ml). Signs of cytotoxicity were not observed in BCSMC after incubation with TNF-alpha. ET-1 was constitutively secreted by BCEC. The production of ET-1 was stimulated by thrombin. TNF-alpha enhanced the release of ET-1 in BCEC, and this enhancement was not modified by the simultaneous addition of interferon-gamma (IFN-gamma). BCSMC did not produce ET-1, either spontaneously or in the presence of TNF-alpha, IFN-gamma, or of both together.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of TNF-alpha on the production of vasoactive substances by cerebral endothelial and smooth muscle cells in culture. 759 52

Nitric oxide plays an important role as an intercellular messenger in the CNS. In the present work we measured NADPH-diaphorase activity, which is considered to be a marker of cells producing nitric oxide, in homogenates of the developing chick retina. The enzyme activity can be detected beginning in 8-day-old embryonic retinas with no further quantitative variations throughout development. Arginine analogues inhibit approximately 65% of the activity in embryonic retinas and 50% in posthatched retinas. The enzyme is stimulated 50% by 2 mM calcium chloride in retinas from 8 to 14 embryonic days, but this effect decreases to 20% in 17-day embryonic retinas and practically disappears in posthatched animals. The stimulation by calcium is completely blocked by arginine analogues. The decrease in enzyme activity at posthatched retinas is not due to stimulation by endogenous calcium or the presence of insufficient amounts of calmodulin, because addition of EGTA or calmodulin, respectively, did not restore the stimulation to levels observed at embryonic stages. Inhibition of NADPH-diaphorase activity by NG-nitro-L-arginine or L-NG-(iminoethyl) ornithine is concentration dependent with IC50 values of approximately 1 mM at all stages studied. However, in the presence of calcium, the inhibition by both analogues is shifted to the left and is apparently biphasic at all developmental stages, including in posthatched animals, with IC50 values in the low micromolar range. NADPH-diaphorase was also detected by histochemistry in specific groups of cells in the early embryonic retina and in subsets of amacrine and ganglion cells, as well as in photoreceptors, in more developed retinas. The results indicate that different isoforms of nitric oxide synthase are present in the chick retina and that a calcium-dependent isoform is predominant in early periods of development.
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PMID:Development of NADPH-diaphorase in the avian retina: regulation by calcium ions and relation to nitric oxide synthase. 875 13

Intracellular concentrations of L-citrulline (Cit) and its metabolites are related to nitric oxide synthase (NOS) activity, an enzyme producing the intercellular messenger NO in animal tissues including the nervous system. A capillary electrophoresis system using laser-induced fluorescence detection is described, and methods are developed to monitor the levels of L-arginine (Arg), Cit, and related molecules in identified neurons of the marine slugs, Pleurobranchaea californica and Aplysia californica. The limits of detection for Arg, Cit, L-arginino-succinate, L-ornithine, and L-arginine phosphate range from 50 amol to 17 fmol (5 nM to 17 microM in the neurons under study); these detection limits are significantly lower than actual intracellular levels of the metabolites, allowing the direct assay of single cells. The levels of NOS metabolites in individual neurons varied form 6 (Arg) and 4 mM (Cit) in putative NOS-containing neurons down to < 1 microM (undetectable) levels in many putative NOS-negative cells. The Arg/Cit ratio is independent of cell volume, correlates with NADPH-diaphorase staining, and appears to be a characteristic parameter for the presence of NOS activity in identified neurons.
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PMID:Capillary electrophoresis analysis of nitric oxide synthase related metabolites in single identified neurons. 962 98

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [3H]arginine to [3H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor NG-methyl-L-arginine (L-NMA), but not the arginase inhibitors L-valine and L-ornithine. NOS activity was predominantly cytosolic, and was characterised by a Km for arginine of 19.05 microM and a Vmax of 2.96 pmol/min per microgram protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.
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PMID:Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida. 1090 61

Less nitric oxide (NO)-dependent vasodilation and excess formation of reactive oxygen species could explain poor placenta perfusion in preeclampsia, but the pathways involved are unknown. We tested the hypothesis that reduced NO activity and increased oxidative stress in preeclamptic placenta is related to a low bioavailability of l-arginine. Placental endothelial NO synthase (ecNOS) expression (by immunoperoxidase) and activity (by diaphorase and [(3)H]L-citrulline formation) were comparable in normotensive pregnancy and in preeclampsia, whereas nitrotyrosine staining, a marker of peroxynitrite, was stronger in preeclamptic villi, confirming previously reported data. Oxidative tissue damage was documented in preeclamptic villi by strong 4-hydroxynonenal-lysine staining (by immunoperoxidase), which closely colocalized with nitrotyrosine. Concentration of the NO precursor l-arginine (by HPLC) in umbilical blood and in villous tissue was lower in preeclampsia than in normotensive pregnancy. This was not caused by a defective l-arginine transport, because gene expression of the CAT-1, 4F2hc, and LAT-1 cationic amino acid transporters (by real-time reverse-transcription polymerase chain reaction [RT-PCR]) was normal. Instead, gene expression (by real-time RT-PCR) and protein tissue content (by immunoperoxidase and Western blot) of arginase II-the enzyme that degrades arginine to ornithine-were higher in preeclamptic villi than in normotensive pregnancy. These results provide a biochemical explanation for defective NO activity and increased oxidative stress in preeclamptic placenta. In normal placenta, adequate concentration of l-arginine orients ecNOS toward NO. In preeclampsia, a lower than normal l-arginine concentration caused by arginase II overexpression redirects ecNOS toward peroxynitrite.
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PMID:L-arginine depletion in preeclampsia orients nitric oxide synthase toward oxidant species. 1521 85

We tested the hypothesis that the decrease in arterial pressure induced by adrenomedullin (ADM) in the hypothalamic paraventricular nucleus (PVN) is mediated by nitric oxide (NO) and/or GABA. Unilateral microinjections of ADM into the PVN of anesthetized rats caused a significant decrease in mean arterial pressure (MAP). The ADM-induced decrease in MAP was significantly attenuated by pretreatment with N(psi)-nitro-L-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), 7-nitroindazole sodium salt (7-NiNa, a selective neuronal NOS inhibitor), N5-(1-Iminoethyl)-L-ornithine (L-NIO, a selective endothelial NOS inhibitor) or bicuculline methiodide, but pretreatment with S-methylisothiourea (SMIT, a selective inducible NOS inhibitor) had no effect on this ADM-induced effect. In addition, coronal sections of rat brains were processed for combined NADPH-diaphorase (a marker of neuronal NOS-containing neurons) histochemistry and in situ hybridization for the receptor-activity-modifying protein 2 (a specific ADM receptor component). Double-labeled neurons were found in both parvocellular and magnocellular subdivisions of the PVN, confirming that NO-producing neurons in the PVN are capable of mediating ADM's effects. Thus, our data provide evidence that the ADM-induced decrease in MAP in the PVN is mediated by NO from neuronal and endothelial NOS, and by GABA.
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PMID:Decrease in arterial pressure induced by adrenomedullin in the hypothalamic paraventricular nucleus is mediated by nitric oxide and GABA. 1509 93

Voluntary exercise such as running induces a dramatic increase in adult stem cell proliferation within the dentate gyrus, and endothelial nitric oxide synthase helps regulate cell proliferation. The role of endothelial nitric oxide synthase in exercise-induced cell proliferation in the brain, however, has not been examined. In the present study, exercise for 1 week increased endothelial nitric oxide synthase and nicotinamide adenine dinucleotide phosphate-diaphorase immunoreactivity in the microvessels of the dentate gyrus. In addition, blocking endothelial nitric oxide synthase activity (via a daily injection of 20 mg/kg L-nitroimidazole ornithine) during exercise reduced the number of cells within the dentate gyrus that were immunoreactive for Ki-67 protein and doublecortin. This study provides the first evidence that endothelial nitric oxide synthase upregulation may modulate exercise-induced granule cell proliferation within the dentate gyrus.
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PMID:L-nitroimidazole ornithine limits exercise-induced increases in cell proliferation in the hippocampus of adult mice. 1683 39

In this study, the role of nitric oxide (NO) in regulation of the pulmocutaneous vasculature of the toad, Bufo marinus was investigated. In vitro myography demonstrated the presence of a neural NO signaling mechanism in both arteries. Vasodilation induced by nicotine was inhibited by the soluble guanylyl cyclase (GC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, and the NO synthase (NOS) inhibitor, N(omega)-nitro-l-arginine (l-NNA). Removal of the endothelium had no significant effect on the vasodilation. Furthermore, pretreatment with N(5)-(1-imino-3-butenyl)-l-ornithine (vinyl-l-NIO), a more specific inhibitor of neural NOS, caused a significant decrease in the nicotine-induced dilation. In the pulmonary artery only, a combination of l-NNA and the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP((8-37)), completely blocked the nicotine-induced dilation. In both arteries, the vasodilation was also significantly decreased by glibenclamide, an ATP-sensitive K(+) (K(+)(ATP)) channel inhibitor. Levcromakalim, a K(+)(ATP) channel opener, caused a dilation that was blocked by glibenclamide in both arteries. In the pulmonary artery, NO donor-mediated dilation was significantly decreased by pretreatment with glibenclamide. The physiological data were supported by NADPH-diaphorase histochemistry and immunohistochemistry, which demonstrated NOS in perivascular nerve fibers but not the endothelium of the arteries. These results indicate that the pulmonary and cutaneous arteries of B. marinus are regulated by NO from nitrergic nerves rather than NO released from the endothelium. The nitrergic vasodilation in the arteries appears to be caused, in part, via activation of K(+)(ATP) channels. Thus, NO could play an important role in determining pulmocutaneous blood flow and the magnitude of cardiac shunting.
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PMID:Neurally-derived nitric oxide regulates vascular tone in pulmonary and cutaneous arteries of the toad, Bufo marinus. 1875 69

The nitric oxide synthase (NOS)/nitric oxide (NO) system integrates cellular biochemical machinery and energetics. In heart microenvironment, dynamic NO behaviour depends upon the presence of superoxide anions, haemoglobin (Hb), and myoglobin (Mb), being hemoproteins are major players disarming NO bioactivity. The Antarctic icefish, which lack Hb and, in some species, also cardiac Mb, represent a unique model for exploring Hb and Mb impact on NOS/NO function. We report in the (Hb(-)/Mb(-)) icefish Chaenocephalus aceratus the presence of cardiac NOSs activity (NADPH-diaphorase) and endothelial NOS (eNOS)/inducible NOS (iNOS) zonal immuno-localization in the myocardium. eNOS is localized on endocardium and, to a lesser extent, in myocardiocytes, while iNOS is localized exclusively in myocardiocytes. Confronting eNOS and iNOS expression in Trematomus bernacchii (Hb(+)/Mb(+)), C. hamatus (Hb(-)/Mb(+)) and C. aceratus (Hb(-)/Mb(-)) is evident a lower expression in the Mb-less icefish. NO signaling was analyzed using isolated working heart preparations. In T. bernacchii, L-arginine and exogenous (SIN-1) NO donor dose-dependently decreased stroke volume, indicating decreased inotropism. L-arginine-induced inotropism was NOSs-dependent, being abolished by NOSs-inhibitor NG-monomethyl-L-arginine (L-NMMA). A SIN-1-induced negative inotropism was found in presence of SOD. NOS inhibition by L-N5-N-iminoethyl-L-ornithine (L-NIO) and L-NMMA confirmed the NO-mediated negative inotropic influence on cardiac performance. In contrast, in C. aceratus, L-arginine elicited a positive inotropism. SIN-1 induced a negative inotropism, which disappeared in presence of SOD, indicating peroxynitrite involvement. Cardiac performance was unaffected by L-NIO and L-NIL. NO signaling acted via a cGMP-independent mechanism. This high conservation degree of NOS localization pattern and signaling highlights its importance for cardiac biology.
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PMID:Morphological and physiological study of the cardiac NOS/NO system in the Antarctic (Hb-/Mb-) icefish Chaenocephalus aceratus and in the red-blooded Trematomus bernacchii. 1902 84


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