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)

We recently demonstrated that reactive astrocytes express NADPH diaphorase activity, a marker for Nitric Oxide Synthase, following transient global ischemia (Neuroscience Letters 154: 125-128). There has been little evidence that astrocytes express Nitric Oxide Synthase or produce NO (nitric oxide) in vivo; although in vitro experiments have shown that cultured astrocytes can produce NO. To determine whether reactive astrocytes express inducible form of NOS (iNOS) in vivo, we studied the pathological changes of rat hippocampus by immunohistochemistry after 10 minutes of transient global ischemia, which results in the selective delayed death of CA1 pyramidal cells and marked gliosis in the CA1 subfield. In the normal hippocampus, astrocytes express neither NADPH diaphorase activity nor iNOS. After ischemia, the temporal and spatial pattern of iNOS, NADPH diaphorase, and GFAP are very similar, indicating that reactive astrocytes express iNOS. Double staining for NADPH diaphorase and GFAP, or iNOS and GFAP confirmed that reactive astrocytes express both NADPH diaphorase activity and iNOS immunoreactivity. These changes were observed three day after ischemia and increased in prominence from one week to one month. The staining pattern of OX42, an antibody that recognizes both microglia and macrophages, is spatially and temporally distinct from the pattern of NADPH diaphorase and iNOS staining. Thus, we conclude that transient global ischemia induces iNOS primarily in reactive astrocytes. This increase in NOS expression and, presumably, NO production by reactive astrocytes may play a role in the process of delayed neuronal death or in the remodeling responses that occur after ischemic damage.
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PMID:Expression of the inducible form of nitric oxide synthase by reactive astrocytes after transient global ischemia. 752 35

Preconditioning of the brain with sublethal ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). In this study, we used NADPH-diaphorase histochemistry to investigate the postischemic changes of nitric oxide synthase (NOS) in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by 4-vessel occlusion for 3 min as an ischemic preconditioning. Three days after the preconditioning or sham operation, second ischemia was induced for 6 min. A transient increase in NADPH-diaphorase activity, beginning after 2 h and maximal after 1 day, was observed in CA1 pyramidal neurons of rats subjected to 3 min of preconditioning ischemia as well as 6 min of subsequent ischemia both with and without preconditioning. In addition, expression of NADPH-diaphorase activity was seen in reactive glial cells in the damaged CA1 region of animals subjected to 6 min of ischemia without preconditioning. Thus, direct involvement of increased NADPH-diaphorase activity in ischemic tolerance was not suggested because the increased NADPH-diaphorase activity preceded the induction of ischemic tolerance which takes place 1-7 days after preconditioning. However, the present findings suggest that the induction of neuronal NADPH-diaphorase activity occurs in response to cerebral ischemia.
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PMID:Induction of NADPH-diaphorase activity in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance. 752 21

Nitric oxide synthase-containing neurons are presumed to be resistant to neurodegeneration and neurotoxicity, however this resistance has not been demonstrated after focal cerebral ischemia. We therefore measured the temporal profile of neuronal nitric oxide synthase (NOS-I) mRNA and immunoreactivity and NADPH-diaphorase reactivity over a one week period after permanent middle cerebral artery (MCA) occlusion in 48 male Wistar rats and compared these data to ischemic cell damage as evaluated on hematoxylin and eosin (H & E) stained sections by light microscopy. NOS-I mRNA increased as early as 15 min after MCA occlusion in the ipsilateral striatum and maximal expression of NOS-I was found in the ipsilateral cortex and striatum 1 h after MCA occlusion. The numbers of NOS-I-containing neurons in the ipsilateral cortex and striatum were significantly greater (P < 0.05) than NOS-I-containing neurons in the contralateral hemisphere at 2-48 h after the onset of ischemia. The number of NOS-I-containing neurons peaked at 4 h after MCA occlusion. Neurons exhibited shrinkage or were swollen at 1 to 4 h after MCA occlusion. At 24-48 h after ischemia, neurons in the ischemic lesion appeared to be eosinophilic or ghost like on H & E stained sections. However, some of these neurons retained morphological integrity on the NOS-I immunohistochemical sections. At 168 h after ischemia, all neurons within the lesion appeared necrotic on H & E stained sections; however, scatterred neurons expressed NOS-I and NADPH-diaphorase. The rapid upregulation of NOS-I and mRNA in the ischemic lesion suggests that NOS-I is involved in focal cerebral ischemic injury; the expression of NOS-I by neurons that retain their morphological structure in the area of the infarct suggests that NOS-I-containing neurons are more resistant to the ischemic insult. Our data also indicate a close association of NOS-I immunoreactivity and NADPH-diaphorase reactivity in ischemic brain.
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PMID:Upregulation of neuronal nitric oxide synthase and mRNA, and selective sparing of nitric oxide synthase-containing neurons after focal cerebral ischemia in rat. 752 66

The effects of cerebral metabolism-improving drugs on NADPH diaphorase activity in the mouse brain were studied, and we found that diaphorase activity in the post-mitochondrial fraction of brain homogenate was enhanced by idebenone in a concentration-dependent manner. Histochemical studies also indicated that diaphorase staining was intensified by idebenone at the same concentration. These results suggest that idebenone may stimulate the production of nitric oxide, probably through its direct action on nitric oxide synthase, thus producing its protective action on neurological disorders due to cerebral hypoxia or ischemia as a consequence of dilating the cerebral blood vessels.
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PMID:Biochemical and histochemical studies of the effects of cerebral metabolism-improving drugs on NADPH diaphorase activity in mouse brain. 752 86

Nitric oxide can act as a neurotransmitter and a retrograde modulator of synaptic transmission, but uncontrolled nitric oxide synthase activity has been associated with neural degeneration. Although earlier studies using immunohistochemistry, in situ hybridization, and NADPH-diaphorase staining had suggested that nitric oxide synthase is not expressed in the CA1 neurons of the hippocampus, we have recently demonstrated that NADPH-diaphorase activity can be detected in CA1 neurons of the hippocampus. To confirm that this diaphorase activity reflects nitric oxide synthase, we have developed a more sensitive in situ hybridization procedure, and an RNase protection assay to detect message for constitutive nitric oxide synthase, the form constitutively expressed in many neurons. Message for constitutive nitric oxide synthase is expressed in the hippocampus, and it is localized to neural cell layers CA1, CA3, the dentate gyrus and some displaced neurons, but not to CA2. Expression of constitutive nitric oxide synthase message in the CA1 region was lost when pyramidal neurons died due to transient forebrain ischemia, supporting the conclusion that CA1 pyramidal cells express constitutive nitric oxide synthase. Although constitutive nitric oxide synthase message is strongly expressed in CA3 and the dentate gyrus, there is little diaphorase activity in these cells, suggesting that there may be post-transcriptional controls that limit constitutive nitric oxide synthase expression in some cells. Message for constitutive nitric oxide synthase is also present in a number of other regions, including the amygdala, several hypothalamic nuclei, the cerebellum, the olfactory bulb, two distinct regions of the perirhinal cortex, the subthalamic nuclei, a neuronal layer in the retrosplenial granular cortex, the lateral geniculate nucleus, the presubiculum, the inferior colliculus, the superior colliculus, the pedunculopontine tegmental nucleus, and scattered individual neurons in the cortex, hippocampus and brainstem. These studies support a role for nitric oxide in multiple regions of the central nervous system. In particular, nitric oxide synthase, the enzyme responsible for the synthesis of nitric oxide, is expressed in the CA1 region of the hippocampus, where there is evidence that nitric oxide may play a major role in long-term potentiation. CA1 hippocampal neurons are an example of a population of neurons that express constitutive nitric oxide synthase but are very sensitive to excitotoxicity and ischemic insults.
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PMID:Expression of the neural form of nitric oxide synthase by CA1 hippocampal neurons and other central nervous system neurons. 753 83

In the hippocampus, ten minutes of transient global ischemia results in the death of CA1 pyramidal cells after a period of one to three days. The neurons in the CA1 region constitutively express NADPH-D (NADPH diaphorase activity). In contrast, astrocytes in the hippocampus do not normally express NADPH-D; but a population of reactive astrocytes (GFAP+ cells) begin to express of NADPH-D one day after transient global ischemia. NADPH-D is thought to be a histological marker for Nitric Oxide Synthase (NOS), the enzyme that is responsible for the synthesis of NO, a potent neurotoxin. We suggest that this increase in NADPH-D/NOS expression is an important element in the sequence of changes that occurs after ischemia, and that NO derived from reactive astrocytes or from neurons may play a causal role in neural cell death after ischemia in the hippocampus.
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PMID:Reactive astrocytes express NADPH diaphorase in vivo after transient ischemia. 768 11

Copper Fenton systems (Cu(II)/H2O2 and Cu(II)/Asc) inactivated the lipoamide reductase and enhanced the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). Cupric ions alone were less effective. As a result of Cu(II)/H2O2 treatment, the number of titrated thiols in LADH decreased from 6 to 1 per subunit. NADH and ADP (not NAD+ or ATP) enhanced LADH inactivation by Cu(II). NADH also enhanced the effect of Cu(II)/H2O2. Dihydrolipoamide, dihydrolipoic acid, Captopril, acetylcysteine, EDTA, DETAPAC, histidine, bathocuproine, GSSG and trypanothione prevented LADH inactivation. 100 microM GSH, DL-dithiothreitol, N-(2-mercaptopropionylglicine) and penicillamine protected LADH against Cu(II)/Asc and Cu(II), whereas 1.0 mm GSH and DL-dithiothreitol also protected LADH against Cu(II)/H2O2. Allopurinol provided partial protection against Cu(II)/H2O2. Ethanol, mannitol, Na benzoate and superoxide dismutase failed to prevent LADH inactivation by Cu(II)/H2O2 or Cu(II). Catalase (native or denaturated) and bovine serum albumin protected LADH but that protection should be due to Cu binding. LADH inhibited deoxyribose oxidation and benzoate hydroxylation by Cu(II)/H2O2. It is concluded that site-specifically generated HO, radicals were responsible for LADH inactivation by Cu(II) Fenton systems. The latter effect is discussed in the context of ischemia-reoxygenation myocardial injury.
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PMID:Inactivation of heart dihydrolipoamide dehydrogenase by copper Fenton systems. Effect of thiol compounds and metal chelators. 775

DT diaphorase is a flavoprotein that enzymatically transfers two electrons from quinones as intermediate substrates and has been reported to increase its activity in the liver after exposure to toxicants. In this series of experiments, we tested the hypothesis that DT diaphorase also increases its activity after exposure to oxidants following gradient ischemia in skin. Using dorsal rat flaps, oxidant stress was induced immediately or during a 7-day period of preconditioning as a bipedicle flap before the distal attachment was divided. DT diaphorase activity (delta Abs/min/100 g) or expression of message was measured during the period of preconditioning to determine the relationship between skin survival, enzyme activity, and expression of message. There was 4.7 +/- 0.8 cm of skin necrosis in the distal end of acute flaps while the preconditioned flaps had no skin necrosis after the distal attachment was divided. In the acute flaps, the DT diaphorase activity was equal throughout the flap for the first 6 hr. After 24 hr of ischemia, the DT diaphorase activity was significantly higher in the proximal end of the flap (1.83 +/- 0.21 delta Abs/min/100 g) than that in the distal end (0.005 +/- 0.01 delta Abs/min/100 g), which was significant (P < 0.05). In the preconditioned flaps, enzyme activity did not increase but there was as 50-fold increase in DT diaphorase activity at the distal end 24 hr after they were divided (P < 0.05). Maximal enzyme induction of DT diaphorase activity occurred after 4 days of preconditioning and correlated with the maximal expression of mRNA. These studies provide the first evidence that DT diaphorase enzyme activity is inducible after oxidant stress. The data also suggests that DT activity remains elevated for at least 6 hr of ischemia and may be a potential source of anti-oxidant activity in ischemic skin.
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PMID:dT diaphorase: increased enzyme activity and mRNA expression in oxidant stress of skin. 815 25

The striatum is vulnerable to hypoxic-ischemic injury during development. In a rodent model of perinatal hypoxia-ischemia, it has been shown that striatal neurons are not uniformly vulnerable. Cholinergic neurons and NADPH-diaphorase-positive neurons are relatively spared. However, it is unknown what classes of striatal neurons are relatively sensitive. One of the major classes of striatal neurons uses enkephalin as a neurotransmitter. We have studied the effect of early hypoxic-ischemic injury on this class of neurons using a quantitative solution hybridization assay for preproenkephalin mRNA in conjunction with in situ hybridization. Hypoxia-ischemia results in an early (up to 24 h) decrease in striatal preproenkephalin mRNA, which is shown by in situ hybridization to occur mainly in the dorsal portion of the striatum. By 14 days, whole striatal preproenkephalin mRNA and total enkephalin-containing peptide levels are normal. However, at 14 days, in situ hybridization reveals that regions of complete preproenkephalin mRNA-positive neuron loss remain in the dorsal region. Normal whole striatal levels are due to an up-regulation of preproenkephalin mRNA expression in the ventrolateral region of the injured striatum. Given the important role that the enkephalin-containing striatal efferent projection plays in regulating motor function, its relative loss may be important in the chronic disturbances of motor control observed in brain injury due to developmental hypoxic-ischemic injury.
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PMID:Acute and persistent suppression of preproenkephalin mRNA expression in the striatum following developmental hypoxic-ischemic injury. 815 36

The striatum is especially vulnerable to hypoxic-ischemic injury, both in adulthood and during development. Striatal injury is likely to play a major role in the chronic abnormalities of motor control which occur as a consequence of developmental hypoxia-ischemia. Previous studies have shown that two striatal neuron phenotypes, cholinergic and NADPH-diaphorase-positive, are resistant to developmental hypoxia-ischemia, but little is otherwise known of patterns of vulnerability among other striatal neurons. In particular, there has been no data available about patterns of vulnerability within the major striatal neuron group, the medium-sized neurons. Since a major anatomical and functional organization of these neurons is in their localization to either the striosome or the matrix compartments, we have examined the effect of developmental hypoxia-ischemia on these compartments using a quantitative morphologic analysis of immunostaining for the calcium-binding protein calbindin-D28k. We have found that there is a predominant loss of the striosome compartment; in the presence of a mean loss of 33% of total striatal area, there was a 49% decrease in striosomal area. There was also a 41% reduction in the number of striosomes, and a small (14%) but significant decrease in the mean area of individual striosomes. The striosome loss was uniform in the rostrocaudal dimension. At a cellular level, the density of calbindin-positive neurons, expressed as number per unit area, was preserved. While there are several possible explanations for the selective loss of the striosome compartment, one hypothesis is that the lower level of calbindin within these neurons makes them more vulnerable to increases in intracellular calcium, which has been postulated to play a role in hypoxic-ischemic injury. The predominant loss of the striosome compartment following hypoxic-ischemic injury may lead to an imbalance with the functionally distinct matrix system. Such an imbalance may contribute to the abnormalities of motor control observed after this form of injury.
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PMID:Relative loss of the striatal striosome compartment, defined by calbindin-D28k immunostaining, following developmental hypoxic-ischemic injury. 824 62


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