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)

The three isoforms of nitric oxide synthase (NOS), neuronal (nNOS), endothelial (eNOS), and inducible (iNOS), can be visualized in cells and tissues by NADPH-diaphorase (NADPH-d) histochemistry, immunocytochemistry and in situ hybridization. Histochemical demonstration of NADPH-d shows the formazan final reaction product as a solid blue deposit. The ultrastructural localization of NADPH-d in the rat hippocampus showed an electron-dense deposit on membranes predominantly of the endoplasmic reticulum. The immunohistochemical demonstration of nNOS, using the nickel enhancement technique, shows positive reaction product over the dendrites and the soma of the nerve cell in the rat brain. Ultrastructural localization of nNOS in whole mount preparations of myenteric plexus and circular smooth muscle from guinea-pig ileum shows that NOS immunoreactivity was patchily distributed in myenteric neurones and was not specifically associated with any intracellular organelles or with plasma membranes. In situ hybridization, using radio-labelled probes, was used to study nNOS mRNA in lumbar dorsal root ganglia after peripheral transection of the sciatic nerve in rats. Labelling of the NOS mRNA-positive neurones is observed as a series of dense granules over the entire cell. NADPH-d histochemistry, immunocytochemistry and in situ hybridization each have a significant role to play in the localization of NOS. NADPH-d detects an enzyme associated with the NOS molecule, immunocytochemistry detects the NOS molecule, and in situ hybridization detects mRNA for NOS. Therefore, if each of these techniques is applied in carefully controlled experiments, consideration of the accumulated data should be valuable in revealing insights into the biology of NOS.
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PMID:Histochemical methods for detecting nitric oxide synthase. 857 39

The salivary glands of the blood-sucking bug Rhodnius prolixus are formed by a single layer of binucleated epithelial cells surrounded by a double layer of transversely oriented smooth muscle cells. The epithelial cells are rich in rough endoplasmic reticulum and mitochondria and have abundant microvillar projections towards the gland lumen. This cell layer surrounds a relatively large cavity where abundant secretory material is stored. Epithelial cells produce an intense and generalized NADPH diaphorase reaction, in contrast to other tissues such as brain, Malpighian tubules and skeletal muscle. Ultrastructural analysis of the osmiophilic reaction product indicates that it is localized within cytoplasmic vacuoles, a similar location to that of NADPH diaphorase (NO synthetase) activity in neuronal cells of vertebrates. Measurements of the time course of protein accumulation, NADPH diaphorase activity and the degree of nitrosylation of hemoproteins (nitrophorins) in the salivary glands of Rhodnius prolixus nymphs after a blood meal indicate that the nitrophorins are synthesized and accumulate when NO production is low (with a 25% loading of the nitrophorins during the fourth- to fifth-instar molt). NO loading of the nitrophorins increases to 90% after the molt, concomitant with a large increase in the salivary NADPH diaphorase activity. It is concluded that synthesis of NO occurs within the epithelial cells while the nitrophorins are stored extracellularly. It is hypothesized that the luminally oriented microvilli may serve as a diffusion bridge to direct intracellularly produced NO into the luminal cavity, where the nitrophorins are stored.
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PMID:Nitric oxide loading of the salivary nitric-oxide-carrying hemoproteins (nitrophorins) in the blood-sucking bug Rhodnius prolixus. 862 44

The activity and distribution of reduced nico-tinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) in the nodose ganglion of normal and vagotomized guinea-pigs were examined by light and electron microscopy. Light microscopy confirmed a remarkable increase in the number of NADPH-diaphorase-reactive neurons in the nodose ganglion following unilateral cervical vagotomy. The increase was present at 5 days but became more prominent at 10 days and was sustained until at least 30 days after vagotomy when compared with the non-lesioned side. The NADPH-diaphorase reaction product was associated with the membrane of the rough endoplasmic reticulum, Golgi apparatus, mitochondria and nucleus of the nodose neurons. In animals killed 5 days post-operation, there was no noticeable degeneration in the nodose neurons. However, at 10 days, the mitochondria in some neurons appeared swollen and vacuolated with disrupted cristae. These changes were accentuated in some nodose neurons 20 and 30 days after vagotomy but there was no evidence of cell death. All the degenerating neurons exhibited NADPH-diaphorase activity. The increase in NADPH-diaphorase activity in the neuronal somata after vagotomy suggests that the enzyme is involved in either the retrograde degeneration or the recovery of the lesioned neurons.
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PMID:NADPH-diaphorase activity in the nodose ganglion of normal and vagotomized guinea-pigs. 876 67

Induction of nitric oxide synthase and increased production of nitric oxide in microglia may play a crucial role in neuronal damage and neurodegenerative disorders. In the present study we have used light and electron microscopical NADPH-diaphorase histochemistry as the visualization procedure for nitric oxide synthase to investigate the time-course and subcellular patterns of NADPH-diaphorase expression in microglia/macrophages of quinolinic acid-lesioned rat striatum. For light microscopy, NADPH-diaphorase histochemistry sections were stained with nitroblue tetrazolium, while for ultrastructural analysis the tetrazolium salt 2-(2'-benzothiazolyl)-5-styryl-3(4'-phthalhydrazidyl) tetrazolium chloride (BSPT) was applied. Light microscopical inspection revealed a progressively increasing number of positive cells with increasing intensity of NADPH-diaphorase staining in microglia/macrophages from day 1 after quinolinic acid injection onward. Electron microscopical examination revealed a membrane bound NADPH-diaphorase in quiescent microglia as well as in activated microglia/macrophages through all stages of the lesion studied. Predominantly membranes of the nuclear envelope and the endoplasmic reticulum were labeled with BSPT-formazan, while in advanced stages selective membrane portions of mitochondria, Golgi apparatus and plasmalemma were also stained. From day 5 onward after lesion induction, a very distinctive type of NADPH-diaphorase was observed, forming accumulations of electron-dense grains that were distributed differentially throughout cytoplasmic areas and phagocytic vacuoles. Dynamics of expression, unique cytosolic localization and occurrence exclusively in activated microglia/macrophages suggest that this particular NADPH-diaphorase activity probably reflects the inducible isoform of nitric oxide synthase, whereas the membrane-bound precipitate may represent the neuronal and/or the endothelial isoform of the enzyme.
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PMID:Induction of cytosolic NADPH-diaphorase/nitric oxide synthase in reactive microglia/macrophages after quinolinic acid lesions in the rat striatum: an electron and light microscopical study. 882 9

The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) was examined in the upper thoracic segment of the spinal cord in rat. Under the light microscope, NADPH-d positive cell bodies and fibers were readily detected in the following areas: 1) the dorsal horn; 2) the dorsolateral funiculus and lateral spinal neurons; 3) spinal autonomic region, consisting of the nucl. intermediolaterialis pars funicularis, nucl. intermediolateralis pars principalis, nucl. intercalatus spinalis and nucl. intercalatus pars paraependymalis; and 4) in the white matter lateral to the nucl. intermediolateralis pars funicularis. In the nucl. intermediolateralis pars principalis, the positive dendrites, running in bundles, were directed medially in the gray matter towards the central canal as well as laterally in the white matter towards the pia mater. The medially-directed positive dendrites fomed a subependymal plexus around the central canal. A dense bundle of NADPH-d positive fibers were also observed running longitudinally. Combined retrograde tracing with fluorogold and NADPH-d histochemistry study revealed that some of the NADPH-d positive neurons, due to their fluorescence labelling, were sympathetic preganglionic neurons that innervated the superior cervical ganglion. Under the electron microscope, the reaction products in the neurons of the nucl. intermediolateralis pars principalis were deposited in their nuclear envelope, rough endoplasmic reticulum, mitochondria and Golgi apparatus. In the neuropil, three types of synaptic configurations were observed: between NADPH-d negative axon terminals and NADPH-d positive dendrites, between NADPH-d positive axon terminals and NADPH-d negative dendrites, and between NADPH-d positive axons terminals and NADPH-d positive dendrites. These synaptic configurations suggest that the neurons are regulated by nitric oxide released from both pre- and post-synaptic elements. The sources of the NADPH-d positive axon terminals associated with the neurons remain unclear although the possibility of their being derived from supraspinal origins has to be considered. The ultrastructural demonstration of NADPH-d reaction product in the three major types of glial cells suggests that nitric oxide might be produced by these cells, but its functional significance awaits further investigation.
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PMID:Light and electron microscopic studies of the distribution of NADPH-diaphorase in the rat upper thoracic spinal cord with special reference to the spinal autonomic region. 884 31

The distribution of the enzyme nitric oxide synthase (NOS) was investigated at the ultrastructural level in synaptic structures of the hippocampal formation in relation to long-term potentiation (LTP), based on the histochemical NADPH-diaphorase (NADPH-d) staining with the tetrazolium salt BSPT. BSPT-formazan, the osmiophilic reaction product, was found to be selectively distributed and predominantly attached to membranes of the endoplasmic reticulum. In synaptic regions mainly the presynaptic sides showed labeling. Although several groups have demonstrated a principal involvement of NO in the LTP-mechanism, we found only a low, statistically insignificant increase in NADPH-d stained presynaptic areas of the dentate gyrus, where LTP was evoked. Postsynaptic elements also did not show any noticeable differences. Based on the present results, the predominantly presynaptic localization of NOS should be preferably considered in models describing a functional role of NO in LTP formation, despite the fact that we failed to reveal any indications for an LTP-related change in synaptically located NADPH-d.
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PMID:Ultrastructural distribution of NADPH-diaphorase in the normal hippocampus and after long-term potentiation. 887 65

We have previously reported that stimulation of astrocyte cultures by particular agonists and calcium ionophores induces cyclic GMP formation through activation of a constitutive nitric oxide synthase (NOS) and that astrocytes from cerebellum show the largest response. In the present work we have used rat cerebellar astrocyteenriched primary cultures to identify and characterise the isoform of NOS expressed in these cells. The specific NOS activity in astrocyte homogenates, determined by conversion of [3H]arginine to [3H]citrulline, was ten times lower than in homogenates from cerebellar granule neurons. Upon centrifugation at 100,000 g, the astroglial activity was recovered in the supernatant, whereas in neurons around 30% of the activity remained particulate. The cytosolic NOS activities of both astrocytes and granule neurons displayed the same Km for L-arginine, dependency of calcium, and sensitivity to NOS inhibitors. Expression of NOS-I in astrocyte cytosolic fractions was revealed by Western blot with a specific polyclonal antiserum against recombinant NOS-I. Double immunofluorescence labelling using anti-glial fibrillary acidic protein (GFAP) and anti-NOS-I antibodies revealed that a minor population of the GFAP-positive cells, usually in clusters, presented a strong NOS-I immunostaining that was predominantly located around the nuclei and had a granular appearance, indicating association with the endoplasmic reticulum-Golgi system. Astrocytes of stellate morphology also showed immunoreactivity in the processes. Similar staining was observed with the avidin-biotin-peroxidase complex using different anti-NOS-I antisera. With this method the majority of cells showed a weak NOS-I immunoreactivity around the nuclei and cytosol. A similar pattern was observed with the NADPH-diaphorase reaction. These results demonstrate that the NOS-I expressed in astrocytes presents the same biochemical characteristics as the predominant neuronal isoform but may differ in intracellular location.
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PMID:Characteristics of nitric oxide synthase type I of rat cerebellar astrocytes. 891 54

The ultrastructural localization of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), which has been considered to be a neuronal nitric oxide synthase (NOS), was explored in the vascular endothelial cells and perivascular nerves of the cerebral arteries in the rat. In order to detect NADPH-d activity, 2-(2'-benzothiazolyl)-5-styryl-3-4-(4'-phthalhydrazidyl) tetrazolium chloride was utilized as a substrate for NADPH-d histochemistry at the electron microscopic level. In vascular endothelial cells, NADPH-d positive deposits were observed on the nuclear envelope and the endoplasmic reticulum (smooth or rough surfaced). Positive deposits were seen on distinct membrane portions of the endoplasmic recticulum (ER) in the perivascular nerves (axons), but no positive materials were observed either in the cytoplasm of the endothelial cells or in the axoplasm of the perivascular nerves. It was concluded that NOS is located on the membranes of the ER and the nuclear envelope, and that NOS may play substantial roles in the regulation of the cerebral vessels.
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PMID:The ultrastructural localization of NADPH-diaphorase in the cerebral arteries of rats. 902 50

Recognition of the role of nitric oxide (NO) in cardiovascular regulations raised an acute interest in NO-generating enzymes-nitric oxide synthases (NOS). Nevertheless, the subcellular localization of inducible isoform of NOS (NOS II) and regulation of its expression in the cardiomyocyte still remains to be elucidated. Therefore, we focused this study on the subcellular localization of NOS II in cultured neonatal rat cardiomyocytes using immunocytochemical techniques at the light and electron microscopic level as well as the demonstration of NADPH-diaphorase activity and the Griess assay for NO measurement. Cultivation of neonatal cardiomyocytes during 2 and more days induced a moderate increase in the NOS II immunolabeling in defined cytoplasmic structures and a nuclear NOS II staining in some cells. Exposure of the cell cultures to exogenous cAMP markedly stimulated NO production with a concomitant enhancement of NOS II immunolabeling of cardiomyocytes. cAMP-induced changes were significantly attenuated by dexamethasone. This report provides evidence for the localization of NOS II in the perinuclear space, Golgi complex, mitochondria, plasma membrane and along contractile fibers of cardiomyocytes, as well as for the appearance of NOS II staining of the cell nuclei in the course of cultivation. In non-cardiomyocytes contaminating the cell culture, positive immunoreaction was detected in the Golgi complex and endoplasmic reticulum. Our data point to a notable constitutive expression of NOS II in rat cardiomyocytes apparently dependent on the developmental stage.
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PMID:Intracellular localization of inducible nitric oxide synthase in neonatal rat cardiomyocytes in culture. 924 81

The NOS-related NADPH-diaphorase activity was studied by transmission electron microscopy in the peritubular myoid cells and fibroblasts of normal mouse testis. The reaction product was observed on the membranes of the endoplasmic reticulum, on the Golgi apparatus and nuclear envelope. The peritubular myoid cells and fibroblasts showed similar ultracytochemical features; the intensity of the enzymatic reaction was suggestive of an important role of the NOS/cGMP enzymatic system in these cells. Some hypotheses on the role of NO in the peritubular myoid cells and fibroblasts are proposed.
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PMID:Ultrastructural demonstration of the NADPH-diaphorase activity in peritubular myoid cells and fibroblasts of the lamina propria of the mouse seminiferous tubules. 929 98


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