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:1.5.1.19 (NOS)
7,285 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied steps that make up the initial and steady-state phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0. 13 to 86 s(-1) upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N(omega)-hydroxy-l-arginine (0.005 and 0.014 s(-1), respectively, at 10 degrees C). O(2) binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nm), which were easily achieved with N(omega)-hydroxy-l-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K(m) for O(2) to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology.
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PMID:Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric-oxide synthase. 1074 53

To date few reports have discussed the presence and function of nitric oxide (NO) in structures of the facial nerve. We performed nicotinamide adenine dinucleotide phosphate (NADPH-d)-diaphorase-histochemistry and immunohistochemistry on the intratemporal portion of the facial nerve, including the geniculate ganglion, of guinea pigs using specific antibodies to the three known isoforms of NO synthase and soluble guanylyl-cyclase (sGC). Normal facial nerves were compared to those treated intratympanically with bacterial lipopolysaccharides (LPS) and tumor necrosis factor-alpha (TNF-alpha). Both constitutive NOS isoforms and sGC could be detected in the bipolar ganglion cells of normal animals, while the inducible isoform (iNOS or NOS II) was not found. Endothelial NOS (NOS III) and sGC were present in blood vessels and were predominantly found in the perineurial sheath and less in the endoneurium. sGC could be detected in all fibers in a cross section of the facial nerve. LPS and TNF treatment led to the detection of iNOS in the perikaryia of the geniculate ganglion and the perineural sheath. These findings imply that NO may be involved in neurotransmission at least in the visceroafferent system. NO regulates vascular tone of nutrient blood vessels in the perineural sheath and endoneurium. The presence of sGC indicates that NO acts via its second messenger cGMP. NOS II expression may be a contributing factor to facial nerve palsy via two different mechanisms: NOS II-generated NO may lead to an overstimulation of the visceroefferent nerve fibers and motor fibers of the facial nerve. Dysregulation in facial nerve blood vessels could lead to edema and elevated pressure on the nerve within its osseous canal.
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PMID:Involvement of nitric oxide synthase in the physiology and pathophysiology of facial nerve function and dysfunction. 1086 32

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

The phenotypic expression and anatomic distribution of nitrergic and peptidergic innervation in the developing rat heart was localized by reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and immunohistochemistry using antibodies against neuronal isoform of nitric oxide synthase (nNOS), neuropeptide Y (NPY) and calcitoningene-related peptide (CGRP). NPY-immunoreactive nerve fibers showed the earliest expression by 16 days of gestation, with preferential innervation of the nodal and perinodal areas, followed by the innervation of the valves and ventricles by postnatal day 7. NPY immunoreactivity was also localized to a large proportion of the intrinsic cardiac ganglia from 16 days of gestation onwards with a progressive increase in the number of neuronal cell bodies per ganglia with age. CGRP-positive nerve fibers appeared by 19 days of gestation and were less dense during the gestational and early postnatal periods, and showed a quantitative increase in density by 7 days, followed by a decrease by 3 weeks postnatal. None of the intrinsic ganglia were stained positive for CGRP, indicating the extrinsic sensory origin of these stained fibers. Nitrergic innervation paralleled the sensory innervation, with the cardiac ganglia and nerve fibers showing a positive labeling from 19 days of gestation onwards. NADPH-d and nNOS were partially co-localized. Double-label immunohistochemistry showed that a considerable proportion of sensory CGRP-immunopositive fibers were also immunoreactive for NOS. The results of the present study show that neuropeptides and nitric oxide are expressed by the late gestational period and that autonomic efferent innervation precedes sensory and nitrergic innervation in the developing heart.
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PMID:Nitrergic and peptidergic innervation in the developing rat heart. 1090 3

Oxidative stress and massive production of nitric oxide (NO) have been implicated in the neuropathogenesis following peripheral nerve injury. This study was aimed to ascertain whether melatonin would exert its neuroprotective effect on the lesioned hypoglossal neurons after peripheral axotomy, since it is known to reduce the oxidative damage in a variety of experimental neuropathologies in which NO is involved. Right-sided hypoglossal nerve transection was performed in adult rats following which the animals were given two different doses of melatonin administered intraperitoneally for 3, 7, 14, 21 and 30 successive days. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were carried out to detect the neuronal NADPH-d/NOS expression in the hypoglossal nucleus (HN). At various time intervals following axotomy, the neurons in the affected HN were induced to express NADPH-d/NOS reactivity on the lesioned side peaking at 14 days. However, the enzyme expression was markedly depressed by melatonin treatment in a dose-dependent manner in terms of frequency of labelled neurons and staining intensity. It is suggested that the suppressive effect of melatonin on NADPH-d/NOS expression may be attributed to its antioxidant properties. Hence, in consideration of therapeutic strategies for reducing the oxidative stress following peripheral nerve injury, melatonin may prove to be beneficial.
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PMID:Melatonin attenuates neuronal NADPH-d/NOS expression in the hypoglossal nucleus of adult rats following peripheral nerve injury. 1093 May 50

The present study was initiated to investigate neuronal degeneration, microglial reactivity and possible roles of NO in the lateral geniculate nucleus (LGN) of glaucomatous rats. An experimental one-eye glaucoma model was created by cauterization of the limbal-derived veins. Neuronal cell viability was studied by immunostaining with antibody against neuronal nuclei. Changes of expressions of nitric oxide synthase I (NOS I), NOS II, ED 1, OX6 and OX42 in the LGN were studied by immunohistochemistry. NADPH-d histochemistry was also employed. In the experimental glaucomatous rats, the number of NeuN labelled neurons was significantly decreased in both the ipsi- and contra-lateral sides of the ventral LGN (vLGN) but not the dorsal LGN (dLGN) at 1 month post-operation and beyond. Expressions of NOS I and NADPH-d were notably increased from 1 week post-operation in the ipsilateral vLGN. In the contralateral side of the vLGN, however, this change was only observed from 1 month post-operation. No NOS II immunoreaction was observed in LGN of both the normal control and glaucomatous rats. Increased microglial reactivity as indicated by OX-42 immunoreactivity was first observed in both sides of the LGN at 1 week post-operation, and this was most significant especially at 1 and 2 months post-operation. The present results suggest that NO and microglial cells may play some important roles in the pathologic processes of neuronal degeneration in the LGN of glaucomatous rats.
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PMID:Nitric oxide, microglial activities and neuronal cell death in the lateral geniculate nucleus of glaucomatous rats. 1099 44

This study examined the effect of suckling on nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, a histochemical marker for nitric oxide synthase, NOS) reactivity and neuronal NOS mRNA expression in the paraventricular (PVN) and supraoptic (SON) nuclei of lactating rats. Freely nursing (non-separated) dams and those separated from pups for 12 h and then reunited for 0, 15, 30, 60, 90, 120 and 180 min were used for the study. Dams separated from pups and sacrificed at time zero (without reunion) showed a significant decrease in NADPH-d staining and NADPH-d positive cells as well as in the NOS mRNA expression in the PVN and SON compared to that observed in non-separated dams. Reunion with pups and restoration of suckling significantly increased NADPH-d reactivity after 15, 30, 60 min, but not after 90, 120 and 180 min compared to non-reunited pups-deprived dams. A pattern of NADPH-d reactivity and neuronal NOS mRNA expression indistinguishable from that observed during free lactation was reinstated shortly (15 min) after the restoration of suckling stimulus, suggesting that the NADPH-d reactivity in lactation depends on the presence of the suckling stimulus. These results show that suckling stimulus may play a modulatory role in the regulation of NOS reactivity in the magnocellular neurones of the hypothalamic PVN and SON during lactation.
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PMID:Effect of suckling on NADPH-diaphorase (Nitric oxide synthase, NOS) reactivity and NOS gene expression in the paraventricular and supraoptic nuclei of lactating rats. 1101 41

After initiating NO synthesis a majority of neuronal NO synthase (nNOS) quickly partitions into a ferrous heme-NO complex. This down-regulates activity and increases enzyme K(m,O(2)). To understand this process, we developed a 10-step kinetic model in which the ferric heme-NO enzyme forms as the immediate product of catalysis, and then partitions between NO dissociation versus reduction to a ferrous heme-NO complex. Rate constants used for the model were derived from recent literature or were determined here. Computer simulations of the model precisely described both pre-steady and steady-state features of nNOS catalysis, including NADPH consumption and NO production, buildup of a heme-NO complex, changes between pre-steady and steady-state rates, and the change in enzyme K(m,O(2)) in the presence or absence of NO synthesis. The model also correctly simulated the catalytic features of nNOS mutants W409F and W409Y, which are hyperactive and display less heme-NO complex formation in the steady state. Model simulations showed how the rate of heme reduction influences several features of nNOS catalysis, including populations of NO-bound versus NO-free enzyme in the steady state and the rate of NO synthesis. The simulation predicts that there is an optimum rate of heme reduction that is close to the measured rate in nNOS. Ratio between NADPH consumption and NO synthesis is also predicted to increase with faster heme reduction. Our kinetic model is an accurate and versatile tool for understanding catalytic behavior and will provide new perspectives on NOS regulation.
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PMID:A kinetic simulation model that describes catalysis and regulation in nitric-oxide synthase. 1103 56

Effects of nicotine on arterial endothelium-dependent relaxations mediated by nitric oxide are controversial. Experiments were designed to test the hypothesis that nicotine can directly alter activity of endothelial nitric-oxide synthase (eNOS). NOS from aortic endothelial cells of untreated dogs and recombinant eNOS, neuronal NOS, and inducible NOS were used for these experiments. NOS activity was determined as conversion of L-[(3)H]arginine to L-[(3)H]citrulline in the absence or presence of nicotine (10(-7)-10(-3) M) in vitro. In separate assays, concentrations of cofactors NADPH, FAD, and tetrahydrobioprotein were reduced by half to assess for possible interaction with nicotine. With enzyme from aortic endothelial cells, total and calcium-dependent accumulation of citrulline increased by 30% in the presence of 10(-5) M nicotine. Nicotine dose dependently also increased citrulline accumulation by recombinant eNOS and neuronal NOS but not inducible NOS. Effects of nicotine on accumulation of citrulline by isolated eNOS and recombinant eNOS were further modulated by changes in the concentration of NADPH in the incubation solution. Our data demonstrate a significant effect of nicotine on eNOS-mediated citrulline accumulation. These results suggest that effects of nicotine on production of nitric oxide may depend on NADPH or oxygen radical interactions with NOS and thus may explain, in part, inconsistent findings of changes in production of endothelium-derived nitric oxide with nicotine administration.
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PMID:Modulation of nitric-oxide synthase by nicotine. 1104 94

The histochemistry of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and immunoreactivity of neuronal nitric oxide synthase (nNOS-IR) can be demonstrated in various cell types of the vertebrate retina. In this study, we have focused on characterizing the different NADPH-d-positive amacrine cell types in turtle retina. Cryostat sections were examined by confocal laser scanning microscopy for double immunofluorescence with antibodies against nNOS and either GABA or glycine, or by combining histochemistry with immunocytochemistry to obtain triple labeling with NADPH-d, GABA, and glycine. Forty-eight percent of the NADPH-d-labeled amacrine cells colocalized GABA, 52% glycine. Here we show that two morphologically different types of amacrine cell are nNOS/glycine-IR and three types are nNOS/GABA-IR. Antibodies against calretinin, parvalbumin, somatostatin, tyrosine hydroxylase, and choline acetyltransferase did not colocalize with nNOS-IR or NADPH-d-labeled amacrine cells, but 15% of the NOS-labeled amacrine cells showed immunoreactivity against calbindin. Only GABA has been seen to colocalize with NADPH-d in amacrine cells in previous reports in other species. The finding here of glycine colocalizing with NO-containing cells is novel. We suggest that NO, apart from its well known function in gap junction regulation, can also modulate the release of both GABA and glycine in the turtle retina.
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PMID:Morphological and neurochemical diversity of neuronal nitric oxide synthase-positive amacrine cells in the turtle retina. 1107 11


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