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:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hydrogen peroxide is the final electron acceptor for the biosynthesis of thyroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocytes. Pig and human thyroid plasma membrane contain a Ca(2+)-dependent NAD(P)H oxidase that generates H(2)O(2) by transferring electrons from NAD(P)H to molecular oxygen. We purified from pig thyroid plasma membrane a flavoprotein which constitutes the main, if not the sole, component of the thyroid NAD(P)H oxidase. Microsequences permitted the cloning of porcine and human full-length cDNAs encoding, respectively, 1207- and 1210-amino acid proteins with a predicted molecular mass of 138 kDa (p138(Tox)). Human and porcine p138(Tox) have 86.7% identity. The strongest similarity was to a predicted polypeptide encoded by a Caenorhabditis cDNA and with rbohA, a protein involved in the Arabidopsis NADPH oxidase. p138(Tox) shows also similarity to the p65(Mox) and to the gp91(Phox) in their C-terminal region and have consensus sequences for FAD- and NADPH-binding sites. Compared with gp91(Phox), p138(Tox) shows an extended N-terminal containing two EF-hand motifs that may account for its calcium-dependent activity, whereas three of four sequences implicated in the interaction of gp91(Phox) with the p47(Phox) cytosolic factor are absent in p138(Tox). The expression of porcine p138(Tox) mRNA analyzed by Northern blot is specific of thyroid tissue and induced by cyclic AMP showing that p138(Tox) is a differentiation marker of thyrocytes. The gene of human p138(Tox) has been localized on chromosome 15q15.
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PMID:Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cdnas. 1060 Dec 91

The flavoprotein inhibitor, diphenyleneiodonium (DPI), inhibits the action of glyceryl trinitrate (GTN) and the D-enantiomer of isoidide dinitrate (IIDN), but not the L-enantiomer (L-IIDN), in isolated rat aorta via inhibition of the bioactivation of these prodrugs. Paradoxically, a vascular NAD(P)H oxidase, which also is inhibited by DPI, has been proposed to generate superoxide that quenches nitric oxide (NO) produced during GTN biotransformation, and increased oxidase levels are proposed to contribute to the phenomenon of organic nitrate tolerance. We examined the effect of DPI on isolated rat aorta using an in vivo model of organic nitrate tolerance. The EC(50) values for GTN-, D-IIDN-, and L-IIDN-induced relaxation of aorta from GTN-tolerant rats were increased 4.5- to 7.5-fold. Treatment of blood vessels with DPI (0.3 microM) increased the EC(50) values for GTN and D-IIDN by the same magnitude in control and tolerant aortae, a result that would not be predicted if DPI and GTN tolerance affected common targets. The expression of NADPH-cytochrome P450 reductase (CPR) during in vivo tolerance was assessed by NADPH-dependent cytochrome c reductase activity of aortic microsomes, immunoblotting, and Northern analysis. By all three determinants, CPR expression was unchanged in aorta from GTN-tolerant rats. Superoxide dismutase-inhibitable NADPH-dependent cytochrome c reductase activity (a measure of superoxide generation) of tolerant rat aortic microsomes was not different from that of controls. Superoxide dismutase-inhibitable NADH-dependent cytochrome c reductase activity was detected only in microsomes from tolerant animals. DPI caused a modest increase in the sensitivity for relaxation by the NO donor DEA NONOate to an equal extent in tolerant and nontolerant tissues, whereas the superoxide scavenger, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), had no effect on the sensitivity for relaxation by GTN. These results would not be expected if tolerance-induced increases in superoxide were a causative factor for the reduced relaxation response in tolerance. We conclude that neither reduced flavoprotein-dependent metabolic activation of organic nitrates, such as that mediated by CPR, nor increased superoxide due to increased NAD(P)H oxidase activity can account for the development of in vivo tolerance to GTN.
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PMID:Effects of the flavoprotein inhibitor, diphenyleneiodonium sulfate, on ex vivo organic nitrate tolerance in the rat. 1077 30

Nerve growth factor (NGF) stimulation of pheochromocytoma PC12 cells transiently increased the intracellular concentration of reactive oxygen species (ROS). This increase was blocked by the chemical antioxidant N-acetylcysteine and a flavoprotein inhibitor, diphenylene iodonium. NGF responses of PC12 cells, including neurite outgrowth, tyrosine phosphorylation, and AP-1 activation, was inhibited when ROS production was prevented by N-acetylcysteine and diphenylene iodonium. The expression of dominant negative Rac1N17 blocked induction of both ROS generation and morphological differentiation by NGF. The ROS produced appears to be H(2)O(2), because the introduction of catalase into the cells abolished NGF-induced neurite outgrowth, ROS production, and tyrosine phosphorylation. These results suggest that the ROS, perhaps H(2)O(2), acts as an intracellular signal mediator for NGF-induced neuronal differentiation and that NGF-stimulated ROS production is regulated by Rac1 and a flavoprotein-binding protein similar to the phagocytic NADPH oxidase.
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PMID:Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species. 1078 20

The effects of known leukocyte NADPH oxidase inhibitors on general cellular oxidant production in cultured human endothelial cells (EC) has been investigated. EC were stimulated with 10 nM phorbol 12-myristate 13-acetate and cellular oxidant production measured in the presence and absence of inhibitors that act on various substituents of the oxidase complex and its activation pathways. The effects of the cytosolic oxidase subunit translocation inhibitors, catechols (3,4-dihydroxybenzaldehyde, caffeic acid, and protocatechuic acid), ortho-methoxy-substituted catechols (apocynin, vanillin, and 4-nitroguaiacol), and quinone, 1,4-naphthoquinone; flavoprotein inhibitors, diphenylene iodonium and quinacrine; haem ligands, imidazole and pyridine; directly acting thiol reagents, disulfiram and penicillamine; NADPH analogue, Cibacron Blue; redox active inhibitors, quercetin and esculetin; intracellular calcium antagonist, TMB-8; and calmodulin antagonists, W-7 and trifluoperazine, were determined. All compounds reduced oxidant production in stimulated EC. These findings add to previous observations suggesting the presence of a functionally active NADPH oxidase in EC. Identifying the major cellular reactive oxygen species source in perturbed EC will provide new insights into our understanding of endothelial dysfunction, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.
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PMID:Endothelial cell oxidant production: effect of NADPH oxidase inhibitors. 1086 39

Angiotensin II (AngII) induces G(1) phase arrest and hypertrophy of cultured renal proximal tubular cells. In previous studies, it was shown that these effects depend on oxygen radical-mediated induction of p27(Kip1), an inhibitor of cyclin-dependent kinases. The present study was undertaken to investigate whether mitogen-activated protein (MAP) kinases serve as signaling intermediates between AngII-induced oxidative stress and induction of p27(Kip1). AngII (10(-7) M) induces a biphasic phosphorylation pattern of p44/42 MAP kinase with an early phosphorylation after 2 min and a later, second phosphorylation peak after prolong incubation (12 h) in cultured proximal tubular cells from two different species (MCT and LLC-PK(1) cells). Total protein expression of MAP kinase was not changed by AngII. These phosphorylation patterns of p44/42 MAP kinase caused activation of the enzyme, as detected by phosphorylated MAP substrate Elk-1 after immuno-precipitation of MAP kinase. Exogenous H(2)O(2) also stimulates a biphasic phosphorylation of p44/42 MAP kinase. The flavoprotein inhibitor diphenylene iodinium, as well as the antioxidant N-acetylcysteine, prevented AngII-induced p44/42 MAP kinase phosphorylation, indicating involvement of reactive oxygen species generated by membrane-bound NAD(P)H oxidase. The MAP kinase kinase inhibitor PD98059 completely inhibits AngII-induced p27(Kip1) expression and (3)[H]leucine incorporation into proteins as a previously established marker of cell hypertrophy. PD98059 did not attenuate AngII-stimulated intracellular synthesis of oxygen radicals. Transient transfection with p44/42 MAP kinase antisense, but not sense, phosphorothioate-modified oligonucleotides also prevented AngII-induced MAP kinase phosphorylation, p27(Kip1) expression, and cell hypertrophy. Furthermore, induction of p27(Kip1) by H(2)O(2) was also abolished in the presence of PD98059. Although AngII induces phosphorylation of the stress-activated p38 MAP kinase, inhibition of this enzyme with SB203580 failed to attenuate induced p27(Kip1) expression and hypertrophy. These data provide evidence that AngII- mediated oxygen stress leads to the phosphorylation of p44/42 MAP kinase in proximal tubular cells. Activation of this enzyme is essential for p27(Kip1) expression, G(1) phase arrest, and hypertrophy of proximal tubular cells. These findings may lead to new concepts concerning interference of the development of proximal tubular hypertrophy, which may eventually turn into a maladaptive process in vivo leading ultimately to tubular atrophy and tubulointerstitial fibrosis.
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PMID:Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27(Kip1): role in angiotensin II-mediated hypertrophy of proximal tubular cells. 1090 52

An NADPH oxidase complex composed of a membrane-bound flavocytochrome b558 consisting of two subunits (p22phox and gp91phox) and cytosolic activating factors (p47phox and p67phox) generates superoxide anions from oxygen in the respiratory burst of phagocytic cells. Inconsistent results have been previously obtained concerning its additional occurrence in pulmonary artery endothelial cells (PAEC), and this issue was addressed in the present study. PAEC isolated from porcine pulmonary trunk contained mRNA for p22phox and gp91phox as demonstrated by reverse transcription-polymerase chain reaction. Immunohistochemistry demonstrated cytochrome subunits, p22phox, gp91phox, p47phox, and p67phox, both in vitro in isolated PAEC and in situ in endothelial cells in tissue sections of the pulmonary trunk. Isolated PAEC generated reactive oxygen species (ROS; measured by lucigenin-induced chemiluminescence and conversion of dihydrorhodamine 123 into rhodamine 123) in response to stimulation with phorbol 12-myristate 13-acetate. This stimulated ROS production was sensitive to the flavoprotein inhibitor diphenylene-iodonium, and reduced when the superoxide scavenger superoxide dismutase was added. Chemiluminescence measurements of superoxide generation by stimulated PAEC accounted for approximately 1% of that generated by stimulated peritoneal macrophages. The data demonstrate a low-output NADPH oxidase system in porcine PAEC sharing several components with that identified in phagocytic cells.
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PMID:NADPH oxidase subunits and superoxide production in porcine pulmonary artery endothelial cells. 1095 20

A cDNA encoding an NADPH oxidase flavoprotein was isolated from the rat thyroid gland. The predicted 1517-residue polypeptide was 82.5% identical to the human THOX2/DUOX2 and 74% similar to THOX1/DUOX1. Rat THOX2 lacks a stretch of 30 residues, corresponding to one exon in the human gene sequence. THOX2 mRNA was found to be expressed in cultured FRTL-5 cells. The level of THOX2 mRNA was increased by cAMP in these cells and it was decreased in the thyroids of rats treated with the antithyroid drug methimazole, unlike the TPO and NIS mRNAs. Since it was found in the intestine, duodenum, and colon, in addition to thyroid, we suggest that it be called LNOX, the new family of long homologs of NOX flavoproteins rather than THOX and/or DUOX.
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PMID:Thyroid oxidase (THOX2) gene expression in the rat thyroid cell line FRTL-5. 1103 19

Inflammatory processes involve both synthesis of inflammatory cytokines, such as interleukin-6 (IL-6), and the activation of their distinct signaling pathways, eg, the janus kinases (JAKs) and signal transducers and activators of transcription (STAT). Superoxide (O(2)(-)) anions activate this signaling cascade, and the vasoconstrictor angiotensin II (Ang II) enhances the formation of O(2)(-) anions via the NAD(P)H oxidase system in rat aortic smooth muscle cells. Ang II activates the JAK/STAT cascade via its type 1 (AT(1)) receptor and induces synthesis and release of IL-6. Therefore, we investigated the role of O(2)(-) anions generated by the NAD(P)H oxidase system on the Ang II activation of the JAK/STAT cascade and its impact on IL-6 synthesis. Ang II stimulation of rat aortic smooth muscle cells induced a rapid increase in O(2)(-) anions determined by laser fluoroscopy, which can be abolished by DPI, a flavoprotein inhibitor. Ang II-induced phosphorylation of JAK2, STAT1alpha/ss, STAT3, and IL-6-synthesis can be abolished by DPI, as determined by immunoprecipitations and Northern blot analysis. Electroporation of neutralizing antisera targeted against p47(phox), a NAD(P)H oxidase subunit, abolished Ang II-induced JAK/STAT activation and IL-6 synthesis. Inhibition of JAK2 by its inhibitor AG490 (10 micromol/L) blocked not only JAK2 activation but also IL-6 synthesis. These results suggest that stimulation of the JAK/STAT cascade by Ang II requires O(2)(-) anions generated by the NAD(P)H oxidase system, and O(2)(-) anion-dependent activation of the JAK/STAT cascade seems to be additionally involved in Ang II-induced IL-6 synthesis. Thus, Ang II-induced inflammatory effects seem to require O(2)(-) anions generated by the NAD(P)H oxidase system.
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PMID:Role of NAD(P)H oxidase in angiotensin II-induced JAK/STAT signaling and cytokine induction. 1111 Jul 59

A growing body of evidence has suggested that a membrane-bound NADH/NADPH oxidase is the predominant source of reactive oxygen species (ROS) in vascular cells. Prior studies have used indirect assessments of superoxide including lucigenin-enhanced chemiluminescence, cytochrome c, and fluorescent dye techniques. The present study was performed to determine if NADH/NADPH oxidase function could be detected human endothelial cells using electron spin resonance. Human umbilical vein endothelial cells (HUVEC) were homogenized and fractionated into cytosolic and membrane components. Cell fractions were incubated in buffer containing either NADH or NADPH (100 microM for each) and the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO). EPR signals were obtained in a Bruker EMX spectrometer. Cytoplasmic fractions were devoid of activity. In contrast, incubation of membrane fractions with NADH produced a signal with a total peak intensity of 1,038 +/- 64, which was significantly greater than that observed with NADPH (540 +/- 101). The signal was completely inhibited by either manganese superoxide dismutase (MnSOD, 100 U/ml) or the flavoprotein inhibitor diphenylene iodinium (DPI, 100 microM). Rotenone (100 microM) did not significantly alter the signal intensity, (833 +/- 88). These data demonstrate direct evidence for a functional NADH/NADPH oxidase in human endothelial cells and show that electron spin resonance is a useful tool for study of this enzyme system.
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PMID:Evidence for a NADH/NADPH oxidase in human umbilical vein endothelial cells using electron spin resonance. 1121 82

It has been suggested that human spermatozoa contain an NADPH oxidase that could generate reactive oxygen species involved in signalling pathways to promote fertility. The proposal depends on observations that the addition of NADPH to purified human spermatozoa stimulates chemiluminescence by the superoxide (O2-) probe, lucigenin. We confirmed these observations, but demonstrated that lucigenin increases NADPH consumption by spermatozoa and stimulates artefactual O2- production via a diphenyleneiodonium (DPI) sensitive flavoprotein. In the absence of cytochrome c, DPI-inhibitable NADPH oxidation by permeabilized spermatozoa was 8 times too small to account for the rate of NADPH-stimulated cytochrome c reduction. Thus NADPH can directly reduce cytochrome c by a flavoprotein dependent mechanism making this O2- assay also unreliable in sperm suspensions. We were unable to observe O2- production by 40 x 10(6) spermatozoa/ml using electron paramagnetic resonance spectroscopy but could identify O(2)(-) generation from 2000 4beta-phorbol-12-myristate-13-actetate (PMA)-stimulated leukocytes. Using spectrophotometry, we did not detect the reduced cytochrome b(558) component of the neutrophil NADPH oxidase in human spermatozoa. No hydrogen peroxide generation was observed using a sensitive Amplex Red assay. We conclude that human spermatozoa do not possess significant NADPH oxidase activity and that the mechanism by which NADPH promotes capacitation must be re-evaluated.
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PMID:A critical investigation of NADPH oxidase activity in human spermatozoa. 1122 43


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