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)

It is well established that the central cardiovascular effects of angiotensin II (Ang II) involve superoxide production. However, the intracellular mechanism by which reactive oxygen species (ROS) signaling regulates neuronal Ang II actions remains to be elucidated. In the present study, we have used neuronal cells in primary cultures from the hypothalamus and brain stem areas to study the role of ROS on the cellular actions of Ang II. Ang II increases neuronal firing rate, an effect mediated by the AT(1) receptor subtype and involving inhibition of the delayed rectifier potassium current (I(Kv)). This increase in neuronal activity was associated with increases in NADPH oxidase activity and ROS levels within neurons, the latter evidenced by an increase in ethidium fluorescence. The increases in NADPH oxidase activity and ethidium fluorescence were blocked by either the AT(1) receptor antagonist losartan or by the selective NAD(P)H oxidase inhibitor gp91ds-tat. Extracellular application of the ROS scavenger, Tempol, attenuated the Ang II-induced increase in neuronal firing rate by 70%. In addition, gp91ds-tat treatment resulted in a 50% inhibition of Ang II-induced increase in firing rate. In contrast, the ROS generator Xanthine-Xanthine oxidase significantly increased neuronal firing rate. Finally, Ang II inhibited neuronal I(Kv,) and this inhibition was abolished by gp91ds-tat treatment. These observations demonstrate, for the first time, that Ang II regulates neuronal activity via a series of events that includes ROS generation and inhibition of I(Kv). This signaling seems to be a critical cellular event in central Ang II regulation of cardiovascular function.
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PMID:NAD(P)H oxidase inhibition attenuates neuronal chronotropic actions of angiotensin II. 1574 42

Dihydrocalcein (H2-calcein) is recommended as a superior probe for intracellular radical (ROS) detection as different to dichlorodihydrofluorescein (H2-DCF), its oxidation product calcein is thought not to leak out of cells. We determined whether H2-calcein is a useful tool to measure ROS in vascular smooth muscle cells. In vitro, both compounds were oxidized by peroxynitrite, hydroxyl radicals and peroxidase, but not hydrogen peroxide or nitric oxide. The intracellular half-life of calcein was several hours whereas that of DCF was approximately 5 min. Intracellular ROS, as generated by the angiotensin II (Ang II)-activated NADPH oxidase, did not increase the oxidation of H2-calcein but increased the oxidation of H2-DCF by approximately 50%. Similar changes were detected using electron spin resonance spectroscopy. Inhibition of the NADPH oxidase using gp91ds-tat prevented the Ang II-induced increase in DCF fluorescence, without affecting cells loaded with H2-calcein. Diphenylene iodonium (DPI), which inhibits all flavin-dependent enzymes, including those in the respiratory chain, had little effect on the basal but prevented the Ang II-induced oxidation of H2-DCF. In contrast, DPI inhibited H2-calcein oxidation in non-stimulated cells by almost 50%. Blockade of respiratory chain complex I inhibited H2-calcein oxidation, whereas inhibitors of complex III were without effect. Calcein accumulated in the mitochondria, whereas DCF was localized in the cytoplasm. In submitochondrial particles, H2-calcein, but not H2-DCF inhibited complex I activity. These observations indicate that H2-DCF is an indicator for intracellular ROS, whereas the oxidation of H2-calcein most likely occurs as a consequence of direct electron transfer to mitochondrial complex I.
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PMID:Analysis of dichlorodihydrofluorescein and dihydrocalcein as probes for the detection of intracellular reactive oxygen species. 1576 50

In this study we identified the involvement of reactive oxygen species (ROS) in signaling and biological effects of the angiopoietin-1 (Ang-1)/tie-2 receptor pathway. Exposure of human umbilical vein endothelial cells to Ang-1 (50 ng/ml) induced rapid and transient production of ROS, particularly superoxide anions. ROS production was attenuated by preincubation with a peptide (gp91ds-tat) that inhibits the association of the gp91(phox) subunit with the p47(phox) subunit of NADPH oxidase and by the expression of a dominant-negative form of Rac-1 (Rac1N17). These results suggest that ROS production in response to Ang-1 exposure originates mainly from a Rac-1-dependent NADPH oxidase. Overexpression of antioxidants (superoxide dismutase and catalase) and Rac1N17, as well as preincubation with selective inhibitors of NADPH oxidase augmented basal p38 phosphorylation, inhibited Ang-1-induced PAK-1 phosphorylation and potentiated Ang-1-induced Erk1/2 phosphorylation but had no influence on AKT and SAPK/JNK phosphorylation by Ang-1. Exposure to Ang-1 (100 ng/ml) for 5 h induced a threefold increase in endothelial cell migration, a response that was strongly inhibited by overexpression of antioxidants, Rac1N17, and selective NADPH oxidase inhibitors. We conclude that activation of tie-2 receptors by Ang-1 triggers the production of ROS through activation of NADPH oxidase and that ROS generation by Ang-1 promotes endothelial cell migration while negatively regulating Erk1/2 phosphorylation.
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PMID:Roles of reactive oxygen species in angiopoietin-1/tie-2 receptor signaling. 1604 36

Because oxidative stress has been strongly implicated in up-regulation of vascular endothelial growth factor (VEGF) expression in ischemic retinopathy, we evaluated the role of NAD(P)H oxidase in causing VEGF overexpression and retinal neovascularization. Dihydroethidium imaging analyses showed increased superoxide formation in areas of retinal neovascularization associated with relative retinal hypoxia in a mouse model for oxygen-induced retinopathy. The effect of hypoxia in stimulating superoxide formation in retinal vascular endothelial cells was confirmed by in vitro chemiluminescence assays. The superoxide formation was blocked by specific inhibitors of NAD(P)H oxidase activity (apocynin, gp91ds-tat) indicating that NAD(P)H oxidase is a major source of superoxide formation. Western blot and immunolocalization analyses showed that retinal ischemia increased expression of the NAD(P)H oxidase catalytic subunit gp91phox, which localized primarily within vascular endothelial cells. Treatment of mice with apocynin blocked ischemia-induced increases in oxidative stress, normalized VEGF expression, and prevented retinal neovascularization. Apocynin and gp91ds-tat also blocked the action of hypoxia in causing increased VEGF expression in vitro, confirming the specific role of NAD(P)H oxidase in hypoxia-induced increases in VEGF expression. In conclusion, NAD(P)H oxidase activity is required for hypoxia-stimulated increases in VEGF expression and retinal neovascularization. Inhibition of NAD(P)H oxidase offers a new therapeutic target for the treatment of retinopathy.
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PMID:Inhibition of NAD(P)H oxidase activity blocks vascular endothelial growth factor overexpression and neovascularization during ischemic retinopathy. 1604 43

Recent studies suggest that the superoxide generating enzyme NADPH oxidase may play a functional role in regulating cerebral vascular tone. We tested whether the activity, function, and expression of NADPH oxidase differs between rat cerebral and systemic arteries. Superoxide production by basilar (BA), middle cerebral (MCA), carotid (CA), renal (RA), and mesenteric (MA) arteries and aorta (AO) was measured using lucigenin-enhanced chemiluminescence. Superoxide production from NADPH oxidase was localized and semiquantified using dihydroethidium. Vascular functional responses were assessed in a myograph or organ bath. Vascular Nox4 protein expression was measured using Western blotting. Superoxide production (basal or in response to NADPH or angiotensin II) in the intracranial arteries, BA, and MCA was 10- to 100-fold greater than in AO, CA, RA, or MA. Similar results were found using either intact vessels or arterial homogenates, and were associated with 10-fold greater expression of Nox4 in the BA versus AO, CA, and MA. Superoxide production was attenuated by the NADPH oxidase inhibitors, diphenyleneiodonium, apocynin, and gp91ds-tat. NADPH and H2O2 were strong relaxing stimuli in the BA, where the H2O2 scavenger catalase, as well as apocynin, attenuated these relaxations and also augmented contractions to angiotensin II. NADPH oxidase activity is markedly higher in intracranial versus systemic arteries, in association with higher Nox4 expression. In cerebral arteries, endogenous H2O2 derived from NADPH oxidase activation appears to cause relaxation and is able to offset angiotensin II-induced constriction. These data are consistent with the concept that NADPH oxidase-derived reactive oxygen species modulate cerebral vascular tone under physiological conditions.
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PMID:NADPH oxidase activity and function are profoundly greater in cerebral versus systemic arteries. 1621 May 46

Oxidative stress occurs in remote liver injury, but the origin of the oxidant generation has yet to be thoroughly delineated. Some reports suggest that the source of the distant oxidative stress originates from the site of initial insult [i.e., xanthine oxidase (XO)]; however, it could also be derived from sources such as phagocytic and/or vascular NAD(P)H oxidase (Nox) enzymes. With a murine model of bilateral hindlimb ischemia-reperfusion, we describe here a mechanism for Nox-dependent oxidant production that contributes, at least in part, to remote hepatic parenchymal injury and sinusoidal endothelial cell (SEC) dysfunction. To determine whether Nox enzymes were the source of oxidants, mice were treated immediately after the onset of hindlimb ischemia with specific inhibitors to XO (50 mg/kg ip allopurinol) or Nox (10 mg/kg ip gp91ds-tat and 3 mg/kg ip apocynin). After 1 h of ischemia, hindlimbs were reperfused for either 3 or 6 h. Inhibition of XO failed to provide any improvement in parenchymal injury, SEC dysfunction, neutrophil accumulation, or microvascular dysfunction. In contrast, the inhibition of Nox enzymes prevented the progression (6 h) of parenchymal injury, significantly protected against SEC dysfunction, and completely prevented signs of neutrophil-derived oxidant stress. At the same time, however, inhibition of Nox failed to protect against the early parenchymal injury and microvascular dysfunction at 3 h of reperfusion. These data confirm that microvascular perfusion deficits are not essential for the pathogenesis of remote hepatic parenchymal injury. The data also suggest that Nox enzymes, not XO, are involved in the progression of compromised hepatic parenchymal and endothelial integrity during a systemic inflammatory response.
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PMID:NAD(P)H oxidase contributes to the progression of remote hepatic parenchymal injury and endothelial dysfunction, but not microvascular perfusion deficits. 1633 98

Endothelial dysfunction (ED) complicates hypertension and is a precursor of atherosclerosis. Reduced NO bioactivity, because of increased reduced NAD(P)H oxidase-derived reactive oxygen species (ROS), plays a critical role in ED. gp91phox, predominantly expressed in the endothelium and adventitia, is a subunit of NAD(P)H oxidase important for its activation in response to angiotensin (Ang) II. Human atherosclerotic plaques are heavy laden with gp91phox. We have shown that in Dahl salt-sensitive (DS) rats, a paradigm of low renin salt-sensitive (SS) hypertension in humans, Ang II receptor blockade normalizes ROS production and endothelium-dependent relaxation (EDR) without significantly affecting systolic blood pressure (SBP). To additionally elucidate the mechanisms involved in the functional association of Ang II in SS hypertension, we administered a cell-permeable inhibitor of the assembly of p47phox with gp91phox in NAD(P)H oxidase, gp91ds-tat (10 mg/kg body weight, 3 weeks by minipump), to DS rats fed a 4% salt diet. Control rats received either vehicle or an inactive scramb-tat peptide. Vehicle-treated DS developed hypertension (SBP 168+/-5 mm Hg), left ventricular hypertrophy (LVH), proteinuria, impaired EDR, and increased aortic ROS production (superoxide 115% and peroxynitrite 157%) and expression of the proatherogenic molecules LOX-1 (130%) and MCP-1 (166%). gp91ds-tat, but not scramb-tat, normalized ROS and EDR, as well as LOX-1 and MCP-1, despite nonsignificant effects on SBP (159+/-5 mm Hg; P>0.05), left ventricular hypertrophy, and proteinuria. Our findings support the notion that in SS hypertension, activation of NAD(P)H oxidase promotes ED and atherogenesis via decreased nitric oxide bioactivity and increased LOX-1 and MCP-1, independent of blood pressure.
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PMID:Reduced NAD(P)H oxidase in low renin hypertension: link among angiotensin II, atherogenesis, and blood pressure. 1634 66

Myogenic vasoconstriction, an intrinsic response to elevated transmural pressure (TMP), requires the activation of sphingosine kinase (Sk1) and the generation of reactive oxygen species (ROS). We hypothesized that pressure-induced Sk1 signaling and ROS generation are functionally linked. Using a model of cannulated resistance arteries isolated from the hamster gracilis muscle, we monitored vessel diameter and smooth muscle cell (SMC) Ca2+i (Fura-2) or ROS production (dichlorodihydrofluorescein). Elevation of TMP stimulated the translocation of a GFP-tagged Sk1 fusion protein from the cytosol to the plasma membrane, indicative of enzymatic activation. Concurrently, elevation of TMP initiated a rapid and transient production of ROS, which was enhanced by expression of wild-type Sk1 (hSk(wt)) and inhibited by its dominant-negative mutant (hSk(G82D)). Exogenous sphingosine-1-phosphate (S1P) also stimulated ROS generation is isolated vessels. Chemical (1 micromol/L DPI), peptide (gp91ds-tat/gp91ds), and genetic (N17Rac) inhibition strategies indicated that NADPH oxidase was the source of the pressure-induced ROS. NADPH oxidase inhibition attenuated myogenic vasoconstriction and reduced the apparent Ca2+ sensitivity of the SMC contractile apparatus, without affecting Ca2+-independent, RhoA-mediated vasoconstriction in response to exogenous S1P. Our results indicate a mandatory role for Sk1/S1P in mediating pressure-induced, NADPH oxidase-derived ROS formation. In turn, ROS generation appears to increase Ca2+ sensitivity, necessary for full myogenic vasoconstriction.
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PMID:Sphingosine kinase functionally links elevated transmural pressure and increased reactive oxygen species formation in resistance arteries. 1647 2

Stretch of beta1 integrins activates an outwardly rectifying, tamoxifen-sensitive Cl(-) current (Cl(-) SAC) via AT1 receptors, NADPH oxidase, and reactive oxygen species, and Cl(-) SAC resembles the volume-sensitive Cl(-) current (I(Cl,swell)). Epidermal growth factor receptor (EGFR) kinase undergoes transactivation upon stretch, integrin engagement, and AT1 receptor activation and, in turn, stimulates NADPH oxidase. Therefore, we tested whether Cl(-) SAC is regulated by EGFR kinase signaling and is volume sensitive. Paramagnetic beads coated with mAb for beta1 integrin were attached to myocytes and pulled with an electromagnet. Stretch activated a Cl(-) SAC that was 1.13 +/- 0.10 pA/pF at +40 mV. AG1478 (10 muM), an EGFR kinase blocker, inhibited 93 +/- 13% of Cl(-) SAC, and intracellular pretreatment with 1 muM AG1478 markedly suppressed Cl(-) SAC activation. EGF (3.3 nM) directly activated an outwardly rectifying Cl(-) current (0.81 +/- 0.05 pA/pF at +40 mV) that was fully blocked by 10 muM tamoxifen, an I(Cl,swell) blocker. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase. Wortmannin (500 nM) and LY294002 (100 microM), blockers of PI-3K, inhibited Cl(-) SAC by 67 +/- 6% and 91 +/- 25% respectively, and the EGF-induced Cl(-) current also was fully blocked by LY294002. Furthermore, gp91ds-tat (500 nM), a cell-permeable, chimeric peptide that specifically blocks NADPH oxidase assembly, profoundly inhibited the EGF-induced Cl(-) current. Inactive permeant and active impermeant control peptides had no effect. Myocyte shrinkage with hyperosmotic bathing media inhibited the Cl(-) SAC and EGF-induced Cl(-) current by 88 +/- 9% and 127 +/- 11%, respectively. These results suggest that beta1 integrin stretch activates Cl(-) SAC via EGFR, PI-3K, and NADPH oxidase, and that both the Cl(-) SAC and the EGF-induced Cl(-) currents are likely to be the volume-sensitive Cl(-) current, I(Cl,swell).
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PMID:EGFR kinase regulates volume-sensitive chloride current elicited by integrin stretch via PI-3K and NADPH oxidase in ventricular myocytes. 1650 46

In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation drives part of the excessive superoxide production implicated in the pathogenesis of heart failure. Pacing-induced heart failure was performed in eight chronically instrumented dogs. Seven normal dogs served as control. End-stage failure occurred after 28 +/- 1 days of pacing, when left ventricular end-diastolic pressure reached 25 mm Hg. In left ventricular tissue homogenates, spontaneous superoxide generation measured by lucigenin (5 microM) chemiluminescence was markedly increased in heart failure (1338 +/- 419 vs. 419 +/- 102 AU/mg protein, P < 0.05), as were NADPH levels (15.4 +/- 1.5 vs. 7.5 +/- 1.5 micromol/gww, P < 0.05). Superoxide production was further stimulated by the addition of NADPH. The NADPH oxidase inhibitor gp91(ds-tat) (50 microM) and the NO synthase inhibitor L-NAME (1 mM) both significantly lowered superoxide generation in failing heart homogenates by 80% and 76%, respectively. G6PD was upregulated and its activity higher in heart failure compared to control (0.61 +/- 0.10 vs. 0.24 +/- 0.03 nmol/min/mg protein, P < 0.05), while superoxide production decreased to normal levels in the presence of the G6PD inhibitor 6-aminonicotinamide. We conclude that the activation of myocardial G6PD is a novel mechanism that enhances NADPH availability and fuels superoxide-generating enzymes in heart failure.
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PMID:Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart. 1682 94


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