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)

We tested the hypothesis that angiotensin II-induced hypertension is associated with an increase in vascular .O2- production, and characterized the oxidase involved in this process. Infusion of angiotensin II (0.7 mg/kg per d) increased systolic blood pressure and doubled vascular .O2- production (assessed by lucigenin chemiluminescence), predominantly from the vascular media. NE infusion (2.75 mg/kg per d) produced a similar degree of hypertension, but did not increase vascular .O2- production. Studies using various enzyme inhibitors and vascular homogenates suggested that the predominant source of .O2- activated by angiotensin II infusion is an NADH/NADPH-dependent, membrane-bound oxidase. Angiotensin II-, but not NE-, induced hypertension was associated with impaired relaxations to acetylcholine, the calcium ionophore A23187, and nitroglycerin. These relaxations were variably corrected by treatment of vessels with liposome-encapsulated superoxide dismutase. When Losartan was administered concomitantly with angiotensin II, vascular .O2- production and relaxations were normalized, demonstrating a role for the angiotensin type-1 receptor in these processes. We conclude that forms of hypertension associated with elevated circulating levels of angiotensin II may have unique vascular effects not shared by other forms of hypertension because they increase vascular smooth muscle .O2- production via NADH/NADPH oxidase activation.
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PMID:Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. 862 76

The determinants of the regio- and stereoselective oxidation of fatty acids by cytochrome P450 BM-3 were examined by mutagenesis of residues postulated to anchor the fatty acid or to determine its active site substrate-accessible volume. R47, Y51, and F87 were targeted separately and in combination in order to assess their contributions to arachidonic, palmitoleic, and lauric acid binding affinities, catalytic rates, and regio- and stereoselective oxidation. For all three fatty acids, mutation of the anchoring residues decreased substrate binding affinity and catalytic rates and, for lauric acid, caused a significant increase in the enzyme's NADPH oxidase activity. These changes in catalytic efficiency were accompanied by decreases in the regioselectivity of oxygen insertion, suggesting an increased freedom of substrate movement within the active site of the mutant proteins. The formation of significant amounts of 19-hydroxy AA by the Y51A mutant and of 11,12-EET by the R47A/Y51A/F87V triple mutant, suggest that wild-type BM-3 shields these carbon atoms from the heme bound reactive oxygen by restricting the freedom of AA displacement along the substrate channel, and active site accessibility. These results indicate that binding affinity and catalytic turnover are fatty acid carbon-chain length dependent, and that the catalytic efficiency and the regioselectivity of fatty acid metabolism by BM-3 are determined by active site binding coordinates that control acceptor carbon orientation and proximity to the heme iron.
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PMID:Structural determinants of active site binding affinity and metabolism by cytochrome P450 BM-3. 1136 73

The hemodynamic and anti-ischemic effects of nitroglycerin (NTG) are rapidly blunted due to the development of nitrate tolerance. With initiation of nitroglycerin therapy one can detect neurohormonal activation and signs for intravascular volume expansion. These so called pseudotolerance mechanisms may compromise nitroglycerin's vasodilatory effects. Long-term treatment with nitroglycerin is also associated with a decreased responsiveness of the vasculature to nitroglycerin's vasorelaxant potency suggesting changes in intrinsic mechanisms of the tolerant vasculature itself may also contribute to tolerance. More recent experimental work defined new mechanisms of tolerance such as increased vascular superoxide production and increased sensitivity to vasoconstrictors secondary to an activation of the intracellular second messenger protein kinase C. As potential superoxide producing enzymes, the NADPH oxidase and the nitric oxide synthase have been identified. Nitroglycerin-induced stimulation of oxygen-derived free radicals together with NO derived from nitroglycerin may lead to the formation of peroxynitrite, which may be responsible for the development of tolerance as well as for the development of cross tolerance to endothelium-dependent vasodilators. The oxidative stress concept of tolerance and cross tolerance may explain why radical scavengers such as vitamin C or substances which reduce oxidative stress, such as ACE-inhibitors, AT1 receptor blockers or folic acid, are able to beneficially influence both tolerance and nitroglycerin-induced endothelial dysfunction. New aspects concerning the role of oxidative stress in nitrate tolerance and nitrate induced endothelial dysfunction and the consequences for the NO/cyclicGMP downstream target, the cGMP-dependent protein kinase will be discussed.
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PMID:Mechanisms underlying nitrate-induced endothelial dysfunction: insight from experimental and clinical studies. 1237 19

Anionic amphiphiles such as sodium- and lithium dodecyl sulfate (SDS, LDS), or arachidonate (AA) initiate NADPH oxidase and proton channel activation in cell-free systems and intact neutrophils. To investigate whether these amphiphiles exert allosteric effects on cytochrome b, trisulfopyrenyl-labeled wheat germ agglutinin (Cascade Blue-wheat germ agglutinin, CCB-WGA) was used as an extrinsic fluorescence donor for resonance energy transfer (RET) to the intrinsic heme acceptors of detergent-solubilized cytochrome b. In solution, cytochrome b complexed with the CCB-WGA causing a rapid, saturable, carbohydrate-dependent quenching of up to approximately 55% of the steady-state fluorescence. Subsequent additions of SDS, LDS, or AA to typical cell-free oxidase assay concentrations completely relaxed the fluorescence quenching. The relaxation effects were specific, and not caused by dissociation of the CCB-WGA-cytochrome b complex or alterations in the spectral properties of the chromophores. In contrast, addition of the oxidase antagonist, arachidonate methyl ester, caused an opposite effect and was able to partially reverse the activator-induced relaxation. We conclude that the activators induce a cytochrome b conformation wherein the proximity or orientation between the hemes and the extrinsic CCB fluorescence donors has undergone a significant change. These events may be linked to NADPH oxidase assembly and activation or proton channel induction.
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PMID:Structural changes are induced in human neutrophil cytochrome b by NADPH oxidase activators, LDS, SDS, and arachidonate: intermolecular resonance energy transfer between trisulfopyrenyl-wheat germ agglutinin and cytochrome b(558). 1248 56

The integral membrane protein flavocytochrome b (Cyt b) comprises the catalytic core of the human phagocyte NADPH oxidase complex and serves to initiate a cascade of reactive oxygen species that participate in the elimination of infectious agents. Superoxide production by the NADPH oxidase complex has been shown to be specifically regulated by the enzymatic generation of lipid second messengers following phagocyte activation. In the present study, a Cyt b-specific monoclonal antibody (mAb 44.1) was labeled with Cascade Blue (CCB) and used in resonance energy transfer (RET) studies probing the effects of a panel of lipid species on the structure of Cyt b. The binding of CCB-mAb 44.1 to immunoaffinity-purified Cyt b was both highly specific and resulted in significant quenching of the steady state donor fluorescence. Titration of the CCB-mAb 44.1:Cyt b complex with the anionic amphiphile lithium dodecyl sulfate (LDS) resulted in a saturable relaxation of fluorescence quenching due to conformational changes in Cyt b at concentrations of the amphiphile required for maximum rates of superoxide production by Cyt b in cell-free assays. Similar results were observed for the anionic amphiphile arachidonic acid (AA), although no relaxation of fluorescence quenching was observed for arachidonate methyl ester (AA-ME). Saturable relaxation of fluorescence quenching was also observed with the anionic, 18:1 phospholipids phosphatidic acid (DOPA) and phosphatidylserine (DOPS), while no relaxation was observed upon addition of the neutral 18:1 lipids phosphatidylcholine (DOPC), phosphatidylethanolamine (DOPE) or diacylglycerol (DAG) at similar levels. Further examination of a variety of phosphatidic acid (PA) species demonstrated DOPA to both potently induce conformational changes in Cyt b and to cause more dramatic conformational changes than PA species with shorter, saturated acyl chains. The data presented in this study support the hypothesis that second messenger lipids, such as AA and PA, directly bind to flavocytochrome b and modulate conformational states relevant to the activation of superoxide production.
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PMID:Anionic amphiphile and phospholipid-induced conformational changes in human neutrophil flavocytochrome b observed by fluorescence resonance energy transfer. 1515 22

Angiotensin II (AngII), acting through angiotensin type 1 (AT1) receptors, exerts powerful effects on central autonomic networks regulating cardiovascular homeostasis and fluid balance; however, the mechanisms of AngII signaling in functionally defined central autonomic neurons have not been fully elucidated. In vascular cells, reactive oxygen species (ROS) generated by the enzyme NADPH oxidase play a major role in AngII signaling. Thus, we sought to determine whether NADPH oxidase is present in central autonomic neurons and, if so, whether NADPH oxidase-derived ROS are involved in the effects of AngII on these neurons. The present studies focused on the intermediate dorsomedial nucleus of the solitary tract (dmNTS) because this region receives autonomic afferents via the vagus nerve and is an important site of AngII actions. Using double-label immunoelectron microscopy, we found that the essential NADPH oxidase subunit gp91phox is present in somatodendric and axonal profiles containing AT1 receptors. The gp91phox-labeled dendrites received inputs from large axon terminals resembling vagal afferents. In parallel experiments using patch clamp of dissociated NTS neurons anterogradely labeled via the vagus, we found that AngII potentiates the L-type Ca2+ currents, an effect mediated by AT1 receptors and abolished by the ROS scavenger Mn(III) tetrakis (4-benzoic acid) porphyrin chloride. The NADPH oxidase assembly inhibitor apocynin and the peptide inhibitor gp91phox docking sequence, but not its scrambled version, also blocked the potentiation. The results provide evidence that NADPH oxidase-derived ROS are involved in the effects of AngII on Ca2+ influx in NTS neurons receiving vagal afferents and support the notion that ROS are important signaling molecules in central autonomic networks.
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PMID:NADPH oxidase contributes to angiotensin II signaling in the nucleus tractus solitarius. 1520 24

The integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the human phagocyte NADPH oxidase, an enzyme complex that initiates a cascade of reactive oxygen species important in the elimination of infectious agents. This study reports the generation and characterization of six mAbs (NS1, NS2, NS5, CS6, CS8, and CS9) that recognize the p22(phox) subunit of the Cyt b heterodimer. Each of the mAbs specifically detected p22(phox) by Western blot analysis but did not react with intact neutrophils in FACS studies. Phage display mapping identified core epitope regions recognized by mAbs NS2, NS5, CS6, CS8, and CS9. Fluorescence resonance energy transfer experiments indicated that mAbs CS6 and CS8 efficiently compete with Cascade Blue-labeled mAb 44.1 (a previously characterized, p22(phox)-specific mAb) for binding to Cyt b, supporting phage display results suggesting that all three Abs recognize a common region of p22(phox). Energy transfer experiments also suggested the spatial proximity of the mAb CS9 and mAb NS1 binding sites to the mAb 44.1 epitope, while indicating a more distant proximity between the mAb NS5 and mAb 44.1 epitopes. Cell-free oxidase assays demonstrated the ability of mAb CS9 to markedly inhibit superoxide production in a concentration-dependent manner, with more moderate levels of inhibition observed for mAbs NS1, NS5, CS6, and CS8. A combination of computational predictions, available experimental data, and results obtained with the mAbs reported in this study was used to generate a novel topology model of p22(phox).
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PMID:Site-specific inhibitors of NADPH oxidase activity and structural probes of flavocytochrome b: characterization of six monoclonal antibodies to the p22phox subunit. 1558 59

The nox2-dependent NADPH oxidase was shown to be a major superoxide source in vascular disease, including diabetes. Smooth muscle cells of large arteries lack the phagocytic gp91phox subunit of the enzyme; however, two homologues have been identified in these cells, nox1 and nox4. It remained to be established whether also increases in protein levels of the nonphagocytic NADPH oxidase contribute to increased superoxide formation in diabetic vessels. To investigate changes in the expression of these homologues, we measured their expression in aortic vessels of type I diabetic rats. Eight weeks after streptozotocin treatment, we found a doubling in nox1 protein expression, while the expression of nox4 remained unchanged. This was associated with a significant increase in the NADPH oxidase activity in membrane fractions of diabetic heart and aortic tissue. Furthermore, we observed a decreased sensitivity of diabetic vessels to acetylcholine and nitroglycerin and a decrease in both acetylcholine-stimulated NO production and phosphorylation of VASP, despite an increase in endothelial NO synthase (NOSIII) expression. In addition, xanthine oxidase activity was markedly increased in plasma and 100,000 g supernatant of cardiac tissue of diabetic rats, while myocardial mitochondrial superoxide formation was only weakly enhanced. We conclude that in addition to phagocytic NADPH oxidase, also nonphagocytic, vascular NADPH oxidase subunit nox1, uncoupled NOSIII, and plasma xanthine oxidase contribute to endothelial dysfunction in the setting of diabetes mellitus.
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PMID:Differential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4. 1599 37

Nitrate tolerance is associated with an enhanced superoxide anion production and can be attenuated by statins, which interact with the 2 main [eNOS and NAD(P)H oxidase] pathways involved in producing this oxidative stress. Three groups of normocholesterolemic rats were treated: group 1 received rosuvastatin (10 mg/kg/d PO) for 5 weeks and in the last 3 days cotreatment with nitroglycerin (NTG 50 mg/kg/d, subcutaneous injections BID); group 2 received only NTG (50 mg/kg/d BID for the last 3 days); and group 3 served as control. Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM to 0.1 mM) were determined on phenylephrine-preconstricted rings and O2 production (RLU/10 s/mg dry weight) was assessed by lucigenin and the luminol analogue (L-012) chemiluminescence technique. In group 2 (NTG), the concentration-response curves to NTG were significantly shifted to the right: the pD2 (-log NTG concentration evoking a half-maximal relaxation) was 6.75+/-0.06 (n=7) versus 7.75+/-0.07 (n=7) in group 3 (not exposed to NTG, P<0.05); O2 production was enhanced (10,060+/-1,205, n=7 versus 5,235+/-1,052, n=7; P<0.05). In contrast, in group 1, the rightward shift was attenuated: pD2 value was 7.20+/-0.10 (n=8), P<0.05 versus group 2; O2 production was decreased (5911+/-663; n=9, P<0.05 versus group 2). In addition, before NTG exposure, rosuvastatin treatment decreased p22phox [the essential NAD(P)H oxidase subunit] abundance in the aortic wall and decreased NAD(P)H oxidase activity. In contrast, this treatment did not alter either eNOS abundance or the basal release of endothelium-derived NO. Interestingly, in vivo treatment with apocynin, an NAD(P)H oxidase inhibitor, produced a protection similar to that with rosuvastatin. Long-term rosuvastatin treatment protects against nitrate tolerance in the rat aorta by counteracting NTG-induced increase in O2 production. This protection seems to involve a direct interaction with the NAD(P)H oxidase pathway rather than an up-regulation of the eNOS pathway.
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PMID:Rosuvastatin treatment protects against nitrate-induced oxidative stress. 1604 29

During the last century, nitroglycerin has been the most commonly used antiischemic and antianginal agent. Unfortunately, after continuous application, its therapeutic efficacy rapidly vanishes. Neurohormonal activation of vasoconstrictor signals and intravascular volume expansion constitute early counter-regulatory responses (pseudotolerance), whereas long-term treatment induces intrinsic vascular changes, eg, a loss of nitrovasodilator-responsiveness (vascular tolerance). This is caused by increased vascular superoxide production and a supersensitivity to vasoconstrictors secondary to a tonic activation of protein kinase C. NADPH oxidase(s) and uncoupled endothelial nitric oxide synthase have been proposed as superoxide sources. Superoxide and vascular NO rapidly form peroxynitrite, which aggravates tolerance by promoting NO synthase uncoupling and inhibition of soluble guanylyl cyclase and prostacyclin synthase. This oxidative stress concept may explain why radical scavengers and substances, which reduce oxidative stress indirectly, are able to relieve tolerance and endothelial dysfunction. Recent work has defined a new tolerance mechanism, ie, an inhibition of mitochondrial aldehyde dehydrogenase, the enzyme that accomplishes bioactivation of nitroglycerin, and has identified mitochondria as an additional source of reactive oxygen species. Nitroglycerin-induced reactive oxygen species inhibit the bioactivation of nitroglycerin by thiol oxidation of aldehyde dehydrogenase. Both mechanisms, increased oxidative stress and impaired bioactivation of nitroglycerin, can be joined to provide a new concept for nitroglycerin tolerance and cross-tolerance. The consequences of these processes for the nitroglycerin downstream targets soluble guanylyl cyclase, cGMP-dependent protein kinase, cGMP-degrading phosphodiesterases, and toxic side effects contributing to endothelial dysfunction, such as inhibition of prostacyclin synthase, are discussed in this review.
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PMID:Explaining the phenomenon of nitrate tolerance. 1619 86


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