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)

Salicylates and nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cancer cells, including those of colon, prostate, breast, and leukemia. We examined the effects of sodium salicylate (NaSal) on reactive oxygen species (ROS) production and the association of these effects with apoptotic tumor cell death. We demonstrate that NaSal mediates ROS production followed by a decrease in mitochondrial membrane potential (deltapsi(m)), release of cytochrome c, and activation of caspase-9 and caspase-3. However, expression of Bcl-2 or Bcl-x(L) prevents ROS production and subsequent loss of deltapsi(m), thereby inhibiting apoptotic cell death. The presence of ROS scavengers and an inhibitor of NADPH oxidase or expression of a dominant negative form of Rac1 blocks ROS production, deltapsi(m) collapse, and the subsequent activation of caspases. These observations indicate that NaSal mediates ROS production critical in the triggering of apoptotic tumor cell death through a Rac1-NADPH oxidase-dependent pathway. Our data collectively imply that NaSal-induced ROS are key mediators of deltapsi(m) collapse, which leads to the release of cytochrome c followed by caspase activation, culminating in tumor apoptosis.
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PMID:Molecular ordering of ROS production, mitochondrial changes, and caspase activation during sodium salicylate-induced apoptosis. 1256 69

The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.
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PMID:DPI induces mitochondrial superoxide-mediated apoptosis. 1256 72

Therapy with high oxygen concentrations (hyperoxia) is often necessary to treat patients with respiratory failure. However, hyperoxia may exacerbate the development of acute lung injury, perhaps by increasing lung epithelial cell death. Therefore, interrupting lung epithelial cell death is an important protective and therapeutic strategy. In the present study, hyperoxia (95% O(2)) results in murine lung epithelium cell death by DNA-laddering, terminal deoxynucleotidyltransferase dUTP nick end labeling, and Annexin V-fluorescein isothiocyanate flow cytometry assay. We show that hyperoxia increases superoxide production, as assessed by nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity and flow cytometric assay, and increases phospho-extracellular signal-regulated kinase (ERK)1/2 by Western blot analysis. These processes are inhibited by a reactive oxygen species inhibitor, diphenylene iodonium (DPI), and by an inhibitor of the mitogen-activated protein (MAP) or ERK kinase (MEK)/ERK1/2 pathway, PD98059. ERK1/2 activation in hyperoxia is also inhibited by DPI. Hyperoxia-induced cell death is associated with cytochrome c release, subsequent caspase 9 and 3 activation, and poly (ADP-ribosyl) polymerase cleavage, which can all be suppressed by DPI and PD98059. However, the broad caspase inhibitor z-VAD-FMK protects cells from death without affecting superoxide generation and ERK1/2 activation. Taken together, our data suggest that hyperoxia, by virtue of activating NADPH oxidase, generates reactive oxygen species (ROS), which mediates cell death of lung epithelium via ERK1/2 MAPK activation, and functions upstream of caspase activation in lung epithelial cells.
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PMID:Reactive oxygen species and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase mediate hyperoxia-induced cell death in lung epithelium. 1259 56

Eosinophil respiratory burst is an important event in asthma and related inflammatory disorders. However, little is known concerning activation of the respiratory burst NADPH oxidase in human eosinophils. Conversely, neutrophils are known to assemble NADPH oxidase in intracellular and plasma membranes. We hypothesized that eosinophils and neutrophils translocate NADPH oxidase to distinct intracellular locations, consistent with their respective functions in O(2)(-)-mediated cytotoxicity. PMA-induced O(2)(-) release assayed by cytochrome c was 3.4-fold higher in atopic human eosinophils than in neutrophils, although membrane-permeable dihydrorhodamine-123 showed similar amounts of release. Eosinophil O(2)(-) release was dependent on Rac, in that it was 54% inhibited by Clostridium difficile toxin B (400-800 ng/ml). In eosinophils stimulated with PMA, a pronounced shift of cytosolic Rac to p22(phox)-positive plasma membrane was observed by confocal microscopy, whereas neutrophils directed Rac2 mainly to intracellular sites coexpressing p22(phox). Similarly, ex vivo sputum eosinophils from asthmatic subjects exhibited predominantly plasma membrane-associated immunoreactivity for Rac, whereas sputum neutrophils exhibited cytoplasmic Rac2 staining. Thus, activated sputum eosinophils, rather than neutrophils, may contribute significantly to the pathogenesis of asthma by extracellular release of tissue-damaging O(2)(-). Our findings suggest that the differential modes of NADPH oxidase assembly in these cells may have important implications for oxidant-mediated tissue injury.
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PMID:Divergence of mechanisms regulating respiratory burst in blood and sputum eosinophils and neutrophils from atopic subjects. 1259 96

The phagocyte NADPH oxidase helps kill pathogens by producing superoxide anion, O2-. This enzyme is electrogenic because it translocates electrons across the membrane, generating an electron current, Ie. Using the permeabilized patch voltage-clamp technique, we studied the temperature dependence of Ie in human eosinophils stimulated by phorbol myristate acetate (PMA) from room temperature to >37 degrees C. For comparison, NADPH oxidase activity was assessed by cytochrome c reduction. The intrinsic temperature dependence of the assembled, functioning NADPH oxidase complex measured during rapid temperature increases to 37 degrees C was surprisingly weak: the Arrhenius activation energy Ea was only 14 kcal mol(-1) (Q10, 2.2). In contrast, steady-state NADPH oxidase activity was strongly temperature dependent at 20-30 degrees C, with Ea 25.1 kcal mol(-1) (Q10, 4.2). The maximum Ie measured at 34 degrees C was -30.5 pA. Above 30 degrees C, the temperature dependence of both Ie and O2- production was less pronounced. Above 37 degrees C, Ie was inhibited reversibly. After rapid temperature increases, a secondary increase in Ie ensued, suggesting that high temperature promotes assembly of additional NADPH oxidase complexes. Evidently, about twice as many NADPH oxidase complexes are active near 37 degrees C than at 20 degrees C. Thus, the higher Q10 of steady-state Ie reflects both increased activity of each NADPH oxidase complex and preferential assembly of NADPH oxidase complexes at high temperature. In summary, NADPH oxidase activity in intact human eosinophils is maximal precisely at 37 degrees C.
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PMID:Temperature dependence of NADPH oxidase in human eosinophils. 1275 16

A great number of drugs, toxicants, and growth factors induce the generation of intermediary reactive oxygen species (ROS). The human promyelocytic leukemia HL60 cell line differentiated along the macrophage or neutrophil lineage is a model system that is frequently used for the generation of ROS by various agents. As a primary source of ROS the superoxide anion produced by an enzymatic complex, NADPH oxidase, is well established. The present study shows that nondifferentiated HL60 cells contain NADPH oxidase and can be used as a model for the assessment of oxidant as well as antioxidant compounds. The expression of the multicomponent NADPH oxidase was demonstrated in nondifferentiated HL60 cells at the molecular level by detection of the mRNAs of the components gp91phox, p47phox, and p67phox as well as functionally by phorbol 12-myristate-13-acetate (PMA)-stimulated generation of superoxide, which was susceptible to inhibition by diphenyleneiodonium. The functional assay was performed using the cells in a log growth phase by adapting a standard microplate assay based on the classic superoxide dismutase-inhibitable reduction of cytochrome c. Validation of the microplate assay was carried out both with nonadherent differentiated HL60 cells and the adherent mouse monocyte-macrophage-like RAW 264.7 cell line, as well as with various compounds of oxidant (bleomycin sulfate, cis-diammineplatinum(II), camptothecin, TNF-alpha, IL-1 beta), nonoxidant (4 alpha-PMA, piracetam), and antioxidant (alpha-tocopherol, ascorbic acid) activity. In summary, we established a highly specific, reproducible and--with the aid of the nondifferentiated HL60 cell line--time-saving superoxide microplate assay as a valuable tool for the rapid screening of compounds for oxidative and antioxidative activity.
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PMID:Promyelocytic HL60 cells express NADPH oxidase and are excellent targets in a rapid spectrophotometric microplate assay for extracellular superoxide. 1451 66

A series of isomeric methoxyindazoles has been evaluated as inhibitors of purified recombinant neuronal, inducible, and endothelial nitric oxide synthases (NOS). 7-Methoxyindazole (7-MI) was the most active compound of this series and displayed selectivity toward the constitutive neuronal (NOS I) and endothelial (NOS III) NOS isoforms, the inducible NOS II being almost insensitive to this inhibitor. 6-, 5-, and 4-Methoxyindazoles were almost inactive against all three NOS isoforms. Inhibition of NO and citrulline formation catalyzed by neuronal NOS in the presence of 7-MI appeared to be competitive versus both substrate L-arginine (L-arg) and (6R)-5,6,7,8-tetrahydrobiopterin (BH(4)) cofactor. 7-MI only slightly inhibited NADPH oxidase activity and was inactive against the cytochrome c (cyt c) reductase activity of neuronal NOS at concentrations up to 100-fold higher than its IC(50) value for inhibition of citrulline formation. UV/Vis and EPR studies indicated that 7-MI interacts with the oxygenase domain of neuronal NOS (NOS I(oxy)) in an identical manner but with a much lower affinity than 7-nitroindazole (7-NI). These results demonstrate that an indazole derivative bearing an electron-rich substituent in the 7-position is also a NOS I inhibitor and that such a compound presents strong similarities with the mechanism of inhibition of 7-NI.
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PMID:Inhibitory effects and spectral interactions of isomeric methoxyindazoles on recombinant nitric oxide synthases. 1462 74

Phagocytosis by inflammatory cells is an essential step and a part of innate immunity for protection against foreign pathogens, microorganism or dead cells. Phagocytosis, endocytotic events sequel to binding particle ligands to the specific receptors on phagocyte cell surface such as Fcgamma recptor (FcgammaR), complement receptor (CR), beta-glucan receptor, and phosphatidylserine (PS) receptor, require actin assembly, pseudopod extension and phagosome closure. Rho GTPases (RhoA, Cdc42, and Rac1) are critically involved in these processes. Abrupt superoxide formation, called as oxidative burst, occurs through NADPH oxidase complex in leukocytes following phagocytosis. NADPH oxidase complex is composed of membrane proteins, p22PHOX and gp91PHOX, and cytosolic proteins, p40PHOX, p47PHOX and p67PHOX. The cytosolic subunits and Rac-GTP are translocated to the membrane, forming complete NADPH oxidase complex with membrane part subunits. Binding of imunoglobulin G (IgG)- and complement-opsonized particles to FcgammaR and CR of leukocytes induces apoptosis of the cells, which may be due to oxidative burst and accompanying cytochrome c release and casapase-3 activation.
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PMID:Phagocytosis induces superoxide formation and apoptosis in macrophages. 1464 85

The polymorphonuclear neutrophil (PMN)-respiratory burst plays a key role in host defense and inflammatory reactions. Modulation of this key neutrophil function by endogenous agents and the mechanisms involved are poorly understood. This study was designed to analyze the mechanisms involved in the effect of adrenaline on neutrophil superoxide anions production. Using the superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay, we report here that the beta-adrenergic agonist, adrenaline at physiologic concentrations (5-100 nM) inhibited formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated but not phorbol-myristate-acetate (PMA)-stimulated PMN superoxide anion production. The inhibitory effect of adrenaline runs in parallel with an increase in intracellular levels of cAMP which was reversed by the protein kinase A (PKA) inhibitor H-89, suggesting a role for PKA in mediating the inhibitory effect of adrenaline on fMLP-induced superoxide production. Adrenaline at physiological concentrations did not inhibit the fMLP-stimulated membrane translocation of the NADPH oxidase components p47phox and p67phox, nor the fMLP-stimulated phosphorylation of p47phox. However, adrenaline strongly depressed the activity of the cytosolic isoform of Phospholipase A(2) (cPLA(2)). We suggest that adrenaline inhibits fMLP induced superoxide production upstream of the NADPH oxidase via a mechanism involving PKA and cPLA(2).
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PMID:Inhibition of formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst in human neutrophils by adrenaline: inhibition of Phospholipase A2 activity but not p47phox phosphorylation and translocation. 1466 41

Efficient apoptotic signaling is a function of a permissive intracellular milieu created by a decrease in the ratio of superoxide to hydrogen peroxide and cytosolic acidification. Resveratrol (RSV) triggers apoptosis in some systems and inhibits the death signal in others. In this regard, the inhibitory effect on hydrogen peroxide-induced apoptosis is attributed to its antioxidant property. We provide evidence that exposure of human leukemia cells to low concentrations of RSV (4-8 micro M) inhibits caspase activation, DNA fragmentation, and translocation of cytochrome c induced by hydrogen peroxide or anticancer drugs C2, vincristine, and daunorubicin. Interestingly, at these concentrations, RSV induces an increase in intracellular superoxide and inhibits drug-induced acidification. Blocking the activation of NADPH oxidase complex neutralized RSV-induced inhibition of apoptosis. Furthermore, our results implicate intracellular hydrogen peroxide as a common effector mechanism in drug-induced apoptosis that is inhibited by preincubation with RSV. Interestingly, decreasing intracellular superoxide with the NADPH oxidase inhibitor diphenyliodonium reversed the inhibitory effect of RSV on drug-induced hydrogen peroxide production. These data show that low concentrations of RSV inhibit death signaling in human leukemia cells via NADPH oxidase-dependent elevation of intracellular superoxide that blocks mitochondrial hydrogen peroxide production, thereby resulting in an intracellular environment nonconducive for death execution.
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PMID:Resveratrol inhibits drug-induced apoptosis in human leukemia cells by creating an intracellular milieu nonpermissive for death execution. 2583 31


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