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)

NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3(-/-)) died from presumed sepsis following intravascular catheter placement, whereas Clcn3(+/+) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(-/-) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3(-/-) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acid-treated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3(-/-) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.
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PMID:Anion channels, including ClC-3, are required for normal neutrophil oxidative function, phagocytosis, and transendothelial migration. 1652 34

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O(2)(.-)) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O(2)(.-), pulmonary microvascular endothelial cells were preloaded with the O(2)(.-)-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O(2)(.-) resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O(2)(.-) triggered Ca(2+) release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O(2)(.-) production and cellular apoptosis. These "signaling" effects of O(2)(.-) were prevented by DIDS treatment, by depletion of intracellular Ca(2+) stores with thapsigargin and by chelation of intracellular Ca(2+). This study demonstrates that O(2)(.-) flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca(2+) release, which activates mitochondrial O(2)(.-) generation.
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PMID:Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling. 1736 Sep 69

Chloride anion is essential for myeloperoxidase (MPO) to produce hypochlorous acid (HOCl) in polymorphonuclear neutrophils (PMNs). To define whether chloride availability to PMNs affects their HOCl production and microbicidal capacity, we examined how extracellular chloride concentration affects killing of Pseudomonas aeruginosa (PsA) by normal neutrophils. PMN-mediated bacterial killing was strongly dependent on extracellular chloride concentration. Neutrophils in a chloride-deficient medium killed PsA poorly. However, as the chloride level was raised, the killing efficiency increased in a dose-dependent manner. By using specific inhibitors to selectively block NADPH oxidase, MPO, and cystic fibrosis transmembrane conductance regulator (CFTR) functions, neutrophil-mediated killing of PsA could be attributed to three distinct mechanisms: CFTR-dependent and oxidant-dependent; chloride-dependent but not CFTR- and oxidant-dependent; and independent of any of the tested factors. Therefore, chloride anion is involved in oxidant- and nonoxidant-mediated bacterial killing. We previously reported that neutrophils from CF patients are defective in chlorination of ingested bacteria, suggesting that the chloride channel defect might impair the MPO-hydrogen peroxide-chloride microbicidal function. Here, we compared the competence of killing PsA by neutrophils from normal donors and CF patients. The data demonstrate that the killing rate by CF neutrophils was significantly lower than that by normal neutrophils. CF neutrophils in a chloride-deficient environment had only one-third of the bactericidal capacity of normal neutrophils in a physiological chloride environment. These results suggest that CFTR-dependent chloride anion transport contributes significantly to killing PsA by normal neutrophils and when defective as in CF, may compromise the ability to clear PsA.
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PMID:The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils. 1835 29

The compartmentalized production of superoxide (*O(2)(-)) by endosomal NADPH oxidase is important in the redox-dependent activation of NF-kappaB following interleukin 1beta (IL-1beta) stimulation. It remains unclear how *O(2)(-) produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated *O(2)(-) movement out of IL-1beta-stimulated endosomes and whether SOD1 at the endosomal surface mediates redox-signaling events required for NF-kappaB activation. The relative outward permeability of NADPH-dependent *O(2)(-) from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for *O(2)(-). In these studies, approximately 60% of *O(2)(-) efflux out of endosomes was inhibited by treatment with either of two anion channel blockers, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) or niflumic acid (NFA). Furthermore, radioisotopic electrodiffusion flux assays on endomembrane proteoliposomes suggested that *O(2)(-) and Cl(-) are transported through the same DIDS-sensitive channel(s). Rab5-based immunoaffinity isolation of IL-1beta-stimulated early endosomes demonstrated SOD1 recruitment to endosomes harboring the IL-1 receptor. Finally, SOD1-deficient cells were found to be defective in their ability to activate NF-kappaB following IL-1beta stimulation. Together, these results suggest that *O(2)(-) exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of *O(2)(-) at the endosomal surface may produce the localized H(2)O(2) required for redox-activation of NF-kappaB.
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PMID:Evidence for a superoxide permeability pathway in endosomal membranes. 1837 95

Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I(e)) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I(e). In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 microM), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.
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PMID:Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst. 1872 17

Studies to date indicate that endothelial cells express glycine-activated chloride channels, which promote hyperpolarization of the endothelial plasma membrane. If such channels are expressed by endothelial cells lining conduit arteries, glycine is likely to have anti-atherogenic activity. This reflects the fact that endothelial hyperpolarization promotes calcium influx, activating the endothelial isoform of nitric oxide synthase, while also down-regulating the activity of the membrane-bound NADPH oxidase, chief endothelial source of superoxide. Since macrophages express glycine-activated chloride channels that suppress production of oxidants and cytokines, glycine may also oppose atherogenesis by influencing intimal macrophage function. In rats, supplemental glycine exerts anti-inflammatory and anti-angiogenic effects attributed to chloride channel activation. Administration of large daily doses of glycine would appear to be practical and safe, and has already been shown to inhibit protein glycation in human diabetics.
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PMID:The hyperpolarizing impact of glycine on endothelial cells may be anti-atherogenic. 1923 35

Oxidative stress, characterized by overproduction of reactive oxygen species (ROS), is a major feature of several pathological states. Indeed, many cancers and neurodegenerative diseases are accompanied by altered redox balance, which results from dysregulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In this review, we consider the role of the intracellular chloride channel 1 (CLIC1) in microglial cells during oxidative stress. Following microglial activation, CLIC1 translocates from the cytosol to the plasma membrane where it promotes a chloride conductance. The resultant anionic current balances the excess charge extruded by the active NADPH oxidase, supporting the generation of superoxide by the enzyme. In this scenario, CLIC1 could be considered to act as both a second messenger and an executor.
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PMID:Chloride intracellular channel 1 (CLIC1): Sensor and effector during oxidative stress. 2038 34

Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC-dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport properties, and multiple chloride channels including CFTR are responsible for chloride supply to neutrophil phagosomes.
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PMID:Chloride transport in functionally active phagosomes isolated from Human neutrophils. 2308 27

Cisplatin resistance is a difficult problem in clinical chemotherapy, and the mechanisms involved in cisplatin resistance require further study. In this study, we investigated the role of chloride channel-3 (ClC-3) in cisplatin resistance. Autophagy was demonstrated by accumulation of LC3-II, beclin 1 and Atg12-Atg5. The ultrastructure changes were observed under electron microscope. Chemical staining with acridine orange or MDC was used to detect acidic vesicular organelles. Quantification of apoptosis was detected by PI and Annexin V staining. The mechanisms involved in the Akt pathway and autophagy were studied by western blot analysis. Our results showed that Akt phosphorylation and autophagy were induced by cisplatin in human glioma U251 cells. Specific inhibition of ClC-3 by ClC-3 siRNA sensitized the apoptosis-resistant U251 cells to cisplatin-mediated cell death and downregulated phosphorylated Akt. Interestingly, ClC-3 suppression also inhibited induction of autophagy by cisplatin although the Akt/mTOR pathway was deregulated. Counteracting the autophagic process by 3-methylademine enhanced cytotoxicity of cisplatin, revealing that autophagy plays a key role in chemoresistance. Suppressing the Akt/mTOR pathway by the NADPH oxidase inhibitor diphenyl iodonium (DPI) indicated that cisplatin-induced activation of Akt/mTOR pathway requires generation of reactive oxygen species (ROS) through NADPH oxidase. Collectively, our results suggest that ClC-3 suppression causes the inhibition of Akt and autophagy, which can enhance the therapeutic benefit of cisplatin in U251 cells.
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PMID:Suppression of chloride channel 3 expression facilitates sensitivity of human glioma U251 cells to cisplatin through concomitant inhibition of Akt and autophagy. 2340 63

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function.
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PMID:Cystic fibrosis transmembrane conductance regulator recruitment to phagosomes in neutrophils. 2354 24


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