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:1.6.99.6 (NADPH oxidase)
10,295 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine alveolar macrophages were exposed in vitro to quartz dusts, metal-containing dusts or silica particles coated with a single metal oxide. The release of reactive oxygen intermediates (ROI) was measured in short-term incubations (90 min). The secretion of both ROI was markedly enhanced by silica particles coated with vanadium oxide and lowered by copper oxide-coated particles. The particle-induced ROI release was significantly decreased by the inhibition of protein kinase C (PKC) as well as phospholipase A2, suggesting the involvement of both enzymes in the NADPH oxidase activation. Quartz dusts induced a transient increase of free cytosolic calcium ion concentration, slight intracellular acidification, and depolarization of the plasma membrane. In the presence of EGTA or verapamil the rise of [Ca2+]i was diminished, suggesting an influx of extracellular calcium ions. The PKC inhibitor GF 109203X did not inhibit the quartz-induced calcium rise, while both the cytosolic acidification and depolarization were prevented. BSA-coating of the quartz particles abolished the calcium influx as well as the decrease of pHi, and possibly hyperpolarized the plasma membrane.
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PMID:Mechanisms of particle-induced activation of alveolar macrophages. 892 Jul 26

Previous studies have indicated that nitric oxide (NO) released from Kupffer cells modulates biological viability of cocultured hepatoma cells. This study was designed to evaluate the mechanisms by which Kupffer cells synthesize and release NO in reponse to cocultured hepatoma cells. Kupffer cells isolated from male Wistar rats were cocultured with rat hepatoma cell line, AH70 cells. The sum of nitrite and nitrate levels increased in the culture medium of Kupffer cells with AH70 cells as compared with those of Kupffer cells or AH70 cells alone. Increased expressions of iNOS and iNOS mRNA in Kupffer cells cocultured with AH70 cells were detected by an immunofluorescence staining and a fluorescence in situ hybridization study, respectively. A fluorescence in situ DNA-protein binding assay revealed that NF-kappaB activation occurs in Kupffer cells and activated NF-kappaB moved into the nuclei preceding to an increased production of NO. Oxidative stress indicated by dichlorofluorescein fluorescence was observed in Kupffer cells cocultured with AH70 cells. An increased calcium mobilization indicated as increased fluo-3-associated fluorescence was also induced in Kupffer cells after coculture with AH70 cells. Monoclonal antibodies directed against rat CD18 and ICAM-1, as well as TMB-8, a calcium inhibitor, prevented the calcium mobilization, active oxygen production, and NF-kappaB activation in addition to the increased production of NO. Pyrrolidine dithiocarbamate, an inhibitor of oxidative NF-kappaB activation, diphenylene iodonium, an NADPH oxidase inhibitor, and quinacrine, a phospholipase A2 inhibitor, significantly attenuated the increase in dichlorofluorescein fluorescence, NF-kappaB activation, and NO production. Therefore, this study suggests that CD18/ICAM-1-dependent cell-to-cell interaction with hepatoma cells causes calcium mobilization and oxidative activation of NF-kappaB, which may lead to the increased production of NO in Kupffer cells.
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PMID:CD18/ICAM-1-dependent oxidative NF-kappaB activation leading to nitric oxide production in rat Kupffer cells cocultured with syngeneic hepatoma cells. 906 44

This investigation was undertaken to clarify the mechanisms of superoxide anion (O2-) generation in rat peritoneal mast cells. Compound 48/80, a typical histamine liberator mediated by calcium influx, elicited O2- generation from the mast cells in a dose-dependent fashion. It was demonstrated by immunohistochemical study and Western blot analysis that the mast cells contained the 47-kDa phagocyte oxidase (p47phox) protein, which was one cytosolic component of the NADPH oxidase system. Arachidonic acid stimulated O2- generation in the mast cells, but other unsaturated fatty acids had no effect. On the other hand, 48/80-induced O2- generation was inhibited by phospholipase A2 inhibitors, such as arachidonyl trifluoromethyl ketone and manoalide. Forskolin, isoprenaline, and dibutyryl cyclic AMP inhibited the O2- generation, and KT-5720, a cyclic AMP-dependent protein kinase (A-kinase) inhibitor, markedly enhanced the O2- generation. These findings suggest that O2- is generated by a NADPH oxidase-like enzyme system in mast cells and that this enzyme system is activated by arachidonic acid released by cytosolic phospholipase A2. Thus, it is regulated by the cyclic AMP-A kinase system.
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PMID:The mechanisms of compound 48/80-induced superoxide generation mediated by A-kinase in rat peritoneal mast cells. 923 5

The effects of arachidonic acid metabolism and NADPH oxidase inhibitor on the hydrogen peroxide (H2O2) generation and endocytotic activity of cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein (LDL) levels have been investigated. EC were incubated with 240 mg/dl LDL cholesterol and cellular H2O2 production and endocytotic activity measured in the presence and absence of the arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A, and NADPH oxidase inhibitor, apocynin. All inhibitors, with the exception of indomethacin, markedly reduced high LDL-induced increases in EC H2O2 generation and endocytotic activity. EC exposed to exogenously applied arachidonic acid had cellular functional changes similar to those induced by high LDL concentrations. EC incubated with 1-25 uM arachidonic acid had increased H2O2 production and heightened endocytotic activity. Likewise, EC pre-loaded with [3H]arachidonic acid when exposed to increasing LDL levels (90-330 mg/dl cholesterol) had a dose-dependent rise in cytosolic [3H]arachidonic acid. The phospholipase A2 inhibitors, 4-bromophenacyl bromide and 7,7-dimethyleicosadienoic acid, markedly inhibited H2O2 production in EC exposed to 240 mg/dl LDL cholesterol. These findings suggest that arachidonic acid contributes mechanistically to high LDL-perturbed EC H2O2 generation and heightened endocytosis. Such cellular functional changes add to our understanding of endothelial perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.
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PMID:Low-density lipoprotein stimulated peroxide production and endocytosis in cultured human endothelial cells: mechanisms of action. 927 82

The role of cytosolic phospholipase A2 (cPLA2) and its mode of activation by opsonized zymosan (OZ) was studied in human neutrophils in comparison with activation by PMA. The activation of cPLA2 by 1 mg/ml OZ or 50 ng/ml PMA is evidenced by its translocation to the membrane fractions on stimulation. This translocation is consistent with dithiothreitol (DTT)-resistant phospholipase A2 (PLA2) activity detected in the membranes of activated cells. Neutrophils stimulated by either OZ or PMA exhibited an immediate stimulation of extracellular-signal-regulated kinases (ERKs). The inhibition of ERKs, DTT-resistant PLA2 and NADPH oxidase activities by the MAP kinase kinase inhibitor PD-98059 indicates that ERKs mediate the activation of cPLA2 and NADPH oxidase stimulated by either OZ or PMA. The protein kinase C (PKC) inhibitor GF-109203X inhibited epidermal growth factor receptor peptide kinase activity, the release of [3H]arachidonic acid, DTT-resistant PLA2 activity and superoxide generation induced by PMA, but did not inhibit any of these activities induced by OZ. PKC activity was similarly inhibited by GF-109203X in membrane fractions separated from neutrophils stimulated by either PMA or OZ. In the presence of the tyrosine kinase inhibit orgenistein, ERKs, PLA2 and NADPH oxidase activities were inhibited in cells stimulated by OZ, whereas they were hardly affected in cells stimulated by PMA. The results suggest that the activation of cPLA2 by PMA or OZ is mediated by ERKs. Whereas PMA stimulates ERKs activity through a PKC-dependent pathway, signal transduction stimulated by OZ involves tyrosine kinase activity leading to activation of ERKs via a PKC-independent pathway.
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PMID:Cytosolic phospholipase A2 and its mode of activation in human neutrophils by opsonized zymosan. Correlation between 42/44 kDa mitogen-activated protein kinase, cytosolic phospholipase A2 and NADPH oxidase. 930 39

Glycosphingolipids (GSLs) and their metabolites play important roles in a variety of biological processes. We have previously reported that lactosylceramide (LacCer), a ubiquitous GSL, stimulates NADPH oxidase-dependent superoxide generation by aortic smooth muscle cells and their consequent proliferation. We postulated that LacCer may upregulate adhesion molecules on human polymorphonuclear leukocytes (hPMNs), perhaps also via NADPH oxidase-dependent reactive oxygen metabolite (ROM) generation. Incubation of hPMNs with LacCer upregulated CD11b/CD18 (Mac-1) and CD11c/CD18, as determined by fluorescence-automated cell sorting. LacCer also stimulated these hPMNs to generate superoxide via NADPH oxidase, as determined by lucigenin-enhanced chemiluminescence. However, the upregulation of Mac-1 by LacCer did not itself appear to be mediated by ROMs, since neither an antioxidant nor an NADPH oxidase inhibitor substantially inhibited the Mac-1 upregulation. However, this Mac-1 upregulation was significantly inhibited by two disparate phospholipase A2 (PLA2) inhibitors. Moreover, LacCer induced arachidonic acid metabolism, which was inhibited by the PLA2 inhibitors, but not by an NADPH oxidase inhibitor. To evaluate the effect of LacCer on hPMN adhesion to endothelium, hPMNs stimulated with LacCer were allowed to adhere to unstimulated human endothelial cell monolayers. LacCer stimulated hPMN adhesion to endothelial cells, which was blocked by anti-CD18 and by the PLA2 inhibitors. We conclude that LacCer stimulates both Mac-1 upregulation and superoxide generation in hPMNs but that ROMs are not the upstream signal for Mac-1 upregulation. This mechanism may well be relevant to acute endothelial injury in inflammation and other pathological conditions.
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PMID:Lactosylceramide stimulates human neutrophils to upregulate Mac-1, adhere to endothelium, and generate reactive oxygen metabolites in vitro. 952 58

Intermittent painful crises due to vasoocclusion are the major clinical manifestation of sickle cell disease (SCD), but subclinical episodes may also occur. There is sparse evidence for the involvement of neutrophils in the pathophysiology of SCD, but production of cytokines by the damaged endothelium might influence neutrophil function and modulate responses to subsequent cytokine exposure. In addition, the activation of neutrophils in the microcirculation could itself exacerbate vasoocclusion. To test whether neutrophil inflammatory responses were altered in SCD, neutrophil phospholipase A2 and NADPH oxidase activity in response to in vitro priming by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) were measured both during and between painful crises. Resting levels of neutrophil phospholipase A2 activity in steady-state SCD (4.0% +/- 0. 5% of total cell radioactivity) were raised relative to control values (2.0% +/- 0.2%, n = 10, P = .008). There was no defect of agonist-stimulated phospholipase A2 or NADPH oxidase activity in steady-state SCD; however, the ability of phospholipase A2 to respond to priming with GM-CSF was attenuated to 63% +/- 17% of control values (n = 10, P = .04). Similarly, neutrophil NADPH oxidase activity after priming with GM-CSF and TNF-alpha was, respectively, 65% +/- 11% (n = 7, P = .03) and 57% +/- 7% of control (n = 10, P = .007) in steady-state disease, and was further reduced during painful vasoocclusive crises to 34% +/- 9% and 25% +/- 3% of control for GM-CSF and TNF-alpha, respectively. These data were not explained by poor splenic function or any racial factor, as normal cytokine responses were seen in splenectomized patients in remission from Hodgkin's disease and in healthy Afro-Caribbean subjects. Abnormal neutrophil cytokine priming responses were not observed in either patients with rheumatoid arthritis or iron-deficiency anemia. Our findings are indicative of an ongoing inflammatory state in SCD between painful crises involving neutrophil activation and an abnormality of cytokine-regulated neutrophil function, which may compromise the host defenses against certain microorganisms.
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PMID:Raised neutrophil phospholipase A2 activity and defective priming of NADPH oxidase and phospholipase A2 in sickle cell disease. 955 1

Experiments were designed to investigate whether leukotriene (LTB4) receptors can couple directly to phospholipase A2 (PLA2) in guinea pig eosinophils and the role of endogenous arachidonic acid (AA) in LTB4-induced activation of the NADPH oxidase. LTB4 (EC50 approximately 16 nM) and AA (EC50 approximately 6 microM) generated hydrogen peroxide (H2O2) in a concentration-dependent manner and at an equivalent maximum rate (5-6 nmol/min/10(6) cells). LTB4 stimulated PLA2 over a similar concentration range that activated the NADPH oxidase, although kinetic studies revealed that the release of [3H]AA (t1/2 approximately 2 s) preceded H2O2 generation (t1/2 > 30 s). Pretreatment of eosinophils with pertussis toxin abolished the increase in inositol(1,4,5)trisphosphate mass, [Ca2+]c, [3H]AA release, and H2O2 generation evoked by LTB4. Qualitatively identical results were obtained in eosinophils in which phospholipase C (PLC) was desensitized by 4beta-phorbol 12,13-dibutyrate with the exception that [3H]AA release was largely unaffected. Additional studies performed with the protein kinase C inhibitor, Ro 31-8220, and under conditions in which Ca2+ mobilization was abolished, provided further evidence that LTB4 released [3H]AA independently of signal molecules derived from the hydrolysis of phosphatidylinositol(4,5)bisphosphate by PLC. Pretreatment of eosinophils with the PLA2 inhibitor, mepacrine, abolished LTB4-induced [3H]AA release at a concentration that inhibited H2O2 by only 36%. Collectively, the results of this study indicate that agonism of LTB4 receptors on guinea pig eosinophils mobilizes AA by a mechanism that does not involve the activation of PLC. In addition, although LTB4 effectively stimulated PLA2, a central role for AA in the activation of the NADPH oxidase was excluded.
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PMID:Leukotriene B4 activates the NADPH oxidase in eosinophils by a pertussis toxin-sensitive mechanism that is largely independent of arachidonic acid mobilization. 957 59

The present study was undertaken to investigate the hypothesis that multiple oxygen radical generating systems contribute to the tumor necrosis factor (TNF) alpha-stimulated transcriptional activation of the vascular cell adhesion molecule (VCAM)-1 in endothelial cells. Experimental evidence has implicated the cytochrome P450 monooxygenase and a phagocyte type NADPH-oxidase as a source of oxygen radicals in these cells. We show here that endothelial cells exhibit cytochrome P450 activity by measuring the O-dealkylation of the exogenous substrate 7-ethoxyresorufin, but components of the phagocyte-type NADPH oxidase could not be demonstrated in endothelial cells. In that latter respect it was surprising that the NADPH oxidase inhibitor apocynin completely prevented the accumulation of VCAM-1 mRNA. However, we found that apocynin also acts as an inhibitor of cytochrome P450 activity in endothelial cells. Therefore the inhibitory effect of apocynin on the induction of VCAM-1 may no longer be used to demonstrate a role for the NADPH oxidase in this process. Furthermore, different cytochrome P450 inhibitors Co2+, metyrapone, SKF525a decreased the endothelial VCAM-1 expression stimulated by TNFalpha. Also under hypoxic conditions the expression of VCAM-1 was reduced. On this basis we assume that the oxygen dependent step in the intracellular signalling cascade underlying the TNFalpha stimulated transcriptional activation of VCAM-1 resides in the activity of a cytochrome P450 dependent monooxygenase. The finding that the phospholipase A2 inhibitor bromophenacylbromide inhibited the expression of VCAM-1 may indicate that arachidonic acid serves as a substrate for the cytochrome P450 monooxygenase reaction, but further research is needed to elucidate the particular cytochrome P450 family member mediating the expression of VCAM-1.
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PMID:Evidence against the involvement of multiple radical generating sites in the expression of the vascular cell adhesion molecule-1. 964 91

Peritoneal dialysis effluent (PDE) contains a low-molecular-weight solute that will activate and prime the NADPH oxidase of human neutrophils via a phospholipase A2 (PLA2)-dependent mechanism. Since the products of PLA2 are known to activate and prime the oxidase we have investigated their role in the dialysis effluent-mediated activation and priming of human neutrophils. NADPH oxidase activity of PDE-primed and -unprimed neutrophils was measured by lucigenin-enhanced chemiluminescence in the presence of known inhibitors of the arachidonic acid cascade. Incubation of neutrophils with the nonselective PLA2 inhibitor quinacrine (0 to 100 microM) reduced oxidase activity in both primed and unprimed cells. Furthermore, primed cells were more sensitive to the action of quinacrine than were unprimed cells. We were unable to determine the relative roles of secretory PLA2 (sPLA2) and cytosolic PLA2 (cPLA2) since the selective sPLA2 inhibitor scalaradial (0 to 100 microM) inhibited oxidase activity in both groups of cells by similar degrees, while the specific cPLA2 inhibitor AACO-CF3 (0 to 50 microM) failed to affect activity in either group. Inhibition of platelet-activating factor (PAF), cycloxygenase, and 5-lipoxygenase-activating protein by hexanolamino-PAF (0 to 25 microM), flurbiprofen (0 to 25 microM), and MK886 (0 to 5 microM), respectively, had no effect upon oxidase activity. However, the direct inhibition of 5-lipoxygenase by caffeic acid or lipoxin A4 resulted in a similar concentration-dependent attenuation of oxidase activity in both primed and unprimed cells. Leukotriene B4 (LTB4) release from primed neutrophils was comparable to that from unprimed cells with the exception of phorbol myristate acetate-stimulated cells, which released fivefold more LTB4 than control. Taken together, these results suggest that it is arachidonic acid per se, and not its metabolites, that is important in priming of the neutrophil NADPH oxidase by dialysis effluent.
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PMID:Role of arachidonic acid and its metabolites in the priming of NADPH oxidase in human polymorphonuclear leukocytes by peritoneal dialysis effluent. 972 36


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