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)

The influence of the plant product magnolol on neutrophil superoxide anion (O2-*) generation has been investigated in the rat. Intraperitoneal injection of magnolol (30mg kg(-1)) significantly inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst in rat whole blood ex-vivo. Magnolol also inhibited the 02-* generation with an IC50 (concentration resulting in 50% inhibition) of 15.4+/-1.6 microM and O2 consumption in rat neutrophils in-vitro. Magnolol weakly inhibited the O2-* generation in the xanthine-xanthine oxidase system, decreased cellular cyclic AMP level and had no effect on cyclic GMP levels. It weakly inhibited neutrophil cytosolic protein kinase C activity but did not alter porcine heart protein kinase A activity. Magnolol attenuated fMLP-induced protein tyrosine phosphorylation with an IC50 of 24.0+/-1.9 microM and the phosphorylation of mitogen-activated protein kinase p42/44 with an IC50 of 28.5+/-4.5 microM. However, magnolol alone activated neutrophil phospholipase D activity as determined by the formation of phosphatidic acid and phosphatidyl-ethanol in the presence of ethanol. In the presence of NADPH, the arachidonate-activated NADPH oxidase activity in a cell-free system was weakly suppressed by magnolol. These results suggest that the inhibition of respiratory burst in fMLP-activated neutrophils by magnolol is probably attributable mainly to the attenuation of protein tyrosine phosphorylation and p42/44 mitogen-activated protein kinase activation, and partly to the suppression of protein kinase C and NADPH oxidase activities.
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PMID:Inhibition by magnolol of formylmethionyl-leucyl-phenyl alanine-induced respiratory burst in rat neutrophils. 1034 29

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.
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PMID:A novel protein kinase target for the lipid second messenger phosphatidic acid. 1042 1

Intracellular signaling events that mediate the long-term effects of Ang II in vascular smooth muscle cells are unclear, but oxidative stress may play an important role. This study examined the ability of Ang II to generate reactive oxygen species and investigated the putative role of phospholipase D (PLD)-dependent signaling pathways for its production in human vascular smooth muscle cells. In addition, we assessed whether redox-sensitive pathways influence Ang II-stimulated cell growth. Primary and low-passage cells (passages 1 to 4) derived from resistance arteries of subcutaneous gluteal biopsies from healthy subjects were studied. Oxidative stress was measured with the fluorescent probe 5-(and 6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA) (8 micromol/L), and the role of PLD was assessed with the PLD inhibitor D-erythro-sphingosine, dihydro (sphinganine) (10 micromol/L). To determine whether NADH/NADPH oxidase contributes to production of reactive oxygen species, Ang II-stimulated cells were pretreated with the specific flavoprotein inhibitor diphenylene iodinium (DPI) (10 micromol/L). DNA and protein synthesis were determined by [(3)H]thymidine and [(3)H]leucine incorporation, respectively. Ang II increased CM-H(2)DCFDA fluorescence, and this was inhibited by catalase (350 U/mL), indicating that the fluorescence signal was derived predominantly from H(2)O(2). Ang II dose-dependently increased H(2)O(2) production (E(max)=57.6+/-1.7 nmol/L, pD(2)=7.7+/-0.06) and PLD activation (E(max)=207+/-3.3% of control, pD(2)=7.7+/-0.5). H(2)O(2) effects were evident within 1 hour, and maximal PLD activation occurred within 40 minutes after stimulation. DPI inhibited (P<0.01) Ang II-stimulated responses. PLD inhibition significantly attenuated (P<0.05) Ang II-elicited H(2)O(2) production (E(max)=29+/-5 nmol/L). DPI and sphinganine inhibited Ang II-induced DNA and protein synthesis. These data indicate that in vascular smooth muscle cells from human peripheral resistance arteries, Ang II increases H(2)O(2) generation via PLD-dependent, NADH/NADPH oxidase-sensitive pathways. These cascades may function as second messengers in long-term Ang II-mediated growth-signaling events.
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PMID:Ang II-stimulated superoxide production is mediated via phospholipase D in human vascular smooth muscle cells. 1052 94

Human neutrophils express two different types of phagocytic receptors, complement receptors (CR) and Fc receptors. In order to characterize the different signaling properties of each receptor we have used non-adherent human neutrophils and investigated CR3, FcgammaRIIA and FcgammaRIIIB for their signaling capacity. Selective activation of each receptor was achieved by coupling specific antibodies to heat-killed Staphylococcus aureus particles, Pansorbins, through their Fc moiety. Despite the fact that these particles are not phagocytosed, we show that addition of Pansorbins with anti-CD18 antibodies recognizing CR3 induced prominent signals leading to a respiratory burst. Stimulation with anti-FcgammaRIIIB Pansorbins induced about half of the response induced by anti-CR3 Pansorbins, whereas anti-FcgammaRIIA Pansorbins induced an even weaker signal. However, FcgammaRIIA induced strong phosphorylation of p72(syk) whereas FcgammaRIIIB induced only a very weak p72(syk) phosphorylation. During CR3 stimulation no tyrosine phosphorylation of p72(syk) was seen. Both phospholipase D and NADPH oxidase activities were dependent on intracellular calcium. This is in contrast to tyrosine phosphorylation of p72(syk) that occurred even in calcium-depleted cells, indicating that oxygen metabolism does not affect p72(syk) phosphorylation. Inhibitors of tyrosine phosphorylation blocked the respiratory burst induced by both FcgammaRIIA and FcgammaRIIIB as well as CR3. This shows that tyrosine phosphorylation of p72(syk) is an early signal in the cascade induced by FcgammaRIIA but not by CR3.
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PMID:CR3, FcgammaRIIA and FcgammaRIIIB induce activation of the respiratory burst in human neutrophils: the role of intracellular Ca(2+), phospholipase D and tyrosine phosphorylation. 1052 59

Human neutrophils participate in the host innate immune response, partly mediated by the multicomponent superoxide-generating enzyme NADPH oxidase. A correlation between phosphorylation of cytosolic NADPH oxidase components and enzyme activation has been identified but is not well understood. We previously showed that p22(phox), the small subunit of the membrane-bound oxidase component flavocytochrome b(558), is an in vitro substrate for both a phosphatidic acid-activated kinase and conventional protein kinase C isoforms (Regier, D. S., Waite, K. A., Wallin, R., and McPhail, L. C. (1999) J. Biol. Chem. 274, 36601-36608). Here we show that several neutrophil agonists (phorbol myristate acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine) induce p22(phox) phosphorylation in intact neutrophils. To determine if phospholipase D (PLD) is needed for p22(phox) phosphorylation, cells were pretreated with ethanol, which reduces phosphatidic acid production by PLD in stimulated cells. Phorbol myristate acetate-induced phosphorylation of p22(phox) and NADPH oxidase activity were not reduced by ethanol. In contrast, ethanol reduced both activities when cells were stimulated by N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan. Varying the time of stimulation with opsonized zymosan showed that the phosphorylation of p22(phox) coincides with NADPH oxidase activation. GF109203X, an inhibitor of protein kinase C and the phosphatidic acid-activated protein kinase, decreased both p22(phox) phosphorylation and NADPH oxidase activity in parallel in opsonized zymosan-stimulated cells. Stimulus-induced phosphorylation of p22(phox) was on Thr residue(s), in agreement with in vitro results. Overall, these data show that NADPH oxidase activity and p22(phox) phosphorylation are correlated and suggest two mechanisms (PLD-dependent and -independent) by which p22(phox) phosphorylation occurs.
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PMID:Phosphorylation of p22phox is mediated by phospholipase D-dependent and -independent mechanisms. Correlation of NADPH oxidase activity and p22phox phosphorylation. 1089 20

In activated neutrophils NADPH oxidase is regulated through various signaling intermediates, including heterotrimeric G proteins, kinases, GTPases, and phospholipases. ADP-ribosylation factor (ARF) describes a family of GTPases associated with phospholipase D (PLD) activation. PLD is implicated in NADPH oxidase activation, although it is unclear whether activation of PLD by ARF is linked to receptor-mediated oxidase activation. We explored whether ARF participates in NADPH oxidase activation by formyl-methionine-leucine-phenylalanine (fMLP) and whether this involves PLD. Using multicolor forward angle light scattering analyses to measure superoxide production in differentiated neutrophil-like PLB-985 cells, we tested enhanced green fluorescent fusion proteins of wild-type ARF1 or ARF6, or their mutant counterparts. The ARF6(Q67L) mutant defective in GTP hydrolysis caused increased superoxide production, whereas the ARF6(T27N) mutant defective in GTP binding caused diminished responses to fMLP. The ARF1 mutants had no effect on fMLP responses, and none of the ARF proteins affected phorbol 12-myristate 13-acetate-elicited oxidase activity. PLD inhibitors 1-butanol and 2, 3-diphosphoglycerate, or the ARF6(N48R) mutant assumed to be defective in PLD activation, blocked fMLP-elicited oxidase activity in transfected cells. The data suggest that ARF6 but not ARF1 modulates receptor-mediated NADPH oxidase activation in a PLD-dependent mechanism. Because PMA-elicited NADPH oxidase activation also appears to be PLD-dependent, but ARF-independent, ARF6 and protein kinase C may act through distinct pathways, both involving PLD.
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PMID:A regulatory role for ADP-ribosylation factor 6 (ARF6) in activation of the phagocyte NADPH oxidase. 1093 44

This study investigates the effects of aliphatic (n-heptane, n-nonane), naphtenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)), and aromatic (methylbenzene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on respiratory burst in human granulocytes. The free radical formation was measured as 2,7-dichlorofluorescein diacetate-amplified (DCF) fluorescence, by electron paramagnetic resonance (EPR) spectroscopy and by hydroxylation of 4-hydroxybenzoate. The chemotactic peptide N-formyl-met-leu-phe (fMLP) and phorbol 12-myristate 13-acetate (PMA), a diacylglycerol analogue, were included as positive controls. DCF fluorescence was elevated in a concentration-dependent manner by C9 hydrocarbons. The C7 hydrocarbons did not stimulate respiratory burst in the concentration range examined. The naphtenic hydrocarbon TMCH showed the strongest effect on respiratory burst and was therefore selected for mechanistic studies of this free radical formation. In the absence of extracellular Ca(2+), fluorescence in response to TMCH and fMLP was reduced by 77 and 90%, respectively. Preincubation of the granulocytes with the protein kinase C inhibitor bisindolylmaleimide reduced the DCF fluorescence stimulated with TMCH, fMLP, and PMA by 82, 56, and 90%, respectively. The phospholipase C inhibitor U73122 lowered the TMCH- and fMLP-activated DCF fluorescence by 87 and 76%. In addition, the TMCH- and fMLP-induced DCF fluorescence, after the preincubation with the phospholipase D modulator n-butanol, was lowered by 83 and 52%, respectively. The importance of protein kinase C, phospholipase C, and phospholipase D for elevation of respiratory burst was also demonstrated by the EPR experiments using the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). Preincubation with the NADPH oxidase inhibitor diphenyleneiodonium and diethyldithiocarbamate, which inhibits superoxide dismutase, led to an almost complete reduction of DCF fluorescence in response to TMCH, fMLP, and PMA. Preincubation with diethyldithiocarbamate led to the elevation of superoxide adducts of DEPMPO. The hydrocarbons stimulated formation of mainly the superoxide (O(*-)(2)) adduct of DEPMPO (DEPMPO-OOH) but also small amounts of the hydroxyl adduct ((*)OH) (DEPMPO-OH). Using 4-hydroxybenzoate as a hydroxyl radical trap confirmed formation of (*)OH after stimulation with the hydrocarbons. In conclusion, our findings indicate that TMCH-activated respiratory burst is dependent on the Ca(2+)-dependent phospholipase C, phospholipase D, and protein kinase C prior to activation of the NADPH oxidase.
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PMID:The effects of aliphatic (n-nonane), naphtenic (1,2, 4-trimethylcyclohexane), and aromatic (1,2,4-trimethylbenzene) hydrocarbons on respiratory burst in human neutrophil granulocytes. 1098 13

We examined the effect of cytochalasin B (CB) or granulocyte colony stimulating factor (GCSF) on superoxide radical (O2-) production of neutrophils by phorbor myristate acetate (PMA)-stimulation. It was observed that O2- generation of intact and GCSF-treated neutrophils by PMA-stimulation showed a lag during the early stage, and was largely inhibited by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (200 microm) or GF109203X (GFX) (0.2 microM), but not by ethanol (1%) and wortmannin (100 nM). In contrast, O2- generation of CB-pretreated neutrophils by PMA-stimulation did not show a lag, but was less than that of intact cells, and was only minimally depressed by the above inhibitors, but was markedly depressed by the simultaneous addition of GFX and ethanol or GFX and wortmannin. Although translocation of p47phox and p67phox to the membrane fraction by PMA-stimulation of intact and GCSF-treated neutrophils occurred in parallel with O2- production, that of CB-treated neutrophils by PMA-stimulation was not always proportional to O2- production. These findings suggest that pretreatment of neutrophils with CB dramatically alters the PMA response of the cells; that is, the protein kinase C-dependent pathway is largely depressed, and a phospholipase D-dependent one for NADPH oxidase activation appears in CB-treated cells.
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PMID:Phorbor myristate acetate induces NADPH oxidase activity of cytochalasin B-primed neutrophils through the protein kinase C-independent pathway. 1099 14

The enzyme NADPH oxidase is regulated by phospholipase D in intact neutrophils and is activated by phosphatidic acid (PA) plus diacylglycerol (DG) in cell-free systems. We showed previously that cell-free NADPH oxidase activation by these lipids involves both protein kinase-dependent and -independent pathways. Here we demonstrate that only the protein kinase-independent pathway is operative in a cell-free system of purified and recombinant NADPH oxidase components. Activation by PA + DG was ATP-independent and unaffected by the protein kinase inhibitor staurosporine, indicating the lack of protein kinase involvement. Both PA and DG were required for optimal activation to occur. The drug reduced activation of NADPH oxidase by either arachidonic acid or PA + DG, with IC(50) values of 46 and 25 microm, respectively. The optimal concentration of arachidonic acid or PA + DG for oxidase activation was shifted to the right with, indicating interference of the drug with the interaction of lipid activators and enzyme components. inhibited the lipid-induced aggregation/sedimentation of oxidase components p47(phox) and p67(phox), suggesting a disruption of the lipid-mediated assembly process. The direct effects of on NADPH oxidase activation complicate its use as a "specific" inhibitor of DG kinase. We conclude that the protein kinase-independent pathway of NADPH oxidase activation by PA and DG involves direct interaction with NADPH oxidase components. Thus, NADPH oxidase proteins are functional targets for these lipid messengers in the neutrophil.
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PMID:Phosphatidic acid and diacylglycerol directly activate NADPH oxidase by interacting with enzyme components. 1106 Mar

Activation of phospholipase D (PLD) occurs in response to various stimuli and results from the activity of two isozymes, hPLD1 and hPLD2. PLD activity appears to be involved in several myeloid cell processes during their development and activation, including proliferation of myeloblasts in the bone marrow and secretion, phagocytosis and NADPH oxidase activation, essential functions of differentiated neutrophils. The present work studies PLD characteristics, activity and both isozyme expression during maturation and differentiation of myeloid cells by using three different systems: leukemic myeloblasts at different stages of maturation, terminally differentiated neutrophils ex vivo and four human myeloid cell lines, NB4, HL-60, PLB 985 and U937, induced to differentiate with alltrans retinoic acid (ATRA), a cyclic adenosine monophosphate (cAMP) analogue or both agents together. HL-60, a bipotential cell line has also been differentiated along the granulocytic pathway with DMSO and the monocytic pathway with 1,25-dihydroxy vitamin D3. In all these systems, PLD activity increases with maturation and differentiation whatever the inducer used and the granulocytic or monocytic pathways. Increase in basal activity which reflects the expression during development of both hPLD1 and hPLD2 appears to be mainly related to the former isozyme expression. Association of PLD characteristic changes with maturation and differentiation was also confirmed using two NB4 clones resistant to these processes. Comparison between PLD characteristics in myeloblasts during maturation and differentiation ex vivo and in vitro in the different cell lines demonstrated that NB4 induced to differentiate with ATRA represents the best model for further studies on the specific roles of each PLD isoform in various functions of differentiated myeloid cells.
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PMID:Modifications in phospholipase D activity and isoform expression occur upon maturation and differentiation in vivo and in vitro in human myeloid cells. 1118 1


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