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 formation of oxygen radicals by phagocytic cells occurs through the activation of a multiple-component NADPH oxidase system. An unidentified low molecular weight GTP-binding protein has been proposed to modulate the activity of the NADPH oxidase. The low molecular weight GTP-binding proteins undergo posttranslational processing, including an initial covalent incorporation of an isoprenyl group. To test whether such an isoprenylation reaction might be required for the activity of the oxidase, we utilized compactin and lovastatin as inhibitors of the isoprenylation pathway. Treatment of DMSO-differentiated HL-60 cells with compactin produced a concentration-dependent inhibition of O2- formation in response to FMLP or phorbol myristate acetate. Cell viability was not affected nor was normal differentiation of the HL-60 cells into a neutrophil-like cell. The inhibitory effect of compactin was specifically prevented by addition of exogenous mevalonic acid to the HL-60 cells, indicating that the inhibitory effects of the drug were due to blockade of the pathway leading to isoprenoid synthesis. Addition of cholesterol, ubiquinone, or dolichol, which are also downstream products of the isoprenoid pathway, did not override the inhibitory effects of the drug. Subcellular fractions were prepared from compactin-treated cells, and the location of the compactin-sensitive factor was determined by complementation analysis in a cell-free NADPH oxidase system. The inhibited factor was localized to the HL-60 cytosol. These data suggest that an isoprenoid pathway intermediate is necessary for activation of the phagocyte NADPH oxidase. This is likely to represent the requirement for an isoprenoid moiety in the posttranslational modification of a low molecular weight GTP-binding protein. Our studies provide support for the involvement of such a low molecular weight GTP-binding protein in NADPH oxidase activation.
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PMID:Isoprenoid metabolism is required for stimulation of the respiratory burst oxidase of HL-60 cells. 131 Jun 93

C1q, a plasma glycoprotein and the recognition component of the classical complement pathway, interacts with specific cells of the immune system resulting in the enhancement of cell function. For example, interaction of C1q with its cell-surface receptor on neutrophils induces the activation of the respiratory burst, a finding previously documented using a chemiluminescent assay to detect oxygen radical formation. In an alternative approach we have now used a modified cytochrome c reduction assay to characterize C1q-mediated production of superoxide anion (O2-) in more detail. C1q coated to microtiter wells induced O2- release, which occurred microtiter wells induced O2- release, which occurred after a lag period of 10 to 20 min, and was then sustained over approximately 1 h. O2- production could be triggered by the purified pepsin-resistant, collagen-like fragment of C1q, but not by mannose-binding protein and pulmonary surfactant protein A, proteins that also contain collagen-like domains. Concentrations of C1q which promoted a vigorous O2- generation did not induce release of neutrophil primary granules and caused little or no secondary granule release. Investigation of the biochemical events mediating C1q stimulated O2- production by neutrophils revealed that the response invoked two biochemical pathways with distinct sensitivities to previously described inhibitors. A role for Ca2+ in initiation of the response was suggested by the inhibitory effect of EGTA, the calmodulin antagonist W7, and the intracellular Ca2+ chelator BAPTA. The protein kinase inhibitor staurosporine did not inhibit the induction of the response, but did block that component of the response occurring after approximately 30 min. Neither phase of C1q-mediated O2- production was inhibited by pertussis toxin, a strong inhibitor of the G-protein-coupled FMLP-mediated response. In summary, C1q-triggered O2- production is relatively unique both in terms of the kinetics of the response and the biochemical pathways evoked. These data support the hypothesis that more than one biochemical pathway induced by ligand-receptor interaction can activate the neutrophil NADPH oxidase.
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PMID:Signal transduction mechanisms of C1q-mediated superoxide production. Evidence for the involvement of temporally distinct staurosporine-insensitive and sensitive pathways. 131 35

Nitric oxide provokes vasodilation and inhibits platelet aggregation. We examined the effect of nitric oxide on superoxide anion production by three sources: activated intact neutrophils, xanthine oxidase/hypoxanthine, and the NADPH oxidase. Nitric oxide significantly inhibited the generation of superoxide anion by neutrophils exposed to either FMLP (10(-7)M) or PMA (150 ng/ml) (IC50 = 30 microM). To determine whether the effect of nitric oxide on the respiratory burst was due to simple scavenging of O2+, kinetic studies that compared effects on neutrophils and the cell-free xanthine oxidase system were performed. Nitric oxide inhibited O2+ produced by xanthine oxidase only when added simultaneously with substrate, consistent with the short half-life of NO in oxygenated solution. In contrast, the addition of nitric oxide to neutrophils 20 min before FMLP resulted in the inhibition of O2+ production, which suggests formation of a stable intermediate. The effect of nitric oxide on the cell-free NADPH oxidase superoxide-generating system was also examined: The addition of NO before arachidonate activation (t = -6 min) significantly inhibited superoxide anion production. Nitric oxide did not inhibit O2+ when added at NADPH initiation (t = 0). Treatment of the membrane but not cytosolic component of the oxidase was sufficient to inhibit O2+ generation. The data suggest that nitric oxide inhibits neutrophil O2+ production via direct effects on membrane components of the NADPH oxidase. This action must occur before the assembly of the activated complex.
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PMID:Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase. 132 92

Activation of human neutrophils by PMA causes a post-translational incorporation of 14C-labeled tyrosine into multiple neutrophil (PMN) proteins, that is distinctly different from the enzymatic tyrosinolation of tubulin in FMLP-stimulated PMN. Post-translational incorporation of other radiolabeled amino acids, including the structurally similar amino acid phenylalanine, does not occur under identical conditions of neutrophil activation, suggesting an involvement of the phenolic hydroxyl group of tyrosine in the PMA-mediated reaction. Similar to the stimulation of PMN tubulin tyrosinolation by FMLP, the PMA-induced incorporation of tyrosine into multiple PMN proteins is closely associated with activation of the NADPH oxidase-mediated respiratory burst in stimulated PMN and can be inhibited by a variety of reducing agents, inhibitors of peroxidase-mediated reactions, and intracellular scavengers of oxygen radicals. Moreover, the PMA-induced post-translational incorporation of tyrosine does not occur in PMN from patients with chronic granulomatous disease and is significantly reduced (50%) in PMN of an individual with myeloperoxidase deficiency. A similar stimulus-induced incorporation of tyrosine into multiple PMN proteins is also observed in PMN exposed to various phagocytic stimuli, and the incorporated radioactivity in cells undergoing phagocytosis is substantially enriched (40- to 50-fold) in isolated PMN phagolysosomes. Consistent with this latter observation, HPLC fractionation of stimulated PMN proteins and analysis of the incorporated radioactivity reveal that the 14C label is primarily associated with PMN membrane proteins. Furthermore, this post-translational incorporation of tyrosine, like that associated with PMA stimulation, is associated with production of oxygen radicals and the generation of protein carbonyl derivatives, which are indicative of oxidative protein modifications via mixed function oxidases. Our findings indicate that tyrosine incorporation into membrane proteins of stimulated PMN is functionally relevant to the physiologic host-defense responses of human neutrophils undergoing phagocytosis.
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PMID:A novel post-translational incorporation of tyrosine into multiple proteins in activated human neutrophils. Correlation with phagocytosis and activation of the NADPH oxidase-mediated respiratory burst. 133 Dec 34

The mechanism of cAMP regulation of the respiratory burst was studied with HL-60 cells that had been DMSO-differentiated to a neutrophil-like cell. To evaluate the effects of known cAMP concentrations, cells were permeabilized with streptolysin-O. Chemotactic peptide (FMLP)-stimulated NADPH oxidase activity was inhibited by cAMP at concentrations higher than 3 microM. Because intracellular calcium was buffered, inhibitory actions of cAMP were not mediated by modulation of calcium concentration. Effects of cAMP on chemotactic peptide signal transduction mediated by phospholipase C, phospholipase D, and phospholipase A2 were then determined. Neither inositol phosphate generation (phospholipase C) nor phosphatidylethanol generation (phospholipase D activity in presence of 1.6% ethanol) induced by FMLP were significantly affected by cAMP. In contrast, cAMP potently inhibited FMLP-induced arachidonic acid mobilization (phospholipase A2). NADPH oxidase activity induced by exogenous arachidonic acid was not inhibited by cAMP. These results indicate that cAMP-mediated inhibition of arachidonic acid mobilization may be important in regulation of the respiratory burst.
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PMID:Regulation of the respiratory burst by cyclic 3',5'-AMP, an association with inhibition of arachidonic acid release. 133 10

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.
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PMID:The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with the neutrophil respiratory burst mediated by a signaling pathway independent of PtdInsP2 breakdown and cytosolic free calcium. 165 5

Studies on the role of microtubule integrity in stimulus-response coupling in neutrophils have generated contradictory data. To determine the role of microtubule integrity in stimulus-response coupling elicited by two different mechanisms, i.e., engagement of the Fc receptors (FcR gamma II, FcR gamma III) or engagement of the receptor for FMLP, we utilized colchicine (10 microM), which reduces pericentriolar microtubules to 29% of control, and compared its effect with that of nocodazole (50 microM) and lumicolchicine (10 microM). We now demonstrate that treatment of neutrophils with colchicine but not lumicolchicine, inhibits degranulation elicited by engagement of Fc receptors but augments degranulation in response to FMLP. In contrast to the ligand-specific effect of microtubule-disruption on degranulation, superoxide anion production (assembly of the NADPH oxidase) is unaffected by colchicine regardless of the ligand. To determine whether intact microtubules were required for responses elicited by ligation of Fc gamma RII(CD32) or Fc gamma RIII(CD16), mAb directed against these receptors were employed. Treatment of neutrophils with mAb KuFc79 directed against Fc gamma RII(CD32) or mAb 3G8 directed against Fc gamma RIII(CD16) inhibited degranulation of neutrophils elicited by immune complexes (IC). In contrast, removal of most of Fc gamma RIII by phosphatidylinositol-specific phospholipase C did not significantly alter degranulation in response to IC. We conclude that degranulation elicited by IC results from ligation of both Fc gamma RII and phosphatidylinositol-specific phospholipase C-insensitive Fc gamma RIII. The importance of microtubule integrity on the generation of intracellular signals was also examined. Degranulation of neutrophils proceeds via pertussis toxin-sensitive and insensitive pathways; treatment of cells with colchicine did not augment the action of pertussis toxin. Stimulation of neutrophils by chemoattractants results in a biphasic increase in 1,2-sn-diacylglycerol; a rapid increase ("triggering") secondary to the action of a phosphatidylinositol-specific phospholipase C, and a late increase ("activation") secondary to the action of a phosphatidylcholine-specific phospholipase C. Treatment of cells with colchicine altered the production of both [3H]-arachidonic acid-diacylglycerol and diacyl[14C]glycerol in parallel to its effect on degranulation. These studies indicate that the requirement of intact microtubules for degranulation is ligand-specific. Furthermore, assembly of the respiratory burst oxidase does not require intact microtubules. Microtubules most likely alter the cycling of specific receptors or the generation of specific intracellular signals required for stimulus-response coupling in the course of degranulation. Intact microtubules are not uniformly required for the discharge of granule contents during exocytosis.
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PMID:Differences in signal transduction between Fc gamma receptors (Fc gamma RII, Fc gamma RIII) and FMLP receptors in neutrophils. Effects of colchicine on pertussis toxin sensitivity and diacylglycerol formation. 184 87

The principal sulfatide of a group of acidic lipids from virulent Mycobacterium tuberculosis, sulfolipid-1 (SL-1), stimulates neutrophil superoxide (O2-) generation and, at lower concentrations, primes neutrophil response to several other metabolic agonists including FMLP, and PMA. These responses to SL-1 were examined in relation to diacylglycerol (DAG) generation, Ca2+ availability and activation of guanine nucleotide binding proteins to clarify the signal transduction pathways involved. Pertussis toxin inhibited the ability of SL-1 to both stimulate neutrophils directly and to prime neutrophils for subsequent responses induced by PMA, suggesting a role for one or more guanine nucleotide regulating proteins in both responses. SL-1 induced a rise in neutrophil DAG levels. DAG generation was inhibited by pretreatment of cells with pertussis toxin. Depletion of extracellular Ca2+ ablated O2- release induced by stimulatory levels of SL-1 but did not inhibit the priming effect induced by substimulatory concentrations of the lipid. Investigation of the activation of the neutrophil NADPH oxidase in a cell-free system revealed that the SL-1-priming effect was associated with translocation of the soluble cytosolic factors required for activation of the enzyme. Cytosolic factor translocation was not observed in pertussis toxin pretreated cells. Our results provide evidence for the role of a guanine nucleotide binding protein in both priming and direct activation of neutrophils by SL-1. This G protein regulates both SL-1-induced DAG generation and cytosolic cofactor translocation involved in neutrophil activation and priming. The multiplicity of effects of SL-1 on signal transduction pathways leading to phagocyte activation and priming may exert a profound influence on the pathogenicity of M. tuberculosis.
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PMID:Activation of human neutrophils by Mycobacterium tuberculosis-derived sulfolipid-1. 184 37

Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway.
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PMID:Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase. 186 64

Neutrophils from cystic fibrosis (CF) patients have been shown previously to be defective in their response (beta-glucuronidase exocytosis, NADPH oxidase activation) to the chemotactic peptide FMLP. In this work, we attempted to identify the defective step in this response. We showed that stimulated CF and control neutrophils do not differ in the formation of inositol phosphates. On the other hand, direct stimulation of protein kinase C with phorbol myristate acetate (PMA) revealed a subnormal stimulation of beta-glucuronidase exocytosis in CF neutrophils. Furthermore, retroinhibition exerted by PMA-activated protein kinase C on stimulated inositol phosphates or on beta-glucuronidase exocytosis was marginal or absent in CF neutrophils, whereas it was significant in the case of control neutrophils. Our observations suggest that the CFTR gene is expressed in neutrophils and is involved in protein kinase C-mediated actions.
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PMID:Defective protein kinase C-mediated actions in cystic fibrosis neutrophils. 189 35


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