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)

An agonist-activated phospholipase D/phosphatidic acid phosphohydrolase (PAH) pathway was recently demonstrated in human neutrophils, and evidence suggests that phosphatidic acid (PA) and/or diradylglycerol (DG) generated from this pathway participates in activation of the O2(-)-generating respiratory burst. We have used a series of cationic amphiphilic compounds (sphingosine, propranolol, chlorpromazine, and desipramine) and antibiotics (clindamycin, trimethoprim, and roxithromycin) all of which inhibit the respiratory burst, to investigate the role of the phospholipase D/PAH pathway in neutrophil activation. The phosphatidylcholine (PC) pool in intact cells was first labeled using [3H]-1-O-alkyl-lysoPC; released [3H]-PA and [3H]-DG were then quantified after the addition of either chemo-attractant or PMA. Using either agonist, all compounds showed a dose-dependent inhibition of [3H]-DG generation which correlated with inhibition of O2- generation, but compounds failed to inhibit directly the NADPH oxidase in a cell-free system. For either activator, a plot of the ID50 values for O2- generation vs those for DG generation was linear over four orders of magnitude. In many cases, inhibition of [3H]-DG generation corresponded to an increase in [3H]-PA, implicating PAH as the locus of inhibition. Superoxide generation was inhibited under conditions where PA was either elevated or minimally affected. Neither O2- release nor DG generation showed any selectivity for stereoisomers of propranolol, suggesting that this inhibition does not act via a specific binding site on PAH. No evidence was obtained for an effect of the inhibitors on PA mobility as monitored by electron spin resonance studies of spin-labeled PA in a model membrane system. Data are consistent with an effect of the inhibitors at the level of the interaction of PAH with the membrane and/or its substrate. These data imply that DG produced via the phospholipase D/PAH pathway functions in the activation or maintenance of the respiratory burst.
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PMID:Role of phospholipase D-derived diradylglycerol in the activation of the human neutrophil respiratory burst oxidase. Inhibition by phosphatidic acid phosphohydrolase inhibitors. 132 85

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

Protein kinase C (PKC) appears to have a central role in the O2- response of neutrophils following stimulation of membrane receptors. The second messenger, diacylglycerol (DG), that activates PKC is derived from membrane phospholipids via activation of phosphatidylinositol 4,5-bisphosphate (PIP2)-phospholipase C (PLC) and phospholipase D (PLD), with the latter pathway being more prominent in primed cells. In resting cells receptor coupling to PLD is through a G-protein. Priming brings a cytoplasmic tyrosine kinase into the transducer sequence which, through protein phosphorylation, increases the efficiency of coupling between membrane receptors and PLD. Phosphatidic acid (PA), the initial product of the PLD pathway, also appears to act as a second messenger by directly activating the NADPH oxidase responsible for generating O2-. Interconversion of PA and DG by phosphatidate phosphohydrolase and DG kinase determines which of these second messengers has the dominant role.
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PMID:New pathways of phagocyte activation: the coupling of receptor-linked phospholipase D and the role of tyrosine kinase in primed neutrophils. 133 78

A comparative study of real-time kinetics of respiratory burst, monitored by H2O2-dependent chemiluminescence, and phospholipase D (PLD)-mediated phosphatidylcholine breakdown has been undertaken on human neutrophils stimulated by N-formylmethionyl-leucylphenylalanine in the absence of cytochalasin B. The fungal metabolite 17-hydroxywortmannin (HWT), an inhibitor of NADPH oxidase activation, decreases phosphatidic acid (PA) production by 30% at a concentration of 1 nM. Higher concentrations (10 nM-1 microM) inhibit PA formation maximally by 50% as compared with control. In all cases, the inhibition is delayed by 20-30 s after addition of the agonist. Thus the full PA generation is actually the result of an early (HWT-insensitive) and a late (HWT-sensitive) phosphatidylcholine breakdown. However, under all conditions, alkylacylglycerol remains at the basal level. PLD activity is dependent on Ca2+ influx, but is fully inhibited in cells depleted of Ca2+ with EGTA and Quin 2. The effect of HWT on the respiratory burst was investigated by measuring the kinetics of H2O2-induced chemiluminescence. This method allows to distinguish various phases of superoxide ion production: a lag, an increase in H2O2 formation (early phase), the duration of H2O2 production (late phase) and the termination of the oxidative burst. The lag remains constant for all HWT concentrations. A concentration of 10 nM-HWT, which fully inhibits the HWT-sensitive part of PA production, decreases superoxide ion production with a delay of about 20 s after addition of the agonist. Higher HWT concentrations, which have no additional effect on PLD inhibition, equally affect an early and a late phase of the burst. Thus high doses of HWT have a site of action which decreases the whole burst but does not affect the PLD any more. Therefore HWT and Ca2+ provide evidence for a two-step process for PLD activation. Only the delayed PA generation is functionally linked to a late phase of the oxidative burst.
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PMID:Human neutrophil phospholipase D activation by N-formylmethionyl-leucylphenylalanine reveals a two-step process for the control of phosphatidylcholine breakdown and oxidative burst. 141 92

To determine the role of protein tyrosine phosphorylation in the activation of phospholipase D (PLD), electropermeabilized HL-60 cells labeled in [3H]alkyl-phosphatidylcholine were treated with vanadate derivatives. Micromolar concentrations of vanadyl hydroperoxide (V(4+)-OOH) induced accumulation of tyrosine-phosphorylated proteins. Concomitantly, V(4+)-OOH or a combination of vanadate and NADPH elicited a concentration- and time-dependent accumulation of phosphatidic acid (PtdOH). In the presence of ethanol a sustained formation of phosphatidylethanol was observed, indicating that a type D phospholipase was activated. A good correlation was found to exist between the accumulation of tyrosine-phosphorylated proteins and activation of PLD. The V(4+)-OOH concentration dependence of the two responses was nearly identical, and the time course of activation was similar, with tyrosine phosphorylation preceding PLD activation by approximately 1 min. The ability of V(4+)-OOH to induce both responses was found to be strictly dependent on the presence of ATP and/or Mg2+, suggesting that PLD activation involves phosphotransferase reactions. Accordingly, ST638, a tyrosine kinase inhibitor, reduced concomitantly tyrosine phosphorylation and PLD activation elicited by V(4+)-OOH. The mechanism of action of V(4+)-OOH was investigated. The diacylglycerol kinase inhibitors, dioctanoylethylene glycol and R59022 potentiated PLD stimulation by exogenous diacylglycerol but not by V(4+)-OOH. Moreover, stimulation by V(4+)-OOH and by phorbol esters was synergystic. Therefore, diacylglycerol-induced activation of protein kinase C is unlikely to mediate the effects of V(4+)-OOH. The response of PLD to V(4+)-OOH was larger than that to guanosine 5'-(gamma-thio)triphosphate. Moreover, the effects of GTP gamma S and V(4+)-OOH were additive. Hence, activation of G proteins cannot account for the stimulation of PLD by V(4+)-OOH. V(4+)-OOH also triggers a burst of O2 consumption by the NADPH oxidase. Inhibition of PtdOH accumulation by addition of ethanol or by ST638 abolished this respiratory burst. Together, the results establish a strong correlation between tyrosine phosphorylation, PLD activation, and stimulation of the NADPH oxidase in HL-60 cells, suggesting a causal relationship.
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PMID:Peroxides of vanadate induce activation of phospholipase D in HL-60 cells. Role of tyrosine phosphorylation. 160 60

Tumor necrosis factor alpha (TNF) primes human neutrophils (PMN) for enhanced superoxide (O2-) production if cells are subsequently stimulated with the chemotactic peptide, n-formyl-Met-Leu-Phe (fMLP). fMLP activates phospholipase D to form phosphatidic acid (PA), and a correlation may exist between PA production and O2- generation in PMN. Therefore, we assessed the ability of TNF to prime phospholipase D activation in PMN stimulated with fMLP. TNF (100 units/ml) pretreatment primed enhanced PA production in PMN challenged with 1 microM fMLP, in the absence of cytochalasin B, as demonstrated by increased production of tritiated PA from PMN label with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine ([3H]LPAF) and by increased PA mass. PA was formed via activation of phospholipase D and occurred with minimal production of diglycerides. Production of O2- was also enhanced in identically treated cells, and we demonstrated a direct correlation between enhanced PA formation and O2- production. Conversely, ethanol inhibition of PA formation led to a comparable reduction in O2- generation. This report of priming of phospholipase D by physiological agonists is the only natural system where enhanced PA formation has been dissociated from diglyceride formation. Our results suggest a link between PA production and NADPH oxidase activation in human PMN.
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PMID:Tumor necrosis factor alpha priming of phospholipase D in human neutrophils. Correlation between phosphatidic acid production and superoxide generation. 184 15

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

Activation of human neutrophils by receptor-mediated agonists, the Ca2+ ionophore A23187, or the protein kinase C activator phorbol myristate acetate all stimulated phospholipase D activity. This was demonstrated by the increased formation of phosphatidic acid, and in the presence of ethanol, phosphatidylethanol (PEt) accumulation. EGTA completely inhibited A23187-induced PEt formation, but only one-half of the fMLP-induced PEt accumulation. Staurosporin, an inhibitor of protein kinase C, strongly inhibited PMA-induced PEt formation, but actually stimulated the formation of PEt in response to fMLP by several-fold. Thus, increased cytosolic Ca2+ and activated protein kinase C can each lead to activation of phospholipase D, but neither is required for receptor-mediated activation of phospholipase D activity. Wortmannin is an irreversible inhibitor of the oxidative burst, but does not inhibit NADPH oxidase or known components of signal transduction. Wortmannin inhibited activation of phospholipase D in response to fMPL. It did not directly inhibit phospholipase D, as the response to A23187 was unaffected. Wortmannin did not inhibit other fMPL-stimulated events, such as aggregation or adherence. We conclude that inhibition by wortmannin defines a third pathway to activation of phospholipase D. Further, its effect on phospholipase D correlates with its effect on the respiratory burst.
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PMID:Activation of human neutrophil phospholipase D by three separable mechanisms. 210 52

It is widely accepted that the activation of the NADPH oxidase of phagocytes is linked to the stimulation of protein kinase C by diacylglycerol formed by hydrolysis of phospholipids. The main source would be choline containing phospholipid via phospholipase D and phosphatidate phosphohydrolase. This paper presents a condition where the activation of the respiratory burst by FMLP correlates with the formation of phosphatidic acid, via phospholipase D, and not with that of diacylglycerol. In fact: 1) in neutrophils treated with propranolol, an inhibitor of phosphatidate phosphohydrolase, FMLP plus cytochalasin B induces a respiratory burst associated with a stimulation of phospholipase D, formation of phosphatidic acid and complete inhibition of that of diacylglycerol. 2) The respiratory burst by FMLP plus cytochalasin B lasts a few minutes and may be restimulated by propranolol which induces an accumulation of phosphatidic acid. 3) In neutrophils stimulated by FMLP in the absence of cytochalasin B propranolol causes an accumulation of phosphatidic acid and a marked enhancement of the respiratory burst without formation of diacylglycerol. 4) The inhibition of the formation of phosphatidic acid via phospholipase D by butanol inhibits the respiratory burst by FMLP.
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PMID:Phosphatidic acid and not diacylglycerol generated by phospholipase D is functionally linked to the activation of the NADPH oxidase by FMLP in human neutrophils. 232 8

The vast majority of extracellular signals alters cell function by activating cell surface receptors. The transmembranous signalling process initiated by an activated receptor leads to the generation of an intracellular signal and eventually to a cellular response. In contrast to receptors that are permanently coupled to an enzyme or an ion channel representing the effector, a large number of surface receptors for hormones, neurotransmitters and receptors for exogenous chemical or physical stimuli reversibly interacts with membranous signal transduction components which, in turn, regulate intracellular messenger-generating effectors. The transducer molecules isolated so far form a family of guanine nucleotide-binding proteins (G- or N-proteins). All isolated G-proteins are composed of three different subunits (alpha, beta, gamma). The alpha-subunit, which is specific for the individual G-protein, binds and hydrolyzes GTP and is target of ADP-ribosylating bacterial toxins. Hormone-induced activation of a receptor causes interaction with the alpha-subunit of a G-protein and the exchange of bound GDP with GTP. The GTP-bound form of the alpha-subunit represents the active form of the G-protein, which is capable of stimulating or inhibiting the respective effector. The active state of the alpha-subunit is terminated by its inherent GTPase activity causing hydrolysis of bound GTP. The beta gamma-complexes of G-proteins are structurally very similar and functionally interchangeable; they appear to dissociate from the alpha-subunits during receptor activation of the G-protein. Possible functions of the beta gamma-complex are to anchor the non-activated G-protein in the membrane, to facilitate G-protein-receptor interaction, and to promote the inactive state of the alpha-subunit. G-protein-regulated effectors include enzymes, ion channels and probably transporters. The best studied G-protein-regulated enzyme is the retinal cyclic GMP-phosphodiesterase which is activated by bleached rhodopsin via the tissue-specific G-protein, termed transducin. The ubiquitously occurring membrane-bound adenylate cyclase is under dual control by families of stimulatory and inhibitory receptors, acting via G-proteins called Gs and Gi, respectively. Moreover, the receptor control of phospholipases A2 and C and probably of phospholipase D most likely involves G-proteins which have not yet been identified. Finally, the activity of NADPH oxidase of neutrophils and that of cyclic AMP phosphodiesterases in liver and fat cells may be regulated via G-proteins. Modulations of non-enzymatic effectors are reviewed elsewhere.
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PMID:[Guanidine nucleotide binding proteins as membrane signal transduction components and regulators of enzymatic effectors]. 284 11


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