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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two principal pathways of Ca2+ release from the sarcoplasmic reticulum of excitable and non-excitable cells have been described: one pathway dependent on the second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), and a second pathway sensitive to Ca2+ and regulated by caffeine and ryanodine. It was found that the Ca(2+)-pump activity of vascular smooth muscle sarcoplasmic reticulum is inhibited by superoxide anion radicals (O2.-); however, the effects of reactive oxygen intermediates on sarcoplasmic reticulum Ca2+ release in vascular muscle cells are not well defined. The purpose of the present study was to evaluate the effects of reactive oxygen intermediates generated from the hypoxanthine/xanthine oxidase reaction system on contractions induced by caffeine, Ins(1,4,5)P3 and norepinephrine in staphylococcal alpha-toxin-permeabilized rabbit mesenteric arteries. This system generates O2.-, H2O2, and hydroxyl radicals. We wished to identify which class of reactive oxygen intermediates is responsible for the associated loss of vascular smooth muscle contractile function. Caffeine and Ins(1,4,5)P3 produced a transient contraction when the sarcoplasmic reticulum of the permeabilized, preparations was preloaded with pCa 7.0 solution for 5 min before washing with 0.5 mM EGTA solution; norepinephrine also produced a transient contraction. Exposure of the preparations to hypoxanthine/xanthine oxidase (for 30 min) attenuated caffeine-induced contraction, but was without effect on Ins(1,4,5)P3-induced contraction. The observed effect of hypoxanthine/xanthine oxidase exposure was superoxide dismutase-inhibitable, suggesting O2.- involvement. Hypoxanthine/xanthine oxidase also inhibited norepinephrine-induced contraction. The effect of hypoxanthine/xanthine oxidase on norepinephrine contraction was protected by catalase, but not by superoxide dismutase and dimethyl sulfoxide; exogenously added H2O2 mimicked the effect of hypoxanthine/xanthine oxidase exposure. H2O2, added exogenously, was without effect on Ins(1,4,5)P3-induced contraction. It is suggested that the pathway of Ca2+ release from the sarcoplasmic reticulum dependent on Ins(1,4,5)P3 is insensitive to O2.-. Instead, caffeine-induced Ca2+ release mechanisms may be susceptible to O2.- and H2O2, rather than O2.- and hydroxyl radicals, may be the active agent in the norepinephrine-induced contraction. Our results are also consistent with the view that the attenuation by H2O2 of the norepinephrine-induced contraction may be linked to the receptor-associated pathway of Ins(1,4,5)P3 formation, but not to degradation processes of Ins(1,4,5)P3.
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PMID:Susceptibility of caffeine- and Ins(1,4,5)P3-induced contractions to oxidants in permeabilized vascular smooth muscle. 904 2

The primary objective of this study was to determine the influence of stretch-induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silastic-bottomed tissue culture wells in medium that was usually supplemented with 10 microM unlabeled arachidonate. Cell injury was produced by stretching (5-10 mm) the Silastic membrane with a 50-ms pulse of compressed air. Stretch-induced cell injury increased the incorporation of [3H]choline into PC in an incubation time- and stretch magnitude-dependent manner. PC biosynthesis was increased three- to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch-induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A2 (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch-induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretreating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch-dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n-acetylcysteine). Arachidonate-enriched cells were more susceptible to stretch-induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non-arachidonate-enriched cells. In summary, the data suggest that stretch-induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron-catalyzed Haber-Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cell's membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch-induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch-induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction.
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PMID:Alterations in phosphatidylcholine metabolism of stretch-injured cultured rat astrocytes. 910 16

Cultured human and rat endothelial cells were used to study cellular toxicity and Ca2+ signalling upon exposure to reactive oxygen species. Superoxide and hydrogen peroxide (O2.-/H2O2) were produced by the hypoxanthine/xanthine oxidase system (HX/XO) and caused intracellular Ca2+ concentration ([Ca2+]i) to rise steadily when activities above 2 mU/ml were used. These Ca2+ increases were also measured when the glucose/glucose oxidase (G/GO) system above 5 mU/ml was used to produce hydrogen peroxide (H2O2). Gross morphological changes appeared to parallel elevated [Ca2+]i levels preceding cell death. However, when HX/XO or G/GO were used at non toxic doses rapid and transient changes in [Ca2+]i were measured. These treatments did not alter subsequent receptor mediated Ca2+ signalling induced by ATP (10 microM) or histamine (100 microM). Superoxide dismutase (50 U/ml), which dismutates O2.- into H2O2 also had no influence, whereas catalase (50 U/ml), which removes H2O2, completely diminished transient [Ca2+]i responses. H2O2 added directly was able to induce similar Ca2+ transients when concentrations of at least 500 microM were used. Buffering trace amounts of iron (o-phenanthroline; 200 microM) in order to inhibit .OH radical formation was not effective to alter Ca2+ changes. Experiments performed in Ca(2+)-free buffer showed a similar rise in [Ca2+]i and readdition of Ca2+ to the extracellular medium indicated the activation of store operated Ca2+ entry. Blocking Ca(2+)-ATPases of the endoplasmatic reticulum with thapsigargin (1 microM) inhibited ROS induced transient increases and cells preincubated with pertussis toxin (200 nM) showed unchanged Ca2+ transients after exposure to both enzyme systems. Phospholipase C inhibitor U73122 (2 microM) effectively reduced hydrogen peroxide induced emptying of intracellular stores. Taken together, we demonstrate that enzymatically produced non-toxic H2O2 rather than O2.- or .OH causes calcium signalling from thapsigargin sensitive stores, and activates store operated Ca2+ entry at least partially by activating phospholipase C. These changes clearly differ from pathological 'oxidative stress' associated with a progressive increase in [Ca2+]i.
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PMID:Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species: role of hydrogen peroxide. 920 90

To understand the pathogenesis of vasculitides, we analyzed how cytokine stimulation of HUVEC in vitro activates the cytotoxic capacity of polymorphonuclear (PMN) granulocytes. IL-1beta, IFN-gamma, or TNF-alpha caused highly significant dose and time-dependent HUVEC injury. TNF-alpha-treated HUVEC activated the PMN by means of phospholipase C-related event, since coincubations conferred PMN to react with a rise of cytosolic calcium concentrations, [Ca2+]i. Ab blockade of ICAM-1 on HUVEC inhibited 50 to 70% of the injury induced by these cytokines, whereas a mAb to E-selectin reduced 45 to 65% of IL-1beta- and TNF-alpha-, but not IFN-gamma-induced cytotoxicity. The role of nitric oxide (NO) was of significance since injury induced by each cytokine was reduced by 60 to 87% by specific NO-synthase inhibitors, as well as by scavenging extracellular NO by oxyhemoglobin. In contrast, injury induced by TNF-alpha was inhibited by neither superoxide dismutase or catalase, alpha1-antitrypsin, alpha2-macroglobulin, nor the platelet-activating factor receptor antagonist WEB-2086. Moreover, PMN from a patient with chronic granulomatous disease were fully capable of mediating cytotoxicity. The possibility that IL-8, produced by HUVEC in response to TNF-alpha, mediated activation of PMN was not corroborated since addition of an IL-8-blocking mAb did not modify HUVEC injury. Nonetheless, the IL-8 mAb (but not WEB-2086) blocked the rise of [Ca2+]i. Thus, in this in vitro model of vasculitis, the effect of IL-1beta, IFN-gamma, and TNF-alpha as promotors of cytokine-mediated neutrophil-dependent injury to HUVEC is a process dependent on expression of adhesion molecules and probably associated with NO produced in the system.
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PMID:Cytokine-induced neutrophil-mediated injury of human endothelial cells. 921 11

The role of polymorphonuclear leukocytes (PMN) in stemming systemic infection is executed mainly by the utilization of molecular O2 leading to the production of reactive oxygen intermediates (ROI). PMN-derived ROI also serve as intra- and extracellular second messengers providing both positive and negative feedback on cellular autoregulation. We investigated the effect of endogenous ROI on two signal transducing pathways: the receptor (R)-G-protein-phospholipase D (PLD) and receptor (R)-G-protein-phospholipase C pathways responsible for the subsequent interleukin-8 (IL-8)-induced PMN respiratory burst. Purified human PMN were primed with LPS adhered to plastic surfaces and stimulated with IL-8 with or without the presence of each of five different selective ROI scavengers/antioxidants: DMSO, N(a)N3, L-alanine, catalase, or superoxide dismutase. Total IL-8 surface receptor expression was assessed by 125I-IL-8 and 125I-labeled mAbs against IL-8R type A and B binding assays; PLD activation was assessed by measuring formation of phosphatidyl ethanol (PEt) in the presence of ethanol; PLC activation was measured by quantitative conversion of [32P]ATP-labeled phosphatidic acid (PA) into diacylglycerol (DAG); expression of G alpha-inhibitory subunit was assessed by SDS-PAGE and immunoblotting with polyclonal Abs against this subunit. Production of O2-, H2O2, HClO, and myeloperoxidase (MPO) in the experimental model was confirmed in a separate set of experiments. The overall impact of antioxidants on each component of the transducing tripartite complex was stimulatory; however, N(a)N3 and SOD exhibited the most ubiquitous effect with consistent up-regulation by N(a)N3 of IL-8R expression, whereas even trace amounts of externally added authentic MPO significantly down-regulated the functional activity of both effector enzymes. These results demonstrate a multiple site-specific targeting of the signal-transducing complex by endogenous PMN-derived ROI and an overall protective effect of ROI scavengers/antioxidants.
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PMID:Endogenous PMN-derived reactive oxygen intermediates provide feedback regulation on respiratory burst signal transduction. 926 41

In this study, we investigated whether (1) collagen-induced platelet aggregation is associated with a burst of H2O2, (2) this oxidant species is involved in the activation of platelets, and (3) the pathways of platelet activation are stimulated by H2O2. Collagen-induced platelet aggregation was associated with production of H2O2, which was abolished by catalase, an enzyme that destroys H2O2. H2O2 production was not observed when ADP or thrombin were used as agonists. Catalase inhibited dose-dependently thromboxane A2 production, release of arachidonic acid from platelet membrane, and Inositol 1,4,5P3 (IP3) formation. In aspirin-treated platelets stimulated with high concentrations of collagen, catalase inhibited platelet aggregation, calcium mobilization, and IP3 production. This study suggests that collagen-induced platelet aggregation is associated with a burst of H2O2 that acts as a second messenger by stimulating the arachidonic acid metabolism and phospholipase C pathway.
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PMID:Hydrogen peroxide is involved in collagen-induced platelet activation. 942 1

N-acetyl-L-cysteine (NAC), which is known as a multipotential agent; an antioxidant, a thiol reagent, or a tyrosine kinase inhibitor, inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced phospholipase D (PLD) activation in HL60 cells in a concentration-dependent manner (IC50 = 2 mM). Its inhibitory mechanism was examined in this study to gain insight into the regulation of PLD activity. NAC had no direct effect on membrane PLD activity in an in vitro assay system. fMLP-induced formation of inositol phosphates via phospholipase C (PLC) was not affected by the drug, suggesting that the receptor-G protein coupling was not inhibited. H2O2, which is known to induce PLD activation in several types of cells, failed to activate PLD in HL60 cells. Pretreatment of 3-amino-1,2,4-triazole (ATZ), a catalase inhibitor, did not enhance fMLP-induced PLD activation. NAC inhibited fMLP-induced tyrosine phosphorylation of several protein bands (42, 44, 64, and 138 kDa) in a concentration-dependent manner. The temporal and concentration-dependent inhibitory profiles for tyrosine phosphorylation of 64- and 138-kDa proteins were well correlated with PLD activation. However, thiol reagents, 1 mM 2,3-dimercapto-l-propanol (2,3-DMP), 1 mM dithiothreitol (DTT), and 2 mM cysteine also did not suppress protein tyrosine phosphorylation or PBut formation by fMLP. Wortmannin, a selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, inhibited these two tyrosine phosphorylation bands. These results suggest that NAC inhibits fMLP-induced PLD activation through blockage of protein tyrosine phosphorylation, which is located at the downstream of PI-3 kinase.
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PMID:Involvement of tyrosine phosphorylation in inhibition of fMLP-induced PLD activation by N-acetyl-L-cysteine in differentiated HL60 cells. 962 Jun 73

The enzyme activities of listeriolysin O (LLO), phosphatidylinositol-specific phospholipase C (PI-PLC), catalase (CA), and superoxide dismutase (SOD) from Listeria monocytogenes SLCC 5764 were examined after heat treatment, growth at elevated temperatures, and anaerobic growth. Growth at elevated temperatures may influence virulence as expressed by virulence enzyme activity. The enzymes were heated for 0 and 10 min at temperatures ranging from 40 to 100 degrees C. The production of LLO was examined when the bacterium was grown at elevated temperatures, and the production of LLO, CA, and SOD were examined after anaerobic growth. LLO was the most heat-labile factor, losing almost all activity when heated above 50 degrees C. CA was more heat stable, showing no decrease in activity until it was heated at 55 degrees C. The PI-PLC enzyme was most heat resistant: the activity decreased between 40 and 50 degrees C and continued to decrease when heated between 65 and 100 degrees C. When L. monocytogenes was grown at elevated temperatures, it produced less LLO than when grown at the optimum growth temperature of 37 degrees C. When the organism was grown under anaerobic conditions, the levels of CA and LLO decreased, while the SOD level remained unchanged.
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PMID:Heat stability of virulence-associated enzymes from Listeria monocytogenes SLCC 5764. 967 77

The role of the inflammatory cytokine interleukin 1beta (IL-1beta) as potent agonist of the PMN respiratory burst signal transduction cascade has been described. We hypothesized that this phenomenon is self-limiting and that polymorphonuclear leukocyte (PMN)-derived reactive oxygen intermediates (ROI) might provide feedback regulation on the IL-1beta surface receptor (IL-1betaR)-G-protein-effector enzyme transducing tripartite complex that ultimately leads to NADPH oxidase activation. Therefore, we separately assessed either baseline or IL-1beta-induced activation of each member of the IL-1betaR-G-protein-phospholipase D (PLD) or IL-1betaR-G-protein-phospholipase C (PLC) signaling systems in the presence or absence of one of several specific ROI scavengers/antioxidants. Purified human PMN were lipopolysaccharide primed, adhered for 2 h, and stimulated with 100 ng/mL IL-1beta with or without 1% v/v dimethyl sulfoxide, 10 mM NaN3, 30 mM L-alanine, 200 U catalase, or 300 U superoxide dismutase (SOD). To validate the use of these antioxidants, the production of O2-, H2O2, hypochlorous acid, or myeloperoxidase (MPO) in the employed experimental model was confirmed in a separate set of experiments. The expression of IL-1betaR type I or II was assessed by binding with corresponding 125I-labeled monoclonal antibodies and corrected for nonspecific binding. PLD activation was assessed by measuring phosphatidyl ethanol formation in the presence of ethanol. PLC activation was determined by quantitative measurement of diacylglycerol. The level of Galpha stimulatory and inhibitory subunits was assessed by Western blotting. IL-1betaR type I expression was significantly up-regulated in the presence of catalase and SOD. PLD activation was increased by dimethyl sulfoxide and NaN3, and PLC activation was up-regulated by NaN3, L-alanine, SOD, and catalase. After 5 min of stimulation with IL-1beta, Gialpha expression was significantly down-regulated by NaN3 and SOD, whereas SOD had an up-regulating effect on the expression of Gs alpha. Increasing concentrations of externally added authentic MPO progressively down-regulated both PLD and PLC activity. Thus, PMN-derived ROI, in addition to their role as antibacterial/fungal agents, serve as second messengers in IL-1beta signal transduction, with MPO having the most ubiquitous role as a modulator of PMN second messenger pathways.
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PMID:The role of neutrophil-derived oxidants as second messengers in interleukin 1beta-stimulated cells. 968 92

Cis-unsaturated fatty acids (c-UFAs) have been shown to be capable of decreasing the survival of macrophage tumor (AK-5) cells in vitro. This cytotoxic action of c-UFAs was found to be associated with an increase in free radical generation and lipid peroxidation process and a simultaneous decrease in cellular anti-oxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase, glutathione and vitamin E. In the present study, it was observed that c-UFAs such as gamma linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can activate phospholipase C (PLC) and enhance diacylglycerol formation; all the fatty acids except alpha linolenic acid (ALA) increased the binding of phorbol dibutyrate acetate (PDBu) suggesting translocation of protein kinase C (PKC) and at the same time these fatty acids (especially GLA, AA, EPA and DHA) also enhanced PKC activity. AA, EPA and DHA decreased the activity of protein kinase A (PKA) both in the cytosol and particulate fractions whereas ALA and GLA enhanced the PKA activity in the particulate fractions; all the fatty acids except ALA reduced cyclic AMP levels and an enhanced phosphorylation of about 13 proteins of the nuclear fraction and about eight proteins of the plasma membrane fraction was noted in c-UFA treated AK-5 cells in vitro. These results suggest that c-UFAs can alter the activities of second messenger systems such as diacylglycerol and protein kinases and can phosphorylate both plasma membrane and nuclear proteins which are likely to be components of NADPH oxidase. Based on these results, it is suggested that fatty acids may mediate their cytotoxic action in part by modulating the expression of PKC. Activated PKC may then intensify the pro-oxidant state by augmenting NADPH oxidase, so inducing superoxide anion generation which may ultimately lead to cytolysis.
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PMID:Effect of cis-unsaturated fatty acids on the activity of protein kinases and protein phosphorylation in macrophage tumor (AK-5) cells in vitro. 1031 18


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