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)

Platelets primed by exposure to subthreshold concentrations of arachidonic acid or collagen are known to be activated by nanomolar levels of hydrogen peroxide. We here demonstrate that this effect is mediated by hydroxyl radicals (OHzero) formed in an extracellular Fenton-like reaction. H2O2-induced platelet aggregation, serotonin release and thromboxane A2 productions were inhibited by OHzero scavengers and by the iron chelator desferrioxamine; hydroxyl radicals were detected directly by ESR measurements of the spin-trapped OHzero adduct. The role of OHzero was confirmed in experiments with exogenously added iron; free or EDTA-bound ferrous iron activated platelets in a process blocked by deoxyribose, mannitol or catalase, whereas ferric iron was without effect unless reductants were included. The activation by OHzero depended on concomitant release of arachidonic acid and was blocked by the phospholipase A2 inhibitors mepacrine and aristolochic acid, and by the Na+/K+ antiporter inhibitor ethylisopropylamiloride. In contrast, neomycin and staurosporin were without effects, indicating that phospholipase C and protein kinase C were not involved in the initial phase of activation. Neither radical formation nor arachidonic acid release was blocked by aspirin. In whole blood aggregation of platelets could be induced by H2O2 generated upon specific stimulation of neutrophils by N-formyl-methionyl-leucyl-phenylalanine; platelet activation and radical formation were blocked by the NADPH oxidase inhibitor diphenyliodonium as well as by catalase and mannitol. These results suggest that reactive oxygen species act as 'second messengers' during the initial phase of the platelet activation process.
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PMID:Role of hydroxyl radicals in the activation of human platelets. 817 49

The respiratory burst oxidase from human neutrophils is a membrane-associated enzyme that catalyzes the reduction of oxygen to O2- at the expense of NADPH. The oxidase is dormant in resting neutrophils, but comes to life when the cells are exposed to certain activating agents. Activation requires GTP or a GTP analog and is associated with the transfer of cytosolic oxidase polypeptides to the plasma membrane. Treatment of resting neutrophil membranes with phospholipase C caused an eightfold rise in their diacylglycerol content, increased the sodium dodecyl sulfate-mediated transfer of cytosolic polypeptides to the membrane, and enhanced O2- production by the membranes after treatment with cytosol and sodium dodecyl sulfate. Use of phospholipase C-treated membranes in the cell-free system caused only a minor change in the Km and Vm for NADPH as compared with the same system containing untreated membranes, but caused the Km for the nonhydrolyzable GTP analog GTP gamma S to fall from 200 to 34 nM. Similar kinetic changes were observed with membranes treated with dioctanoylglycerol. These findings are consistent with the idea that the activity of a G protein can be regulated by diacylglycerol.
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PMID:Effects of diacylglycerol on the activation and kinetics of the respiratory burst oxidase in a cell-free system from human neutrophils: evidence that diacylglycerol may regulate nucleotide uptake by a GTP-binding protein. 821 91

Previous studies have shown that intracellular killing of bacteria by monocytes is stimulated by interaction between IgG and Fc gamma receptors (Fc gamma R) in the membrane of these cells. In the present study anti-Fc gamma R monoclonal antibodies (mAb) were used to investigate the relative contributions of the various classes of Fc gamma R to the intracellular killing of Staphylococcus aureus by human monocytes and the biochemical pathways involved. Anti-Fc gamma RI or anti-Fc gamma RII mAb, but not anti-Fc gamma RIII mAb, efficiently stimulated the intracellular killing of bacteria by monocytes. Cross-linking Fc gamma RI or Fc gamma RII, but not Fc gamma RIII, on monocytes with mouse anti-Fc gamma R mAb followed by bridging with F(ab')2 fragments of goat anti-mouse IgG enhanced this process. Since the NADPH oxidase inhibitor diphenyleneiodonium blocked the Fc gamma R-mediated intracellular killing of S. aureus, oxygen-dependent bactericidal mechanisms are most probably involved. Cross-linking Fc gamma RI or Fc gamma RII but not binding of the mAb to the Fc gamma R on monocytes activated phospholipase C, as demonstrated by the increase in the intracellular concentration of inositol-(1,4,5)-triphosphate. The enhanced intracellular killing stimulated by cross-linking Fc gamma R on monocytes was completely blocked by U-73122, an inhibitor of phospholipase C-dependent processes. Protein kinase C activity, but not the rise in the cytosolic free Ca++ concentration or pertussis toxin-sensitive G proteins, is essential for the Fc gamma R-mediated intracellular killing of bacteria by monocytes. Together, these results demonstrate that cross-linking Fc gamma RI or Fc gamma RII is equally effective in stimulating the intracellular killing of bacteria by monocytes and that this stimulation is a phospholipase C-dependent process.
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PMID:Stimulation of the intracellular killing of Staphylococcus aureus by human monocytes mediated by Fc gamma receptors I and II. 822 59

Incubation of human keratinocytes with nanomolar concentrations of Staphylococcus aureus alpha-toxin leads to irreversible depletion of cellular ATP. The toxin forms hexamers in the target cell membranes, and rapid transmembrane flux of K+, Na+, and 86Rb+ is observed. Unexpectedly, pores formed in keratinocytes through application of low but lethal doses of alpha-toxin appeared to be considerably smaller than those formed in erythrocyte membranes. They permitted neither rapid influx of Ca2+ or propidium iodide, nor efflux of carboxyfluorescein. Larger pores allowing flux of all three markers did form when the toxin was applied at high concentrations. Flux of monovalent ions and reduction in cellular ATP levels evoked by low toxin doses correlated temporally with a fall in oxygen consumption, which was interpreted to reflect breakdown of mitochondrial respiration. The lethal event could not be thwarted by manipulating the extracellular K+ or Ca2+ concentrations. Realization that alpha-toxin may form very small pores in nucleated cells is important for future research on cellular toxin effects and membrane repair processes.
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PMID:Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions. 822 71

It has previously been shown that human umbilical artery (HUA) smooth muscle produces thromboxane A2 in response to increasing oxygen levels and that this thromboxane promotes contraction. To investigate the intracellular action of thromboxane A2, strips of HUA longitudinal smooth muscle were permeabilized using alpha-toxin from the bacterium Staphylococcus aureus. This treatment rendered the surface membrane permeable to low-molecular-weight substances but left functional thromboxane A2 receptors. Tension measurements were used to investigate the effect of the stable thromboxane A2 analogue, U-46619, on the Ca2+ sensitivity of smooth muscle contractile proteins. U-46619 (1 nM to 1 microM) potentiated submaximal Ca(2+)-activated force (generated by [Ca2+], 50 nM to 3 microM) but not maximal Ca(2+)-activated force (generated by [Ca2+], 10-100 microM). The specific thromboxane A2 receptor antagonist, GR-32191B (1 microM), inhibited the action of U-46619 (0.1 microM). The potentiation of submaximal Ca(2+)-activated force produced by the muscle in response to U-46619 (0.1 microM) was antagonized by guanosine 5'-O-(2-thiodiphosphate) (1 mM), the nonhydrolyzable analogue of GDP, and mimicked by guanosine 5'-O-(3-thiotriphosphate) (100 microM), the nonhydrolyzable analogue of GTP. These results suggest that U-46619 acts via the previously identified thromboxane A2 receptor to promote Ca2+ sensitivity of tension production in HUA smooth muscle. Furthermore, this effect appears to be mediated via a G protein.
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PMID:Thromboxane A2 analogue, U-46619, potentiates calcium-activated force in human umbilical artery. 832 17

There are multiple mechanisms whereby ACE inhibitors could be beneficial during myocardial ischemia and reperfusion, including: i) reduced formation of angiotensin II, ii) decreased metabolism of bradykinin, iii) antioxidant activity, and iv) possibly other unknown mechanisms. Reduced formation of angiotensin II should be beneficial because this peptide exerts several actions that are potentially detrimental to the ischemic/reperfused myocardium, including vasoconstriction, increased release of norepinephrine, stimulation of phospholipase C and/or A2, and increased afterload with an attendant increase in oxygen demands. Reduced metabolism of bradykinin could be beneficial by increasing myocardial glucose uptake, by causing vasodilation, and by stimulating production of endothelium-derived relaxing factor and prostacyclin. Although earlier studies suggested that sulfhydryl-containing ACE inhibitors scavenge superoxide anions, recent data have shown that these drugs scavenge hydroxyl radical and hypochlorous acid with no effect on superoxide anion. Studies in isolated hearts have demonstrated that ACE inhibitors attenuate the metabolic, arrhythmic, and contractile dearrangements associated with ischemia and reperfusion, and have suggested that such beneficial effects are mediated by potentiation of bradykinin and/or increased synthesis of prostacyclin. Studies in models of myocardial stunning after brief (15-min) ischemia in vivo (anesthetized dogs) suggest that ACE inhibitors enhance the recovery of contractile function after a single brief ischemic episode. No data are available regarding the effect of these drugs on myocardial stunning after a prolonged, partly reversible episode, after multiple consecutive brief ischemic episodes, and after global ischemia. The mechanism for the salutary effects of ACE inhibitors on stunning remains a mystery. It may involve an antioxidant action (in the case of thiol-containing molecules) or potentiation of prostaglandins (in the case of non-thiol-containing molecules). What is clear is that the enhanced recovery of function effected by these drugs is not due to hemodynamic effects, inhibition of the converting enzyme per se, or an "antischemic" action (since the drugs were effective when given at the time of reperfusion). The effects of ACE inhibitors on myocardial infarct size remain controversial. Further studies will be necessary to conclusively establish whether ACE inhibitors can protect against the detrimental effects of myocardial ischemia and reperfusion. Nevertheless, the evidence provided thus far is encouraging and warrants an in-depth assessment of the role of these drugs in attenuating myocardial ischemia/reperfusion injury.
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PMID:Effect of angiotensin-converting enzyme inhibitors on myocardial ischemia/reperfusion injury: an overview. 835 31

The response to a number of agents has been compared in two short-term assays used for the detection of virus inducers and tumor promoters: (i) induction of the EBV-DR-promoter in Raji cells, as measured by DR-CAT induction (DR-CAT test) and (ii) induction of the oxidative burst in human PMN, as measured by chemiluminescence in the presence of luminol or lucigenin (CL test). In order to validate the two assays, we have investigated the responses to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), 1,2-dioctanoylglycerol (DAG), phospholipase C (PLC EC-3-1-4-30) and ionophore A23187, which are active in both systems: arachidonic acid, linoleic acid and NaCl were found active only in the CL test. Staurosporine (protein kinase inhibitor), tamoxifen (estrogen antagonist and protein kinase C inhibitor), forskolin (protein kinase A activator), R59949 (diacylglycerol kinase inhibitor), curcumin and N-acetyl-L-cysteine (scavengers of reactive oxygen species) and NaCl acted as inhibitors. A good concordance of the EC50 values of inducing substances was found between the two assays, except for A23187 and DAG, which were required at much higher concentrations in the DR-CAT test. The inhibition patterns by the panel of inhibitors revealed similarities and discrepancies in the induction pathways between the two systems, providing information on their mode of action. The two assays, which complement each other, were shown to detect a number of known or suspected EBV inducers or tumor promoters, and thus appear useful for screening of new compounds or mixtures as well as of potential antiviral and antipromoting substances.
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PMID:Validation of two test systems for detecting tumor promoters and EBV inducers: comparative responses of several agents in DR-CAT Raji cells and in human granulocytes. 839 78

Activated phagocytes produce large amounts of reactive oxygen intermediates, including peroxides. In addition to their microbicidal effect, it has recently been suggested that reactive oxygen species play a role as intracellular messengers. The mechanism of action remains unknown, but peroxides have been reported to increase tyrosine phosphorylation, an effect potentiated by vanadate. In this report we studied the effects of a combination of H2O2 and vanadate on Ca2+ homeostasis in granulocytic HL60 cells. The peroxides induced a transient elevation of cytosolic [Ca2+] associated with release from internal stores. Ca2+ mobilization was accompanied by increased generation of inositol 1,4,5-trisphosphate, implicating phospholipase C (PLC). A sizable increase in phosphotyrosine accumulation by several polypeptides in the M(r) 20,000 to 250,000 range preceded the [Ca2+] changes. We therefore considered the possibility that tyrosine phosphorylation of a phospholipase mediates the observed effects. Differentiated (granulocytic) HL60 cells did not have detectable levels of PLC gamma 1 but had substantial PLC gamma 2. Immunoprecipitation and immunoblotting experiments demonstrated that PLC gamma 2 becomes tyrosine-phosphorylated upon treatment of the cells with peroxides of vanadate. If associated with activation, such phosphorylation of PLC gamma 2 can account for the rise in [Ca2+]. Although capable of mobilizing internal Ca2+ stores, the peroxides failed to produce the sustained [Ca2+] increase predicted by the "capacitative" model. Mn2+ influx determinations indicated that this is due to impairment of divalent cation entry by the peroxides, uncoupling the plasma membrane from the internal stores. Changes in [Ca2+] homeostasis could mediate some of the messenger actions of reactive oxygen species.
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PMID:Cytosolic [Ca2+] homeostasis and tyrosine phosphorylation of phospholipase C gamma 2 in HL60 granulocytes. 842 12

To determine whether bacterial luciferase is expressed in the anaerobe Clostridium perfringens to produce an oxygen-requiring bioluminescence reaction, a suitable plasmid vector possessing the luxA and luxB genes of Vibrio fischeri was constructed and introduced into C. perfringens cells. luxAB were placed under the transcriptional control of the C. perfringens alpha-toxin gene promoter region. Suitable ribosome binding sites were introduced upstream of both genes. Bioluminescence was strongly expressed in C. perfringens transformants. Comparisons of in vivo and in vitro bioluminescence measurements demonstrated that in vivo data constituted a quantitative measure of gene expression. This is the first study to show that luxA and luxB genes can be expressed in an anaerobic bacterium and that bioluminescence can be used as a quantitative reporter system in future in vivo studies of gene expression in C. perfringens.
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PMID:Bioluminescence (lux) expression in the anaerobe Clostridium perfringens. 845 91

Hippocampal slices were transiently exposed to an oxygen- and glucose-free environment which causes a pronounced drop of both ATP and creatine phosphate, an anoxic depolarization, and an incomplete recovery of synaptically evoked population spike in the CA1 region after 1 h (48.5 +/- 3.6% of baseline values). This recovery could be markedly enhanced by the application of N-methyl-D-aspartate receptor antagonists. To examine the influence of metabotropic glutamate receptors on neuronal recovery from hypoxia/hypoglycemia, we applied various antagonists and agonists of the metabotropic glutamate receptors to the bath during the interval from 20 min before to 10 after hypoxia/hypoglycemia. The metabotropic glutamate receptor antagonists (+)-alpha-methyl-4-carboxyphenylglycine and L-2-3- amino-phosphonopropionic acid were both able to enhance the population spike recovery significantly. However, the mixed metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid also exhibited a protective effect on population spike recovery, leaving the anoxic depolarization and N-methyl-D-aspartate responses during the hypoxia/hypoglycemia untouched. With the help of more subtype-specific agonists, we found that an activation of phospholipase C coupled (class 1) metabotropic glutamate receptors prior to hypoxia/hypoglycemia may be responsible for the protective effect seen with 1S, 3R-1-aminocyclopentane-1,3-dicarboxylic acid, because the specific class 1 metabotropic glutamate receptor agonist trans-azetidine-2,4-dicarboxylic acid appeared to be highly protective, but only if it was applied 20 min before the hypoxia/hypoglycemia. An activation of class 2 metabotropic glutamate receptors by (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine, which inhibits adenylyl cyclase activity, led to a marked deterioration of the population spike recovery and even to a total prevention of the protective effect of the N-methyl-D-aspartate agonist D-2-amino-5-phosphonopentanoic acid. Our data suggest that prior activation of class 1 metabotropic glutamate receptors is beneficial, while their activation during hypoxia/hypoglycemia is detrimental. Furthermore, the activation of class 2 metabotropic glutamate receptors decreases the recovery from hypoxia/hypoglycemia.
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PMID:Metabotropic glutamate receptor subtypes differentially influence neuronal recovery from in vitro hypoxia/hypoglycemia in rat hippocampal slices. 854 5


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