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 are released into the peripheral circulation from the bone marrow where they arise as fragments of megakaryocyte cytoplasm. To characterize the effects of platelet agonists on megakaryocytes, we examined calcium signaling and desensitization to thrombin, the thromboxane A2 (TxA2) mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619), and platelet-activating factor (PAF) in cultured CHRF-288-11 megakaryocytic cells. Initially, we compared agonist-stimulated calcium transients in fura-2-loaded CHRF-288-11 cells and human platelets. The 50% effective concentration values for the agonists to increase free cytosolic calcium were as follows: thrombin (0.11 +/- 0.02 U/ml in CHRF, 0.19 +/- 0.03 U/ml in platelets), U46619 (147 +/- 33 nM in CHRF, 157 +/- 5 nM in platelets), and PAF [15 +/- 2 nM in CHRF, 16 +/- 2 nM in platelets (n = 4 each)]. CHRF-288-11 thrombin, TxA2, and PAF receptors were demonstrated to be coupled to phospholipase C because each of the agonists stimulated phosphatidylinositol hydrolysis in myo-[3H]inositol-loaded CHRF-288-11 cells and pharmacological inhibition of phospholipase C-blunted agonist-stimulated calcium signaling. CHRF-288-11 cells exposed to the three agonists for 1 h showed different patterns and extent of homologous and heterologous desensitization. Protein kinase C activation appeared to be necessary but not sufficient for desensitization because 1) activation of protein kinase C with phorbol 12-myristate 13-acetate inhibited the calcium responses to all three agonists, 2) inhibition of protein kinase C with staurosporine attenuated subsequent desensitization to each agonist, and 3) each agonist increased protein kinase C activity in CHRF-288-11 cell homogenates.
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PMID:Differential megakaryocytic desensitization to platelet agonists. 141 71

The turnover of choline-containing phosphoglycerides (PC) in response to agonist stimulation is well documented in human neutrophils. We have now compared the enzymic pathways of N-formylmethionyl-leucylphenylalanine (fMLP)-, A23187- and phorbol-12-myristate 13-acetate (PMA)-induced diglyceride (DG) and phosphatidic acid (PA) generation in these cells. In order to distinguish between phospholipase C- and D-mediated PC breakdown, human neutrophils were radiolabelled with 1-O-[3H]alkyl-2-acyl-glycero-3-phosphocholine and stimulated in the presence of ethanol or propranolol. The addition of 0.5% ethanol to the incubation mixture resulted in the production of phosphatidylethanol, indicative of phospholipase D activation, in response to all three stimuli. Concomitant with phosphatidylethanol formation was a partial block of PA production. The production of DG was also partially blocked by addition of ethanol. Propranolol (200 microM) was also used to assess the contributions of phospholipases C and D toward DG generation. Inhibition of PA phosphohydrolase by propranolol resulted in the complete abolition of DG generation when neutrophils were stimulated with fMLP. In contrast, propranolol only partially inhibited DG generation in response to A23187 and PMA. These results suggested that DG production in response to fMLP stimulation is mediated via the activation of phospholipase D, whereas A23187- or PMA-induced DG generation may involve more than one pathway. However, examination of the water-soluble choline metabolites produced indicated that phospholipase D was responsible for the production of PA and DG in response to all three stimuli.
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PMID:Comparison of diglyceride production from choline-containing phosphoglycerides in human neutrophils stimulated with N-formylmethionyl-leucylphenylalanine, ionophore A23187 or phorbol 12-myristate 13-acetate. 141 27

Staphylococcus aureus alpha-toxin is a pore-forming exotoxin that probably represents a significant virulence factor in staphylococcal infections. Previous studies demonstrated that this agent stimulated arachidonate metabolism with subsequent formation of prostacyclin in endothelial cells and leukotriene B4 in granulocytes. We now examined the effect of alpha-toxin on the synthesis of platelet-activating factor (PAF) in cultured porcine pulmonary artery endothelial cells. PAF was labeled by bioincorporation of tritiated acetate and separated/quantitated using thin-layer chromatography, straight-phase-HPLC, or post-HPLC-bioassay. Alpha-toxin induced synthesis of small amounts of PAF in a time- and dose-dependent manner. The maximal amount of PAF elicited by alpha-toxin was approximately 7% of that observed after application of the ionophore A23187. Staphylococcal alpha-toxin but not the ionophore induced a rapid fall in cellular ATP-content. On kinetic grounds, the decrease in ATP-levels did not explain the differences in stimulated PAF-synthesis. Low amounts of PAF induced by the action of alpha-toxin on endothelial cells may contribute to the development of inflammatory lesions in infectious disease.
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PMID:Stimulation of PAF-synthesis in pulmonary artery endothelial cells by Staphylococcus aureus alpha-toxin. 144 May 26

Experiments were performed to immunologically identify protein kinase C (PKC) in cultured IEC-6 cells. Polyclonal antibodies specific to PKC revealed an immunoreactive band of approximately 84 kDa in both cytosolic and solubilized particulate fractions. Treatment with phorbol 12-myristate 13-acetate (PMA; 10 nM x 60 min) increased the intensity of the 84-kDa band by 25% in the solubilized particulate fraction while decreasing it by 36% in the cytosolic fraction. Prolonged 24-h treatment with 300 nM PMA completely abolished the 84-kDa band in both fractions. Isoform-specific antisera demonstrated that alpha- and epsilon-isoforms of PKC were expressed in IEC-6 cells. Treatment of quiescent cultures with PMA induced a maximal 400% increase in ornithine decarboxylase (ODC) activity. Similarly, addition of exogenous phospholipase C (PLC) to quiescent cells stimulated ODC activity. Downregulation of PKC with 300 nM PMA x 24 h inhibited basal, serum, and PLC-stimulated ODC activity by 70%. Northern analysis revealed that PKC downregulation was correlated with a marked reduction in ODC mRNA levels, suggesting regulation of ODC enzyme at this level. Despite their ability to modulate ODC activity in quiescent cultures, neither PMA nor PLC induced [3H]thymidine incorporation at 24 h. Furthermore, downregulation of PKC did not attenuate thymidine incorporation. However, chronic PMA treatment caused the cells to contact-inhibit at a 30% lower cell density, 3.16 x 10(6) vs. 2.1 x 10(6) cells/35-mm plate, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein kinase C regulation of IEC-6 cell ornithine decarboxylase. 144 49

We describe here and partially characterize a Ca(2+)-independent phospholipase A2 that acts on phosphatidylinositol in normal human peripheral blood neutrophils. Neutrophils incubated with myo-[3H]inositol to form [3H]phosphatidylinositol and then stimulated with the calcium ionophore A23187 produced [3H]lysophosphatidylinositol. This deacylation was further characterized in cell sonicates by the specific release of [3H]arachidonic acid from exogenous [1-14C]stearoyl-2-[3H]arachidonyl-phosphatidylinositol. This phospholipase A2 is Ca2+ independent, retaining full activity in the presence of 10 mM EDTA, and is optimally active at alkaline pH (pH 9). A phosphatidylinositol-hydrolyzing phospholipase C activity was characterized by the production of [3H]-/[14C]-diglycerides. This phospholipase C activity is dependent on the presence of exogenous Ca2+ and is optimally active at neutral pH (pH 7.5). The lipoxygenase/cyclooxygenase inhibitors eicosatetraenoic acid and nordihydroguaiaretic acid and the calmodulin antagonist trifluoperazine were the only compounds tested that showed significant inhibition of phospholipase A2 activity. However, none of these phosphatidylinositol-hydrolyzing phospholipase A2 inhibitory compounds resulted in the accumulation of any radiolabeled diglyceride, monoglyceride, or phosphatidic acid intermediates. Following subcellular fractionation on sucrose density gradients, it was found that the plasma membrane-enriched fractions contained the highest specific activity for phospholipase A2; however, the cytosolic fraction contained a large part of the total phospholipase A2 activity. Furthermore, when neutrophils were first exposed to several agents, including lipopolysaccharide, phorbol myristate acetate, or N-formyl-methionyl-leucyl- phenylalanine, and then subfractionated, there was a significant translocation of the enzyme activity from the cytosolic fraction to the membrane-enriched fractions. These data suggest that this Ca(2+)-independent, phosphatidylinositol-hydrolyzing phospholipase A2 may play an important role in early cell activation, providing free arachidonic acid for subsequent metabolism into biologically active eicosanoids.
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PMID:Phosphatidylinositol hydrolysis by phospholipase A2 and C activities in human peripheral blood neutrophils. 146 38

This paper provides evidence that central sensitization and persistent nociception following formalin-induced tissue injury in rats is dependent on the production of protein kinase C. Persistent nociceptive behavior in rats induced by subcutaneous formalin injection was significantly reduced by intrathecal pretreatment with a phospholipase C inhibitor (neomycin), and an inhibitor of protein kinase C (W-7), and was significantly enhanced by a phorbol ester (phorbol 12-myristate 13-acetate, PMA) and a stimulator of protein kinase C (SC-10). It is expected that noxious inputs associated with tissue injury produce a release of aspartate and glutamate within the spinal dorsal horn which by acting at ionotropic (NMDA) and metabotropic excitatory amino acid receptors produce an increase in intracellular messengers such as calcium and diacylglycerol which stimulate protein kinase C.
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PMID:Contribution of protein kinase C to central sensitization and persistent pain following tissue injury. 150 74

We have recently described a novel glycoprotein, Kp43, expressed on the surface of human natural killer (NK) cells that appears to regulate their functional activity. In this report, signaling mechanisms through the Kp43 surface antigen have been studied. Incubation of interleukin 2 (IL-2)-treated NK cells with anti-Kp43 monoclonal antibody F(ab')2 fragments resulted in the time- and dose-dependent stimulation of NK cell phospholipase D. Phospholipase D activation through the Kp43 surface antigen was found to take place in the absence of polyphosphoinositide turnover and appeared not to depend on the presence of Ca2+ in the extracellular medium. On the other hand, signaling mechanisms through the CD16 receptor (FcR-III) on NK cells were comparatively studied. Stimulation of IL-2-treated NK cells with anti-CD16 monoclonal antibody F(ab')2 fragment also resulted in time- and dose-dependent activation of phospholipase D. However, CD16-triggered phospholipase D activation took place concomitant to phospholipase C-mediated polyphosphoinositide breakdown and showed a strong dependence on extracellular Ca2+. These results provide, to our knowledge, the first evidence for the presence of activatable phospholipase D in NK cells, as well as the first indication that distinct receptor-modulated pathways exist for activation of phospholipase D within the same cell type. On the other hand, phosphatidic acid, the physiologic product of phospholipase D action on phospholipids, was found to mimic the effect of anti-Kp43 monoclonal antibody regarding tumor necrosis factor alpha (TNF-alpha) biosynthesis and secretion by NK cells. Addition of phosphatidic acid vesicles to IL-2-treated NK cell cultures stimulated a TNF-alpha production that was abolished when the cells were previously treated with actinomycin D. Other phospholipids, including lysophosphatidic acid, were ineffective. However, phosphatidic acid-induced TNF-alpha production was strongly inhibited by the presence of propranolol, an inhibitor of phosphatidic acid phosphohydrolase. Moreover, in cells responding to phorbol myristate acetate, a compound that triggers activation of phospholipase D, TNF-alpha synthesis was also inhibited by propranolol. Thus, these data suggest a second messenger role for phosphatidic acid-derived diradylglycerol in the induction of TNF-alpha gene expression.
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PMID:Phospholipase D activation in human natural killer cells through the Kp43 and CD16 surface antigens takes place by different mechanisms. Involvement of the phospholipase D pathway in tumor necrosis factor alpha synthesis. 153 70

The effects of endothelin, a novel vasoconstrictive peptide, on the delayed rectifier K+ current (IK) were examined in single dialyzed cells from guinea pig ventricles. Either big endothelin or endothelin-1 enhanced IK at a dissociation constant of 2 nM with L-type Ca2+ current being unaffected. Under intracellular perfusion with pCa 7.6 solution, 3 nM big endothelin increased IK by 55 +/- 38.5%. Either pretreatment with 10 microM 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H 7) or a low Ca2+ [10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and minus CaCl2] internal solution diminished the enhancement. Preceding stimulation of protein kinase C (PKC) by 10-20 nM 12-O-tetradecanoylphorbol-13-acetate also reduced the degree of enhancement. When Na+ was eliminated from the solutions, endothelin increased IK distinctively in cells internally dialyzed with a low Ca2+ solution. This enhancement was not abolished by either pretreatment with H 7 or by removal of Ca2+ from the external perfusate but by increasing the internal EGTA concentration to 40 mM. Preincubation with ryanodine or internal perfusion with heparin also reduced the IK enhancement under Na(+)-free conditions. Intracellular application of 200 microM guanosine 5'-O-(3-thiotriphosphate) effectively attenuated the effect of endothelin. It is concluded that endothelin enhances IK via phospholipase C-mediated PKC activation and intracellular Ca2+ mobilization. GTP-binding protein is involved in these reactions.
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PMID:Endothelin enhances delayed potassium current via phospholipase C in guinea pig ventricular myocytes. 153 93

In perfused rat liver stimulation of the hepatic nerve plexuses increased via alpha 1-receptors glucose and lactate output decreased flow and caused an overflow of noradrenaline into the hepatic vein. Infusion of noradrenaline and adrenaline also elicited similar metabolic and hemodynamic alterations via alpha 1-receptors, whereas infusion of isoproterenol via beta 2-receptors enhanced glucose output and slightly reduced lactate release without affecting flow. The influence of circulating catecholamines on the nerve stimulation-dependent changes was investigated. Noradrenaline (100 nmol/L) or adrenaline (40 nmol/L) but not isoproterenol (1 mumol/L), which themselves caused about half-maximal alterations, strongly inhibited the nerve stimulation-induced increase in glucose and lactate output and decrease in flow but had no effect on noradrenaline overflow. The protein kinase C activator (4 beta)phorbol 12-myristate, 13-acetate (100 nmol/L) but not its analog (4 alpha)phorbol 12,13-didecanoate (100 nmol/L) strongly inhibited the metabolic and hemodynamic changes caused by nerve stimulation or noradrenaline infusion. The protein kinase C inhibitor H7 (20 mumol/L) partially prevented the inhibition of the nerve actions by noradrenaline. The results lead us to conclude that noradrenaline and adrenaline inhibited the metabolic and hemodynamic nerve actions by means of a mechanism involving protein kinase C rather than presynaptic alpha-receptors or beta-receptors. The catecholamines apparently increased via alpha 1-receptors inositol 1,4,5-trisphosphate, which in turn enhanced cytosolic Ca2+ and thus altered metabolism and in part hemodynamics, and diacylglycerol, which in turn activated protein kinase C and thus feedback inhibited the signal chain from alpha 1-receptors via G proteins to phospholipase C.
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PMID:Inhibition by noradrenaline and adrenaline of the increase in glucose and lactate output and decrease in flow after sympathetic nerve stimulation in perfused rat liver: possible involvement of protein kinase C. 154 30

The lipid mediator platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC) has been shown to elicit several important biochemical signaling responses in mammalian cells, including polyphosphoinositide hydrolysis, arachidonic acid release/eicosanoid production, and protein tyrosine phosphorylation. In the present study, the roles of Ca2+ and protein kinase C (PKC), two signaling components of the phospholipase C pathway, in AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation, were investigated in cultured rat Kupffer cells. AGEPC at nanomolar concentrations induced an increase in intracellular calcium concentration ([Ca2+]i), stimulated membrane PKC activity, and resulted in protein tyrosine phosphorylation. The maximal increase in [Ca2+]i and membrane PKC activity in response to AGEPC were observed within 30-50 s, whereas the AGEPC-induced protein tyrosine phosphorylation reached maximal levels within 2-5 min. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) but not 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of calcium release from intracellular compartments, nearly abolished the AGEPC-induced increase in [Ca2+]i suggesting involvement of extracellular calcium influx in this event. Both EGTA and TMB-8 abolished or inhibited AGEPC-stimulated protein tyrosine phosphorylation and eicosanoid formation, respectively. The calcium ionophore A23187 alone stimulated eicosanoid production and protein tyrosine phosphorylation with an identical pattern to that of AGEPC. Phorbol myristate acetate (PMA), an activator of PKC, which did not affect [Ca2+]i, mimicked the actions of AGEPC, stimulating eicosanoid production and promoting tyrosine phosphorylation of a set of proteins similar to those phosphorylated following AGEPC stimulation. AGEPC-enhanced tyrosine phosphorylation of some of the protein substrates and eicosanoid production were inhibited in cells "down-regulated" for PKC. Furthermore, both PMA- and AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation were attenuated or abolished by at least one of the PKC inhibitors, staurosporine, and calphostin C. Taken together, these results are consistent with the conclusions that: (a) AGEPC stimulates the phospholipase-mediated arachidonic acid release/eicosanoid synthesis cascade and protein tyrosine phosphorylation through extracellular Ca(2+)-dependent and PKC-dependent and -independent mechanism(s) and (b) the Ca(2+)-PKC interaction determines the efficacy of the AGEPC-stimulated cellular events.
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PMID:Platelet-activating factor-stimulated protein tyrosine phosphorylation and eicosanoid synthesis in rat Kupffer cells. Evidence for calcium-dependent and protein kinase C-dependent and -independent pathways. 155 80


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