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)

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.
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PMID:Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase. 312 81

Addition of pertussis toxin to rabbit neutrophils inhibits the fMet-Leu-Phe- induced increases in Na+ influx and in intracellular pH. In addition, pretreatment of the cells with the toxin inhibits the decrease in the levels of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and the enhanced production of phosphatidic acid produced by the chemotactic factor fMet-Leu-Phe. Furthermore, the fMet-Leu-Phe-induced changes in the phosphorylation of a 46-kDa protein and of several other proteins are also inhibited by the toxin. On the other hand, the phorbol 12-myristate 13-acetate (PMA)-induced increases in the phosphorylation of several proteins are not inhibited by the toxin. PMA, but not its inactive analogue 4 alpha-phorbol 12,13-didecanoate, was also found to stimulate Na+ influx and to increase the intracellular pH in rabbit neutrophils. These ionic effects, like those produced by fMet-Leu-Phe, are inhibited by amiloride. The stimulated Na+ influx and H+ efflux produced by the phorbol ester, on the other hand, are not inhibited by pertussis toxin. The results reported here suggest that the activity of the Na+/H+ antiport in neutrophils is regulated by protein kinase C; that the G-protein system, either directly or indirectly, is involved in the stimulus-response coupling sequence in these cells; and that the toxin acts at, or prior to, the steps responsible for the activation of phospholipase C, and it does not affect the sequence of reactions initiated by the activation of the protein kinase C.
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PMID:Pertussis toxin inhibits fMet-Leu-Phe- but not phorbol ester-stimulated changes in rabbit neutrophils: role of G proteins in excitation response coupling. 315 93

Using the [3H]inositol-labeled plasma membranes isolated from the differentiated human leukemic (HL-60) cells, the mode of inhibitory action of the Ca2+/phospholipid-dependent enzyme protein kinase C in the chemotactic peptide, fMet-Leu-Phe (fMLP)-induced, phospholipase C-mediated hydrolysis of phosphoinositides was investigated. In this cell-free membrane system, fMLP in the presence of GTP plus Ca2+, GTP in the presence of Ca2+, or Ca2+ alone could induce the formation of inositol bis- and trisphosphate (IP2 and IP3, respectively). When the intact cells were pre-treated with 12-O-tetradecanoylphorbol-13-acetate, the fMLP- and GTP-induced formation of IP2 and IP3 was markedly reduced but the Ca2+-induced reactions were not reduced in the isolated membranes. This result suggests that protein kinase C impairs the coupling of the GTP-binding protein to the phospholipase C. In another experiment, preincubation of the isolated membranes with pure rat brain protein kinase C inhibited the fMLP-induced formation of IP2, but did not inhibit the GTP- or Ca2+-induced reaction. Under the same conditions, protein kinase C did not inhibit the fMLP-, GTP-, or Ca2+-induced formation of IP3. This result suggests that protein kinase C impairs additionally the coupling of the fMLP receptor to the GTP-binding protein leading to the formation of IP2. The reason for the failure of protein kinase C to inhibit the fMLP-induced formation of IP3 in the cell-free membrane system is unknown, but several possible mechanisms are discussed.
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PMID:Modes of inhibitory action of protein kinase C in the chemotactic peptide-induced formation of inositol phosphates in differentiated human leukemic (HL-60) cells. 347 61

The relationship between receptor binding of the formylated peptide chemoattractant formylmethionylleucylphenylalanine (fMet-Leu-Phe), lysosomal enzyme secretion and metabolism of membrane phospholipids was evaluated in both human polymorphonuclear leucocytes (PMN) and the dimethyl sulphoxide (Me2SO)-stimulated human myelomonocytic HL-60 leukaemic cell line. In both cell types, exposure to fMet-Leu-Phe (100 nM) induced rapid lysosomal enzyme secretion (maximal release less than 30 s) and marked changes in the 32P-labelling of the inositol lipids phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as well as phosphatidic acid (PtdA). Specifically, levels of [32P]PtdIns and [32P]PtdIns(4,5)P2 decreased rapidly (peak decrease at 10-15s), with a subsequent increase at 30 s and later. PtdIns4P and PtdA showed only an increase. In Me2SO-differentiated HL-60 cells prelabelled with [3H]inositol for 20 h, fMet-Leu-Phe caused a net increase in the cellular content of [3H]inositol phosphates, including a rapid increase in [3H]inositol 1,4,5-trisphosphate, suggesting that PtdIns(4,5)P2 breakdown occurs by a phospholipase C mechanism. Both lysosomal enzyme secretion and changes in phospholipid metabolism occur over the same agonist concentration range with a similar time course. Binding of [3H]fMet-Leu-Phe, although occurring over the same concentration range, exhibited markedly slower kinetics. Although depletion of extracellular Ca2+ had no effect on ligand-induced polyphosphoinositide turnover, PtdIns turnover, PtdA labelling and lysosomal enzyme secretion were severely curtailed. These studies demonstrate a receptor-mediated enhancement of phospholipid turnover that correlates with a specific biological response to fMet-Leu-Phe. Further, the results are consistent with the idea that phospholipase C-mediated degradation of PtdIns(4,5)P2, which results in the formation of inositol trisphosphate, is an early step in the stimulus-secretion coupling pathway of the neutrophil. The lack of correlation between these two responses and the equilibrium-binding condition suggests that either these parameters are responsive to the rate of ligand-receptor interaction or only fractional occupation is required for a full biological response.
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PMID:Secretagogue-induced phosphoinositide metabolism in human leucocytes. 608 66

In a previous study it was found that the expression of the exogenous fMet-Leu-Phe-receptor (NFPR) in the insulin-secreting cell line RINm5F mediates inhibition of hormone release and additionally raises cytosolic calcium concentration ([Ca2+]i) by activating phospholipase C (PLC) in a pertussis-toxin (PTX)-sensitive manner. We investigated whether an endogenous receptor could elicit similar effects and examined the interaction with PTX-insensitive signalling pathways. The hormone galanin inhibited insulin release at subnanomolar concentrations and increased [Ca2+]i, mainly by a PTX-sensitive mechanism with an EC50 (50 nM) comparable with that for hyperpolarization of membrane potential. The effect of galanin or fMet-Leu-Phe on [Ca2+]i was inhibited by pre-activation of the P2-receptor by ATP, which mobilizes calcium in a PTX-insensitive fashion. Simultaneous activation of the P2- and peptide receptors caused additive increases in [Ca2+]i saturating at a calcium concentration corresponding to the optimal ATP response. This suggests a specific convergence of PTX-sensitive and -insensitive pathways. In contrast, galanin and FMLP inhibited the insulin secretion induced by ATP (1-100 microM), but only when added prior to the nucleotide. In permeabilized cells, FMLP added after the calcium stimulus still inhibited secretion, indicating that the inefficacy observed in intact cells was not due to the rapid ATP-evoked rise in [Ca2+]i. Thus, (i) insulin-secreting cells possess an endogenous PTX-sensitive pathway mobilizing [Ca2+]i, (ii) inhibitory hormones preferentially activate different effectors depending on the agonist concentration and (iii) activation of NFPR or galanin receptor reveals an unusual dissociation between [Ca2+]i rises and insulin secretion, pointing towards an overriding inhibitory control of exocytosis.
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PMID:Regulation of cytosolic calcium and insulin secretion by galanin and ATP receptors: interactions of pertussis-toxin-sensitive and -insensitive signalling pathways. 752 49

Differentiated HL-60 cells acquire responsiveness to fMet-Leu-Phe (fMLP), which activates phospholipase C and O2- generation in a pertussis toxin-sensitive manner. Addition of retinoic acid (RA) for the last 24 h during dimethyl sulfoxide (Me2SO)-induced differentiation enhanced fMLP-dependent signals and interaction between fMLP receptor and G(i). RA modifies both the function and subunit composition of G(i)2, the predominant G(i) of HL-60 membranes, as shown by comparing purified G(i)2 from membranes of Me2SO-treated cells (D-G(i)2) to G(i)2 from membranes of cells treated with both Me2SO and RA (DR-G(i)2). As compared to D-G(i)2, DR-G(i)2 induced more fMLP binding when added to membranes of pertussis toxin-treated HL-60 cells and, in the presence of GTP gamma S, stimulated beta gamma-sensitive phospholipase C in extracts of HL-60 cells to a much greater extent at a lower concentrations. Immunoblasts revealed that RA induced expression of the gamma 2 subunit, which was otherwise undetectable in G(i)2 purified from HL-60 cells or in HL-60 membranes. Possibly by inducing expression of gamma 2, RA alters two functions of the G(i) beta gamma subunit, modulation of fMLP receptor-G(i)2 coupling and activation of the effector, Phospholipase C.
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PMID:Potentiation of Gi-mediated phospholipase C activation by retinoic acid in HL-60 cells. Possible role of G gamma 2. 789 Jul 21

The hypothesis that protein kinase C (PKC) participates in agonist-mediated desensitization of formyl peptide receptors in HL-60 granulocytes was tested. fMet-Leu-Phe and leukotriene B4(LTB4) produced homologous desensitization of agonist-stimulated intracellular calcium transients. Pre-treatment with the PKC activator, phorbol myristate acetate (PMA; 10 nM), abolished both fMet-Leu-Phe and LTB4-stimulated calcium transients. Membranes prepared from control HL-60 granulocytes (NM) or cells treated with 10 nM PMA (PMA-M) demonstrated increased formyl peptide receptor and G protein density, as determined by radioligand binding and pertussis toxin- and cholera toxin-catalysed ADP ribosylation. fMet-Leu-Phe stimulation of guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S) binding and GTP hydrolysis and GDP inhibition of fMet-Leu-Phe binding were not different between NM and PMA-M. Pre-treatment with 10 nM PMA did not inhibit subsequent fMet-Leu-Phe-stimulated superoxide generation or phospholipase D activation. We conclude that PKC desensitizes fMet-Leu-Phe-stimulated phospholipase C, but not phospholipase D, responses and that PKC activation does not mediate agonist-induced desensitization of formyl peptide receptors.
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PMID:Desensitization by protein kinase C activation differentially uncouples formyl peptide receptors from effector enzymes in HL-60 granulocytes. 813 77

When cytochalasin B-treated neutrophils were stimulated with fMet-Leu-Phe (fMLP) in the presence of Ca2+, phospholipase C (PLC) activity, as measured by inositol-1,4,5-triphosphate (IP3) formation, preceded phospholipase D (PLD)-catalyzed breakdown of choline-containing phosphoglycerides to form choline and diradyl-sn-glycero-3-phosphate (phosphatidic acid), suggesting a possible link between PLC and PLD. However, in the absence of cytochalasin B or extracellular Ca2+, PLC was fully activated by fMLP with minimal activation of PLD, indicating that PLC activation alone is not sufficient for PLD activation. Full activation of PLD by fMLP required the simultaneous presence of both Ca2+ and cytochalasin B, a condition that caused no further enhancement of PLC. This result suggests that PLD products are not involved in the regulation of PLC activation. Furthermore, under conditions of complete inhibition of PLC by phorbol 12-myristate 13-acetate (PMA), there was no inhibition of PLD, showing that fMLP can activate PLD in the absence of PLC. Treatment of intact neutrophils with pertussis toxin inhibited both PLC and PLD, with PLC inhibition occurring at lower concentrations that PLD inhibition. These differential effects of pertussis toxin and the observed lack of inhibition of fMLP-stimulated PLD by PMA, which is believed to inactivate G-proteins involved in PLC activation, imply that PLC and PLD are linked to fMLP receptors through distinct G-proteins. Taken together, these observations suggest that, in fMLP-stimulated neutrophils, PLC and PLD are activated through independent mechanisms.
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PMID:Phospholipase C and phospholipase D are activated independently of each other in chemotactic peptide-stimulated human neutrophils. 831 46

In neutrophils fMet-Leu-Phe activates phospholipase C via a pertussis toxin sensitive G-protein and induces granule secretion. We have transfected a human cDNA sequence encoding the fMet-Leu-Phe receptor into the insulin secreting cell line RINm5F to study receptor-effector coupling with special regard to secretion. Stable overexpression resulted in membrane hyperpolarization, reduction of cAMP accumulation and inhibition of insulin secretion upon exposure of cells to fMet-Leu-Phe with EC50 values in the pmol range. As in the neutrophil, nanomolar concentrations of ligand induced membrane depolarization and activation of phospholipase C, with subsequent mobilization and influx of calcium. In permeabilized cells the inhibitory effect of fMet-Leu-Phe on secretion was partially retained indicating a direct action of the fMet-Leu-Phe receptor on exocytosis. Pertussis toxin abolished the effects of fMet-Leu-Phe. Our results suggest conserved coupling from fMet-Leu-Phe receptor to pertussis toxin sensitive transducers analogous to the mechanism in neutrophils. However, the net biological effect of receptor activation is determined by additional factors intrinsic to the host cell.
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PMID:Conserved transducer coupling but different effector linkage upon expression of the myeloid fMet-Leu-Phe receptor in insulin secreting cells. 839 32

Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PTx)-sensitive activation of phospholipase C (PLC) have been investigated using a cotransfection assay system in COS-7 cells. The abilities of the receptors for C5a and fMLP to activate PLC beta2 and PLC beta3 through the Gbetagamma subunits of endogenous Gi proteins in COS-7 cells were tested because both PLC beta2 and PLC beta3 were shown to be activated by the betagamma subunits of G proteins in in vitro reconstitution assays. Neither of the receptors can activate endogenous PLC beta3 or recombinant PLC beta3 in transfected COS-7 cells. However, both receptors can clearly activate PLC beta2 in a PTx-sensitive manner, suggesting that the receptors may interact with endogenous PTx-sensitive G proteins and activate PLC beta2 probably through the Gbetagamma subunits. These findings were further corroborated by the results that PLC beta3 could only be slightly activated by Gbeta1gamma1 or Gbeta1gamma5 in the cotransfection assay, whereas the Gbetagamma subunits strongly activated PLC beta2 under the same conditions. PLC beta3 can be activated by Galphaq, Galpha11, and Galpha16 in the cotransfection assay. In addition, the Ggamma2 and Ggamma3 mutants with substitution of the C-terminal Cys residue by a Ser residue, which can inhibit wild type Gbetagamma-mediated activation of PLC beta2, were able to inhibit C5a or fMLP-mediated activation of PLC beta2. These Ggamma mutants, however, showed little effect on m1-muscarinic receptor-mediated PLC activation, which is mediated by the Gq class of G proteins. These results all confirm that the Gbetagamma subunits are involved in PLC beta2 activation by the two chemoattractant receptors and suggest that in COS-7 cells activation of PLC beta3 by Gbetagamma may not be the primary pathway for the receptors.
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PMID:Pertussis toxin-sensitive activation of phospholipase C by the C5a and fMet-Leu-Phe receptors. 866 41


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