Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To examine the role of Rho family proteins in prostaglandin F2 alpha (PGF2 alpha)-mediated phospholipase D (PLD) activation of osteoblast-like cell line MC3T3-E1 cells, we used Toxin-B from Clostridium difficile, which inhibits Rho family proteins by monoglucosylation. Pretreatment of [3H]myristic acid-labeled MC3T3-E1 cells with Toxin B induced rounding-up of the cells and inhibited the PGF2 alpha-induced PLD activation by 60%, but not the phospholipase C (PLC) activation. Cytochalasin D also induced rounding the cells, but showed a small inhibition in the PLD activation. Brefeldin A (BFA) had marginal inhibitory effect on the PGF2 alpha-induced PLD activation. In digitonin-permeabilized MC3T3-E1 cells, [3H]P But formation was stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or 4 beta-phorbol 12-myristate 13-acetate (PMA) in the presence of Ca2+ (1 microM) and ATP (1 mM), and phosphatidylinositol 4,5-bisphosphate (PIP2) was also required for its full PLD activation. Pretreatment of the digitonin-permeabilized MC3T3-E1 cells with Toxin B reduced the GTP gamma S- and PMA-stimulated PLD activities by 80% and 60%, respectively. On the other hand, C3 toxin which inhibits Rho by ADP-ribosylation, exerted a partial inhibitory effect on the GTP gamma S-stimulated PLD activity. These results suggest that Cdc42 as well as RhoA appear to be involved in the PLD activation mediated by PGF2 alpha and also that the PLD activation may be independent of actin cytoskeleton in MC3T3-E1 cells.
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PMID:Involvement of Rho family proteins in prostaglandin F2 alpha-induced phospholipase D activation in the osteoblast-like cell line MC3T3-E1. 927 85

To determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on phospholipase D (PLD) activity in osteoblast-like UMR-106 cells, we used cells prelabeled with [3H] myristic acid or [3H] arachidonic acid, which were preferentially incorporated to phosphatidylcholine. The treatment of [3H] myristate-labeled cells with TPA in the presence of 1% ethanol caused a dose-dependent formation of [3H] phosphatidylethanol (PEt), a product specific to PLD, suggesting an activation of this enzyme. Pretreatment of the cells with protein kinase C (PKC) inhibitors (GF109203X, staurosporine or H-7) abolished the TPA-dependent formation of PEt. The PEt formation in response to TPA treatment was not observed after the pretreatment of the cells with TPA to downregulate PKC. These results suggest the involvement of PKC in the TPA-induced activation of PLD. With [3H] arachidonate-labeled cells, TPA treatment in the absence of ethanol resulted in the liberation of [3H] arachidonic acid, which was gradually converted to prostaglandin E2 (PGE2), but the accumulations of [3H] phosphatidic acid (PA) and [3H] diacylglycerol (DAG) were very small and temporary. In contrast, PA was linearly accumulated following TPA treatment, when the cells were pretreated with an inhibitor of phosphatidate phosphohydrolase (PAP), propranolol, with no accumulation of either DAG or arachidonic acid. The TPA treatment of the cells pretreated with a DAG lipase inhibitor, RHC-80267, caused the generation of DAG after a lag period of approximately 5 min, with a very small and temporary accumulation of PA. The TPA treatment of cells pretreated with a cyclooxygenase (COX) inhibitor, indomethacin, blocked the PGE2 production. The TPA-induced PGE2 production was not affected by the pretreatment of cells with a phospholipase A2 inhibitor, p-bromophenacylbromide, or with a phospholipase C inhibitor, D-609. TPA also stimulated PGE2 production in osteoblastic cells that were enzymatically isolated from adult rat calvaria, and the experiments with lipid metabolizing enzyme inhibitors gave the same profile of inhibition of TPA-induced PGE2 production as was observed in UMR-106 cells. These results suggest that PA formed as a consequence of the activation of PLD by TPA is rapidly converted to arachidonic acid via a PAP/DAG lipase pathway, followed by a gradual conversion of arachidonic acid to PGE2 by COX in both UMR-106 cells and isolated adult osteoblastic cells, and that neither phospholipase A2 nor phospholipase C is involved in the TPA-induced PGE2 production. To the best of our knowledge, this is the first report that shows that the activation of PKC in osteoblastic cells leads to the production of PGE2 via a PLD/PAP/DAG lipase/COX pathway.
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PMID:Phorbol ester-induced production of prostaglandin E2 from phosphatidylcholine through the activation of phospholipase D in UMR-106 cells. 973 43

A new, simple, and very sensitive assay for phospholipase A and C is described. The assay is based on the bioluminescence developed by the mutant of the bacterium Beneckea harveyi as a response to myristic acid released from dimyristoyl phosphatidylcholine by either phospholipase A or by a phospholipase C-lipase coupled system. It is possible to assay these enzymes at a rate corresponding to a release of as little as 1 to 2 pmol of myristic acid per minute.
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PMID:A new, fast, and very sensitive bioluminescence assay for phospholipases A and C. 976 27

Covalent modification with lipid can target cytosolic proteins to biological membranes. With intrinsic membrane proteins, the role of acylation can be elusive. Herein, we describe covalent lipid modification of an integral membrane glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from the kinetoplastid Trypanosoma brucei. Myristic acid was detected on cysteine residue(s) (i.e. thiomyristoylation). Thiomyristoylation occurred both co- and post-translationally. Acylated GPI-PLC was active against variant surface glycoprotein (VSG). The half-life of fatty acid on GPI-PLC was 45 min, signifying the dynamic nature of the modification. Deacylation in vitro decreased activity of GPI-PLC 18-30-fold. Thioacylation, from kinetic analysis, activated GPI-PLC by accelerating the conversion of a GPI-PLC.VSG complex to product. Reversible thioacylation is a novel mechanism for regulating the activity of a phospholipase C.
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PMID:S-myristoylation of a glycosylphosphatidylinositol-specific phospholipase C in Trypanosoma brucei. 1002 18

The unicellular eukaryote Tetrahymena is a popular model for the study of lipid metabolism. Less attention, however, has been given to the inositol phospholipids of the cell, although it is known that this class of lipids plays an important role in eukaryotic cell signaling. Tetrahymena pyriformis phosphatidylinositol was isolated, purified, and characterized by proton nuclear magnetic resonance analysis and [2-(3)H]myoinositol labeling. Labeling was also used for polyphosphoinositide (phosphatidylinositol phosphate and phosphatidylinositol bisphosphate) identification. Tetrahymena inositol phospholipids were found to belong to the diacylglycerol group, although major Tetrahymena phospholipids, phosphatidylcholine and aminoethylphosphonoglycerides, have been found to be mainly alkylacylglyceroderivatives. Further characterization of Tetrahymena phosphatidylinositol by gas chromatographic analysis indicated that 80% of fatty acids were myristic acid and palmitic acid. This is also in contrast to the fatty acid profile of Tetrahymena phosphatidylcholine and phosphatidylethanolamine, with respect both to the fatty acid length and degree of unsaturation, and may indicate that specific diacylglycerol species are connected with the phosphatidylinositol metabolism in this cell. Treatment of [3H]inositol-labeled Tetrahymena cells with mastoparan, a G-protein-activating peptide, induced changes in the polyphosphoinositide levels, suggesting that inositol phospholipids may form in Tetrahymena a functional signaling system similar to that of higher eukaryotes. Addition of 10 microM mastoparan resulted in a rapid and transient increase in [3H]phosphatidylinositol phosphate followed by a decrease in [3H]phosphatidylinositol bisphosphate. Similar changes in lipids have been reported when phosphoinositide-phospholipase C pathway is activated in both animal and plant cells.
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PMID:Characterization of inositol phospholipids and identification of a mastoparan-induced polyphosphoinositide response in Tetrahymena pyriformis. 1090 87

Progesterone, the physiological inducer of amphibian meiosis, acts within minutes at plasma membrane receptors of the Rana pipiens oocyte to release 1,2-diacylglycerol (DAG) from plasma and intracellular membranes. High-performance liquid chromatography (HPLC) analysis of lipid extracts of uninduced oocytes indicates the presence of at least three classes of DAG with a total DAG content of about 150 micromol/kg wet weight. Within 3-5 min after exposure to progesterone, there was a differential increase in all three DAG classes with a twofold increase in total DAG by 10 min. The fatty acid composition of the DAGs in uninduced and progesterone-stimulated oocytes was compared using thin layer chromatographic analysis of lipid extracts from oocytes double-labeled with [14C] or [3H]glycerol and [14C] or [3H]fatty acids. The ratio of labeled fatty acid/labeled glycerol was measured in phosphatidylcholine (PC), phosphatidylinositol (PI) and DAG. The linoleic (18:2) or arachidonic (20:4) acid/glycerol ratios in basal DAG were low compared to that in PC or PI. In contrast, the myristic (14:0), palmitic (16:0) or oleic (18:1) acid/glycerol ratios in basal DAG were relatively high compared to the ratio in PC and PI. A transient increase in both linoleic and palmitic acid labeling of DAG occurred within the first 1-2 min in progesterone-treated oocytes, followed by a return to or below the basal level. Arachidonic and myristic acid labeling of DAG fall within the first minute after progesterone treatment, followed by a sustained rise over the next 10 min. The [3H]oleic acid/[14C]glycerol ratio of DAG does not change significantly following exposure to progesterone. Pretreatment with a phospholipid N-methylation inhibitor (2-methylaminoethane) precluded the rise in linoleic and palmitic acid-rich DAG, whereas pretreatment with a diglyceride kinase inhibitor (D102) produced a sustained elevation of linoleic and palmitic acid-rich DAG. These results indicate that the DAG released in response to progesterone is composed of multiple new molecular species of DAG and that both the palmitate and linolate-rich forms are rapidly phosphorylated to form phosphatidic acid (PA). The newly formed DAG species differ from the basal DAG species and reflect sequential activation of sphingomyelin (SM) synthase, PC-specific phospholipase D (PLD) and PI-specific phospholipase C in response to progesterone, which we have described previously.
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PMID:Molecular species analysis of 1,2-diacylglycerol released in response to progesterone binding to the amphibian oocyte plasma membrane. 1115 65

The membrane-associated form of the variable surface glycoprotein (mfVSG) from African trypanosomes is a potent macrophage activator capable of inducing production of tumor necrosis factor alpha (TNFalpha) in both bovine and murine models. Using a bovine model, we have re-investigated the hypothesis that the diacylglycerol moiety of the glycosylphosphatodylinositol (GPI) anchor is involved in macrophage activation and might be the actual parasite toxin. The anchor of the variable surface glycoprotein (VSG) was labeled with (3)H-myristic acid and VSG purified in its membrane-associated form. The dimyristylglycerol moiety of the anchor was released by phospholipase C cleavage. Integrity of the anchor and efficiency of cleavage was verified by autoradiography and methanol:hexane extraction. For analysis of biological function, bovine monocytes were used which had been incubated with bovine interferon gamma (primed) or with culture medium (unprimed). The VSG purified in its membrane-associated form was found to stimulate both primed and unprimed cells to secrete TNFalpha. The same preparation from which the dimyristylglycerol moiety had been cleaved was no longer able to stimulate unprimed cells but could still stimulate primed cells. Our data indicate that the presence of the dimyristylglycerol is not an absolute requirement for induction of TNFalpha production but can substitute for the interferon gamma priming. Therefore, we favor the hypothesis that stimulation of macrophages to secrete TNFalpha by the mfVSG is mediated by an as yet unknown trigger moiety and is facilitated by the dimyristylglycerol anchor.
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PMID:An accessory role for the diacylglycerol moiety of variable surface glycoprotein of African trypanosomes in the stimulation of bovine monocytes. 1129 33

Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD). This stimulates production of phosphatidic acid (PA), which increases expression of COX-2 mRNA and protein. Inhibition of PC-PLC by D609 (tricyclodecanoyl xanthogenate), and of PC-PLD activity by 1-butanol, reduced LPS-dependent over-production of PA and suppressed the increase of COX-2 mRNA and protein. Activation of PKC, normally seen in LPS-treated cells, was mimicked with phorbol myristic acid (PMA), and this also increased PA production and enhanced COX-2 expression. Propranolol inhibition of phosphatidic acid phosphohydrolase (PPH) increased PA accumulation and enhanced LPS-dependent COX-2 protein synthesis. These results suggest that inhibitors of PC-PLC, PKC and PC-PLD, or activators of PPH could be useful in the management of LPS-induced overproduction of prostaglandins and of vascular dysfunction in septic shock.
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PMID:Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide. 1287 87

In this paper the effect of N-terminal parathyroid hormone-related protein (PTHrp) and PTHrp-engaged pathways on MCF-7 breast cancer cell migration/invasivity and matrix metalloproteinases (MMPs) production were investigated. We found that: a) migration is not affected by PTHrp and Forskolin (FK)-activated PKA, while Phorbol Myristate Acetate (PMA)-activated PKC strongly stimulates MCF-7 cells motility. b) MMPs production was unaffected by PTHrp, but FK reduced membrane-type (MT)-1 MMP expression. Conversely, PMA induced a marked increase of MT1-MMP and MMP-9. c) Chemical activation of PKC is not sufficient, by itself, to confer invasive ability to MCF-7 cells, unless they were provided with additional factors, supplied by fibroblasts. d) Matrix invasion likely occurs through an activation cascade, involving at least three components: pro-MMP-9 and MT-1 MMP (supplied by PMA-stimulated MCF-7 cells) and pro MMP-2 (supplied by fibroblasts). e) The selective chemical inhibition of the adenylylciclase (AC)/PKA and phospholipase C (PLC)/PKC pathways confirmed that MCF-7 cells invasivity is not affected by exogenous PTHrp, which can only modulate their growth. However, the PTHrp responsibility in breast cancer invasion cannot be completely excluded. Indeed, fibroblasts are known to respond to PTHrp (which is a normal product of MCF-7 as well as other breast cancer cells) with enhanced release of MMP-2. On the basis of the documented requirement of fibroblast-derived MMP-2 for MCF-7 cell invasivity, a novel humoral fibroblast-breast cancer cell interaction, mediated by PTHrp, can be recognised.
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PMID:Role of PTHrp and PTHrp-engaged pathways in MCF-7 cells migration/invasion. 1645 37


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