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)

We sought to relate norepinephrine (NE) stimulation of phosphatidic acid (PA) production to functional responses of rat aorta and pathways for PA production. The time course for changes in PA was closely related to Ca-dependent tonic responses in 42K efflux and contraction. NE (30 microM for 1 min) increased PA and reduced phosphatidylcholine (PC) and phosphatidylinositol (PI) based on Pi analyses and 32P labeling of phospholipids. The 32P-to-Pi ratio in PA (0.8 +/- 0.2, n = 13) was similar to PC (0.8 +/- 0.1, n = 14) but was significantly lower (P < 0.001) than PI (4.6 +/- 0.5, n = 14). The 32P-to-Pi ratio in PA was also lower (P < 0.02) than phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. NE also increased [3H]PA twofold (P < 0.05) when PC was selectively labeled with [3H]myristic acid. These observations are more consistent with PA being formed from the hydrolysis of PC by phospholipase D (PLD) than by the phosphorylation of diacylglycerol produced by the action of phospholipase C. PLD was assayed by the formation of phosphatidylethanol (PEt) via a transphosphatidylation reaction with ethanol (half-maximal stimulation at 0.4-0.5% vol/vol). The time course for PLD stimulation by NE was similar to PA, with significant increases (P < 0.002) during 10 s to 30 min exposure. Once formed, PEt was degraded slowly, with a half time > 3 h. It is concluded that NE stimulates PLD in rat aorta, which forms a significant amount of PA from the hydrolysis of PC.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stimulation of phospholipase D activity and phosphatidic acid production by norepinephrine in rat aorta. 846 Jun 67

Glycosylphosphatidylinositol (GPI)-anchored proteins occur widely, perhaps universally, on the surface of animal cells, where they perform a variety of important functions. However, the existence of GPI-anchored proteins on plant cells has never been established. Evidence is presented in this communication for the occurrence of a 50 kDa GPI-anchored alkaline phosphatase (AP) induced in the duckweed Spirodela oligorrhiza by phosphate deprivation. Triton X-114 partitioning of the Spirodela proteins yielded two forms of AP activity. The detergent-associated form was labeled prominently by [3H]ethanolamine, [3H]myristic acid and [3H]palmitic acid. This amphiphilic form of AP, like authentic GPI-anchored AP from mammals, was clearly resolved from the remaining, water-soluble AP activity by two types of incompletely-denaturing polyacrylamide gel electrophoresis. Lipid covalently bound to the solvent-delipidated amphiphilic AP was resistant to cleavage by phosphatidylinositol-specific phospholipase C. Strong acid or alkaline hydrolysis of the 3H-fatty acid-labeled amphiphilic AP yielded radioactive fatty acids and a radioactive lipid tentatively identified as a long chain base. The more abundant water-soluble AP was also radioactive in plants incubated with [3H]ethanolamine and was labeled to a lesser extent by 3H-fatty acids. The water-soluble AP, unlike its amphiphilic counterpart, could be freed of all fatty acid radioactivity by mild alkaline hydrolysis, indicating the continued presence of an ester-linked fatty acid. All evidence supports the conclusion that Spirodela AP is synthesized as an amphiphilic protein with a ceramide-containing GPI anchor.
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PMID:Evidence for a glycosylinositolphospholipid-anchored alkaline phosphatase in the aquatic plant Spirodela oligorrhiza. 864 7

The present study examines whether nitrogen dioxide (NO2)-induced activation of protein kinase C (PKC) is associated with increased expression of specific PKC isoforms and/or with enhanced generation of phosphatidylcholine(PC)-derived diacylglycerol (DAG) in pulmonary artery endothelial cells (PAEC). Western blot analysis revealed that exposure to 5 ppm NO2 resulted in increased expression of PKC alpha and epsilon isoforms in both cytosol and membrane fractions in a time-dependent fashion compared with controls. A time-dependent elevated expression of PKC isoform beta was observed in the cytosol fraction only of N02-exposed cells. PKC isoform gamma was not detectable in either the cytosolic or membrane fractions from control or N02-exposed cells. Scatchard analysis of [3h]phorbol 12,13-dibutyrate (PDBu) binding showed that exposure to N02 for 24 h increased the maximal number of binding sites (Bmax) from 15.2 +/- 2.3 pmol/mg (control) to 42.3 +/- 5.3 pmol/mg (p < 0.01, n = 4) (NO2-exposed). Exposure to NO2 significantly increased PC specific-phospholipase C and phospholipase D activities in the plasma membrane of PAEC (p < 0.05 and p < 0.001, respectively). When [3H]-myristic acid-labeled cells were exposed to NO2, significantly increased radioactivity was associated with cellular DAG. These results show for the first time that exposure of PAEC to NO2 results in elevated expression of specific PKC isoforms and in enhanced generation of cellular DAG, and the latter appears to arise largely from the hydrolysis of plasma membrane PC.
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PMID:NO2-induced expression of specific protein kinase C isoforms and generation of phosphatidylcholine-derived diacylglycerol in cultured pulmonary artery endothelial cells. 876 15

1. Pharmacological characterization of different lysophosphatidylcholines was performed based on their effect on the Ca2+ sensitivity of contraction in alpha-toxin-permeabilized rat mesenteric arteries. Furthermore, the effect of noradrenaline on [3H]-myristate-labelled lysophosphatidylcholine levels was assessed, to investigate whether lysophosphatidylcholines could be second messengers. 2. Palmitoyl or myristoyl L-alpha-lysophosphatidylcholine increased the sensitivity to Ca2+, whereas lysophosphatidylcholines containing other fatty acids had less or no effect. 3. L-alpha-phosphatidylcholine, L-alpha-glycerophosphorylcholine, palmitic acid, myristic acid and choline, potential metabolites of lysophosphatidylcholines, did not affect contractions. 4. Noradrenaline (GTP was required) and GTP gamma S increased the sensitivity to Ca2+, and GDP-beta-S inhibited the effect of noradrenaline. Lysophosphatidylcholines, however, had no requirement for GTP and caused sensitization in the presence of GDP-beta-S. 5. Calphostin C, a relatively specific protein kinase C inhibitor, did not affect contraction induced by Ca2+, but abolished the sensitizing effect of lysophosphatidylcholine. 6. Noradrenaline caused no measurable changes in the levels of [3H]-myristate-labelled phosphatidylcholine and lysophosphatidylcholine at 30 s and 5 min stimulation. 7. These results suggest that lysophosphatidylcholines can increase Ca2+ sensitivity through a G-protein-independent, but a protein kinase C-dependent mechanism. However, the role for lysophosphatidylcholines as messengers causing Ca2+ sensitization during stimulation with noradrenaline remains uncertain because no increase in [3H]-myristate labelled lysophosphatidylcholine could be measured during noradrenaline stimulation.
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PMID:Increase by lysophosphatidylcholines of smooth muscle Ca2+ sensitivity in alpha-toxin-permeabilized small mesenteric artery from the rat. 888 21

The metabolic fate of endogenous diacylglycerol (DAG) in cultured A10 smooth muscle cells was determined. Preincubation of A10 cells with [3H]myristic acid or [3H]arachidonic acid resulted in preferential labeling of phosphatidylcholine (PC) or phosphatidylinositol (PI), respectively. Addition of PC-specific phospholipase C (PC-PLC) to [3H]myristate-labeled A10 cells resulted in a 10-fold increase in radiolabeled DAG, which was converted to monoacylglycerol (MG) and fatty acid (FA). DAG degradation and MG formation was inhibited by tetrahydrolipstatin, a DAG lipase inhibitor. PC-derived DAG was not converted to phosphatidic acid; in addition, PC resynthesis or triacylglycerol synthesis was not observed. Addition of PI-specific PLC (PI-PLC) to [3H]arachidonate-labeled A10 cells resulted in a modest increase in radiolabeled DAG that was also hydrolyzed to MG and FA. Therefore, the principal metabolic fate of endogenous DAG generated from membrane phospholipids by treatment of A10 cells with PC-PLC and PI-PLC was hydrolysis by a DAG lipase pathway.
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PMID:Diacylglycerols derived from membrane phospholipids are metabolized by lipases in A10 smooth muscle cells. 889 25

Stimulation of rat thymocytes by concanavalin A (Con A) results in a very early increase of the cellular level of phosphatidic acid (PA), while that of diacylglycerol (DAG) was not affected. As the biological activity of PA is very likely to be determined by its molecular species composition, the present study aims to investigate the pathways leading to the production of PA in Con A-stimulated rat thymocytes. Prelabeling the cells with [3H]arachidonic acid, [3H]myristic acid, [3H]choline, or [14C]lysophosphatidylcholine allowed us to determine that PA is formed by both phosphoinositide (PIs) and phosphatidylcholine (PC) hydrolysis. We then investigated whether PA derived from PC was formed by phospholipase C (PLC) or phospholipase D (PLD) hydrolysis. In the presence of 1-butanol, the production of phosphatidylbutanol was only observed in tetradecanoyl phorbol acetate (TPA)-stimulated cells. The use of a specific PC phospholipase C inhibitor resulted in a decrease of Con A-stimulated PA production in cells labeled with [3H]myristate. When cells were labeled with [3H]choline, only TPA stimulation induced a release of labeled choline. All together, these experiments suggest that PA is originated from two phospholipid sources, predominantly PI via PLC hydrolysis and to a lesser extent PC, by PLC hydrolysis also. Molecular species analyses by reverse phase HPLC are in agreement with this hypothesis, as diacyl-GP molecular species composition is similar to that of diacyl-GPC and DAG in resting cells, but resembles that of diacyl-GPI in Con A-treated cells. Thus, in stimulated cells, the amount of 18:0/20:4 species doubled while those of saturated and monounsaturated species decreased.
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PMID:Contribution of phosphoinositides and phosphatidylcholines to the production of phosphatidic acid upon concanavalin A stimulation of rat thymocytes. 890 87

The inhibitory effect of cromakalim on the mediator release from mast cells caused by antigenantibody reactions was in controversy with the specific antigen used. However, it has recently been observed that cromakalim inhibits the release of mediators from superfused tracheal and parenchymal strips or lung mast cells after passive sensitization with the IgG1 antibody. An attempt, therefore, was made to determine the inhibitory mechanisms of cromakalim on the release of mediators such as histamine and leukotriene released by the activation of enzymes during mast cell activation. Guinea pig lung mast cells were purified through enzyme digestion, rough percoll and continuous percoll density gradients. The purified mast cells were prelabeled with [3H]palmitic acid. PLD activity was assessed more directly by the production of labeled phosphatidylethanol by PLD-mediated transphosphatidylation in the presence of ethanol. In the cells labelled with [3H]myristic acid, [3H] DAG production was measured. The methyltransferase activity was assessed by measuring the incorporation of [3H]methyl moiety into phospholipids in sensitized mast cells labelled with L-[3H] methylmethionine. cAMP level was measured by radioimmunoassay. Cromakalim resulted in a decrease in the amount of histamine and leukotrienes releases by 30% in the ovalumin-induced mast cell. Cromakalim had little effect on phospholipase D activity enhanced by the activated mast cell. Cromakalim inhibited the initial increase of diacylglycerol production during mast cell activations. Cromakalim inhibited the phospholipid methylation increased in the activated mast cell. These results show that cromakalim decreases histamine release by inhibiting the initial increase of 1,2-diacylglycerol during the mast cell activation, which is mediated via the phosphatidylinositide-phospholipase C system rather than the phosphatidylcholine-phospholipase D system. Furthermore, cromakalim reduces phosphatidylcholine production by inhibiting the methyltransferase, which decreases the conversion of phosphatidylcholine into arachidonic acid and inhibits the production of leukotrienes.
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PMID:The effects of cromakalim on the mediator releases from guinea pig lung mast cell activated by specific antigen-antibody reactions. 899 65

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


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