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)

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-sn-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent, starved Swiss 3T3 fibroblasts. We utilized exogenous dioleoylglycerol as substrate for the kinase. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C (Ca2+/phospholipid-dependent enzyme) by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on Swiss 3T3 membrane-bound diacylglycerol kinase nor does it directly effect cytosolic diacylglycerol kinase. When phorbol ester is added to Swiss 3T3 membranes in the presence of ATP, magnesium, and calcium, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Reconstitution studies show that the soluble rat brain diacylglycerol kinase binds to diacylglycerol-enriched membranes, produced by treatment of red cell ghosts with phospholipase C or calcium, suggesting that cytosolic diacylglycerol kinase may be capable of translocation to the membrane in response to elevated substrate concentration in the intact cell. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, by 5 min, also suggesting that the translocation of diacylglycerol kinase activity is regulated primarily by substrate concentration.
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PMID:Phorbol ester-induced translocation of diacylglycerol kinase from the cytosol to the membrane in Swiss 3T3 fibroblasts. 253 15

The effect of interference with diacylglycerol metabolism was investigated in pancreatic mouse islets. In the presence of the diacylglycerol lipase inhibitor RHC 80,267, glucose-induced insulin secretion was reduced 50-60%; whereas carbacholin-induced insulin secretion was unaffected. Addition of the diacylglycerol kinase inhibitor R 59,022 did not change glucose-stimulated insulin secretion but abolished the inhibition seen in the presence of RHC 80,267. RHC 80,267 increased islet glucose utilisation, measured as formation of tritiated water from 5-[3H]-glucose, 3-fold but did not affect glucose oxidation to CO2, lactate production or islet ATP levels. Glucose utilisation in leucocytes and hepatocytes was not increased by addition of RHC 80,267. Islet lipid production from glucose was augmented 4-fold in the presence of RHC 80,267 but only accounted for about 5% of the increase in glucose utilisation. The activity of adenylate cyclase and phosphoinositide-specific phospholipase C was unaffected by RHC 80,267. Concentrations of RHC 80,267 below 35 mumol/l did not alter the activity of phospholipase A2; whereas higher concentrations of the drug inhibited phospholipase A2 activity approx 25%. The data support the hypothesis that production of arachidonic acid from diacylglycerol may be involved in regulation of insulin secretion.
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PMID:Effect of diacylglycerol lipase inhibitor RHC 80267 on pancreatic mouse islet metabolism and insulin secretion. 265 50

Bovine aortic endothelial cells were grown on microcarrier beads and were perfused with Krebs-Ringer solution. Endothelium-derived relaxing factor (EDRF) was bioassayed on a cascade of four strips of rabbit aorta, and prostacyclin was analyzed by RIA of 6-oxo-prostaglandin F1 alpha. The endothelial cells released EDRF and prostacyclin when stimulated with bradykinin and its analogues, or with ADP, ATP, arachidonic acid, and phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3). The detection of EDRF was potentiated by superoxide dismutase, and the relaxation of rabbit aortic strips induced by EDRF was antagonized by methylene blue. The release of EDRF and prostacyclin was inhibited by phorbol myristate acetate, R59022 (a diacylglycerol kinase inhibitor), and gentamycin. We suggest that the release of EDRF and prostacyclin is coupled and the initial common step is activation of a phospholipase C.
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PMID:Receptor-mediated release of endothelium-derived relaxing factor and prostacyclin from bovine aortic endothelial cells is coupled. 283 51

R59 022 has been suggested to function as a selective inhibitor of diacylglycerol kinase in platelets and erythrocyte membranes. In the present study we have studied the effect of this drug on [3H]diacylglycerol and [3H]phosphatidic acid formed in response to FMLP in human neutrophils. Our results indicate that R59 022 (50 microM) itself (without any stimulus) caused a significant hydrolysis of [3H]phosphatidylinositol (6-7%), which resulted in an accumulation of [3H]diacylglycerol and [3H]phosphatidic acid. On the other hand, R59 022 at lower concentrations (10 microM) exhibited a biphasic response on the time-dependent formation of [3H]phosphatidic acid in response to FMLP. [3H]phosphatidic acid formed at 30 sec and 60 sec after stimulation with FMLP was neither inhibited nor stimulated whereas the amount of [3H]phosphatidic acid formed at 2 min and 3 min was significantly higher than that obtained with FMLP alone. Our results demonstrate that the increased formation of diacylglycerol and phosphatidic acid in response to FMLP in the presence of R59 022 is likely due to the activation of phospholipase C and/or D rather than the inhibition of DG kinase. We therefore conclude that R59 022 is relatively nonspecific and can affect several other enzymes involved in the agonist-stimulated turnover of phospholipids.
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PMID:[3H]phosphatidic acid formed in response to FMLP is not inhibited by R59 022, a diacylglycerol kinase inhibitor. 283 68

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.
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PMID:Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187. 289 66

Diacylglycerol-induced translocation of diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from the soluble to the membrane-bound compartments was demonstrated both in crude tissue homogenates and in a reconstituted enzyme-membrane model system. In homogenates of either rat brain or liver, incubation with diacylglycerol or phospholipase C, but not phospholipase A2 or phospholipase D, resulted in the translocation of diacylglycerol kinase activity from the soluble to the particulate fraction. This observation formed the basis for the first step in a two-step purification of diacylglycerol kinase. Enzyme extracted in 1 M salt from membranes of rat brain homogenates made in the presence of phospholipase C was purified further by affinity chromatography on a column containing phosphatidylserine, diacylglycerol, and cholesterol immobilized in polyacrylamide. This step yielded an enzyme preparation (step 2 enzyme) that was 500- to 750-fold purified (relative to the tissue homogenate) and required phosphatidylserine for stability. All other lipids tested failed to stabilize the enzyme. The properties of the enzyme preparation were similar to those of mammalian diacylglycerol kinases described by others. Reconstitution experiments showed that the soluble step 2 enzyme bound to inside-out vesicles of human erythrocytes only in the presence of diacylglycerol or phospholipase C but not phospholipase A2 or D. Redistribution of the kinase from soluble to vesicle-bound forms occurred rapidly and was dependent on the concentration of phospholipase C used to treat the vesicles. Physiological concentrations of calcium (50-1000 nM) did not enhance the phospholipase C-mediated translocation of the kinase. Thus, diacylglycerol kinase can translocate from cytosol to membranes in a manner dependent on the content of membrane-bound diacylglycerol but independent of the ambient concentration of calcium.
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PMID:Diacylglycerol-induced translocation of diacylglycerol kinase: use of affinity-purified enzyme in a reconstitution system. 302 39

The inositol phospholipid metabolism is one of the main pathways of signal transduction in cells. We measured the activities of its key enzymes in v-Ha-ras-transformed 208F rat fibroblasts. In the ras-transformed clones, incorporation of [32P]Pi into intermediates of the inositol phospholipid metabolism was stimulated. The activities of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in the transformed clones were about 35-50% more than in untransformed cells, indicating increased inositol phospholipid metabolism. However, the activity of diacylglycerol kinase in their membrane fraction was 25-35% less than that of untransformed cells, although the total diacylglycerol kinase activity did not change. The imbalance of these kinases could constitute one of the main reasons leading to the increased level of inositol phosphates and the accumulation of diacylglycerol to 2-2.2 times that in control 208F cells. Phosphatidylinositol-4,5-bisphosphate-phospholipase C activity did not change on the transformation when assayed under various conditions. The increased level of diacylglycerol caused intracellular translocation, activation, and down-regulation of protein kinase C changes which may be one of the essential events in transformation by the v-Ha-ras gene.
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PMID:Enhancement of inositol phospholipid metabolism and activation of protein kinase C in ras-transformed rat fibroblasts. 305 34

Isolated rat hepatocytes responded to a variety of Ca2+-mobilizing agents (vasopressin, angiotensin II, epinephrine, epidermal growth factor, ATP, and ADP) with a rapid increase in phosphatidate mass, as measured by a sensitive new method. When hepatocytes were incubated with vasopressin (10(-8) M), phosphatidate levels increased 2-3-fold in 2 min, but there was no significant increase in diacylglycerol at this time. Changes in the fatty acid composition of phosphatidate also preceded those in diacylglycerol. De novo synthesis of phosphatidate from [3H]glycerol was unaffected by vasopressin in short-term incubation. Incubation of washed rat liver plasma membranes with GTP gamma S caused a time-dependent increase in phosphatidate. When membranes were incubated with GTP gamma S and [gamma-32P]ATP, no incorporation of 32P into phosphatidate was observed. This excludes the phospholipase C-diacylglycerol kinase pathway and suggests that a phospholipase D activity produced the phosphatidate. At submaximal concentrations of GTP gamma S, ATP and ADP stimulated membrane phosphatidate formation, presumably by acting through P2-purinergic receptors. Only phosphatidylcholine, among the major phospholipids, decreased in the membranes in response to GTP gamma S. The fatty acid composition of the phosphatidate produced in response to vasopressin in hepatocytes also suggests that phosphatidylcholine may be the source of hormonally elicited phosphatidate. We conclude that Ca2+-mobilizing hormones mainly increase phosphatidate levels in hepatocytes by a mechanism that does not involve phosphorylation of diacylglycerol or de novo synthesis but involves a guanine nucleotide-binding protein coupled to phospholipase D.
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PMID:Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D mechanism. 311 99

Addition of Ca2+ to a plasma-membrane fraction derived from human or rabbit neutrophils led to the specific breakdown of polyphosphoinositides. The degradation products were identified as diacylglycerol and inositol bis- and tris-phosphate, thus demonstrating the presence of a Ca2+-activated phospholipase C. The newly generated diacylglycerol resembled the polyphosphoinositides in its fatty acid composition, and in the presence of MgATP2- it was converted into phosphatidate. These results therefore demonstrate the presence in neutrophil plasma membranes not only of polyphosphoinositide phosphodiesterase but also of diacylglycerol kinase.
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PMID:The Ca2+-activated polyphosphoinositide phosphodiesterase of human and rabbit neutrophil membranes. 608 40

Previous studies have demonstrated that [3H]arachidonic acid is released from prelabeled human neutrophil phospholipids when the cells are stimulated by calcium ionophore A23187 or by opsonized zymosan. Neither lysophospholipid generated by phospholipase A2 activity, diacylglycerol nor monoacylglycerol produced via phospholipase C/diacylglycerol lipase action have been identified following neutrophil challenge. The inability to detect any intermediates during the release of arachidonate is due to either rapid reacylation of lysophospholipid or conversion of diacylglycerol (monoacylglycerol) to cellular acylglycerols. The addition of exogenous [14C]fatty acid at the time of challenge was employed to determine the involvement of either phospholipase A2 or phospholipase C activities. Neutrophil stimulation with calcium ionophore A23187 resulted in an incorporation of exogenous [14C]arachidonate into phosphatidylinositol and phosphatidylcholine, those phospholipids which specifically release arachidonate. When the saturated fatty acid, [14C]stearate, replaced [14C]arachidonate, very little [14C]fatty acid was incorporated into any of the phospholipid species. Lipid phosphorus measurements revealed no significant mass change in any phospholipid class following ionophore challenge. Production of [14C]phosphatidic acid was not detected, as would be expected if diacylglycerol kinase and de novo phospholipid metabolism were significantly involved.
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PMID:Mechanism of arachidonic acid release in human polymorphonuclear leukocytes. 640 27


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