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 activation of membrane-bound phospholipase D (PLD) resulting in the generation of phosphatidic acid (PA) is increasingly recognized as an integral event in the initiation of a variety of cellular responses. We explored whether alpha-thrombin is a physiologic agonist for PLD activation in human umbilical vein endothelial cells (HUVEC). HUVEC monolayers were labeled with [32Pi] and PLD activity determined by formation of the PLD metabolite [32P] phosphatidylethanol (PEt) in the presence of 5 g/L ethanol by thin-layer chromatography. alpha-Thrombin rapidly (1 minute) increased PA and PEt formation in a dose-dependent manner (10(-6) to 10(-10)) with maximal PLD stimulation achieved with 10 nmol/L alpha-thrombin producing a threefold to fourfold increase in PA and a sixfold to eightfold increase in PEt over controls at 15 minutes. Esterolytically active zeta-thrombin (10 nmol/L) and gamma-thrombin (1 mumol/L), but not inactive DIP-alpha-thrombin (1 mumol/L) also increased PLD activity. The role of Ca2+ flux in human endothelial cell PLD activation was investigated and PEt formation was significantly enhanced by Ca2+ ionophores A23187 and ionomycin (1 mumol/L, three-fold to fourfold increase in PEt). Alpha-Thrombin-stimulated PEt formation was abolished (greater than 90% inhibition) with chelation of intracellular calcium (Ca2+i) by pretreatment with BAPTA-AM (25 mumol/L, 30 minutes) but only mildly attenuated (30% inhibition) by removal of extracellular calcium (Ca2+E) with EGTA (5 mmol/L). The protein kinase C (PKC) inhibitor staurosporine reduced alpha-thrombin-induced PEt formation in a dose-dependent manner (10 mumol/L, 78% inhibition) and PKC downregulation with chronic PMA treatment (18 hours) also resulted in marked inhibition of alpha-thrombin-induced PEt formation. Neither pertussis nor botulinum C bacterial toxins significantly altered alpha-thrombin-induced PLD responses. In contrast, similar pretreatment with cholera toxin (1 microgram/mL, 60 minutes) consistently augmented alpha-thrombin-stimulated PLD activity by 50% to 90%. Comparable results were observed with agents which increased cAMP such as forskolin, 8-bromo cAMP, or dibutyryl cAMP and cholera toxin augmentation was abolished by 2-dideoxyadenosine, a competitive inhibitor of adenylyl cyclase activity. These studies demonstrate that alpha-thrombin is a potent stimulus for human PLD-mediated PA formation and that cyclic adenosine nucleotides modulate agonist-induced cellular PLD activity. In this model of PLD activation, alpha-thrombin receptor occupancy leads to the breakdown of phosphatidylinositol 4,5-bisphosphate catalyzed by phospholipase C producing the Ca2+ secretagogue IP3 and DAG.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Thrombin stimulation of human endothelial cell phospholipase D activity. Regulation by phospholipase C, protein kinase C, and cyclic adenosine 3'5'-monophosphate. 131 12

Lucifer yellow (LY) accumulation was used to measure macrophage pinocytosis. The hematopoietic growth factors, macrophage colony-stimulating factor (CSF-1), granulocyte-macrophage CSF (GM-CSF), and interleukin 3, and the macrophage activators, lipopolysaccharide and zymosan, all stimulated LY uptake in both murine bone marrow-derived macrophages (BMMs) and resident peritoneal macrophages (RPMs) without affecting LY efflux. The stimulation of pinocytosis in the poorly cycling RPMs and in BMMs by nonmitogens dissociates stimulation of pinocytosis from subsequent DNA synthesis. Regulation of pinocytosis in BMMs appears to be independent of that of urokinase-type plasminogen activator expression. The increases in CSF-mediated BMM pinocytosis were not inhibited by pertussis toxin, by elevations in intracellular cAMP, or by glucocorticoids and were only partially inhibited by inhibitors of Na+/H+ antiport and Na+/K(+)-ATPase activities. Protein kinase C activation could be involved in regulating BMM pinocytosis because phorbol myristate acetate, oleoylacyglycerol, and exogenously added phospholipase C can all stimulate it. Ca2+ ionophores were inactive, whereas the Na+/H+ ionophore monensin potently inhibited BMM pinocytosis.
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PMID:Regulation of pinocytosis in murine macrophages by colony-stimulating factors and other agents. 131 79

This study investigated the cellular mechanisms underlying the endothelin-1 (ET-1)-induced contraction of rat aorta with focus on the involvement of phospholipase D (PLD). Preincubating rat aorta in Ca(2+)-free solution reduced the contraction by 80%, whereas diltiazem (10 microM), a voltage-operated Ca2+ channel blocker, caused only a small reduction (27%, P less than 0.05) of the contraction. In myo-[3H]inositol-labeled aorta, ET-1 stimulated the formation of [3H]inositol bisphosphate and [3H]inositol trisphosphate, indicating the activation of phospholipase C (PLC). In aorta labeled with 32PO4, [3H] myristic acid or [32P]lyso-platelet-activating factor followed by exposure to ethanol (0.5%), ET-1 stimulated phosphatidylethanol (PEt) production, suggesting that ET-1 activates PLD. The PEt response was not attenuated by staurosporine (ST, 0.1 microM), an inhibitor of protein kinase C (PKC) but was inhibited by removal of Ca2+. The ET-1-induced PEt response was at least additive to that induced by phorbol 12-myristate 13-acetate (1 microM). ET-1 also stimulated the release of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) into the tissue medium. Unlike the PEt responses, the 6-keto-PGF1 alpha response could be inhibited by ST. Removal of Ca2+ abolished the response. These results suggest that 1) ET-1 activates multiple cellular mechanisms including PLC, PLD, and the arachidonate cascade; 2) PKC activation may not be essential for the ET-1 activation of PLD but may play an important role in the ET-1 stimulation of 6-keto-PGF1 alpha release; and 3) Ca2+ is an important factor in the ET-1-induced PLD activity and 6-keto-PGF1 alpha release.
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PMID:Activation of multiple mechanisms including phospholipase D by endothelin-1 in rat aorta. 131 92

Fructose-1,6-diphosphate (FDP) is a physiological product which exhibits pharmacological properties. This study shows that FDP (1-3 mM) inhibits platelet aggregation induced by the agonists thrombin, vasopressin, platelet activating factor, ADP, adrenaline, arachidonate and the stable thromboxane analogue U 44069. Thrombin-promoted ATP secretion and cytosolic Ca2+ rise are also drastically inhibited by FDP, which decreases, although to a lesser extent, the protein kinase C-dependent phosphorylation of the 47 kDa protein. The inhibition on thrombin-induced aggregation is shared, albeit less efficiently, by glucose-1,6-diphosphate and fructose-2,6-diphosphate but not by other phosphorylated monosaccharides (fructose-1:2 cyclic,6-diphosphate, glucose-1- and glucose-6-phosphate, fructose-1- and fructose-6-phosphate, mannose-6-phosphate and 5-phosphoryl ribose-1-pyrophosphate). FDP does not affect platelet activation induced by the protein kinase C activators dioctanoylglycerol or phorbol 12-myristate 13-acetate. No increase of cAMP concentration is observed in FDP-treated platelets. Altogether, these results indicate that FDP inhibits platelet activation at a level preceding phospholipase C. The data are consistent with a general inhibitory action of FDP on signal transmission.
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PMID:Fructose-1,6-diphosphate inhibits platelet activation. 131 5

The cDNAs encoding the murine LH receptor (LHR) and the human beta 2-adrenoceptor (h beta 2AR) were cloned and RNAs complementary to their sense strands (cRNAs) were injected into defolliculated Xenopus oocytes. This led to expression, respectively, of LH- and isoproterenol-stimulable adenylyl cyclase activities, indicating that functionally active receptor cDNAs had been cloned. In oocytes injected with LHR cRNA, but not in control or h beta 2AR cRNA-injected oocytes, human CG and LH increased a Ca(2+)-activated Cl- current, as measured by the two-microelectrode voltage-clamp method. This effect was not seen with isoproterenol in control or h beta 2AR cRNA-injected oocytes, it was also not observed in response to forskolin or (Bu)2cAMP. The response to human CG could be obtained in the absence of extracellular Ca2+ but was abolished by injection of EGTA, indicating that it was caused by mobilization of Ca2+ from intracellular stores. The response was unaffected by overnight treatment with 1 microgram/ml pertussis toxin. The experiments show that a glycoprotein hormone receptor can be expressed as a functionally active molecule in Xenopus oocytes, and that the LHR has the ability of activating two separate intracellular signaling pathways: one forming the second messenger cAMP, and the other mobilizing Ca2+ from intracellular stores. It is proposed that the latter is secondary to a primary activation of phospholipase C by the LHR, which elevates intracellular Ca2+ via intermediary elevation of inositol phosphates, presumably (1,4,5)inositol trisphosphate.
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PMID:Ca2+ mobilization by the LH receptor expressed in Xenopus oocytes independent of 3',5'-cyclic adenosine monophosphate formation: evidence for parallel activation of two signaling pathways. 131 58

We have investigated the effects of isoproterenol (ISO) and forskolin on carbachol(CCh)- and fluoroaluminate (AlF4-)-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, myo-inositol 1,4,5-trisphosphate (IP3) production, 1,2-diacylglycerol, measured as phosphatidic acid (PA) formation, and contraction in the bovine iris sphincter smooth muscle. The data from these studies can be summarized as follows. (1) CCh (20 microM) stimulated significantly PIP2 hydrolysis, IP3 production, PA formation, and contraction. (2) Addition of ISO (0.1-25 microM), which raises the tissue cAMP level, to muscle precontracted with CCh attenuated PIP2 hydrolysis, IP3 production, PA formation and contraction in a time- and dose-dependent manner. (3) AlF4- (10 microM) induced a slow but progressive hydrolysis of PIP2, accompanied by parallel production of IP3, formation of PA, and contraction of the smooth muscle. The effects of AlF4- were dose-dependent and inhibited by deferoxamine, an Al3+ ion chelator. (4) Both forskolin (1-25 microM), which directly stimulates adenylate cyclase, and ISO inhibited the responses induced by AlF4- (10 microM) in a dose-dependent manner. (5) NaF (1-5 mM) had no effect on the activity of phospholipase C (PLC), purified from bovine iris sphincter. Furthermore, phosphorylation of the enzyme by catalytic subunit of protein kinase A had no inhibitory effect on PLC activity against PIP2. In conclusion, neither the muscarinic receptor nor PLC are the target sites for cAMP inhibition; instead the putative G-protein, which couples the activated muscarinic receptor to PLC, may be phosphorylated by cAMP-dependent protein kinase. This could attenuate the stimulation of PLC by the G-protein, thus resulting in inhibition of PIP2 hydrolysis and consequently leading to muscle relaxation. These results demonstrate cross-talk between the cAMP and IP3-Ca2+ second messenger systems and suggest that this could constitute a regulatory mechanism for the process of contraction-relaxation in smooth muscle.
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PMID:Effects of isoproterenol and forskolin on carbachol- and fluoroaluminate-induced polyphosphoinositide hydrolysis, inositol trisphosphate production, and contraction in bovine iris sphincter smooth muscle: interaction between cAMP and IP3 second messenger systems. 131 46

The effects of phorbol esters on phospholipase C (PLC) activity towards phosphoinositides and phosphatidylcholine (PC) in bovine corneal epithelial cells were examined. The cells were labeled with 32Pi, myo[3H]inositol or methyl[14C]choline, and PLC stimulated by incubation of the cells with Ca2+ ionophore, ionomycin. The PLC activity was assessed by monitoring the loss of radioactivity from the labeled phospholipids or the accumulation of their radioactive metabolites. The data from this study can be summarized as follows: Addition of 20 microM ionomycin to the prelabeled cells resulted in a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and somewhat slower hydrolysis of phosphatidylinositol (PI) and phosphatidylcholine (PC) with concomitant several-fold increase in phosphatidic acid (PA). The effects of the ionophore were time- and dose-dependent. Incubation of the cells with phorbol 12,13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) caused increased radioactivity in PC and PA, whereas the radioactivity in PI and PIP2 remained unchanged. The effects of PDBu were inhibited by staurosporine and H-7, and inactive derivatives of phorbol esters failed to exert any effect on phospholipid metabolism. Pretreatment of the corneal epithelial cells with PDBu or PMA abolished the ionomycin-induced hydrolysis of phosphoinositides and PC. The data suggest that activation of protein kinase C by phorbol esters in corneal epithelial cells results in inhibition of PLC activity towards phosphoinositides and PC through a mechanism probably involving phosphorylation of the enzyme.
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PMID:Phorbol esters inhibit ionomycin-induced hydrolysis of phosphoinositides and phosphatidylcholine in bovine corneal epithelial cells. 131 39

[3H]Inositol ([3H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [3H]Ins was incorporated into a common lipid pool through both CMP-dependent and independent mechanisms. These are as follows: (1) a reverse reaction catalyzed by phosphatidyl-inositol (PtdIns) synthase, and (2) the reaction performed by the PtdIns headgroup exchange enzyme, respectively. Membrane phosphoinositides prelabeled in either CMP-dependent or independent fashions were hydrolyzed by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and carbachol-stimulated phospholipase C. Unlike CMP-dependent labeling, however, CMP-independent incorporation of [3H]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxycholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [3H]Ins was incorporated into lipids primarily through the PtdIns synthase-catalyzed reaction because of the presence of deoxycholate required to observe carbachol-stimulation of phospholipase C. Little direct breakdown of [3H]PtdIns was detected because production of myo-[3H]inositol 1-monophosphate was minimal and myo-[3H]inositol 1,4-bisphosphate was the predominant product. Although PtdIns labeling and 3H-polyphosphoinositide formation were unaffected by GTP gamma S and carbachol and had no or little lag period, GTP gamma S- and carbachol-stimulated appearance of 3H-Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [3H]Ins to 3H-Ins phosphates was restricted to a narrow range of free calcium concentrations (10-300 nM). These results show the concerted activities of PtdIns synthase, PtdIns 4-kinase, and phospholipase C, and constitute a simple assay for guanine nucleotide-dependent agonist stimulation of phospholipase C in a brain membrane system using [3H]Ins as labeled precursor.
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PMID:Concerted CMP-dependent [3H]inositol labeling of phosphoinositides and agonist activation of phospholipase C in rat brain cortical membranes. 131 77

We have identified a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C activity in Dictyostelium discoideum. Addition of Ca2+ (20 microM) results in the rapid formation of Ins(1,4,5)P3 within 5 s and leads to sustained inositol phosphate production for up to 40 min in membranes prepared from [3H]inositol-labelled cells. The phospholipase C activity is primarily membrane-bound under the conditions used to lyse the cells. In addition to this activity we also identified a family of Ca(2+)-regulated phospholipase activities active on a range of phospholipid substrates, using [3H]palmitate labelling. Inositol-specific phospholipase C activity is highest in vegetatively growing cells and in starved cells during the first 6 h in development, during which time Ca2+ elicited a 5-fold stimulation of inositol phosphate formation. After this time, total activity decreased progressively until 15 h, after which the activity remained constant up until 24 h. During this period, Ca2+ was able to stimulate a 2-fold increase in inositol phosphates.
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PMID:Characterization of phospholipase activity in Dictyostelium discoideum. Identification of a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C. 131 14

Rat 6 fibroblasts that stably overexpress cDNA for the beta 1 isozyme of protein kinase C (PKC3 cells) were used to determine the effect of protein kinase C (PKC) overexpression on hormonal stimulation of phospholipid hydrolysis. In control Rat 6 cells, inositol trisphosphate levels (InsP3) were increased 9-fold in 15 s in response to 10 nM alpha-thrombin, compared with only a 2-fold increase in PKC3 cells. PKC overexpression also inhibited thrombin-stimulated production of 1,2-diacylglycerol, the other product of phosphatidylinositol 4,5-bisphosphate hydrolysis, by 73% at 15 s. In permeabilized cells, PKC overexpression greatly reduced guanosine thiotriphosphate-stimulated InsP3 accumulation, but did not affect InsP3 stimulation by increased free calcium concentration. These data suggest that desensitization of thrombin-stimulated phosphoinositide-phospholipase C is enhanced by PKC-beta 1 overexpression and may involve modulation of G-protein/phospholipase C coupling. In contrast, thrombin was 4.5-fold more effective in stimulation of phosphatidylcholine-phospholipase D activity in PKC3 cells than in control cells, as determined by phosphatidylethanol formation. In permeabilized cells, guanosine thiotriphosphate also stimulated phospholipase D activity more effectively in PKC3 cells than in control cells, suggesting that upregulation of phospholipase D activity by PKC overexpression occurs distal to the thrombin receptor. These results suggest that PKC may act as a switch to up-regulate phosphatidylcholine-phospholipase D and down-regulate phosphoinositide-phospholipase C stimulations.
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PMID:Differential regulation of phosphoinositide and phosphatidylcholine hydrolysis by protein kinase C-beta 1 overexpression. Effects on stimulation by alpha-thrombin, guanosine 5'-O-(thiotriphosphate), and calcium. 131 71


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