Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stimulation of [3H]inositol-prelabelled rat cerebral-cortex slices with carbachol results in the accumulation of four [3H]inositol bisphosphate isomeric species, Ins(1,3)P2, Ins(1,4)P2, Ins(3,4)P2 and Ins(4,5)P2. Although the last isomer ran as a minor peak on h.p.l.c., its accumulation was dramatically enhanced in the presence of Li+ (1 mM), such that at 30 min it represented almost 35% of the total bisphosphate fraction. The accumulation of Ins(4,5)P2 appeared to be very sensitive to Li+ (EC50 = 94 +/- 3 microM), strongly implicating a Li(+)-sensitive metabolism. Evidence for this is provided from the rapid but Li(+)-sensitive decay of Ins(4,5)P2 when muscarinic-receptor stimulation is antagonized by atropine at a time when accumulations have reached a new steady state. Manipulation of phospholipase D by activators and inhibitors of protein kinase C did not suggest a role for phospholipase D hydrolysis of PtdInsP2 in the formation of Ins(4,5)P2. Attempts to reveal Ins(4,5)P2 metabolism, or indeed its synthesis from Ins(1,4,5)P3, were not successful with broken cell preparations and strongly suggest discrete compartmentation of inositol phosphate metabolism in the intact cell.
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PMID:Evidence for lithium-sensitive inositol 4,5-bisphosphate accumulation in muscarinic cholinoceptor-stimulated cerebral-cortex slices. 144 2

We have used microphysiometry and antisense methodology to show that the epsilon isoenzyme of protein kinase C (PKC) is involved in the signal transduction pathway of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a human bone marrow cell line, TF-1. These cells require GM-CSF or a related cytokine for proliferation. When the cells are appropriately exposed to GM-CSF, they exhibit a burst of metabolic activity that can be detected on the time scale of minutes in the microphysiometer, a biosensor-based instrument that measures the rate at which cells excrete protons. These cells express PKC alpha and -epsilon, as determined by Western blot analysis. Treatment with isoenzyme-specific antisense oligonucleotides inhibits expression appropriately, but only inhibition of PKC epsilon appreciably diminishes the burst of metabolic activity induced by GM-CSF. Consistent with the involvement of PKC epsilon, GM-CSF appears to activate phospholipase D and does not cause a detectable increase in cytosolic [Ca2+].
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PMID:PKC epsilon is involved in granulocyte-macrophage colony-stimulating factor signal transduction: evidence from microphysiometry and antisense oligonucleotide experiments. 144 33

Previously, the protein kinase C (PKC) inhibitor sphingosine was found to stimulate phospholipase D (PLD)-mediated hydrolysis of both phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) in NIH 3T3 fibroblasts [Kiss & Anderson (1990) J. Biol. Chem. 265, 7345-7350]. Here we examined the possible relationship between the opposite effects of sphingosine on PKC-mediated protein phosphorylation and PLD activation. After treatments for 3-5 min, sphingosine (25 microM) and the PKC activators phorbol 12-myristate 13-acetate (PMA) (100 nM), bryostatin (100 nM) or platelet-derived growth factor (50 ng/ml) synergistically stimulated the hydrolysis of both PtdEtn and PtdCho in NIH 3T3 fibroblasts prelabelled with [14C]ethanolamine or [14C]choline. Inhibition of PMA-induced phospholipid hydrolysis could also be elicited by sphingosine, but this process required prolonged (60 min) treatments of fibroblasts with 40-60 microM-sphingosine. Similarly to sphingosine, the protein phosphatase inhibitor okadaic acid also had either potentiating or inhibitory effects on PMA-stimulated PLD activity, depending on the length of incubation time and the concentration of PMA. Consistent with the presence of an inhibitory component in the overall action of PKC, the PKC inhibitor staurosporine and down-regulation of PKC activity by prolonged (24 h) treatment with PMA similarly enhanced PLD activity. Data suggest that (a) sphingosine may enhance PMA-mediated phospholipid hydrolysis by neutralizing the action of an inhibitory PKC isoform, and that (b) the stimulatory PKC isoform is less sensitive to the inhibitory action of sphingosine.
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PMID:Regulation of phospholipase D by sphingosine involves both protein kinase C-dependent and -independent mechanisms in NIH 3T3 fibroblasts. 147

Calcium ionophore exposure generates diglycerides (DAG) from phosphatidylcholine (PC) hydrolysis in Madin-Darby canine kidney (MDCK) epithelial cells. This study compares calcium ionophore-activated PC hydrolysis with the previously described phorbol ester-stimulated PC hydrolysis pathway using MDCK cells labeled with [14C]-linoleic acid. Lipid species were measured using thin-layer chromatography. DAG resulted in part from PC hydrolysis because DAG increased in cells labeled with [palmitoyl-2-14C]phosphatidylcholine. Neither protein kinase C (PKC) inhibitors nor PKC depletion affected the ionomycin (IONO)-induced increase in DAG. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the increased DAG after IONO but not after phorbol 12,13-dibutyrate (PDBu) exposure. The EGTA effect was reversed by adding excess calcium but was not reversed by adding excess Mg2+. IONO exposure also increased phosphatidic acid (PA) production. The PA was produced by phospholipase D (PLD) because phosphatidylethanol was produced when IONO was added to the cells in the presence of ethanol. Although increasing concentrations of ethanol resulted in progressively less PA, it had no effect on increased DAG after IONO exposure at any time point tested. These data are consistent with both increased phospholipase C (PLC) and increased PLD activity following ionomycin. In contrast to IONO exposure, ethanol completely prevented the increase in DAG after PDBu exposure, consistent with DAG produced by PLD activation. These results demonstrate that calcium activates both PC-specific PLC and PLD in MDCK cells and that the calcium-activated pathway is independent of the previously described PKC activation pathways.
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PMID:Calcium-activated phosphatidylcholine-specific phospholipase C and D in MDCK epithelial cells. 147 64

Evidence for a general role of phospholipase D in signal transduction is accumulating. In the present study, the activity of the enzyme was investigated in heart tissue under basal conditions and after addition of phorbol esters or aluminum fluoride (AlF-4; 10 mM NaF plus 10 microM AlCl3). Atria of rats and chickens were incubated with [3H]-myristic acid in order to label preferentially phosphatidylcholine. Under basal conditions, the tissues generated choline and phosphatidic acid (PtdOH), the primary catalytic products of phospholipase D. When 0.5 or 2.0% ethanol was present, [3H]-phosphatidylethanol (PETH) was rapidly formed at the expense of [3H]-PtdOH. This transphosphatidylation reaction is specific for phospholipase D activity. The basal formation of PETH was not inhibited by a Ca(2+)-free, EGTA-containing medium. The phorbol ester 4 beta-phorbol-12 beta, 13 alpha-dibutyrate (PDB), which is known to activate protein kinase C, enhanced the net formation of choline, whereas the inactive 4 beta-phorbol-13 alpha-acetate (PAc) was ineffective. PDB (0.2 microM), in contrast to PAc, also increased the formation of [3H]-PtdOH and, in the presence of ethanol, of [3H]-PETH. The PDB-evoked formation of PETH occurred again at the expense of PtdOH. Treshold and maximum effective concentrations of PDB were 10 nM and 0.2-0.6 microM, respectively. The effects of PDB on either choline efflux and generation of PETH showed the same Ca(2+)-dependency, i.e., both effects were blocked by a Ca(2+)-free, EGTA-containing medium, but not by a Ca(2+)-free medium without EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phospholipase D in heart: basal activity and stimulation by phorbol esters and aluminum fluoride. 148 61

The stimulation of phospholipase D (PLD) activity by endothelin-1 (ET1) was investigated in rabbit iris sphincter prelabelled with [3H]myristic acid. In the presence of 0.5% ethanol, ET1 caused a time- and dose-dependent increase in the production of [3H]phosphatidylethanol ([3H]PEt). Within 30 s the peptide increased PEt formation by 30% and after 5 min increased it by 140%. The EC50 value for ET1-stimulated PEt formation was found to be 30 nM. This value is appreciably lower than the EC50 we previously obtained for ET1-induced inositol trisphosphate production (45 nM), but considerably higher than that for arachidonic acid release (1 nM). PEt formation was significantly stimulated by prostaglandin F20, phorbol 12,13-dibutyrate (PDBu), chloroform, A23187 and A1F4-, but it was not affected by carbachol or the platelet-activating factor. PDBu-stimulated PEt formation was blocked by staurosporine and it was not potentiated by A23187. Staurosporine had no effect on ET1-stimulated PEt formation. Our data indicate that ET1 stimulation of PLD occurs independently of protein kinase C activation, phospholipase C activation and intracellular Ca2+ mobilization, and phospholipase A2 activation. In this tissue the ET1 receptor is probably coupled to the three phospholipases through several G-proteins, and this appears to be species and receptor type specific.
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PMID:Activation of phospholipase D by endothelin-1 and other pharmacological agents in rabbit iris sphincter smooth muscle. 148 66

Sphingosine inhibits protein kinase C activity in vitro and has been used to implicate this enzyme in signal transduction and cell function. We report that sphingosine directly inhibits phospholipases A2 and D. Sphingosine inhibits Ca(2+)-dependent phospholipases A2 from Naja naja, porcine pancreas, Crotalus adamanteus, human disc and neutrophil in a dose-dependent manner with IC50 values ranging from 5-40 microM using [1-14C]oleate-labelled autoclaved E. coli (20 microM) as substrate. Inhibition is comparable using the same concentrations (20 microM) of [1-14C]oleate-labelled C. albicans or E. coli, or aqueous dispersions of 1-acyl-2-[1-14C]linoleoylglycerophosphoethanolamine or -choline. Sphinganine and stearylamine are as inhibitory as sphingosine; monoolein is less inhibitory (IC50 = 70 microM), while octylamine, N-acetylsphingosine, sphingomyelin and ceramide have no effect. Inhibition is relieved by increasing concentrations of substrate phospholipid. The molar ratio of sphingosine to phospholipid required for 50% inhibition ranges from 0.5 to 1.0 with 2-100 microM E. coli phospholipid. In contrast, sphingosine has a biphasic effect on the hydrolysis of E. coli by S. chromofuscus phospholipase D; concentrations less than or equal to 25 microM stimulate activity while concentrations greater than 25 microM are inhibitory. Addition of Triton X-100 eliminates both the stimulatory and inhibitory effects of sphingosine on phospholipase D activity.
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PMID:Sphingolipid metabolism and signal transduction: inhibition of in vitro phospholipase activity by sphingosine. 150 2

This study was performed to determine the effects of phorbol esters and ionomycin on phospholipase D (PLD) activity in bovine corneal epithelial cells (BCEC). The cells were prelabeled with [3H]myristic acid and incubated for specific time intervals with various test agents in the presence and absence of ethanol. The PLD activity was assayed by monitoring the formation of labeled phosphatidylethanol ([3H]PEt) in [3H]myristate labeled cells. In the absence of ethanol, 1 microM phorbol 12-myristate 13-acetate (PMA) increased the formation of labeled phosphatidic acid ([3H]PA) with no significant effect on the radioactivity of [3H]PEt. In the presence of 85 mM ethanol, whereas there was only a small further increase in [3H]PA, the formation of [3H]PEt was increased by several-fold, demonstrating activation of PLD by the phorbol ester. The effects of PMA were time- and dose-dependent, and were mimicked by phorbol 12,13-dibutyrate. The inactive phorbol derivatives, 4-alpha-phorbol, 4-alpha-phorbol 12,13-didecanoate, 4-alpha-phorbol 12-myristate 13-acetate and 4-alpha-phorbol 12,13-dibutyrate, were without effect. Short-time (30 min) incubation of BCEC with staurosporine or H-7, or prolonged (20 hours) incubation with PMA rendered the cells less sensitive to subsequent treatment with PMA, suggesting that activation of PLD in the cells is mediated by protein kinase C (PKC). Addition of 20 microM ionomycin in the presence of ethanol also increased the formation of [3H]PA and [3H]PEt in a time- and dose-dependent manner. Co-presence of ionomycin and PMA at submaximal concentrations in the incubation medium resulted in increased formation of [3H]PA and [3H]PEt which was less than their individual effects combined, indicating a lack of synergism between Ca2+ and PMA in activating PLD. Incubation of BCEC with staurosporine resulted in significant inhibition of ionomycin-induced production of [3H]PEt, suggesting that in addition to direct activation of PLD by Ca2+, the enzyme is probably stimulated by sequential activation of PLC (producing diacylglycerol) and PKC following the ionomycin addition. We conclude that BCEC possess PLD which is stimulated by PKC as well as elevated intracellular Ca2+.
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PMID:Stimulation of phospholipase D by phorbol esters and ionomycin in bovine corneal epithelial cells. 150 99

We have previously reported that endothelin-1 stimulates phospholipase C-induced hydrolysis of phosphatidylinositol-4,5-bisphosphate. Other signal transduction pathways that hydrolyze alternative phospholipids through phospholipase D may also mediate endothelin-stimulated cellular responses. We initially evaluated endothelin-dependent generation of 32P-phosphatidic acid as an indirect indication of phospholipase D activity in rat mesangial cells. Endothelin (10(-7) M) induced an elevation of phosphatidic acid that was maximal at 15 min and persisted upward of 60 min. Pretreatment with the diacylglycerol-kinase inhibitor, R59022, did not reduce formation of endothelin-stimulated 32P-phosphatidic acid, demonstrating that the sequential actions of phospholipase C/diacylglycerol kinase do not contribute to endothelin-stimulated phosphatidic acid formation. We next conclusively identified a role for phospholipase D in the generation of phosphatidic acid by assessing the formation of 3H-phosphatidylethanol from 3H-alkyl lyso glycerophosphocholine and exogenous ethanol. Endothelin stimulated 3H-alkyl phosphatidylethanol formation in the presence but not the absence of 0.5% ethanol. Also, endothelin induced a concomitant elevation of 3H-alkyl-phosphatidic acid that was significantly reduced when the cells were exposed to exogenous ethanol, reflecting the formation of phosphatidylethanol. In addition, endothelin stimulated the release of 3H-choline and 3H-ethanolamine, demonstrating that additional phospholipids may serve as substrates for phospholipase D. Phorbol esters and synthetic diglycerides mimicked the effects of endothelin to stimulate phospholipase D and inhibitors of protein kinase C significantly reduced endothelin-stimulated phospholipase D. In addition, endothelin did not stimulate phosphatidylethanol formation in protein kinase C down-regulated cells. The calcium ionophore, ionomycin, did not stimulate phospholipase D and mesangial cells pretreated with BAPTA to chelate cytosolic calcium did not show a diminished endothelin-stimulated phospholipase D. Thus these data demonstrate that mesangial cells possess a protein kinase C-regulated phospholipase D activity that can be stimulated with endothelin.
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PMID:Endothelin stimulates phosphatidic acid formation in cultured rat mesangial cells: role of a protein kinase C-regulated phospholipase D. 153 86

We have studied the effects of the vasoactive agents phorbol 12-myristate 13-acetate (PMA) and vasopressin (VP) on phosphatidylcholine metabolism in cultured rat glomerular mesangial cells. PMA and VP stimulate the incorporation of [3H]choline into phosphatidylcholine and the release of [3H]choline into the culture medium. VP, but not PMA, also increases the release of phosphorylcholine into the medium. This suggests that PMA specifically stimulates phospholipase D, whereas VP stimulates phospholipases C and D. Experiments were also conducted to look for production of phosphatidic acid and diacylglycerol, products of phospholipase D- and C-mediated breakdown of phosphatidylcholine. Treatment of cells prelabeled with [3H]myristic acid for 2.5 min with PMA or VP increases the content of [3H]myristic acid in diacylglycerol and phosphatidic acid. A dual labeling study ([3H]myristic acid and [14C]arachidonic acid) suggests that phosphatidylcholine is an important source of diacylglycerol in cells treated with VP and PMA. When PMA or VP are added to [3H]myristic acid-labeled cells in the presence of ethanol, increased labeling of phosphatidylethanol is seen as early as 2.5 min. Desensitization of protein kinase C by overnight treatment of cells with PMA blocked subsequent VP-stimulated formation of phosphatidylethanol and release of [3H]choline. When cells were simultaneously treated with VP and PMA, additive effects on phosphatidylethanol formation and [3H]choline release were observed.
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PMID:Vasopressin and phorbol ester-stimulated phosphatidylcholine metabolism in mesangial cells. 153 83


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