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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)

Post-translational modifications such as phosphorylation and palmitoylation play important roles for the function and regulation of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins. Here we demonstrate that the human endothelin receptor A (ETA) incorporates [3H]palmitate. Mutation of a cluster of five cysteine residues present in the cytoplasmic tail of ETA into serine or alanine residues completely prevented palmitoylation of the receptor. The ligand binding affinity of the non-palmitoylated ETA mutants was essentially unchanged as compared to the palmitoylated wild type ETA suggesting that the replacement of the cysteine residues did not alter the overall structure of the receptor. Furthermore, the ligand-induced stimulation of adenylyl cyclase by the mutant ETA was unaffected by the mutation. In contrast, the mutated non-palmitoylated receptors but not the wild type receptor failed to stimulate phosphatidylinositol hydrolysis by phospholipase C activation upon challenge by endothelin-1. Furthermore, the mutant receptors failed to stimulate the ligand-induced transient increase in the cytoplasmic calcium seen with the wild type ETA. Endothelin-1 induced mitogenic stimuli via the wild type receptors but not through the mutated receptors suggesting an important role for phospholipase C in this signal transduction pathway. The differential regulation of distinct signal transduction pathways by post-translational modification suggests that palmitoylation of the ETA provides a novel mechanism of modulating ETA receptor activity.
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PMID:Palmitoylation of endothelin receptor A. Differential modulation of signal transduction activity by post-translational modification. 870 36

Two major intermediaries in signal transduction pathways are pp60v-sre family tyrosine kinases and heterotrimeric guanine nucleotide-binding proteins. In Rat-1 fibroblasts transformed by the v-src oncogene, endothelin-1 (ET-1)-induced inositol 1,4,5-trisphosphate accumulation is increased 6-fold, without any increases in the numbers of ET-1 receptors or in the response to another agonist, thrombin. This ET-1 hyperresponse can be inhibited by an antibody directed against the carboxyl terminus of the Gq/G11 alpha subunit, suggesting that the Gq/G11 protein couples ET-1 receptors to phospholipase C (PLC). While v-src transformation did not increase the expression of the Gq/G11 alpha subunit, immunoblotting with anti-phosphotyrosine antibodies and phosphoamino acid analysis demonstrated that the Gq/G11 alpha subunit becomes phosphorylated on tyrosine residues in v-src-transformed cells. Moreover, when the Gq/G11 protein was extracted from control and transformed cell lines and reconstituted with exogenous PLC, AIF*4-stimulated Gq/G11 activity was markedly increased in extracts from v-src-transformed cells. Our results demonstrate that the process of v-src transformation can increase the tyrosine phosphorylation state of the Gq/G11 alpha-subunit in intact cells and that the process causes an increase in the Gq/G11 alpha-subunit's ability to stimulate PLC following activation with AIF-4.
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PMID:Transformation of Rat-1 fibroblasts with the v-src oncogene increases the tyrosine phosphorylation state and activity of the alpha subunit of Gq/G11. 871 Aug 57

A multitude of agonists like e.g. endothelin-1, angiotensin-II, serotonin, thrombin, histamine and vasopressin as well as alpha 1-adrenergic and muscarinic stimulation lead to stimulation of the phosphoinositide cycle in the heart. Besides the seven membrane spanning-domain receptor-coupled stimulation of the key enzyme of the phosphoinositide cycle, phospholipase C-beta, another class of hormones, growth factors, also couple to the phosphoinositide cycle, now through receptors with intrinsic tyrosine kinase activity that can phosphorylate and stimulate the phospholipase C-gamma isozyme. In this review we summarize the multitude of receptor (sub)types, G-protein-subunit- and phospholipase C-isozymes that are present in the heart. Furthermore, generation of second messengers and cellular responses are described together with the (patho)physiological implications for the heart of phosphoinositide cycle activation and second messenger accumulation.
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PMID:Phosphoinositide-generated messengers in cardiac signal transduction. 873 23

There is now clear evidence that receptor-dependent phospholipase D is present in myocardium. This novel signal transduction pathway provides an alternative source of 1,2-diacylglycerol, which activates isoforms of protein kinase C. The members of the protein kinase C family respond differently to various combinations of Ca2+, phosphatidylserine, molecular species of 1,2-diacylglycerol and other membrane phospholipid metabolites including free fatty acids. Protein kinase C isozymes are responsible for phosphorylation of specific cardiac substrate proteins that may be involved in regulation of cardiac contractility, hypertrophic growth, gene expression, ischemic preconditioning and electrophysiological changes. The initial product of phospholipase D, phosphatidic acid, may also have a second messenger role. As in other tissues, the question how the activity of phospholipase D is controlled by agonists in myocardium is controversial. Agonists, such as endothelin-1, atrial natriuretic factor and angiotensin II that are shown to activate phospholipase D, also potently stimulate phospholipase C-beta in myocardium. PMA stimulation of protein kinase C inactivates phospholipase C and strongly activates phospholipase D and this is probably a major mechanism by which agonists that promote phosphatidyl-4,5-bisphosphate hydrolysis secondary activate phosphatidylcholine-hydrolysis. On the other hand, one group has postulated that formation of phosphatidic acid secondary activates phosphatidyl-4,5-bisphosphate hydrolysis in cardiomyocytes. Whether GTP-binding proteins directly control phospholipase D is not clearly established in myocardium. Phospholipase D activation may also be mediated by an increase in cytosolic free Ca2+ or by tyrosine-phosphorylation.
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PMID:Regulation and functional significance of phospholipase D in myocardium. 873 27

The influence of increased incorporation of linoleic acid (18:2n-6) and eicosapentaenoic acid (20:5n-3) in membrane phospholipids on receptor-mediated phospholipase C beta (PLC-beta) activity in cultured rat ventricular myocytes was investigated. For this purpose, cells were grown for 4 days in control, stearic acid (18:0)/oleic acid (18:1n-9), 18:2n-6 and 20:5n-3 enriched media, and subsequently assayed for the basal- and phenylephrine- or endothelin-1-induced total inositol phosphate formation. The various fatty acid treatments resulted in the expected alterations of fatty acid composition of membrane phospholipids. In 18:2n-6-treated cells, the incorporation of this 18:2n-6 in the phospholipids increased from 17.1 mol % in control cells to 38.9 mol %. In 20:5n-3-treated cells, incorporation of 20:5n-3 and docosapentaenoic acid (22:5n-3) in the phospholipids increased from 0.5 and 2.7 mol % in control cells to 23.2 and 9.7 mol %, respectively. When 20:5n-3-treated cells were stimulated with phenylephrine or endothelin-1, the inositolphosphate production decreased by 33.2% and increased by 43.4%, respectively, as compared to cells grown in control medium. No effects were seen in 18:2n-6-treated cells. When 18:0/18:1n-9-treated cells were stimulated with endothelin-1, inositolphosphate formation increased by 26.4%, whereas phenylephrine-stimulated inositolphosphate formation was not affected. In saponin-permeabilized cells, that were pre-treated with 20:5n-3, the formation of total inositolphosphates after stimulation with GTP gamma S, in the presence of Ca2+, was inhibited 19.3%. This suggests that the 20:5n-3 effect on intact cardiomyocytes could be exerted either on the level of agonist-receptor, receptor-GTP-binding-protein coupling or GTP-binding-protein-PLC-beta interaction. Investigation of the time course of saponin-induced permeabilization of the cardiomyocytes, measured by the release of lactate dehydrogenase, unmasked a slight decrease in the rate of permeabilization by 20:5n-3 pretreatment, indicating a protective effect. This led the authors to measure the cholesterol/phospholipid molar ratio, the double bond index of membrane phospholipids, and the membrane fluidity; the latter by using a diphenylhexatriene probe. In 20:5n-3-pretreated cells, a strong increase in the cholesterol/phospholipid molar ratio (from 0.23 to 0.39), a marked increase in the double bond index (from 1.76 to 2.33), and a slight decrease in fluidity (steady-state anisotropy rss of the diphenylhexatriene probe increased from 0.196 to 0.217) were observed. Thus, treatment of cardiomyocytes for 4 days with 20:5n-3, but not with 18:2n-6, causes alterations of receptor-mediated phospholipase C beta activity. A causal relationship may exist between the 20:5n-3 causes alterations of the physicochemical properties in the bilayer and of the agonist-stimulated phosphatidylinositol cycle activity.
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PMID:Eicosapentaenoic acid incorporation in membrane phospholipids modulates receptor-mediated phospholipase C and membrane fluidity in rat ventricular myocytes in culture. 876 46

1. We have investigated the effect of propofol, an intravenous anaesthetic, on the intracellular calcium concentration ([Ca2+]i), Ca2+ entry pathways and on inositol phosphate formation in vascular smooth muscle cells. [Ca2+]i and Ca2+ flux were monitored with the Ca(2+)-sensitive fluorescent dye, fura-2, and by 45Ca2+ uptake. Production of labelled inositol phosphates was analysed by anion-exchange chromatography. 2. Treatment of the cells with endothelin-1 (ET-1) increased formation of inositol phosphates and elevated [Ca2+]i due to both release of Ca2+ from intracellular pools and prolonged entry of Ca2+ from outside the cell. Propofol reduced production of inositol phosphates mediated by ET-1 and arginine vasopressin which activate phospholipase C. 3. The sustained Ca2+ entry stimulated by ET-1 was found to occur through the activation of L-type Ca channels. This was inhibited by propofol in a dose-dependent manner. 4. Activation of protein kinase C (PKC) by phorbol esters activated a pharmacologically-similar channel and produced a similar change in [Ca2+]i due to Ca2+ entry. The entry was blocked by an L-type channel antagonist, nicardipine and by the anaesthetic drug, propofol. 5. Treatment of the cells with thapsigargin, a selective inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, also elevated [Ca2+]i by inducing the release of intracellular Ca2+ and the continued entry of extracellular Ca2+ through a nicardipine-insensitive Ca channel. Neither release nor entry induced by thapsigargin was affected by propofol. 6. These findings suggest that propofol selectively inhibits Ca2+ entry through the L-type channel induced by ET-1 and phorbol esters but has no effects on Ca2+ entry via the nicardipine-insensitive channel and on Ca2+ release from intracellular pools initiated by thapsigargin. This may represent one of the mechanisms responsible for propofol-induced vasodilatation.
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PMID:Propofol regulation of calcium entry pathways in cultured A10 and rat aortic smooth muscle cells. 882 36

Cultured astrocytes express bradykinin (BK) receptors, which are coupled to phospholipase C (PLC) through G-protein to mediate phosphoinositide (PI) hydrolysis. The regulation of this BK receptor-G protein-PLC pathway by cAMP and endothelin-1 (ET-1) was explored by short-term (20 min) and long-term (24 h) treatment with 100 mu M dibutyryl cyclic AMP (dBcAMP) or 10 nM ET-1. Short-term treatment of cells with dBcAMP had no effect on BK-induced PI hydrolysis; however, long-term treatment resulted in potentiation of the BK response. Similar effects were seen after 10 mu M forskolin pretreatment of the cells. We further explored the site of action of 24 h dBcAMP pretreatment and found that AlF(4)-, ionomycin- or A3187-induced PI hydrolysis was not affected but (3H)BK binding was increased. These results indicate that the site of action of dBcAMP is the BK receptor and Scatchard plot analysis showed that the Bmax was increased but the Kd decreased. Cycloheximide (0.5 mu M) blocked the increase in (3H)BK binding, indicating that new synthesis of receptor protein might occur during 24 h pretreatment with dBcAMP. Twenty minutes pretreatment of cells with ET-1 resulted in desensitization of the ET-1 induced P1 response, while the BK response was unaffected. After 24 h pretreatment with ET-1, desensitization to ET-1 still occurred, while BK-induced PI hydrolysis was markedly potentiated. (3H)BK binding and AlF(4)--induced but not A23187- or ionomycin-induced PI hydrolysis were increased, indicating that the site of action of long-term ET-1 treatment was the BK receptor and G protein; Scatchard analysis showed an increase in Bmax but no effect on Kd. These effects were blocked by cycloheximide, indicating that new synthesis of both receptor protein and G protein might occur during 24 h pretreatment with ET-1. (3H)Thymidine uptake was inhibited or potentiated by dBcAMP and ET-1, respectively. Possible dBcAMP-induced differentiation and ET-1-induced proliferation may contribute to the increased expression of receptor proteins.
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PMID:Potentiation of bradykinin-induced inositol phosphates production by cyclic AMP elevating agents and endothelin-1 in cultured astrocytes. 883 91

We investigated the vascular effects and mechanism of action of endothelin-1 (ET-1) in the skin by intra-arterial infusion of ET-1 and its precursor Big ET-1 via a direct cutaneous artery in isolated perfused pig skin flaps (6 x 16 cm). The vascular contractivity was studied by monitoring the perfusion pressure in the skin flap. There was evidence to indicate local conversion of Big ET-1 to ET-1 in the pig skin. It was also observed that ET-1 was a potent long-lasting vasoconstrictor with a potency of approximately 10- and 300-fold higher than those of Big ET-1 and norepinephrine, respectively. The vasoconstrictor action of ET-1 was blocked (P < 0.01) by a selective ETA-receptor antagonist (BQ-123 or BQ-610; 10(-7) M) and enhanced (P < 0.05) by a nitric oxide synthase inhibitor (NG-monomethyl-L-arginine or N omega-nitro-L-arginine methyl ester; 10(-5) M). ET-1-induced increase in perfusion pressure was attenuated (P < 0.05) by an L-type Ca(2+)-channel antagonist (nitrendipine, verapamil, or nifedipine; 10(-5) M) and by removal of Ca2+ from the perfusate. ET-1-induced increase in perfusion pressure was also attenuated (P < 0.05) by a phospholipase C inhibitor (neomycin; 10(-2) M), a protein kinase C (PKC) inhibitor (chelerythrine or H-7; 10(-5) M), and an intracellular Ca2+ chelator [1,2-bis(2-aminophenoxy)]ethane-N,N,N',N'-tetraacetic acid (BAPTA); 10(-5) M]. Furthermore, it was observed that the concentration-dependent (5 x 10(-8) to 10(-5) M) increase in perfusion pressure induced by phorbol 12,13-dibutyrate, a PKC activator, was not affected by verapamil (10(-5) M) or removal of Ca2+ from the perfusate. Taken together, these observations suggest that the vasoconstrictor mechanism of ET-1 in the pig skin involved activation of ETA receptors, L-type Ca2+ channels, phospholipase C, and PKC and that the vasoconstrictor effect caused by activation of PKC was independent of L-type Ca2+ channels.
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PMID:Vascular effects and mechanism of action of endothelin-1 in isolated perfused pig skin. 884 80

1. Brain capillary endothelial cells responded to uridine 5'-triphosphate (UTP) and adenosine 5'-triphosphate (ATP) by activation of phospholipase C and by large changes in [Ca2+]i. These cells expressed mRNA sequences identical to the sequence of the P2Y2-purinoceptor of rat pituitaries. 2. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) at 100 microM did not prevent UTP and ATP induced accumulations of total [3H]-inositol (poly)phosphates. It inhibited UTP and ATP induced intracellular Ca2+ mobilization (IC50 = 30 microM) by non competitive mechanism. 3. PPADS (100 microM) inhibited endothelin-1 induced accumulation of total [3H]-inositol (poly)phosphates by less than 20% and prevented most of endothelin-1 induced intracellular Ca2+ mobilization (IC50 = 30 microM). 4. PPADS (100 microM) had no action on ionomycin induced intracellular Ca2+ mobilization. 5. Microinjection of inositol (1,4,5)trisphosphate (InsP3) into Xenopus oocytes induced large Ca2+ activated Cl- currents that were prevented by heparin and by PPADS. 6. It is concluded that PPADS does not recognize rat P2Y2-purinoceptors and prevents UTP and ATP induced intracellular Ca2+ mobilization by a non-specific mechanism that could involve the inhibition of InsP3 channels.
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PMID:The effect of PPADS as an antagonist of inositol (1,4,5)trisphosphate induced intracellular calcium mobilization. 888 21

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (IP3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC50 = 1.2 nM) than in activating PLC (EC50 = 1.5 nM) or PLA2 (EC50 = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA2 was stimulated first (t1/2 = 12 s), followed by PLC (t1/2 = 48 s) and lastly PLD (t1/2 = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ETB receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ETA receptor subtype to activate, via G proteins, phospholipases A2, C and D in a temporal sequence.
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PMID:Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells. 890 57


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