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 have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.
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PMID:Stimulation of phospholipase D activity in human neuroblastoma (LA-N-2) cells by activation of muscarinic acetylcholine receptors or by phorbol esters: relationship to phosphoinositide turnover. 200 44

A major biochemical pathway that has been implicated in the control of normal and malignant growth involves phosphoinositide metabolism. In this pathway, receptor-mediated activation of a phosphoinositide-specific phospholipase C causes the hydrolysis of phosphatidylinositol-4,5-bisphosphate which generates two putative second messengers, inositol-1,4,5-trisphosphate and diacylglycerol (DAG). Since DAG has been shown to be elevated in many transformed cells, we sought to determine if the levels of PKC isoenzymes are also increased. Northern blot analysis of mRNAs from 46 human tumour cell lines was performed using probes for the human PKC-I (gamma), PKC-II (beta) and PKC-III (alpha) genes. PKC-II mRNAs were significantly increased in 4 out of 12 sarcoma lines and 1 malignant melanoma cell line. PKC-III was increased in 2 out of 12 sarcoma cell lines and 1 kidney carcinoma cell line. In contrast, in the majority of carcinoma-derived cell lines tested, there was a decreased or moderate expression of either PKC-II or PKC-III mRNAs or both. It is interesting that tumour cell lines which overexpressed one isoenzyme (e.g. PKC-II), did not contain detectable levels of another isoenzyme (e.g. PKC-III), as determined by Northern blotting. Altogether, these results suggest that the overexpression of distinct PKC isoenzymes may be involved in abnormal growth regulation in some human tumours, especially in sarcomas.
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PMID:[Overexpression of protein kinase-C-isoenzymes in human tumor cell lines]. 203 50

We have identified two distinct cellular responses that occur in human astrocytes in the presence of angiotensin (Ang) peptides and are linked to specific receptor subtypes. Ang II and the N-terminal heptapeptide Ang-(1-7) stimulated release of prostaglandin (PG) E2 and PGI2 (measured as the stable metabolite 6-keto-PGF1 alpha). In contrast, only Ang II but not Ang-(1-7) activated phosphoinositide-specific phospholipase C, leading to mobilization of intracellular calcium. The Ang II-induced PGE2 and PGI2 syntheses were attenuated by [Sar1,Ile8]Ang II but not by [Sar1,Thr8]Ang II. Ang-(1-7)-induced PGE2 and PGI2 syntheses were not inhibited by either of these two classical antagonists. DuP 753, a subtype 1-selective Ang receptor antagonist, blocked the Ang II-induced release of PGE2 but not PGI2. In contrast, CGP 42112A, the subtype 2-selective antagonist, totally blocked the Ang II-induced PGI2 release and partially attenuated the PGE2 release. Ang-(1-7)-induced PGE2 and PGI2 release was not altered by DuP 753; however, CGP 42112A totally blocked the effects of Ang-(1-7) on PG stimulation. Calcium mobilization in response to Ang II was blocked by [Sar1,Thr8]Ang II, [Sar1,Ile8]Ang II, and DuP 753 but not by CGP 42112A. These data suggest that human astrocytes contain both Ang receptor subtypes. The subtype 1 Ang receptor participates both in the release of PGs and in the mobilization of calcium, whereas the subtype 2 receptor is coupled to the release of PGs only. In addition, PG release coupled to subtype 2 Ang II receptors occurs through a calcium-independent mechanism and responds uniquely to Ang-(1-7).
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PMID:Subtype 2 angiotensin receptors mediate prostaglandin synthesis in human astrocytes. 204 58

Ligand-mediated perturbation of the T-cell antigen receptor (TCR) triggers a rapid increase in phosphoinositide-specific phospholipase C (PLC) activity in resting T-cells. Although the mechanism by which TCR ligation regulates PLC activity is unknown, recent studies suggest that coupling of this receptor complex to PLC activity is dependent on an intermediate protein tyrosine phosphorylation event(s). In the present study, we demonstrate that antibody-mediated TCR cross-linkage results in the tyrosine phosphorylation of PLC-gamma 1. Stimulation of the TCR for 30 s induced a 4-5-fold increase in the level of PLC activity recovered in anti-phosphotyrosine (Tyr(P)) antibody immunoprecipitates from stimulated Jurkat cells. The appearance of PLC activity in the immunoprecipitates preceded the onset of phosphoinositide hydrolysis in vivo, which began 30-60 s after TCR ligation. Furthermore, the TCR-mediated increase in anti-Tyr(P) antibody-bound PLC activity was inhibited by staurosporine at drug concentrations identical with those required for in vivo inhibition of TCR-dependent phosphoinositide breakdown. Immunoblot analyses demonstrated that TCR ligation dramatically increased the level of tyrosine-phosphorylated PLC-gamma 1 present in anti-Tyr(P) antibody immunoprecipitates from stimulated Jurkat cells. These results strongly suggest that the TCR complex expressed by Jurkat cells is functionally coupled to the phosphoinositide-dependent signaling pathway through the tyrosine phosphorylation of PLC-gamma 1.
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PMID:T-cell antigen receptor ligation induces tyrosine phosphorylation of phospholipase C-gamma 1. 206 1

Several classes of growth factors can be distinguished that act through different signal transduction pathways. One class is constituted by the peptide growth factors that bind to receptors with ligand-dependent protein tyrosine kinase activity. Another class of mitogens activates a phosphoinositide-specific phospholipase C via a receptor-linked G protein. An intriguing member of this class is lysophosphatidic acid (LPA). LPA mitogenicity is not dependent on other mitogens and is blocked by pertussis toxin. LPA evokes at least three separate signalling cascades: (i) activation of a pertussis toxin-insensitive G protein mediating phosphoinositide hydrolysis; (ii) release of arachidonic acid in a GTP-dependent manner, but independent of prior phosphoinositide hydrolysis; and (iii) activation of a pertussis toxin-sensitive Gi protein mediating inhibition of adenylate cyclase. The peptide bradykinin mimics LPA in inducing responses (i) and (ii), but fails to activate Gi and to stimulate DNA synthesis. Our results suggest that the mitogenic action of LPA occurs through Gi or a related pertussis toxin substrate and that, unexpectedly, the phosphoinositide hydrolysis pathway is neither required nor sufficient, by itself, for mitogenesis.
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PMID:Growth factor-like action of lysophosphatidic acid: mitogenic signalling mediated by G proteins. 211 27

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization.
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PMID:Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C. 211 42

Cholate-solubilized extracts from bovine liver plasma membranes preincubated with the nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) displayed enhanced phosphoinositide-specific phospholipase C activity compared with extracts from membranes incubated without nucleotide or with ATP or GDP analog. Resolution of the GTP gamma S-elicited activator of phospholipase C was achieved using heparin-Sepharose which bound the phospholipase C activity. Recombination of non-adsorbed extract with salt-eluted phospholipase C activity resulted in a stimulation of enzyme activity. The GTP gamma S-dependent activator was purified, on the basis of its ability to activate partially purified phospholipase C, by sequential chromatography on Q-Sepharose, Sephacryl S-300, octyl-Sepharose, and Mono Q. The presence of G-protein beta subunits and the alpha subunits of Gi1, Gi2, and Gi3 was detected, by immunoblot analysis, in Mono Q-purified phospholipase C activator preparations. Resolution of the activator from these alpha subunits was achieved by incubation with pertussis toxin in the presence of millimolar NAD+ followed by rechromatography on Mono Q. The phospholipase C activator, thus resolved from ADP-ribosylated alpha i subunits, possessed an approximate Mr of 42 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and copurified with a substoichiometric amount of beta subunit. Immunoblot analysis of fractions from the final Mono Q column revealed cross-reactivity of the 42-kDa phospholipase C activator with antipeptide antibodies raised against residues 160-169 of alpha i1 and a region of sequence common to all known G-protein alpha subunits. The 42-kDa activator was not recognized by other alpha subunit-specific or common antibodies. These findings identify the purified phospholipase C activator as a novel G-protein alpha subunit. This may represent the active subunit of the pertussis toxin-insensitive G-protein mediating receptor-stimulated phosphoinositide breakdown in mammalian liver.
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PMID:Purification from bovine liver membranes of a guanine nucleotide-dependent activator of phosphoinositide-specific phospholipase C. Immunologic identification as a novel G-protein alpha subunit. 212 Feb 13

The deacylated forms of the phosphoinositides were used to determine whether the guinea pig uterus phosphoinositide-specific phospholipase C (PI-PLC I, Mr 60,000) required fatty acids at the sn-1 and sn-2 positions for the hydrolysis of the sn-3 phosphodiester bond. L-alpha-Glycerophospho-D-myo-inositol 4-phosphate (Gro-PIP), but not glycerol 3-phosphate (Gro-3-P), L-alpha-glycerophospho-D-myo-inositol (Gro-PI), or L-alpha-glycerophospho-D-myo-inositol 4,5-bisphosphate (Gro-PIP2), inhibited PI-PLC I in a concentration-dependent manner. Assays performed with 10 microM [3H]phosphatidylinositol ([3H]PI), 10 microM [3H]phosphatidylinositol 4-phosphate ([3H]PIP) or 10 microM [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) as substrates, with increasing [Gro-PIP] revealed an IC50 = 380 microM. Kinetic studies with increasing [3H]PI substrate concentrations in the presence of 100 microM and 300 microM Gro-PIP demonstrated that Gro-PIP exhibited competitive inhibition; Kis = 40 microM. Ca2+ concentrations over the range 1.1 microM to 1 mM did not effect inhibition, suggesting that Gro-PIP inhibition of [3H]PI hydrolysis was calcium-independent. To determine whether Gro-PIP was a substrate, 20 microM and 500 microM [3H]Gro-PIP were incubated with PI-PLC I. Anion-exchange HPLC analysis revealed no [3H]IP2 product formation, indicating that [3H]Gro-PIP was not hydrolyzed. Assays performed with [3H]PI and [3H]PIP substrates in the presence of 500 microM [3H]Gro-PIP revealed approx. 75% less [3H]inositol 1-phosphate ([3H]IP1) and [3H]inositol 1,4-bisphosphate ([3H]IP2) product formation, respectively, indicating that [3H]Gro-PIP inhibited the hydrolysis of the substrates by PI-PLC I. These data suggest that Gro-PIP does not serve as a substrate, and that it inhibits PI-PLC I by competitive inhibition in a Ca2(+)-independent fashion.
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PMID:Glycerol-3-phospho-D-myo-inositol 4-phosphate (Gro-PIP) is an inhibitor of phosphoinositide-specific phospholipase C. 215 9

It has been suggested that K+, Li+ and Fl- affect the function of G proteins coupled to signal transducing enzymes. Lithium, at concentrations which were found to reduce forskolin-stimulated adenylate cyclase activity, was without effect on either membrane [3H]phosphatidylinositol-4,5-bisphosphate ([3H]PIP2) hydrolysis measured in the absence or presence of 5'-guanylyl-imidodiphosphate (Gpp(NH)p), or (at greater than or equal to 2.3 mM Li+) upon the stimulation of rat cerebral cortical inositol phospholipid breakdown by either carbachol, noradrenaline or NaF measured at either 6 or 18 mM K+. The increase in assay [K+] greatly enhanced the inositol phospholipid response to carbachol but not to NaF. The inhibitory effect of carbachol upon forskolin-stimulated adenylate cyclase was not affected by raising the [K+] from 6 to 18 mM. At 6 mM K+ (both in the absence and presence of 15 microM AlCl3), the effects of carbachol and NaF upon inositol phospholipid breakdown were essentially additive, whereas at 18 mM K+, the breakdown response to carbachol (antagonised by pirenzepine with a pA2 value of 7.6) was similar in the absence and presence of NaF. It is concluded that in the rat cerebral cortex: (a) Li+ does not affect the function of either the phosphoinositide-specific phospholipase C enzyme itself or the Gp coupled to this enzyme; (b) the difference between the additivity between NaF and carbachol seen at different assay [K+] may reflect the K(+)-dependent changes in the tetrodotoxin-resistant and tetrodotoxin-sensitive pathways of carbachol stimulation of inositol phospholipid breakdown reported by Gurwitz and Sokolovsky (1987, Biochemistry 26, 633); and (c) the effect of K+ on muscarinic receptor-coupled inositol phospholipid breakdown is not found for muscarinic receptors inhibitorily coupled to adenylate cyclase. Evidence is also presented to suggest that NaF affects the dephosphorylation of the formed [3H]inositol polyphosphates.
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PMID:Effect of monovalent ions upon G proteins coupling muscarinic receptors to phosphoinositide hydrolysis in the rat cerebral cortex. 215 22

Profilin is generally thought to regulate actin polymerization, but the observation that acidic phospholipids dissociate the complex of profilin and actin raised the possibility that profilin might also regulate lipid metabolism. Profilin isolated from platelets binds with high affinity to small clusters of phosphatidylinositol 4,5-bisphosphate (PIP2) molecules in micelles and also in bilayers with other phospholipids. The molar ratio of the complex of profilin with PIP2 is 1:7 in micelles of pure PIP2 and 1:5 in bilayers composed largely of other phospholipids. Profilin competes efficiently with platelet cytosolic phosphoinositide-specific phospholipase C for interaction with the PIP2 substrate and thereby inhibits PIP2 hydrolysis by this enzyme. The cellular concentrations and binding characteristics of these molecules are consistent with profilin being a negative regulator of the phosphoinositide signaling pathway in addition to its established function as an inhibitor of actin polymerization.
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PMID:The actin-binding protein profilin binds to PIP2 and inhibits its hydrolysis by phospholipase C. 215 83


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