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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)

Inositol phospholipids play a crucial role in the intracellular signal transduction in most cell types. Activation of an enzyme called phospholipase C or PIP2-phosphodiesterase (PIP2-PDE) leads to the production of two second messenger molecules, diacylglycerol (DG) and inositol 1,4,5-triphosphate (IP3). DG activates a kinase called protein kinase C, whereas IP3 mediates the release of Ca2+ from intracellular storage sites. The measurement of IP3 and its degradation products, inositol diphosphate (IP2) and inositol monophosphate (IP1) provides a way of assessing the extent to which this complex system has been activated. In the central nervous system (CNS) most of the studies on the neurotransmitter stimulated formation of inositol phosphates (IPs) have been performed on brain slices, a mixture of mainly neurons and glial cells. The recent development of pure neuronal cultures provides a means of determining which of these responses were of neuronal origin. The purpose of this review is to summarize the results obtained in neurons in primary culture together with a brief appraisal of the possible function of this second messenger system in neurons.
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PMID:Putative role of inositol phospholipid metabolism in neurons. 282 May 14

In this study we have used a density perturbation method to isolate anti-Thy-1 antibody-induced Thy-1 caps from mouse T-lymphoma cells in the absence of detergents, and then compared the phospholipid composition of these capped membranes with that of uncapped membranes. Initial phospholipid analysis by two-dimensional thin layer chromatography (2-D TLC) reveals a significant increase in the amount of 32P-labeled phosphatidylcholine in the Thy-1 capped membrane. In contrast, no significant changes are observed in the labeling of phosphatidylserine, phosphatidylethanolamine, or the sphingomyelins. Therefore, it is suggested that phosphatidylcholine may be involved in the organization and/or regulation of Thy-1 antigen redistribution. The composition of phosphoinositide in uncapped and capped membranes was analysed separately using one-dimensional thin layer chromatography (1-D TLC) to resolve phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PlP), and phosphatidylinositol 4, 5-bisphosphate (PIP2) from all other phospholipids. This analysis reveals a significant reduction in levels of PIP and PIP2, but not PI, in Thy-1 caps. Through the use of ion exchange column chromatography, we have found an increased production of all three species of inositol phosphates during anti-Thy-1 antibody-induced capping. Inositol 1, 4, 5-triphosphate (IP3) shows the most significant increase, compared to the much smaller increases in inositol 4, 5-bisphosphate (IP2) and inositol monophosphate (IP). These results suggest that the binding of anti-Thy-1 antibody to Thy-1 antigen activates phospholipase C which, in turn, initiates polyphosphoinositide turnover and IP3 production. It is proposed that these observed effects are the result of early signal transducing events which are prerequisite steps in Thy-1 receptor cap formation.
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PMID:Isolation of Thy-1 caps and analysis of their phospholipid composition in mouse T-lymphoma cells. 282 3

Platelet-activating factor (PAF) receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) was studied in platelets that were made refractory, by short-term pretreatments, to either PAF or thrombin. Generation of [3H]inositol triphosphate ( [3H]IP3) was monitored specifically for this purpose. [3H]Inositol-labeled rabbit platelets that were incubated (10 min) with increasing concentrations of PAF and subsequently challenged by the same concentration of PAF had greatly diminished PLC activity ( [3H]IP3 production) as compared to controls. Platelets incubated (10 min) with fixed concentrations of PAF and then challenged with increasing concentrations of PAF had log-dose response curves of [3H]IP3 production progressively shifted to the right (i.e., to higher concentrations) and were depressed as the PAF pretreatment with 10 nM PAF became completely refractory to further PAF stimulation of PLC. Washing the pretreated platelets with either buffer or buffer containing 0.5% bovine serum albumin did not restore the PAF for 10 min), platelets remained fully responsive to thrombin (2 units/ml)-stimulated production of [3H]IP3. Platelets pretreated with increasing concentrations of thrombin (0.15-2 units/ml) for different times (5-40 min) became refractory to both thrombin and PAF. It is concluded that PAF receptor-coupled activation of PLC becomes refractory (desensitized) in platelets preexposed to PAF, whereas platelets pretreated with thrombin are desensitized to both thrombin and PAF. It is proposed that thrombin has two transmembrane pathways leading to the activation of PLC, one shared with PAF and another utilizing separate mechanistic inputs.
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PMID:Desensitization of receptor-coupled activation of phosphoinositide-specific phospholipase C in platelets: evidence for distinct mechanisms for platelet-activating factor and thrombin. 282

The metabolism of D-glucose is believed to initiate and regulate insulin secretion by islet beta-cells, although the identity of the metabolite which couples glucose metabolism to the cellular events involved in insulin secretion is unknown. An alternative hypothesis involves the presence of a glucoreceptor for which there has been no biochemical evidence. We have investigated whether glucose recognition by the beta-cell is coupled to phospholipase C. We have used digitonin-permeabilized, [3H]inositol-prelabeled islets to study glucose and carbachol activation of phospholipase C. In this model, carbachol recognition by its muscarinic receptor was coupled to phospholipase C activation. D-Glucose (but not L-glucose) also stimulated phospholipase C activity in these permeabilized islets. This effect was not due to glucose metabolism since glucose 6-phosphate did not affect phospholipase C activity and since phosphorylation of [3H]glucose was not detectable in digitonin-permeabilized islets. Glucose had no effect on the myo-inositol-1,4,5-trisphosphate-5-phosphatase or 3-kinase activities. In the absence of agonist, free Ca2+ concentrations between 0.1 and 1 microM (as determined with a Ca2+-specific electrode) did not influence phospholipase C activity. Stimulation of phospholipase C activity by either carbachol or glucose required Ca2+ in the submicromolar range and was optimal at 0.5 microM free Ca2+.myo-Inositol-1,3,4,5-tetrakisphosphate production from permeabilized islets was synergistically augmented by Ca2+ (0.5-10 microM) and glucose. Phospholipase C activity in islets is therefore not directly activated by free Ca2+ concentrations in the submicromolar range. Furthermore, glucose per se activates phospholipase C activity independently of glucose metabolism. A working hypothesis based on these findings is that glucose is recognized by a site which is coupled to phospholipase C in islets.
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PMID:Studies of the Ca2+ requirements for glucose- and carbachol-induced augmentation of inositol trisphosphate and inositol tetrakisphosphate accumulation in digitonin-permeabilized islets. Evidence for a glucose recognition site in insulin secretion. 283 Nov 91

This essay attempts to summarize some of the best evidence for the role of inositol trisphosphate as a second messenger in signal transduction processes. The following aspects are addressed in the essay: (a) The synthesis of inositol trisphosphate and other inositol lipids, (b) Receptor-phosphatidylinositol bisphosphate phospholipase C coupling and the N-ras protooncogene, (c) Inositol trisphosphate and intracellular calcium, (d) Cell growth and oncogenes, (e) Receptors linked to the phosphatidylinositol cycle, (f) Phototransduction and (g) Interactions between inositol trisphosphate and other second messengers.
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PMID:Role of inositol trisphosphate as a second messenger in signal transduction processes: an essay. 283 62

[3H]Inositol-labelled GH3 rat anterior pituitary tumour cells were permeabilized with digitonin and were incubated at 37 degrees C in the presence of ATP and Mg2+. [3H]Polyphosphoinositide breakdown and [3H]inositol phosphate production were stimulated by hydrolysis-resistant GTP analogues and by Ca2+. Of the nucleotides tested, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) was the most effective stimulus. Activation by GTP gamma S appeared to be mediated by a guanine nucleotide-binding (G) protein as GTP gamma S-stimulated [3H]inositol phosphate production was inhibited by other nucleotides with a potency order of GTP = GDP = guanosine 5'-[beta-thio]diphosphate greater than ITP greater than GMP greater than UTP = CTP = adenosine 5'-[gamma-thio]triphosphate. The stimulatory effects of 10 microM-GTP gamma S on [3H]inositol phosphate levels were reversed by spermine and spermidine with IC50 values of approx. 0.25 and 2 mM respectively. Putrescine was inhibitory only at higher concentrations. Similarly, GTP gamma S-induced decreases in [3H]polyphosphoinositide levels were reversed by 2.5 mM-spermine. The inhibitory effects of spermine were not overcome by supramaximal concentrations of GTP gamma S. In contrast, [3H]inositol phosphate production stimulated by addition of 0.3-0.6 mM-Ca2+ to incubation media was only partially inhibited by spermine (5 mM), and spermine was not inhibitory when added Ca2+ was increased to 1 mM. These data show that polyamines, particularly spermine, inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis with a marked selectivity towards the stimulatory effects of GTP gamma S.
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PMID:Polyamines inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis. Studies with permeabilized GH3 cells. 285 Jul 92

A rat hepatocyte cell line was cultured in Higuchi's medium with fetal calf serum and insulin and labeled with 35SO2/4-. The cells were treated with a number of ligands to displace the heparan 35SO4 proteoglycan (HSPG) from the pericellular matrix. Maximum release was obtained with D-mannose-6-PO4 (50 mM), D-glucose-6-PO4 (50 mM), myo-inositol-2-PO4 (2-5 mM), myo-inositol hexaphosphate (2-5 mM), and DL-myo-inositol-1-PO4 (1-2 mM). D-myo-Inositol-1,3,4-(PO4)3 (1 mM) and L-myo-inositol-1-PO4 (2 mM) were intermediate in their ability to release the cell surface HSPG, whereas heparin (2 mg/ml), yeast phosphomannan (4 mg/ml), D-xylose-1-PO4 (50 mM), D-glucose-6-SO4 (50 mM), and myo-inositol hexasulfate (5 mM) were ineffective. When 35SO2/4- was added to cell cultures, the total cell surface HSPG increased linearly, but the percentage of the total cell surface [35SO4]HSPG that was released by myo-inositol-PO4 increased with time during the labeling period, reaching a maximum of 65% after 5 h. When cells were labeled for 12 h without insulin in the medium, the maximum amount of cell surface HSPG that was released by myo-inositol-PO4 was reduced to 30%. However, when cells labeled in the absence of insulin were treated with phosphatidylinositol-specific phospholipase C and then myo-inositol-PO4, the release of the cell surface [35SO4]HSPG was increased to 73%. When the [35SO4]HSPG that was released from the cell surface by treatment with myo-inositol-PO4 was added to cultures of unlabeled hepatocytes, it was taken up very rapidly and a portion of the internalized HSPG was converted to free heparan SO4 chains which appeared in the nucleus. Uptake was Ca2+- and Mg2+-independent. The amount of [35SO4]HSPG taken up was markedly reduced when the myo-inositol-PO4-releasable [35SO4]HSPG was pretreated with trypsin, thermolysin, alkaline borohydride, or alkaline phosphatase. When the cells were grown in inositol-deficient medium or in the presence of myo-inositol-PO4, the amount of heparan SO4 found in the nucleus was markedly reduced, and the cells no longer exhibited contact inhibition. These effects of myo-inositol deficiency on the growth and nuclear heparan SO4 were accentuated by addition of LiCl to the cultures to prevent phosphatidylinositol synthesis from the endogenous myo-inositol-PO4.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Involvement of phosphatidylinositol and insulin in the coordinate regulation of proteoheparan sulfate metabolism and hepatocyte growth. 295 71

Inositol trisphosphate (IP3) formed by phospholipase C-mediated breakdown of triphosphoinositide (PIP2) may be a ubiquitous second messenger for a number of Ca2+-mobilizing receptor agonists. Using [3H]inositol-labeled rabbit peritoneal neutrophils, we report that radiolabeled inositol phosphates are generated in response to the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). fMet-Leu-Phe-stimulated formation of [3H]IP3 occurs with a rapid time course and a concentration dependence which closely parallels that of stimulated lysosomal enzyme secretion. The synthetic peptide methionyl-leucyl-phenylalanine, which is unable to promote secretion, failed to elevate [3H]IP3 accumulation, and the competitive antagonist t-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe depressed the stimulant action of fMet-Leu-Phe on [3H]IP3 levels and secretion. The Ca2+ ionophore ionomycin, which promotes secretion, was unable to enhance IP3 levels, confirming that polyphosphoinositide hydrolysis is a specific receptor-mediated event that precedes calcium mobilization during neutrophil activation. The ability of leukotriene B4 to also promote a rapid accumulation of [3H]IP3 suggests that there exists in the neutrophil an interaction between phospholipase A2 and C-mediated events. These findings support the hypothesis that IP3 may be a pivotal messenger for signal transfer by Ca2+-mobilizing receptor agonists.
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PMID:Characterization of formylmethionyl-leucyl-phenylalanine stimulation of inositol trisphosphate accumulation in rabbit neutrophils. 298 3

Previous studies have shown that metabolism of phosphatidylinositol by phospholipase C produces a mixture of two water-soluble products: inositol 1-phosphate and inositol 1,2-(cyclic)phosphate. In the present study, we demonstrate that the water-soluble products of phosphatidylphosphoinositol (polyphosphoinositide) cleavage by purified ram seminal vesicle phospholipase C enzymes also contain cyclic phosphates. Inositol cyclic phosphates were detected by 18O labeling. In the presence of acid, cyclic phosphates are rapidly hydrolyzed to phosphomonoesters, and when the hydrolysis is carried out in H2 18O, the resultant phosphomonoesters will contain 18O. The 18O content of the phosphomonoesters was measured following alkaline phosphatase treatment and conversion of the inorganic phosphate to a volatile derivative for gas chromatography/mass spectrometry. Inositol cyclic phosphates were found in the phospholipase C cleavage products of all three phosphoinositides, but the ratio of cyclic to noncyclic product was found to decrease in the order phosphatidylinositol greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol 4,5-bisphosphate. The formation of myo-inositol 1,2(cyclic)-4-bisphosphate was further substantiated by anion-exchange HPLC of the water-soluble products of [32P]phosphatidylinositol 4-phosphate metabolism by phospholipase C. Two peaks were detected one of which, on acid treatment, incorporated 18O from H2 18O into phosphate groups, consistent with this peak containing the cyclic phosphate product. These results suggest that polyphosphoinositide breakdown in stimulated cells may occur via a cyclic phosphate intermediate, as has been described for phosphatidylinositol. These cyclic phosphates contain a reactive bond that may play a role in phosphoinositide-derived signal transduction.
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PMID:Inositol cyclic phosphates are produced by cleavage of phosphatidylphosphoinositols (polyphosphoinositides) with purified sheep seminal vesicle phospholipase C enzymes. 298 59

5-Methyltryptamine, through a GTP-dependent mechanism, stimulated breakdown of endogenous [3H]inositol-labeled phosphoinositides in membranes prepared from blowfly salivary gland homogenates through a phospholipase C exhibiting a pH optimum of approximately 7.0. Unlabeled membranes, prepared from salivary gland homogenates, hydrolyzed exogenous [3H]phosphatidylinositol 4,5-bisphosphate substrate with generation of labeled inositol phosphates. Inositol trisphosphate formation was increased approximately 200% by 10 microM guanosine 5'-(O-thio)-trisphosphate (GTP gamma S) within 30 s. 5-Methyltryptamine, in the presence of 10 microM GTP gamma S, increased the rate of inositol trisphosphate formation by approximately 500% within 30 s. Half-maximal activation of hormone-stimulated breakdown of exogenous substrate required approximately 0.05 microM GTP gamma S. [3H]Phosphatidylinositol was also hydrolyzed during incubation with membranes, resulting in the generation of inositol, glycerol phosphoinositol, and inositol monophosphate. Formation of inositol monophosphate was stimulated approximately 30% by 10 microM GTP gamma S and 10 microM 5-methyltryptamine. Neither inositol nor glycerol phosphoinositol formation was affected by hormone. These results indicate that in a cell-free system from blowfly salivary glands, 5-methyltryptamine, through a GTP-dependent mechanism, directly activates a phospholipase C which mediates phosphoinositide hydrolysis.
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PMID:5-Methyltryptamine stimulates phospholipase C-mediated breakdown of exogenous phosphoinositides by blowfly salivary gland membranes. 299 46


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