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 microinjected a mAb specifically directed to phosphatidylinositol 4,5-bisphosphate (PIP2) into one blastomere of two-cell stage Xenopus laevis embryos. This antibody binds to endogenous PIP2 and reduces its rate of hydrolysis by phospholipase C. Antibody-injected blastomeres undergo partial or complete arrest of the cell cycle whereas the uninjected sister blastomeres divided normally. Since PIP2 hydrolysis normally produces diacylglycerol (DG) and inositol 1,4,5-triphosphate (Ins[1,4,5]P3), we attempted to measure changes in the levels of DG following stimulation of PIP2 hydrolysis in antibody-injected oocytes. The total amount of DG in antibody-injected oocytes was significantly reduced compared to that of water-injected ones following stimulation by either acetylcholine or progesterone indicating that the antibody does indeed suppress PIP2 hydrolysis. We also found that the PIP2 antibodies greatly reduced the amount of intracellular Ca2+ released in the egg cortex during egg activation. As an indirect test for Ins(1,4,5)P3 involvement in the cell cycle we injected heparin which competes with Ins(1,4,5)P3 for binding to its receptor, and thus inhibits Ins(1,4,5)P3-induced Ca2+ release. Microinjection of heparin into one blastomere of the two-cell stage embryo caused partial or complete arrest of the cell cycle depending upon the concentration of heparin injected. We further investigated the effect of reducing any [Ca2+]i gradients by microinjecting dibromo-BAPTA into the blastomere. Dibromo-BAPTA injection completely blocked mitotic cell division when a final concentration of 1.5 mM was used. These results suggest that PIP2 turnover as well as second messenger activity influence cell cycle duration during embryonic cell division in frogs.
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PMID:Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability, or Ca2+ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres. 130 10

In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P3 affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins but with no Ins(1,4,5)P3 metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the delta-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P3-recognizing proteins hitherto examined. Ins(1,4,5)P3 at concentrations greater than 1 microM strongly inhibited 85-kDa phospholipase C delta activity, without changing its dependence on the concentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of which is the delta isozyme of phospholipase C.
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PMID:Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol. 131 9

[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

Recent studies have indicated two major mechanisms for the release of arachidonic acid (20:4) from membrane phospholipids: 1) activation of phospholipase A2 and 2) stimulated hydrolysis of poly-phosphoinositides (PI) and diacylglycerols (DG) through phospholipase C and diacylglycerol lipase, respectively. In mammalian brain both mechanisms seem to be operable, although the relative contributions by these two pathways have not been carefully assessed. In this study three experimental protocols were used to examine 20:4 release in brain due to ischemia and agonist stimulation, as well as the metabolic relationship between this release and the increase in diacylglycerols, lysophospholipids, and inositol phosphates. The preferential release of arachidonic acid during the initial phase after decapitation was attributed mainly to the sequential hydrolysis of poly-PI to DG. During the second phase, the release of 20:4 along with other free fatty acids (FFA) correlated well with the increase in labeled lysophospholipids, suggesting the involvement of phospholipase A2. Diacylglycerols in brain are enriched in 18:0 and 20:4. Decapitation induced a rapid increase in the level of DG, which remained elevated during the 30 min period under study. Between 5 sec and 5 min, the increase in FFA lagged behind that of DG. The parallel increases in 18:0 and 20:4 in the FFA pool further support the notion that, during the early phase, 20:4 could be derived from the sequential hydrolysis of poly-PI and DG. Decapitation also induced a sequential appearance of Ins(1,4,5)P3, Ins(1,4)P2, and Ins(4)P, which peaked at 30 sec, 1 min, and 2 min, respectively. The level of 20:4 in brain was also examined with respect to poly-PI turnover due to stimulation by cholinergic agonists. Administration of pilocarpine to lithium-treated mice resulted in increased accumulation of labeled inositol monophosphate (IP1) compared to the amount in controls receiving lithium alone, as well as a less obvious increase in 20:4. Both pilocarpine-mediated increases (IP1 and 20:4) could be blocked by atropine. These results point to the presence of an active mechanism for poly-PI turnover and for the recycling of 20:4 in brain.
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PMID:Contributions to arachidonic acid release in mouse cerebrum by the phosphoinositide-phospholipase C and phospholipase A2 pathways. 132 24

The human monocytic cell line U937 possesses two classes of the IgG Fc receptor (Fc gamma R), a high-affinity 72-kDa Fc gamma R (Fc gamma RI) and a low-affinity 40-kDa Fc gamma R (Fc gamma RII). Cross-linking of either class of Fc gamma R in U937 cells elicits an increase in the concentration of free intracellular Ca2+. A rapid rise in the concentration of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and of several other inositol phosphates derived from Ins-1,4,5-P3 was observed after cross-linking of Fc gamma Rs in U937 cells. This result suggests that Ins-1,4,5-P3, generated by the action of phospholipase C (PLC), acts as a second messenger by which Fc gamma Rs mobilize intracellular Ca2+ in U937 cells. The mechanism by which the cross-linking of Fc gamma Rs triggers activation of PLC was studied. Cross-linking of Fc gamma RI or Fc gamma RII resulted in a rapid and transient phosphorylation of PLC-gamma 1 on tyrosine residues. It has previously been shown that phosphorylation of PLC-gamma 1 on tyrosine residues activates its enzymatic activity in cells. Prior incubation of U937 cells with a protein tyrosine kinase inhibitor, herbimycin A, prevented the tyrosine phosphorylation of PLC-gamma 1 and the hydrolysis of phosphatidylinositol 4,5-bisphosphate induced by the cross-linking of Fc gamma Rs. Thus, Fc gamma RI and Fc gamma RII appear to be functionally coupled to a nonreceptor tyrosine kinase that phosphorylates PLC-gamma 1 after receptor cross-linking, thereby causing activation of PLC-gamma 1.
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PMID:Tyrosine phosphorylation of phospholipase C-gamma 1 induced by cross-linking of the high-affinity or low-affinity Fc receptor for IgG in U937 cells. 137 7

The effects of quisqualate and kainate on synaptosomal inositol phosphate (InsP) labelling, 45Ca influx and intrasynaptosomal free calcium ([Ca2+]i) were investigated. Each agonist caused a concentration-dependent increase in both [Ca2+]i and InsP labelling: quisqualate, however, produced significantly larger responses in both parameters and at lower EC50 values. Neither quisqualate or kainate significantly affected 45Ca influx into synaptosomes, indicating that the observed increases in [Ca2+]i were due to mobilisation from intracellular stores. The concentration-dependent increases in [Ca2+]i promoted by quisqualate and kainate were monophasic, whereas the increases in InsP formation fitted well to a biphasic curve. The EC50 values suggest that both kainate and quisqualate initially mobilise calcium from inositol 1,4,5-trisphosphate (Ins 1,4,5-P3)-sensitive stores and that the resultant increases in [Ca2+]i will, above a certain threshold, promote further increases in InsP production by stimulation of Ca(2+)-dependent phospholipase C. When saturating concentrations of kainate and quisqualate were used in combination, the effects on both InsP labelling and [Ca2+]i were not additive but were slightly higher than those produced by kainate alone: combined administration of the two agonists had no effect on 45Ca influx. These results suggest that kainate acts as a partial agonist at the presynaptic quisqualate metabotropic glutamatergic receptor.
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PMID:Kainate and quisqualate effects on rat presynaptic cortical receptors are metabotropic and non-additive. 165 93

1. Phosphatidylinositol 4-phosphate (PtdIns4P) is degraded by isolated membranes from Xenopus laevis oocytes. 2. Incubation of [4-32P]PtdIns4P with membranes yields only radioactive inorganic phosphate, indicating the presence of a phosphomonoesterase. 3. Membranes hydrolyze Ptd[2-3H]Ins4P to produce mainly Ptd[2-3H]Ins in the lipid phase. In this incubation [3H]inositol and inositol monophosphate appear in the water phase. 4. Membrane incubations of Ptd[2-3H]Ins4P carried out in the presence of excess non-radioactive Ins(1,4)P2 allows the trapping of small amounts of [3H]Ins(1,4)P2. These results demonstrate the presence of a phospholipase C. 5. Testing several phosphorylated analogs, it is determined that fructose 1,6-bisphosphate and alpha-glycerophosphate are potent inhibitors of the oocyte PtdIns4P phosphomonoesterase.
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PMID:The hydrolysis of phosphatidylinositol 4-phosphate in membranes of Xenopus laevis oocytes: characteristics of a phosphomonoesterase. 166 8

Rat aortic smooth muscle rings without endothelial cells were subjected to alpha 1-adrenoceptor stimulation. We measured the contractile state of the smooth muscle cells and the formation of inositol phosphates (InsPs) on receptor stimulation. Using different extracellular calcium-containing solutions (2.5 mM, 0.1 mM and Ca(2+)-free) enabled us to discriminate three contractile phases after noradrenaline (10(-5) M) stimulation: an initial fast contraction (15 s) and a fast and slow component of the sustained contraction, which was established 10 min after stimulation. Under normal calcium conditions in the presence of 10 mM LiCl the formation of Ins(1,4,5)P3 was increased predominantly after stimulation, while the formation of Ins(1,3,4)P3, Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4 and InsP5/InsP6 was also stimulated. The cAMP-inducing agent forskolin (0.5 microM) induced a relaxation of the basal tone and increased the level of the InsP4 isomers. The noradrenaline-induced contractile responses as well as the formation of InsP fractions mentioned were inhibited by forskolin. Further an increase in the formation of phosphatidylinositol bisphosphate was observed. It is concluded that in rat aorta InsPs and in particular Ins(1,4,5)P3 is involved in the different contractile phases caused by alpha 1-adrenoceptor stimulation. The relaxation induced by forskolin under these circumstances could be explained by an interaction of forskolin, most likely via the formation of cAMP, with InsPs formation at the level of phospholipase C activation.
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PMID:Inositol phosphates formed in rat aorta after alpha 1-adrenoceptor stimulation are inhibited by forskolin. 168 Jul 20

Formation of inositol polyphosphates has been characterized in cultured bovine adrenal chromaffin cells in terms of calcium dependency and isomers of inositol polyphosphates. There are two distinct pathways of generation of InsP3. Stimulants such as high K+ induce InsP3 accumulation by a calcium uptake-dependent mechanism. Stimulants such as Ang II induce InsP3 accumulation by a calcium uptake-independent mechanism. Both mechanisms are involved in nicotinic stimulation. These results suggest that calcium entry as well as receptor-mediated mechanisms play a significant role in phosphoinositides hydrolysis through phospholipase C in adrenal chromaffin cells. Nicotinic receptor stimulation induces a rapid and transient increase in Ins(1,4,5)P3 accumulation followed by a slower accumulation of Ins(1,3,4)P3. Moreover, nicotine induces a large and rapid increase in Ins(1,3,4,5,6)P5 accumulation with an extent and time course similar to Ins(1,4,5)P3, which peaks at 15 sec after stimulation. Nicotine also induced Ins(1,3,4,5)P4 and InsP6 accumulation with a slower time course and a lesser magnitude than Ins(1,3,4,5,6)P5. These results indicate that adrenal chromaffin cells possess fine regulation of inositol polyphosphates metabolism and that inositol polyphosphates are involved with the control of cellular function in these cells.
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PMID:Formation of inositol polyphosphates in cultured adrenal chromaffin cells. 175 3

Although stimulated [3H] inositol phosphate turnover has been demonstrated in isolated, perfused [3H] inositol prelabelled rat hearts, there is still no information regarding Ins (1,4,5)P3 levels in intact cardiac muscle. Using a D-myo-Ins(1,4,5)P3 assay system, Ins(1,4,5)P3 levels were determined in isolated perfused rats hearts during ischaemia, reperfusion and alpha 1-adrenergic stimulation via noradrenaline (3 x 10(-5) M). Control hearts contained +/- 674 pmols Ins(1,4,5)P3/g dry heart weight. Myocardial Ins(1,4,5)P3 levels were significantly decreased (+/- 389 pmols/g dry heart weight) after exposure to 20 mins of normothermic ischaemic cardiac arrest (NICA). Reperfusion produced a marked increase in Ins(1,4,5,)P3 levels (+/- 1,115 pmols/g dry heart weight) after only 30 s. Noradrenaline caused a 3-4 fold increase in tissue Ins(1,4,5)P3 levels within 30 s. After 20 mins stimulation with noradrenaline, the Ins(1,4,5)P3 levels were still significantly elevated. The rise in tissue Ins(1,4,5)P3 levels during reperfusion as well as during noradrenaline administration was counteracted by neomycin (0.5 x 10(-3) M), an inhibitor of phosphoinositidase specific phospholipase C. In both events neomycin restored the Ins(1,4,5)P3 levels to control values. For correlation of tissue Ins(1,4,5)P3 levels with mechanical events, noradrenaline (3 x 10(-5) M), in the presence of 10 mM LiCl, 10(-7) M propranolol and 10(-7) M atropine, was administered to isolated perfused rat hearts and the mechanical performance recorded over a period of 20 mins. Noradrenaline caused a significant increase in peak systolic pressure and work performance which was maintained for at least 10 mins, suggesting that the positive inotropic effects of noradrenaline may be provoked by Ins(1,4,5)P3. Furthermore, the finding that 20 min NICA followed by 30 s reperfusion causes an immediate significant increase in Ins(1,4,5)P3 content suggests a role for the phosphatidylinositol pathway in the intracellular Ca2+ overloading, characteristic of ischaemia-reperfusion.
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PMID:Increased myocardial inositol trisphosphate levels during alpha 1-adrenergic stimulation and reperfusion of ischaemic rat heart. 179 34


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