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)

Interactions between ATP and adenosine on the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and mobilization of intracellular calcium were investigated in the smooth muscle cell line DDT1 MF-2. Activation of adenosine A1 receptors with adenosine or cyclopentyladenosine (CPA) or of nucleotide receptors with ATP increased both Ins(1,4,5)P3 formation and intracellular calcium concentrations. The A1 receptor-induced Ins(1,4,5)P3 formation (EC50 10 nM) was antagonized by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and by pretreatment of the cells with pertussis toxin (PTX). ATP-stimulated Ins(1,4,5)P3 formation (EC50 21 microM) was attenuated, but still present, after PTX treatment. ATP and CPA had supraadditive effects on Ins(1,4,5)P3 accumulation and CPA increased ATP-induced Ins(1,4,5)P3 accumulation in a concentration-dependent manner with an EC50 of 3 nM, a concentration which per se had little or no effect on Ins(1,4,5)P3 accumulation. ATP (EC50 4 microM) and CPA (EC50 4 nM) both increased intracellular calcium levels. The effect of ATP was partially sensitive to PTX treatment, whereas the effect of CPA was blocked both by PTX and by DPCPX. Concentrations of ATP and CPA that by themselves were insufficient to raise intracellular calcium were able to do so when combined. The synergy between ATP and CPA on the mobilization of intracellular calcium was abolished after treatment of cells with PTX or when DPCPX was included in the experiment. Since ATP was metabolized by ecto-enzymes to ADP, AMP, and adenosine, we also examined whether adenosine formed from ATP could enhance the ATP effects on Ins(1,4,5)P3 accumulation. Indeed, the addition of the A1 receptor antagonist DPCPX or removal of endogenous adenosine by inclusion of adenosine deaminase in the experimental medium significantly attenuated the ATP response, and the two treatments did not have additive effects. The present study thus demonstrates that in a clonal cell line two types of receptors increase phospholipase C activity, but via different pathways; nucleotide receptors appeared to act via partially PTX-insensitive, and A1 receptors via PTX-sensitive G-proteins. ATP and CPA are not only able per se to induce formation of Ins(1,4,5)P3 and mobilize intracellular calcium, but they also act synergistically. Finally, it is demonstrated that endogenous adenosine, possibly formed from the rapid breakdown of ATP, can significantly enhance some ATP effects.
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PMID:ATP and its metabolite adenosine act synergistically to mobilize intracellular calcium via the formation of inositol 1,4,5-trisphosphate in a smooth muscle cell line. 132 90

PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.
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PMID:Characterization of ATP receptor which mediates norepinephrine release in PC12 cells. 132 38

We investigated the turnover of polyphosphoinositides in bovine retinal microvascular endothelial cells and rat astrocytes cultured in the presence of high ambient concentrations of glucose in order to study the possible involvement of this pathway in the pathogenesis of diabetic retinopathy. a 35-45% decrease in the amount of 32P incorporated into phosphatidylinositol(4)phosphate (PIP) and phosphatidyl-inositol(4,5)biphosphate (PIP2) occurred in rat astrocytes but not bovine retinal endothelial cells grown for 14 +/- 3 days in a medium with an elevated (28 mM) glucose concentration. Incorporation of 32P into phosphatidylinositol, phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine was not altered by these conditions. A 39-45% decrease in 32P incorporated into PIP/PIP2 was also found in rat astrocytes grown in 28 mM glucose which were detergent solubilized and incubated with [32P]ATP. Exposure to elevated concentrations of glucose decreased the amount of PIP/PIP2 cleaved by ionomycin or fluoroaluminate treatment, but did not disturb phospholipase C activity. Thus, the lower level of PIP/PIP2, induced by exposure to elevated concentrations of glucose, appears due to changes in phospholipid substrate levels, or polyphosphoinositide kinase activity, rather than a decrease in ATP levels or phospholipase C activity. These results suggest that high ambient glucose levels alter second-messenger generation by astrocytes. In turn, cellular interactions dependent upon these second messengers and important for maintenance of normal microvessel function in the retina may be disrupted.
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PMID:Effect of elevated ambient glucose upon polyphosphoinositide turnover in bovine retinal endothelial cells and rat astrocytes. 132 21

Extracellular ATP has been shown to induce intracellular Ca2+ mobilization and adenylate cyclase inhibition via P2 purinoceptors in several species of cells. Now we found that in calf vascular smooth muscle cells the addition of ATP to the medium did not induce inhibition but stimulation of cyclic AMP accumulation, in addition to stimulation of inositol phosphate production. Adenosine and AMP also induced cyclic AMP accumulation but their efficacy was much less than that of ATP. The ATP action was not influenced by the presence of either adenosine deaminase or of an ATP regenerating system, whereas the AMP action was increased by the regenerating system. The results indicate that the cyclic AMP accumulation by ATP is due to ATP itself but neither to adenosine nor to AMP, both of which are produced from ATP. ATP receptor coupled to the cyclic AMP generation was shown to be different from that coupled to phospholipase C based on the difference in the potency order of the receptor agonists and in the sensitivity of P2 receptor agonists to 8-cyclopentyl-1,3-dipropylxanthine (CPX)- and suramin-induced antagonism. We conclude that in the aortic smooth muscle cells a novel P2-type receptor directly coupled to adenylate cyclase activation exists in addition to the previously known P2 receptor linked to phospholipase C activation.
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PMID:P2 purinoceptor-mediated cyclic AMP accumulation in bovine vascular smooth muscle cells. 133 Jun 37

Macrophages express two distinct types of nucleotide (P2 purinergic) receptors for extracellular ATP: one type induces a Ca(2+)-mobilizing response via the activation of phosphatidylinositol-phospholipase C (PI-PLC) while the second type induces the rapid formation of nonselective pores which are permeated by ions and small (< 1 kDa) organic molecules. We have confirmed the presence of these two ATP receptor types in the BAC1.2F5 murine macrophage cell line and have identified 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) as a selective and potent agonist for the so-called P2z or pore-forming ATP receptor type. Several lines of evidence indicated that occupation of these P2z receptors is also accompanied by a rapid and large increase in the activity of a phosphatidylcholine-selective phospholipase D (PLD) effector enzyme. In cells metabolically labeled with [3H]oleic acid or [3H]glycerol and stimulated in the presence of ethanol, ATP and BzATP induced a severalfold increase in the rate and extent of [3H]phosphatidylethanol (PEt) accumulation. These responses were stimulated only by ATP, BzATP, and ATP gamma S (adenosine 5'-O-(3-thiotriphosphate) with the rank order of potency: BzATP >> ATP > ATP gamma A; there was no response to other adenine nucleotides or to non-adenine nucleotides. Significantly, the ability of P2z receptor agonists to stimulate this PLD activity was not dependent on the presence of extracellular [Ca2+] or elevation of cytosolic [Ca2+]. The inability of ionomycin, gramicidin, digitonin, UTP, platelet-activating factor, or phorbol ester to quantitatively mimic these nucleotide effects suggested that activation of this PLD by P2z receptor agonists was not a secondary response due to: 1) enhanced Ca2+ influx; 2) membrane depolarization; 3) nonselective permeabilization of the plasma membrane; 4) stimulation of Ca(2+)-mobilizing ATP receptors; 5) stimulation of a primary PI-PLC pathway; or 6) activation of protein kinase C. These findings suggest that activation of a novel PLD-based signaling pathway may play an important role in the modulation of macrophage function by pore-forming P2z receptors for extracellular ATP.
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PMID:A novel pathway for the activation of phospholipase D by P2z purinergic receptors in BAC1.2F5 macrophages. 133 Oct 96

The vasoactive factors thrombin, bradykinin (BK), and ATP are released in response to tissue damage and inflammation and act on endothelium to modulate vascular perfusion. We have investigated the second messenger response of endothelium activated by these agonists and, in particular, the mechanism of desensitization to BK. Fura-2 fluorescence ratio imaging of calf pulmonary artery endothelial cells (CPAE) revealed 5- to 10-fold increases on intracellular Ca (Cai) in response to these agents. Maximal doses caused Cai to increase from 52 to 248 nM (thrombin), 556 nM (BK), and 643 nM (ATP). Agonists elicited a rapid (within 30 s) increase of Cai due to release of Ca from intracellular stores followed by a secondary elevation of Cai dependent on entry of external Ca. The temporal characteristics of the Cai responses to all agonists were heterogeneous from cell to cell, and, interestingly, repeated stimulation gave identical signature responses from individual cells, although the amplitude of the Cai response decreased to thrombin and especially bradykinin but not for ATP. This decrease was agonist specific because ATP elicited large increases of Cai after thrombin or BK desensitization. Maximal desensitization was obtained with BK applied for 5-10 min followed by a rest of < 10 min before restimulation. Although desensitization primarily reduced the elevation of Cai due to the release of the internal store, entry of extracellular Ca was also reduced. Cells responded heterogeneously to desensitization in that those with prominent extracellular Ca entry responded most strongly upon a second stimulation with BK. Because desensitized cells still responded to ATP with an increase of Cai, the desensitization was controlled at a step prior to the activation of phospholipase C. Desensitization occurred by a reduction of BK receptor number; a 10-min BK pretreatment reduced [3H]BK binding to receptors by 70% (from 14,600 receptors/cell, Km = 5 nM, to 5,300). As surface receptor numbers decreased, internalized receptors increased as assayed by an acetic acid wash. The time course of the receptor internalization was similar to the decrease in Cai response to BK. We conclude that the vasoactive agonists thrombin, BK, and ATP increase the second messenger Cai in endothelial cells and that a desensitized Cai response occurs with BK, but not with ATP, due to downregulation and endocytosis of the BK receptor.
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PMID:Calcium signaling in endothelia: cellular heterogeneity and receptor internalization. 133 90

The hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) by rat sciatic nerve cytosolic phosphoinositidase C [phosphoinositide-specific phospholipase C (PIC)] was studied at neutral pH and at ionic concentrations that approximate intracellular conditions. The principal water-soluble product formed was shown to be inositol trisphosphate by anion exchange chromatography. The maximum hydrolysis rate (2.5 nmol/min/mg protein) was achieved at less than 100 nM Ca2+. Hydrolysis was markedly increased to 15 nmol/min/mg protein by inclusion of K+ in the reaction mixture. In the presence of 200 mM K+, the optimum Ca2+ was increased to approximately 600 nM. Higher Ca2+ concentrations progressively inhibited PIP2 hydrolysis. Mg2+ also inhibited the reaction, but the presence of equimolar amounts of ATP and Mg2+ had no effect. Appreciable degradation of phosphatidylinositol-4-phosphate (PIP) also occurred in the nanomolar Ca2+ range, whereas breakdown of phosphatidylinositol (PI) required millimolar Ca2+. The presence of PIP but not PI inhibited PIP2 hydrolysis. Upon subcellular fractionation of nerve, more than 50% of recovered PIC activity was in the cytosol and about 20% was located in a myelin-enriched fraction. Using PIP2 as substrate, PIC activities in nerves from normal and streptozotocin-induced diabetic animals were not different. However, the myelin-associated enzyme from diabetic animals was more labile to freezing and thawing.
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PMID:Activity and distribution of phosphoinositidase C in rat sciatic nerve. 133 36

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.
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PMID:Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production. 133 83

In rat glioma C6 cells, extracellular ATP stimulated phosphoinositide (PI) hydrolysis in concentration- and time-dependent manners with a median effective dose value of 60 microM. The maximal response was attained at 300 microM ATP. Of adenine nucleotides, ATP and adenosine 5'-O-(3-thiotriphosphate) were most effective, while adenosine, AMP and beta,gamma-methylene ATP were ineffective. Similar results were obtained in cultured rat astrocytes. The stimulatory effects of ATP and ADP were negated by removal of external Ca++ in C6 cells. ATP at 300 microM induced an elevation of intracellular Ca++ concentration in 1-[2-(5-carboxyoxazol-2-yl)-6-amino-benzofuran-5-oxy]-2-(2'-amino- 5'- methylphenoxy)-ethane-N,N,N',N' acid-loaded C6 cells. This response was not blocked by nifedipine (10 microM) and verapamil (10 microM). A Ca++ ionophore A23187 (10 microM) stimulated PI hydrolysis in C6 cells. The responses to ATP (300 microM) and A23187 (10 microM) were additive. In digitonin-permeabilized C6 cells, Ca++ at the concentration of 100 microM evoked PI hydrolysis, and ATP alone did not affect the Ca++ dependence. GTP gamma S (100 microM) stimulated the PI hydrolysis at a range of 0.1 to 10 microM Ca++, and ATP enhanced the GTP gamma S response in the permeabilized cells. These results suggest that activation of P2-purinergic receptors by ATP causes phospholipase C to be activated by subthreshold concentrations of Ca++ via GTP-binding proteins, resulting in an activation of the enzyme in response to stimulated Ca++ influx.
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PMID:Mechanism of extracellular ATP-stimulated phosphoinositide hydrolysis in rat glioma C6 cells. 133 61

Phosphatidylinositol (PtdIns) kinase and phosphatidylinositol 4-phosphate (PtdIns4P) kinase have been studied in a purified sarcolemmal fraction isolated from rat heart. Both enzymes were Mg(2+)-dependent and their activities were maximal at 2.5 mM Mg2+ and pH 7.5. Kinetic analysis of endogenous substrate phosphorylation by ATP showed that the apparent Km and Vmax values for PtdIns kinase were 292 +/- 17 microM and 1390 +/- 80 pmol.mg-1.min-1, respectively, while the apparent Km and Vmax values for PtdIns4P kinase were 398 +/- 25 microM and 382 +/- 24 pmol.mg-1.min-1. Under normal conditions, the activity of PtdIns4P kinase was lower than that of PtdIns kinase; however, the former activity increased several fold in the presence of PtdIns4P as an exogenous substrate. The enzymatic synthesis of intramembranal PtdIns4P and phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P2) was maximally enhanced by 0.1% Triton X-100 and inhibited by micromolar concentrations of Ca2+. Inhibition of PtdIns and PtdIns4P kinase showed IC50 values for Ca2+ of 20 and 6 microM, respectively, and was independent of either Ca(2+)-induced activation of phospholipase C and polyphosphoinositide monophosphoesterases or low ATP concentrations. The results indicate that purified rat heart sarcolemmal membranes contain a very active phosphoinositide phosphorylation system which is regulated by micromolar levels of Ca2+. The Ca2+ effect may contribute to the feedback inhibition of the receptor-activated formation of inositol 1,4,5-trisphosphate.
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PMID:Phosphoinositide kinases in rat heart sarcolemma: biochemical properties and regulation by calcium. 133 11


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