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)

Stimulation of phagocytic cells with micromolar concentrations of extracellular ATP primes the production of toxic oxygen metabolites in response to chemoattractants and independently activates a secretory response in vitro. It is hypothesized that extracellular ATP derived from platelet storage granules and damaged endothelium at sites of localized tissue damage or infection may potentiate the pro-inflammatory effects of phagocytic cells in vivo. ATP-dependent functional responses in the phagocyte appear to be due to stimulation of putative P2 purinoreceptors that are coupled to the activation of a phospholipase C via a pertussis toxin-sensitive G-protein. The existence in nature of at least four subtypes of P2 purinoreceptors has been proposed based on the rank order of potency of nucleotide analogs of ATP studied in a variety of cell types. However, no studies involving the structural identification and characterization of the putative receptors have been reported. We have used the Xenopus oocyte expression system to demonstrate acquired adenosine 5'-(thio) triphosphate (ATP gamma S) responsiveness in oocytes injected with mRNA from the promyelocytic leukemia cell line HL60 by measuring the accelerated efflux of intracellular calcium. Two peaks of ATP gamma S responsiveness (Peak I and Peak II) were detected in sucrose gradient fractionated RNA that corresponded to transcript sizes of 4 and 6 kilobases and that were distinct from a third peak previously shown to be enriched in formyl peptide chemoattractant receptor activity. Peak I and Peak II RNA endowed receptor activity in the oocyte that was pharmacologically indistinguishable: ADP and AMP were inactive whereas UTP and ITP exhibited activity that was similar in potency to that of ATP gamma S. Both Peak I and Peak II ATP gamma S-dependent activity was inhibited by pertussis toxin. These data strongly support the concept of phagocytic cell receptors for extracellular nucleotide triphosphates whose ligand specificity is distinct from all other previously described P2 purinoreceptor subtypes, with the exception of the P2 receptor described in Ehrlich ascites tumor cells, by virtue of the ineffectiveness of ADP as a stimulus. These receptors are most likely composed of a single polypeptide chain that can be expressed in the Xenopus oocyte in a functional form regulated by a pertussis toxin-sensitive G-protein.
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PMID:Characterization of phagocyte P2 nucleotide receptors expressed in Xenopus oocytes. 169 46

Extracellular ATP activates two distinct types of P2 purinoreceptor-mediated signaling pathways in macrophages, 1) the rapid formation of nonselective pores/channels in the plasma membrane and 2) a guanine nucleotide-binding protein-dependent stimulation of phosphotidylinositol-specific phospholipase C, with subsequent mobilization of intracellular Ca2+. Several studies have suggested that the pore-forming, or P2z, purinoreceptor may be involved in the cytolytic effects of ATP on macrophages and other cell types. We have identified 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) and UTP as selective agonists for the P2z purinoreceptor and Ca(2+)-mobilizing nucleotide receptor, respectively, in BAC1.2F5 macrophages. In this paper we demonstrate that BzATP and ATP (but not UTP) activate membrane depolarization in BAC1.2F5 cells and also stimulate appropriate electrophysiological responses, consistent with the expression of the P2z purinoreceptor, in Xenopus oocytes injected with poly(A)+ RNA derived from BAC1.2F5 cells. Micromolar concentrations of BzATP or millimolar concentrations of ATP induced a sustained increase in the membrane holding current in these voltage-clamped oocytes. This response was significantly potentiated in the absence of extracellular divalent cations, consistent with the specificity of the P2z purinoreceptor for tetrabasic nucleotides. The sustained currents induced by BzATP or ATP were distinct from the transient and/or oscillating increases in Ca(2+)-dependent Cl- currents that were stimulated by UTP but not BzATP. UTP-stimulated transient currents and nucleotide-dependent increases in aequorin luminescence in poly(A)+ RNA-injected oocytes were independent of extracellular Ca2+ and were correlated with the mobilization of intracellular Ca2+ stores. Sucrose fractionation of the poly(A)+ RNA from BAC1.2F5 cells resulted in the enrichment of mRNA species encoding the components of the P2z purinoreceptor, as well as the Ca(2+)-mobilizing nucleotide receptor, in fractions containing 2.5-4.0-kilobase species.
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PMID:Expression of the pore-forming P2Z purinoreceptor in Xenopus oocytes injected with poly(A)+ RNA from murine macrophages. 768 70

The role of ATP and ADP as intercellular mediators is now well established. The presence of the nucleotides in extracellular fluids can result from several mechanisms: cell lysis, selective permeabilization of the plasma membrane and exocytosis of secretory vesicles, such as platelet dense bodies. Extracellular adenine nucleotides are rapidly degraded by ectonucleotidases expressed inter alia on the surface of endothelial cells. They act on cells via the family of P2 receptors which encompasses more than 5 subtypes, some of which have been cloned recently. The P2T, P2U and P2Y receptors belong to the superfamily of receptors coupled to G proteins, whereas the P2X receptor is a cation channel and the P2Z receptor a non-selective pore. ATP and ADP stimulate the endothelial production of prostacyclin (PGI2) and nitric oxide (NO), two vasodilators and inhibitors of platelet aggregation, via an increase in cytosolic Ca2+. This action of adenine nucleotides is believed to limit the extent of intravascular platelet aggregation and to help localize thrombus formation to areas of endothelial damage. The endothelial response to nucleotides is mediated by at least two distinct subtypes of P2 receptors, P2Y and P2U, both coupled to phospholipase C.
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PMID:Involvement of multiple receptors in the actions of extracellular ATP: the example of vascular endothelial cells. 775 78

P2Y purinergic receptors previously have been shown to couple either to activation of phospholipase C through a pertussis toxin-insensitive mechanism or to inhibition of adenylyl cyclase through pertussis toxin-sensitive members of the G1 family of G proteins. These and other pharmacological data strongly suggest that multiple P2Y purinergic receptors exist. Webb et al. [FEBS Lett. 324:219-225 (1993)] cloned a cDNA that, when expressed in frog oocytes, displayed the general pharmacological characteristics of a P2Y purinergic receptor but whose second messenger linkage was not resolved. We have now cloned the meleagrid (turkey) homologue of the previously cloned chick P2Y purinergic receptor and have stably expressed it in a heterologous human cell line (1321N1 astrocytoma cells) to establish its signaling properties. The purinergic receptor agonist 2-methylthio-ATP (2MeSATP) stimulated a marked activation of phospholipase C in 1321N1 cells stably expressing the meleagrid receptor. The order of potency of a series of analogues of ATP and ADP for stimulation of phospholipase C by the receptor expressed in 1321N1 cells [2MeSATP = 2-methylthio-ADP > adenosine 5'-O-(2-thio)diphosphate > ADP > 2-chloro-ATP = adenosine 5'-O-(3-thio)triphosphate > or = ATP > adenylyl-imidodiphosphate > UTP] was similar to that observed for P2Y purinergic receptors in turkey erythrocytes and many other tissues and was markedly different from those of the P2U and P2X purinergic receptor subtypes. Stimulation of inositol lipid hydrolysis by P2Y purinergic agonists was not affected by preincubation of cells with pertussis toxin. In contrast to its marked effects on phospholipase C activity, 2MeSATP caused only a small and variable inhibition of cAMP accumulation. Ribonuclease protection analysis of turkey tissues showed that this P2Y purinergic receptor is most highly expressed in blood and brain. Taken together, these results indicate that a phospholipase-C-activating P2Y purinergic receptor has been cloned and stably expressed in 1321N1 astrocytoma cells.
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PMID:Expression of a cloned P2Y purinergic receptor that couples to phospholipase C. 805 61

Study of P2-purinoceptor subtypes has been difficult due to the lack of potent and selective ligands. With the goal of developing high affinity P2-purinoceptor-selective agonist, we have synthesized a series of analogues of adenine nucleotides modified on the purine ring as chain-extended 2-thioethers or as N6-methyl-substituted compounds. Chemical functionality incorporated in the thioether moiety included cyanoalkyl, nitroaromatic, amino, thiol, cycloalkyl, n-alkyl, and olefinic groups. Apparent affinity of the compounds for P2Y-purinoceptors was established by measurement of P2Y-purinoceptor-promoted phospholipase C activity in turkey erythrocyte membranes and relaxation of carbachol-contracted smooth muscle in three different preparations (guinea pig taenia coli, rabbit aorta, and rabbit mesenteric artery). Activity at P2X-purinoceptors was established by measurement of contraction of rabbit saphenous artery and of the guinea pig vas deferens and urinary bladder. All 11 of the 2-thioethers of ATP stimulated the production of inositol phosphates with K0.5 values of 1.5-770 nM, with an (aminophenyl)ethyl derivative being most potent. Two adenosine diphosphate analogues were equipotent to the corresponding ATP analogues. Adenosine monophosphate analogues were full agonists, although generally 4 orders of magnitude less potent. ATP 2-thioethers displayed pD2 values in the range of 6-8 in smooth muscle assay systems for activity at P2Y-receptors. There was a significant correlation for the 2-thioether compounds between the pK0.5 values for inositol phosphate production and the pD2 values for relaxation mediated via the P2Y-purinoceptors in the guinea pig taenia coli, but not for the vascular P2Y-receptors or for the P2X-receptors. At P2X-receptors, no activity was observed in the rabbit saphenous artery, but variable degrees of activity were observed in the guinea pig vas deferens and bladder depending on distal substituents of the thioether moiety. N6-Methyl-ATP was inactive at P2X-receptors, and approximately equipotent to ATP at taenia coil P2Y-receptors. This suggested that hybrid N6-methyl and 2-thioether ATP derivatives might be potent and selective for certain P2Y-receptors, as was shown for one such derivative, N6-methyl-2-(5-hexenylthio)-ATP.
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PMID:Identification of potent, selective P2Y-purinoceptor agonists: structure-activity relationships for 2-thioether derivatives of adenosine 5'-triphosphate. 825 22

Adenine nucleotides inhibited isoproterenol- and forskolin-stimulated cyclic AMP accumulation in C6-2B rat glioma cells. Inhibition occurred in the presence of a phosphodiesterase inhibitor, and no effect of adenine nucleotides was observed in direct measurements of phosphodiesterase activity in intact cells. Pretreatment of C6-2B glioma cells with pertussis toxin blocked the inhibitory effects of P2Y-purinergic receptor agonists. The pharmacological specificity for a series of ATP and ADP analogs (2-methylthioadenosine 5'-triphosphate > or = 2-methylthioadenosine 5'-diphosphate > adenosine 5'-O-(2-thiodiphosphate) > 2-chloro-adenosine 5'-triphosphate = ADP = adenosine 5'-O-(3-thiotriphosphate) > ATP > UTP) was similar to that expected of a P2Y-purinergic receptor; the P2X-purinergic receptor agonists, alpha,beta-methyleneadenosine 5'-triphosphate and beta,gamma-methylene-adenosine 5'-triphosphate, had no effect. Because activation of phospholipase C occurs in response to P2-purinergic receptor activation in many target tissues, the effects of P2Y-receptor agonists on inositol phosphate accumulation were measured in C6-2B cells. No evidence for P2Y-purinergic receptor-mediated regulation of inositol lipid metabolism was observed under conditions where muscarinic cholinergic receptor activation or AIF4-markedly increased inositol phosphate accumulation. These results suggest that a P2-purinergic receptor subtype with distinct signaling properties exists on C6-2B rat glioma cells. Although this receptor expresses the general pharmacological specificity of a phospholipase C-coupled P2Y-purinergic receptor, it may represent a unique receptor subtype since it inhibits adenylyl cyclase.
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PMID:Identification of a P2Y-purinergic receptor that inhibits adenylyl cyclase. 826 74

The effect of extracellular ATP was studied in PC12 cells, a neurosecretory line that releases ATP. The addition of micromolar concentrations of ATP to PC12 cells evoked a transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i), as measured with the Ca(2+)-sensitive dye fura 2. AMP and adenosine were without effect, ruling out the involvement of P1 receptors in mediating this response. The increase in [Ca2+]i was reduced in calcium-free media and virtually eliminated by the addition of EGTA, suggesting that calcium influx was the primary response initiated by extracellular ATP. Nucleotide triphosphates such as UTP and, to a lesser degree, ITP also evoked an increase in [Ca2+]i while GTP and CTP had little effect. In order to identify the receptor subtype mediating this response, the efficacy of ATP and ATP cogeners was assessed. The rank order potency was ATP > adenosine 5'-[gamma-thio]triphosphate > ADP > 2-methylthioadenosine triphosphate (2-MeSATP) approximately adenosine 5'-[beta-thio]diphosphate >> adenosine 5'-[alpha beta-methylene] triphosphate, adenosine 5'[beta gamma-imido]triphosphate. This profile is not characteristic of either the P2X or the conventional P2Y receptors. The Ca2+ response exhibited desensitization to ATP that was dependent on the extracellular metabolism of ATP. UTP was equally effective in desensitizing the response. ATP, UTP, ITP, and to a much lesser extent 2MeSATP increased inositol phosphate production in a dose-dependent manner, suggesting receptor coupling to phosphatidylinositol-specific phospholipase C. These data are consistent with the view that PC12 cells express a class of non-P2Y nucleotide receptors (P2N) that mediate calcium influx and the accumulation of inositol phosphates.
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PMID:Intracellular signalling by nucleotide receptors in PC12 pheochromocytoma cells. 843 8

1. The human P2U-purinoceptor was stably expressed in 1321N1 human astrocytoma cells and the pharmacological selectivity of the expressed receptor was studied by measurement of inositol lipid hydrolysis. 2. High basal levels of inositol phosphates occurred in P2U-purinoceptor-expressing cells. This phenomenon was shown to be due to release of large amounts of ATP from 1321N1 cells, and could be circumvented by adoption of an assay protocol that did not involve medium changes. 3. UTP, ATP and ATP gamma S were full and potent agonists for activation of phospholipase C with EC50 values of 140 nM, 230 nM, and 1.72 microM, respectively. 5BrUTP, 2C1ATP and 8BrATP were also full agonists although less potent than their natural congeners. Little or no effect was observed with the selective P2Y-, P2X-, and P2T-purinoceptor agonists, 2MeSATP, alpha,beta-MeATP, and 2MeSADP, respectively. 4. Diadenosine tetraphosphate, Ap4A, was a surprisingly potent agonist at the expressed P2U-purinoceptor with an EC50 (720 nM) in the range of the most potent P2U-purinoceptor agonists. Ap4A may be a physiologically important activator of P2U-purinoceptors.
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PMID:Pharmacological selectivity of the cloned human P2U-purinoceptor: potent activation by diadenosine tetraphosphate. 856 28

Adenosine-5'trisphosphate (ATP) is stored and co-released with various neurotransmitters but it may also act as a fast excitatory neurotransmitter trough the activation of purinoreceptor(s). In this study the effect of ATP on phospholipase C (PLC) degrading labelled PtdIns(4,5)P2 and PtdIns in brain cortex slices, brain homogenate and subcellular fractions was investigated. It was found that ATP added into brain slices activated significantly and specifically PtdIns(4,5)P2 degradation and this process was inhibited by theophylline. Moreover, ATP maintained a higher level of inositol(1,4,5)P3 radioactivity in total water-soluble inositol metabolites. However, ATP added directly for the assay of PLC into brain homogenate or subcellular fractions inhibits phosphoinositide degradation in a receptor-independent manner and suppresses conversion of Ins(1,4,5)P3 into Ins(1,4)P2. Our results indicate that ATP acting extracellularly through a purinergic receptor(s) activates PtdIns(4,5)P2 degradation and release of Ins(1,4,5)P3. ATP acting directly on PLC inhibits in a receptor-independent manner phosphoinositide degradation, and protects against liberation of lipid-derived second messengers.
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PMID:ATP a potent regulator of inositol phospholipids-phospholipase C and lipid mediators in brain cortex. 876 2

Cultured, proliferating microglial cells can be further activated by lipopolysaccharide (LPS) and, thereby, turned into a non-proliferating state. While proliferating cells exhibit only inwardly rectifying potassium channels, non-proliferating cells express, in addition, outwardly rectifying potassium channels. The characteristics of the two channel populations are markedly different. Inward potassium currents inactivate and can be abolished by extracellular Cs+ and Ba2+. Outward potassium currents only slightly inactivate and can be abolished by 4-aminopyridine, quinine and charybdotoxin. An increase in the intracellular free Ca2+ concentration depresses the outward potassium current. ATP or its structural analogues stimulate two types of P2-purinoceptors on microglial cells, a ligand-activated cationic channel (P2x) which produces depolarization and a G protein coupled receptor (P2Y) which produces hyper-polarization via the opening of K+ channels. Both P2X- and P2Y-receptor stimulation may increase the intracellular Ca2+ concentration. In the former case, Ca2+ enters the cells via non-selective cationic channels. In the latter case, Ca2+ may be released from intracellular stores, owing to activation of the enzyme phospholipase C and subsequent generation of inositol 1,4,5-trisphosphate (IP3). It is assumed that neuronal damage leads to efflux of ATP into the extracellular space with subsequent activation of microglia. ATP-induced excessive depolarizations by P2X-purinoceptor stimulation may be counteracted by outwardly rectifying potassium channels and by hyperpolarizing P2Y-purinoceptors in non-proliferating microglia.
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PMID:Molecular mechanisms of microglial activation. B. Voltage- and purinoceptor-operated channels in microglia. 880 85


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