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)

1. A series of chain-extended 2-thioether derivatives of adenosine monophosphate were synthesized and tested as agonists for activation of the phospholipase C-linked P2Y-purinoceptor of turkey erythrocyte membranes, the adenylyl cyclase-linked P2Y-purinoceptor of C6 rat glioma cells, and the cloned human P2U-receptor stably expressed in 1321N1 human astrocytoma cells. 2. Although adenosine monophosphate itself was not an agonist in the two P2Y-purinoceptor test systems, eleven different 2-thioether-substituted adenosine monophosphate analogues were full agonists. The most potent of these agonists, 2-hexylthio AMP, exhibited an EC50 value of 0.2 nM for activation of the C6 cell receptor. This potency was 16,000 fold greater than that of ATP and was only 10 fold less than the potency of 2-hexylthio ATP in the same system. 2-hexylthio adenosine was inactive. 3. Monophosphate analogues that were the most potent activators of the C6 cell P2Y-purinoceptor were also the most potent activators of the turkey erythrocyte P2Y-purinoceptor. However, agonists were in general more potent at the C6 cell receptor, and potency differences varied between 10 fold and 300 fold between the two receptors. 4. Although 2-thioether derivatives of adenosine monophosphate were potent P2Y-purinoceptor agonists no effect of these analogues on the human P2U-purinoceptor were observed. 5. These results support the view that a single monophosphate is sufficient and necessary for full agonist activity at P2Y-purinoceptors, and provide insight for strategies for development of novel P2Y-purinoceptor agonists of high potency and selectivity.
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PMID:Identification of potent P2Y-purinoceptor agonists that are derivatives of adenosine 5'-monophosphate. 886 29

The functional properties of the G protein-coupled P2Y1 receptor were investigated in Xenopus oocytes. Incubation of oocytes expressing either the human or turkey P2Y1 receptor with adenine nucleotide agonists resulted in an increase in Cl- current and activation of a novel cation current with an inwardly rectifying current-voltage relationship. Activation of either the human P2Y2 (P2U-purinergic) or M1 muscarinic receptor expressed in oocytes resulted in an increase in Cl- current similar to that observed in P2Y1 receptor-expressing oocytes but had no effect on cation current. P2 receptor agonists stimulated both the cation current and Cl- current in P2Y1 receptor-expressing oocytes with EC50 values and an order of potency (2-methylthioadenosine diphosphate > 2-methylthioadenosine triphosphate (2MeSATP) > ATP > UTP) that were similar to those previously observed for activation of phospholipase C in 1321N1 human astrocytoma cells stably expressing the human or turkey P2Y1 receptor. The P2Y receptor antagonists suramin and pyridoxal phosphate 6-azophenyl-2'-4'-disulfonic acid both shifted to the right the concentration-response relationship for 2MeSATP for stimulation of oocyte currents. Although injection of oocytes with either GDPbetaS (guanyl-5'-yl thiophosphate) or GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) resulted in loss of adenine nucleotide-promoted Cl- channel activation, neither guanine nucleotide altered the 2MeSATP-stimulated cation current. These data are consistent with the view that activation of the novel cation current by the P2Y1 receptor does not involve a G protein. Tail current analysis of the novel P2Y1 receptor-associated cation conductance revealed that the open channel current-voltage relationship was outwardly rectifying with a reversal potential of -38 mV for the turkey P2Y1 receptor and -36 mV for the human P2Y1 receptor. Replacement of Na+ with K+ ions in the bathing solution produced a shift in reversal potential to near zero mV, but significant outward rectification remained. The cation current was not permeable to either Ca2+ or Ba2+ and exhibited steady-state inactivation at holding potentials below -60 mV. These results indicate that the P2Y1 receptor exhibits both metabotropic properties and a novel G protein-independent ionotropic response when expressed in Xenopus oocytes.
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PMID:A guanine nucleotide-independent inwardly rectifying cation permeability is associated with P2Y1 receptor expression in Xenopus oocytes. 891 May 62

Although P2 receptors mediate a myriad of physiological effects of extracellular adenine nucleotides, study of this broad class of receptors has been compromised by a lack of P2 receptor-selective antagonist molecules. The adenine nucleotide-promoted inositol lipid hydrolysis response of turkey erythrocyte membranes, which has been used extensively as a model for P2Y receptors, has been applied to identify molecules that competitively block these receptors. Adenosine-3'-phosphate-5' -phosphosulfate (A3P5PS) promoted activation of phospholipase C that was only 10-25% of that observed with the full P2Y receptor agonists ATP, ADP, and 2-methylthio-ATP (2MeSATP). The small stimulatory effects of A3P5PS were saturable. Moreover, these effects were entirely the result of interaction with the P2Y receptor, because A3P5PS had no effect on activation of phospholipase C through the beta-adrenergic receptor and produced a concentration-dependent inhibition of 2MeSATP-promoted activity over the same range of A3P5PS concentrations that alone caused a small activation of phospholipase C. Increasing concentrations of A3P5PS produced a rightward shift of the concentration-effect curve for 2MeSATP, and Schild transformation of these data revealed that A3P5PS is a competitive P2Y receptor antagonist with a pKB of 6.46 +/- 0.17. The presence of a phosphate in the 2'- or 3'-position appears to be crucial for antagonist activity, because adenosine-3' -phosphate-5'- phosphate (A3P5P) and adenosine-2'- phosphate-5'-phosphate also exhibited competitive antagonist/partial agonist activities. Other 3'-substituted analogues, such as 3'-amino-ATP and 3'-benzoylbenzoyl-ATP, were full agonists with no antagonist activity. A3P5PS, A3P5P, and adenosine-2',5'-diphosphate also were competitive antagonists in studies with the cloned human P2Y1 receptor stably expressed in 1321N1 human astrocytoma cells. Moreover, both A3P5PS and A3P5P were devoid of agonist activity at the human P2Y1 receptor. The effects of these 2'- and 3'-phosphate analogues were specific for the phospholipase C-coupled P2Y1 receptor, because no agonistic or antagonistic effects on the adenylyl cyclase-coupled P2Y receptor of C6 glioma cells or on P2Y2, P2Y4, or P2Y6 receptors stably expressed in 1321N1 human astrocytoma cells were observed. These results describe specific competitive antagonism of the P2Y1 receptor by an adenine nucleotide derivative and provide a potential new avenue for P2 receptor drug development.
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PMID:Identification of competitive antagonists of the P2Y1 receptor. 891 64

Mas-7, a mastoparan derivative, induces elevation of intracellular free Ca2+ concentration ([Ca2+]i) along two independent pathways. The minor contribution occurs via phospholipase C activation and is negatively regulated by treatment with phorbol 12-myristate 13-acetate, a protein kinase C activator. The major contribution involves plasma membrane pores allowing not only Ca2+, Mn2+, and Na+ to enter but also the uptake of ethidium bromide (314 Da) and lucifer yellow (457 Da), but not fura-2 (831 Da), Evans blue (961 Da), and fluorescein-conjugate phalloidin (1,175 Da). Mas-7-induced current, as measured in planar lipid bilayers, reveals that Mas-7-induced pores have two slope conductances, 290 and 94 pS, and that the pores are nonselective for cations. The results also indicate that Mas-7 can produce pores by direct interaction with the plasma membrane without the involvement of membrane proteins and cytosolic factors. Besides in human neuroblastoma cells, similar Mas-7 effects were also observed in other cell lines such as HL-60, 1321N1 human astrocytoma, and bovine chromaffin cells. The data suggest that the Mas-7-induced [Ca2+]i elevation is the combined result of Ca2+ release from stores via phosphoinositide turnover and prolonged Ca2+ influx through membrane pores.
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PMID:Induction of cytosolic Ca2+ elevation mediated by Mas-7 occurs through membrane pore formation. 895 10

In U373 MG cells, a line derived from a human astrocytoma, histamine stimulated the release of [3H]gamma-aminobutyric acid ([3H]GABA) in a concentration-dependent manner (286 +/- 23% of basal release at 1 mM histamine). Neither Ca2+ removal nor Cd2+ (100 microM) affected [3H]GABA release evoked by 100 microM histamine but the response was significantly reduced by 10 microM U-73122 ({1-[6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)-amino)-hexyl]-1 H-pyrrole-2,5-dione}), an inhibitor of phospholipase C activation (79 +/- 8% inhibition) and by 10 microM dimethylbenzamil, a selective blocker of plasma membrane Na+/Ca2+ exchange (58 +/- 6% inhibition). In [3H]inositol-labelled cells histamine stimulated [3H]inositol phosphate accumulation (EC50, 17 +/- 2 microM; maximum effect, 203 +/- 4% of basal). Histamine-evoked Ca2+ mobilisation yielded an EC50 of 12 +/- 2 microM and maximum delta[Ca2+]i of 337 +/- 23 nM. Thapsigargin (1 nM) increased [Ca2+]i (delta[Ca2+]i 164 +/- 12 nM) and prevented any further increase by histamine (100 microM). The effects of histamine on [3H]GABA release, [3H]inositol phosphate accumulation and Ca2+ mobilisation were blocked by the selective histamine H1 receptor antagonist mepyramine. Taken together, these results indicate that histamine stimulates [3H]GABA release by increasing [Ca2+]i. The mechanism of release may be related to changes in transmembranal Na+ gradients and reversal of GABA carrier transport due to stimulation of plasma membrane Na+/Ca2+ exchange.
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PMID:Histamine H1 receptor activation stimulates [3H]GABA release from human astrocytoma U373 MG cells. 900 31

In previous studies, we showed that angiotensin II (Ang II) and its congener peptides-angiotensin-(2-8) [Ang-(2-8)] and angiotensin-(1-7) [Ang-(1-7)]-activate 2 distinct signal transduction pathways in a mixed population of human cortical astrocytoma cells. This suggested that different populations of astrocytes could be heterogeneous with respect to their expression of Ang II receptors or the responses to which these receptors are coupled. To compare the responses which are activated by Ang II and its congener peptides in astrocytes from different brain regions, we measured phospholipase C (PLC) activity and prostaglandin release in isolated astrocytes from 4 different areas of neonatal rat brain. In medullary and cerebellar astrocytes, Ang II activated a phosphoinositide-specific PLC in a dose-dependent manner with EC50s of 1.74 and 1.86 nM, respectively. Ang-(2-8) also caused an increase in inositol phosphate release. PLC activity was coupled to an AT1 receptor in both medullary and cerebellar astrocytes, as demonstrated by the inhibition of Ang II-activation of inositol phosphate release by the AT1 antagonist losartan. The AT2 antagonist PD 123319 was ineffective. Ang II and Ang-(2-8) also released prostacyclin from medullary and cerebellar astrocytes, measured as the release of its stable metabolite 6-keto-PGF1 alpha. In contrast, Ang II did not activate PLC or release prostaglandins in astrocytes isolated from the cortex or hypothalamus. In addition, Ang-(1-7) did not stimulate the release of inositol phosphates or prostacyclin in astrocytes from any of the neonatal rat brain regions examined. However, bradykinin (1 microM) activated PLC or released prostacyclin in astrocytes isolated from all 4 brain regions. These results suggest that Ang II receptors on region-specific astrocytes activate distinct signal transduction mechanisms in response to different angiotensin peptides.
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PMID:Angiotensin II activates distinct signal transduction pathways in astrocytes isolated from neonatal rat brain. 909 77

In some cell systems muscarinic receptor stimulation can induce proliferation or transformation. This phenomenon is subtype-specific (only m1 and m3 receptors are effective) and cell type dependent. In 1321N1 astrocytoma cells activation of m3 receptors stimulates phospholipase C, but does not induce DNA synthesis. In contrast the thrombin receptor, which also couples to phospholipase C, is strongly mitogenic and induces AP-1-dependent gene expression. Various experimental findings indicate that this discrepancy is not due to muscarinic receptor desensitization or blockade of growth stimulatory pathways. Muscarinic receptor number may be limiting, in particular for receptor coupling to the pertussis toxin-insensitive G-protein G12. This G-protein is required for thrombin-induced mitogenesis in 1321N1 cells and may couple selectively to the thrombin versus muscarinic receptor. In cardiomyocytes hypertrophic cell growth is induced by heterologously expressed m1 or m3 receptors but not by the endogenous m2 receptors. Studies using chimeric receptors confirm that induction of hypertrophy requires signalling through phospholipase C, but indicate that additional signals are needed to induce the morphological features of this response. We suggest that small G-proteins of the Rho subfamily, in addition to G12, mediate growth responses to G-protein-coupled receptors.
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PMID:Pathways and roadblocks in muscarinic receptor-mediated growth regulation. 912 50

1. Four different phospholipase C (PLC)-activating P2Y receptors have been cloned and stably expressed in 1321N1 human astrocytoma cells. These include the human homologues of the P2Y1, P2Y2 and P2Y4 receptors and the rat homologue of the P2Y6 receptor. 2. The nucleotide selectivities of these four receptors have been compared directly by measuring inositol phosphate accumulation in response to nucleotides under conditions in which the initial purity and stability of agonist was rigidly assured and quantitatively assessed. 3. The P2Y1 receptor is specific for adenine nucleotides and slightly more sensitive to disphosphates than triphosphates. When expressed in 1321N1 astrocytoma cells, it couples selectively to the stimulation of PLC and not to the inhibition of adenylyl cyclase. 4. The P2Y2 receptor is activated by UTP and ATP with similar potency and is not activated by nucleoside diphosphates. Diadenosine terraphosphate is a potent agonist at this receptor. 5. The P2Y4 receptor is highly selective for UTP over ATP and is not activated by nucleoside disphosphates. 6. The P2Y6 receptor is activated most potently by UDP, but weakly or not at all by UTP, ADP and ATP. The P2Y6 receptor appears to be identical to the uridine nucleotide-specific receptor previously characterized in C6-2B rat glioma cells. 7. We have identified a P2Y receptor on C6 glioma cells that inhibits adenylyl cyclase but has no effect on PLC. This receptor exhibits a pharmacological selectivity similar but not identical to that of the P2Y1 receptor. When the P2Y1 receptor was expressed in these C6 cells, it conferred an inositol lipid signalling response to adenine nucleotides that was pharmacologically identical to that of the P2Y1 receptor. Thus, the P2Y receptor of C6 glioma cells represents an additional receptor that exhibits the classical pharmacological selectivity of a P2Y1-R, but which couples to adenylyl cyclase rather than to PLC.
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PMID:Pharmacological and second messenger signalling selectivities of cloned P2Y receptors. 913 7

We investigated the action of histamine on C6-astroglioma cells using patch clamp recording and intracellular calcium measurement. Application of 100 microM histamine hyperpolarized the resting membrane potential and increased free intracellular calcium. Membrane hyperpolarization was accompanied by a decrease in input resistance. The effect of histamine was reversible and responses persisted following repeated applications. In voltage clamp experiments histamine elicited an outward current associated with a conductance increase and a reversal potential near the Nernst potential for potassium. The action of histamine was blocked by mepyramine but not by cimetidine or thioperamide suggesting that a H1 receptor mediated the response. Quinidine and charybdotoxin, but not apamin, blocked the hyperpolarization. Buffering internal calcium with BAPTA diminished the activation of the potassium channel, suggesting a calcium-dependent K(+)-channel, which was also found to be regulated by protein kinase C and phosphatases. The increase in intracellular calcium was not dependent on external calcium or sensitive to pertussis toxin, cholera toxin, forskolin or 8-bromo-cAMP. Both the hyperpolarization and the increase in intracellular calcium were blocked by thapsigargin or the phospholipase C inhibitor U73122. These results indicate that histamine liberates calcium from internal stores by activation of phospholipase C which in turn leads to an increase of intracellular Ca2+ and thereby to the activation of a calcium-dependent potassium channel in C6 glial cells.
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PMID:Histamine H1 receptors in C6 glial cells are coupled to calcium-dependent potassium channels via release of calcium from internal stores. 915 Dec 92

1. The functional activity of deoxyadenosine 5'(alpha-thio)triphosphate (dATP alpha S) was assessed at the cloned human P2Y1 receptor stably expressed in 1321N1 human astrocytoma cells and transiently expressed in Cos-7 cells. 2. Cells expressing the receptor responded to adenine nucleotides with an increase in [3H]-inositol phosphate accumulation. Half-maximal responses were obtained at approximately 30 nM for 2-methylthioadenosine-5'-triphosphate (2MeSATP), 300 nM for dATP alpha S, and 1000 nM for adenosine 5'-triphosphate (ATP). dATP alpha S produced a maximal response that was only 37 +/- 4% of that produced by ATP or 2MeSATP. dATP alpha S also competitively antagonized the phospholipase C response to 2MeSATP with a KB of 644 +/- 14 nM. Thus dATP alpha S acts as a low potency partial agonist at P2Y1 receptors. 3. The selectivity of dATP alpha S for P2Y1 receptors was determined by examining its capacity to activate P2Y2, P2Y4 and P2Y6 receptors also stably expressed in 1321N1 cells. Although dATP alpha S was a partial agonist at P2Y1 receptors it was a full agonist at P2Y2 receptors, albeit with a potency that was two orders of magnitude lower than at P2Y1 receptors. No agonist or antagonist activity was observed at P2Y4 and P2Y6 receptors. 4. Although [35S]-dATP alpha S bound to a relatively high density (ca 10 pmol mg-1 protein) of binding sites in membranes from 1321N1 or Cos-7 cells expressing the P2Y1 receptor, no difference in the total density of sites was observed between membranes from wild-type, empty vector-transfected, or P2Y1 receptor-expressing cells. Moreover, adenine nucleotide analogues inhibited [35S]-dATP alpha S binding with an order of potency that differed markedly from that for the accumulation of inositol phosphates in intact transfected P2Y1 receptor-expressing cells. Saturation binding experiments demonstrated multiple affinity states for [35S]-dATP alpha S binding in wild-type Cos-7 cell membranes. These data from 1321N1 and Cos-7 cells suggest that cellular membranes exhibit a large number of high affinity binding sites for [35S]-dATP alpha S that are not related to P2Y receptor subtypes.
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PMID:An examination of deoxyadenosine 5'(alpha-thio)triphosphate as a ligand to define P2Y receptors and its selectivity as a low potency partial agonist of the P2Y1 receptor. 915 46


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