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)

The effects of adenosine on hippocampal neurons were examined by patch-clamp recording and Ca2+ imaging using fura-2 fluorescence. In the whole-cell patch-clamp configuration, adenosine evoked outwardly rectifying K+ currents in a dose-dependent manner. These currents were not inhibited by a nonselective P1 purinoceptor antagonist or selective adenosine A1, A2A receptor antagonists and moreover, selective adenosine A1, A2A receptor agonists evoked no current. In contrast, P2 purinoceptor agonists produced similar outward currents with the order of potency: ADP > or = 2-methylthio ATP > ATP > adenosine >> AMP. No response was obtained to UTP, alpha, beta-methylene ATP or beta, gamma-methylene ATP. The intracellular perfusion of a broad G-protein inactivator, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), abolished adenosine-evoked currents, whereas a Gi/Go-protein inhibitor, pertussis toxin, had no effect. Furthermore, the currents were blocked by a phospholipase C inhibitor, neomycin, or specific protein kinase C inhibitors, GF109203X (bisindolyl maleimide, C25H24N4O2) and protein kinase C inhibitor peptide. In the cell-attached patch-clamp configuration, adenosine elicited single-channel currents with two major kinds of slope conductances. Likewise, application of adenosine outside the patch electrode again produced single-channel currents with same conductances. A potent protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced single-channel currents in a fashion that mimics the effect of adenosine. The evoked currents were blocked by GF109203X. In addition, adenosine enhanced intracellular free Ca2+ concentration ([Ca2+]i). This [Ca2+]i increase was inhibited by GDP beta S or neomycin, but was not affected by pertussis toxin. These results, thus, suggest that adenosine activates the K+ channel and enhances cytosolic Ca2+ release via a P2Y purinoceptor linked to a pertussis toxin-insensitive G-protein, which is involved in a phospholipase C-mediated phospholipid-signaling pathway.
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PMID:Adenosine activates the K+ channel and enhances cytosolic Ca2+ release via a P2Y purinoceptor in hippocampal neurons. 881 2

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

1. B10 cells, a clonal line of rat brain capillary endothelial cells, exhibit a single P2 purinoceptor, activation of which leads to increases in free intracellular calcium. In the current study the identity of this P2Y receptor was determined by its binding parameters for a range of purinoceptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigated. 2. The radioligand [35S]-dATP alpha S bound with high affinity (Kd = 9.8 nM) to the P2Y purinoceptor expressed on B10 cells, which was found to be extremely abundant (Bmax = 22.5 pmol mg-1 protein). The calculated Ki values of a range of P2 purinoceptor agonists which competitively displaced binding of [35S]-dATP alpha S led to the rank order of affinity: dATP alpha S (Ki 3.4 nM) > 2-chloroATP (2-ClATP) (13 nM), ATP (22 nM) > ATP gamma S (43 nM) > 2-methylthioATP (2-MeSATP) (88 nM) > ADP (368 nM) > > UTP, L-beta,gamma-methyleneATP (both > 10,000 nM). The P2 purinoceptor antagonists, Reactive blue 2 and suramin, were also able to displace binding, with Ki values of 833 and 1358 nM respectively. In contrast pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) was able to displace only 20% of [35S]-dATP alpha S binding at a concentration of 100 microM. 3. 2-ClATP (EC50 = 0.22 microM), 2-MeSATP (0.54 microM), ADP (7.9 microM) and ATP (a partial agonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussis toxin. The purinoceptor antagonist, PPADS, was found to be inactive at a concentration of 100 microM. 4. A P2Y receptor cDNA was derived from mRNA from B10 cells and from C6-2B, a rat glioma cell line known to possess a P2Y receptor that is coupled to the inhibition of adenylate cyclase. Sequence analysis of the entire coding region revealed that both were 100% identical to the rat P2Y1 purinoceptor cDNA. No other P2Y-type receptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2-MeSATP has been shown to increase phospholipase C activity. 5. Since the receptor responsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activity of 2-MeSATP (characteristic of P2Y1 receptors alone among all known P2Y purinoceptors) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y1 receptor but not of any other type resembling the known P2Y receptors, it is concluded that a P2Y1 receptor on rat brain microvascular endothelial cells can account for all of the observations. This single P2Y1 receptor, therefore, appears to couple in different native cell types to either adenylate cyclase inhibition or to phospholipase C activation.
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PMID:The P2Y purinoceptor in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase. 896 47

The effects of diadenosine tetraphosphate (AP4A) diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A) on the cytosolic-free calcium concentration ([Ca2+]i) were evaluated in cultured rat glomerular mesangial cells (MCs) using the fluorescent dye technique. The addition of 10 mumol L-1 AP4A, AP5A or AP6A significantly increased [Ca2+]i in MCs by 57 +/- 9 nmol L-1 n = 17; P < 0.01), 76 +/- 27 nmol L-1 (n = 9; P < 0.01) or 65 +/- 12 nmol L-1 (n = 18; P < 0.01) respectively. In the absence of extracellular calcium, there was no significant change in [Ca2+]i in MCs after administration of diadenosine polyphosphates, indicating that these agents induce transplasma membrane Ca2+ influx. AP6A significantly enhanced the angiotensin II-induced changes in [Ca2+]i in MCs. The AP5A-induced transplasma membrane Ca2+ influx was inhibited by the P2 purinoceptor blockers suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), but was not affected by the adenosine A1 receptor blocker 8-cyclopentyl-1.3-dipro-pylzanthine (CPDPX). Adenosine triphosphate (ATP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma S) increased [Ca2+]i in MCs, whereas alpha, beta-methylene ATP had no effect on [Ca2+]i in MCs. Measurements of diacylglycerol and phosphatidic acid showed that AP5A and AP6A also stimulated phospholipase C, but had no effect on phospholipase D. The inhibition of phosphatidylcholine-specific phospholipase C significantly reduced the AP5A-induced [Ca2+]i increase. In summary, diadenosine polyphosphates induce Ca2+ influx through P2 purinoceptors and may be involved in the local regulation of vascular resistance evoked by the Ca(2+)-dependent contractile response of mesangial cells.
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PMID:Diadenosine polyphosphates induce transplasma membrane calcium influx in cultured glomerular mesangial cells. 901 82

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

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

Although ATP, acting through a P2 purinoceptor, can stimulate a pronounced positive inotropic effect in cardiac ventricular myocytes, the receptor-effector mechanism that underlies this stimulatory cardiac action is not well understood. The objectives of the present study were to develop the cultured chick embryo ventricular myocytes as a novel model for the cardiac P2 purinoceptor and to determine the mechanism underlying its positive inotropic effect. ATP caused an 89 +/- 8.9% (n = 14 cells) increase in the myocyte contractility, with an efficacy and potency order of ATP > ADP > AMP >> adenosine. 2-Methylthio-ATP (2-MeS-ATP) but not alpha,beta-methylene-ATP was able to stimulate myocyte contractility, with a maximal increase of 54 +/- 2.6% (n = 11 cells). Although UTP potently stimulates phosphoinositide hydrolysis, it had an only modest positive inotropic effect (27 +/- 7% maximal increase; n = 8 cells). In contrast to previous suggestions, the 2-MeS-ATP-stimulated positive inotropic response does not require the action of phospholipase C (PLC), such as that of the inositol phosphates; the UTP effect on contractility appears to be mediated via the 2-MeS-ATP-sensitive P2 receptor. The PLC inhibitor U-73122 had no effect on the 2-MeS-ATP-stimulated increase in contractility, providing further evidence against a role for PLC in the inotropic effect of 2-MeS-ATP. An adenosine 3',5'-cyclic monophosphate-independent Ca2+ entry-stimulating mechanism appears to underlie a direct coupling of the receptor to stimulation of the myocyte contractility. This new PLC- and adenosine 3',5'-cyclic monophosphate-independent positive inotropic mechanism represents a target for developing novel positive inotropic therapeutics.
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PMID:A novel phospholipase C- and cAMP-independent positive inotropic mechanism via a P2 purinoceptor. 937 75

ADP is an important platelet agonist causing shape change from smooth discoid shape to spiculated spheres and platelet aggregation. However, the molecular mechanisms involved in ADP-induced platelet activation have not been elucidated. We demonstrated earlier the existence of two distinct ADP receptors on platelets, one coupled to phospholipase C, P2TPLC, and the other to inhibition of adenylyl cyclase, P2TAC (Daniel, J. L., Dangelmaier, C., Jin, J., Ashby, B., Smith, J. B., and Kunapuli, S. P. (1998) J. Biol. Chem. 273, 2024-2029), in addition to the previously described P2X1 receptor. Here we report the cloning of a cDNA clone encoding the P2Y1 receptor from a human platelet cDNA library by homology screening with radiolabeled P2Y1-P2Y6 receptor cDNAs. ADP or 2-methyl(thio)-ADP-induced intracellular calcium increases were inhibited by the P2Y1 receptor-specific antagonists, adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS), adenosine 3'-phosphate 5'-phosphate (A3P5P), and adenosine 2'-phosphate 5'-phosphate (A2P5P), in a concentration-dependent manner, but not by ARL 66096 or alpha, beta-MeATP. A3P5PS, A3P5P, and A2P5P also inhibited the shape change of aspirinated platelets induced by 10 microM ADP or 3 microM 2-methyl-(thio)-ADP in a concentration-dependent manner, with complete inhibition occurring at 300 microM. On the other hand ARL 66096 (100 nM), a potent P2TAC antagonist and alpha, beta-methylene-ATP (40 microM), a P2X1 receptor agonist, had no effect on ADP-induced platelet shape change. On the contrary, ADP-induced inhibition of adenylyl cyclase was blocked by ARL 66096, but not by alpha, beta-MeATP or the P2Y1 receptor-specific antagonists, A3P5PS, A3P5P, or A2P5P. These results demonstrate the role of the P2Y1 receptor in ADP-induced platelet shape change and calcium mobilization and support the idea that several P2 receptors are involved in the regulation of different aspects of platelet stimulus-response coupling.
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PMID:Molecular basis for ADP-induced platelet activation. II. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. 944 40

The antagonist activity of N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate (N6MABP) has been examined at the phospholipase C-coupled P2Y1 receptor of turkey erythrocyte membranes. N6MABP antagonized 2MeSATP-stimulated inositol phosphate hydrolysis with a potency approximately 20 fold greater than the previously studied parent molecule, adenosine 3',5'-bisphosphate. The P2Y1 receptor antagonism observed with N6MABP was competitive as revealed by Schild analysis (pK(B) = 6.99 +/- 0.13). Whereas N6MABP was an antagonist at the human P2Y1 receptor, no antagonist effect of N6MABP was observed at the human P2Y2, human P2Y4 or rat P2Y6 receptors.
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PMID:Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate. 963 Mar 35

Analogues of the P2 receptor antagonists pyridoxal-5'-phosphate and the 6-azophenyl-2',4'-disulfonate derivative (PPADS), in which the phosphate group was cyclized by esterification to a CH2OH group at the 4-position, were synthesized. The cyclic pyridoxine-alpha4, 5-monophosphate, compound 2 (MRS 2219), was found to be a selective potentiator of ATP-evoked responses at rat P2X1 receptors with an EC50 value of 5.9 +/- 1.8 microM, while the corresponding 6-azophenyl-2',5'-disulfonate derivative, compound 3 (MRS 2220), was a selective antagonist. The potency of compound 3 at the recombinant P2X1 receptor (IC50 10.2 +/- 2.6 microM) was lower than PPADS (IC50 98.5 +/- 5.5 nM) or iso-PPADS (IC50 42.5 +/- 17.5 nM), although unlike PPADS its effect was reversible with washout and surmountable. Compound 3 showed weak antagonistic activity at the rat P2X3 receptor (IC50 58.3 +/- 0.1 microM), while at recombinant rat P2X2 and P2X4 receptors no enhancing or antagonistic properties were evident. Compounds 2 and 3 were found to be inactive as either agonists or antagonists at the phospholipase C-coupled P2Y1 receptor of turkey erythrocytes, at recombinant human P2Y2 and P2Y4 receptors, and at recombinant rat P2Y6 receptors. Similarly, compounds 2 and 3 did not have measurable affinity at adenosine A1, A2A, or A3 receptors. The lack of an aldehyde group in these derivatives indicates that Schiff's base formation with the P2X1 receptor is not necessarily required for recognition of pyridoxal phosphate derivatives. Thus, compounds 2 and 3 are relatively selective pharmacological probes of P2X1 receptors, filling a long-standing need in the P2 receptor field, and are also important lead compounds for future studies.
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PMID:A pyridoxine cyclic phosphate and its 6-azoaryl derivative selectively potentiate and antagonize activation of P2X1 receptors. 963 52


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