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. The coding sequence of the P2Y1-purinoceptor was cloned from a human genomic library. 2. The open reading frame encodes a protein of 373 amino acids that is 83% identical to the previously cloned chick and turkey P2Y1-purinoceptor and is > or = 95% homologous to the recently cloned rat, mouse, and bovine P2Y1-purinoceptors. 3. The human P2Y1-purinoceptor was stably expressed in 1321N1 human astrocytoma cells using a retroviral vector. Although the P2Y1-purinoceptor agonist, 2MeSATP, had no effect on inositol phosphate accumulation in cells infected with the P2Y1-purinoceptor virus. No effect of 2MeSATP on cyclic AMP accumulation was observed in P2Y1-receptor-expressing 1321N1 cells. 4. The pharmacological selectively of 18 purinoceptor agonists was established for the expressed human P2Y1-purinoceptor. 2MeSATp was more potent than ATP but less potent than 2MeSADP. ADP also was more potent than ATP. A similar maximal effect was observed with most agonists tested. However, alpha, beta-MeATP had no effect and 3'-NH2-3'-deoxyATP and A2P4 were partial agonists. The order of potency of agonists for activation of the turkey P2Y1-purinoceptor, also stably expressed in 1321N1 cells, was identical to that observed for the human P2Y1-purinoceptor. 5. C6 glioma cells express a P2Y-purinoceptor that inhibits adenylyl cyclase but does not activate phospholipase C. Expression of the human P2Y1-purinoceptor in C6 cells conferred 2MeSATP-stimulated inositol lipid hydrolysis to these cells. The phospholipase C-activating human P2Y1-purinoceptor could be delineated from the endogenous P2Y-purinoceptor of C6 glioma cells by use of the P2-purinoceptor antagonist, PPADS, which blocks the P2Y1-purinoceptor but does not block the endogenous P2Y-purinoceptor of C6 cells. P2-purinoceptor agonists also exhibited differential selectivities for activation of these two P2Y-purinoceptors.
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PMID:Second messenger cascade specificity and pharmacological selectivity of the human P2Y1-purinoceptor. 873 91

1. Previous studies have shown that bovine pulmonary artery endothelium (CPAE) has P2Y and P2U purinoceptors, rat C6 glioma cells have P2U purinoceptors and mouse RAW 264.7 cells have pyrimidinoceptors, all of which are coupled to phosphoinositide-specific phospholipase C (PI-PLC). The dual actions of PPADS, suramin and reactive blue as antagonists of receptor subtypes and ecto-ATPase inhibitors were studied in these three cell types. 2. In CPAE, suramin, at 3-100 microM, competitively inhibited the PI responses induced by 2MeSATP and UTP, with pA2 values of 5.5 +/- 0.3 and 4.4 +/- 0.4, respectively. Reactive blue, at 1-3 microM, produced shifts to the right of the 2MeSATP and UTP curves, but no further right shift at 10 microM. PPADS, at 10 microM, caused a 3 fold right shift of the 2MeSATP curve, but no further shift at concentrations up to 100 microM. In contrast, a dose-dependent shift to the left of the UTP curve and a weak inhibition of the ATP response were seen with PPADS. 3. In RAW 264.7 cells, suramin and reactive blue, but not PPADS, competitively inhibited the UTP response, with pA2 values of 4.8 +/- 0.5 and 5.8 +/- 0.7, respectively. 4. In C6 glioma cells, although suramin and reactive blue inhibited the ATP response, a potentiation effect on ATP and UTP responses was seen with PPADS. 5. The ecto-ATPase inhibitory activity of these three receptor antagonists were determined. All three inhibited ecto-ATPase present in CPAE, C6 and RAW 264.7 cells, with IC50 values of 4, 4.8 and 4.7 for PPADS, 4, 4.4 and > > 4 for suramin, and 4.5, 4.7 and 4.7 for reactive blue. 6. This study indicates that PPADS, suramin and reactive blue ar ecto-ATPase inhibitors. This property, combined with their antagonistic selectivity for receptor subtypes, can result in inhibition of, potentiation of, or lack of effect on agonist-mediated PI responses. Reactive blue is a more potent antagonist than suramin on P2Y, P2U and pyrimidinoceptors, and PPADS is a weak antagonist for P2Y receptors.
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PMID:Inhibition of ecto-ATPase by PPADS, suramin and reactive blue in endothelial cells, C6 glioma cells and RAW 264.7 macrophages. 898 11

The effect of ATP in human and rabbit corpus cavernosum (CC) smooth muscle was investigated. Strips of human CC were vertically mounted in an organ bath and the tonic tension was recorded. ATP (0.1-3 mM) induced a concentration-dependent relaxant effect, with a pD2 value of 3.01+/-0.3. The purine-induced relaxation was not affected by L-NAME (100 microM). In rabbit CC, ATP also induced a concentration-dependent relaxation, which was not influenced by L-NAME or by indomethacin (3 microM), with a pD2 value of 3.1 +/-0.4. The ATP-induced relaxant effect in rabbit CC was increased by both the inhibitor of adenosine reuptake, dipyridamole (3 microM) and by the inhibitor of adenosine deaminase, EHNA (0.3 microM). Moreover CGS 15943 (3 microM), an A2a adenosine antagonist, reduced the ATP-induced relaxation. UTP was not able to produce relaxation. The two ATP analogues 2-methylthioATP and alpha,beta-methylene ATP were able to induce relaxation in rabbit CC, with the following order of potency: 2-methylthioATP > ATP > alpha,beta-methylene ATP thus suggesting a role for P2y receptors. However, reactive blue (500 microM), an unspecific P2y antagonist, did not modify the ATP relaxant response. The inhibition of phospholipase C by U73122 (3 microM) and of the endoplasmic reticulum Ca2+ATPase by thapsigargin (1 microM) did not modify the ATP-induced relaxation. The P2x specific antagonist PPADS (30 microM) and suramine (500 microM) were not able to modify the ATP relaxation either in the absence or presence of CGS 15943 (3 microM). These results confirm that ATP acts as a potent and NO-independent relaxant agent of human and rabbit CC. Our findings also show that the ATP effect is partially attributable to the metabolic breakdown of ATP to adenosine, which acts through A2a receptor stimulation, but is also due to a direct stimulation of P2 receptors that are different from the classical P2y and P2X receptor subtypes for ATP.
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PMID:Studies on the mechanisms involved in the ATP-induced relaxation in human and rabbit corpus cavernosum. 1003 32

The effect of adenosine triphosphate (ATP) on the intracellular Ca2+ concentration ([Ca2+]i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca2+]i increase, which was dose dependent in the 2.5-50 microM range (EC50=4.3 microM). The ATP-evoked [Ca2+]i rise was not modified during the first minute following the removal of external Ca2+. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca2+]i increase. Caffeine (10 mM) and ryanodine (1 microM) did not affect the ATP-induced [Ca2+]i rise. The pituicytes responded to various P2 purinoceptor agonists with the following order of potency: ATP=ATP[gamma-S]=2-MeSATP>/=ADP, where ATP[gamma-S] is adenosine 5'-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5'-triphosphate. Adenosine, AMP, alpha, beta-methylene adenosine-5'-triphosphate (alpha,beta-MeATP), beta, gamma methylene adenosine-5'-triphosphate (beta,gamma-MeATP) and uridine 5'-triphosphate (UTP) were ineffective. The P2 purinoceptor antagonists blocked the ATP-evoked [Ca2+]i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid. The ATP-evoked [Ca2+]i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 microM) abolished the ATP-evoked [Ca2+]i rise, whereas its inactive stereoisomer U-73343 (0.5 microM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca2+]i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P2Y receptor.
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PMID:ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes). 1008 53

1. The mobilization of Ca2+ by purinoceptor activation and the relative contributions of intra- and extracellular sources of Ca2+ were investigated using microfluorimetric measurements of fura-2 loaded in cultured neurones from rat intracardiac ganglia. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expression of mRNA for the G protein-coupled P2Y2 and P2Y4 receptors. 3. Brief application of either 300 microM ATP or 300 microM UTP caused transient increases in [Ca2+]i of 277 +/- 22 nM and 267 +/- 39 nM, respectively. Removal of external Ca2+ did not significantly reduce these [Ca2+]i responses. 4. The order of purinoceptor agonist potency for [Ca2+]i increases was ATP = UTP > 2-MeSATP > ADP >> adenosine, consistent with the profile for P2Y2 purinoceptors. ATP- and UTP-induced rises in [Ca2+]i were completely and reversibly blocked by 10 microM PPADS (a P2 purinoceptor antagonist) and partially inhibited by 100 microM suramin (a relatively non-specific purinoceptor antagonist). 5. In the presence of the endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (10 microM) in Ca2+-free media, the [Ca2+]i responses evoked by ATP were progressively decreased and abolished. 6. ATP- and UTP-induced [Ca2+]i rises were insensitive to pertussis toxin, caffeine (5 mM) and ryanodine (10 microM) but were significantly reduced by U-73122, a phospholipase C (PLC) inhibitor. 7. In fura-2-loaded cells, perforated patch whole-cell recordings show that ATP and UTP evoked slow outward currents at -60 mV, concomitant with the rise in [Ca2+]i, in approximately 30 % of rat intracardiac neurones. 8. In conclusion, these results suggest that in r intracardiac neurones, ATP binds to P2Y2 purinoceptors to transiently raise [Ca2+]i and activate an outward current. The signalling pathway appears to involve a PTX-insensitive G protein coupled to PLC generation of IP3 which triggers the release of Ca2+ from a ryanodine-insensitive Ca2+ store(s).
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PMID:P2Y purinoceptor activation mobilizes intracellular Ca2+ and induces a membrane current in rat intracardiac neurones. 1089 18

Membrane-bound P2-receptors mediate the actions of extracellular nucleotides in cell-to-cell signalling. P2X-receptors are ligand-gated ion channels, whereas P2Y-receptors belong to the superfamily of G-protein-coupled receptors. So far, the P2Y family is composed of eight cloned and functionally defined subtypes. Five of them (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11) are present in human tissues. The P2Y3-, p2y8- and tp2y-receptors may be species orthologues. The principal physiological agonists of the cloned human P2Y-receptors are ADP (P2Y1), UTP/ATP (P2Y2), UTP (P2Y4), UDP (P2Y6) and ATP (P2Y11). The rat P2Y4-receptor is activated by both UTP and ATP. Specific patterns of polar amino acid residues in the exofacial portions of transmembrane domains (TMs) 6 and 7 of the P2Y-receptors may account for the ligand specificity of the subtypes. Suramin acts as an antagonist at most P2Y-receptors with the exception of P2Y4- and tp2y-receptors. PPADS has been shown to block P2Y1-, the human P2Y4- and P2Y6-receptors. The nucleotide analogue 2'-deoxy-N6-methyladenosine-3',5'-bisphosphate (MRS 2179), in contrast, seems to be a potent and selective antagonist at the P2Y1-receptor. All cloned and functionally expressed P2Y-receptors are able to couple to phospholipase C. The P2Y11-receptor mediates in addition a stimulation of adenylate cyclase and the tp2y-receptor an inhibition of this signal transduction pathway. Other functionally defined subtypes, e.g., the receptor mediating an inhibition of adenylate cyclase in blood platelets, are not yet cloned. The distribution of P2Y1 mRNA is widespread. The receptor plays a crucial role in blood platelet aggregation and mediates the adenine nucleotide-induced release of the endothelium-derived relaxing factor nitric oxide. P2Y1-receptors may also be involved in the modulation of neuro-neural signalling transmission. P2Y2 transcripts are abundantly distributed. One important example for its functional role is the control of chloride ion fluxes in airway epithelia. The P2Y4-receptor is highly expressed in the placenta. The distribution of the P2Y6-receptor is widespread including heart, blood vessels and brain. The P2Y11-receptor may play a role in the differentiation of immunocytes.
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PMID:Molecular pharmacology of P2Y-receptors. 1111 26

1. In glioma C6 cells, the stimulation of P2Y receptors by ADP, ATP and UTP initiated an increase in the intracellular Ca2+ concentration, in a process that involved the release of Ca2+ from InsP(3)-sensitive store and the capacitative, extracellular Ca2+ entry. The presence of external Ca2+ was not necessary to elevate Ca(2+). 2. The rank order of potencies of nucleotide analogues in stimulating [Ca2+](i) was: 2MeSADP > ADP > 2MeSATP = 2ClATP > ATP > UTP. alpha,beta-Methylene ATP, adenosine and AMP were ineffective. 3. ADP and UTP effects were additive, while actions of ATP and UTP were not additive on [Ca2+](i) increase. Similarly, cross-desensitization between ATP and UTP but not between ADP and UTP occurred. 4. Suramin, a non-specific nucleotide receptors inhibitor, antagonized ATP-, UTP- and ADP-evoked Ca2+ responses. PPADS, a selective antagonist of the P2Y(1) receptor-generated InsP(3) accumulation, decreased ADP-initiated Ca2+ response with no effect on ATP and UTP. 5. Pertussis toxin (PTX) reduced ADP- and ATP-induced Ca2+ increases. Short-term treatment with TPA, inhibited both ATP and ADP stimulatory effects on [Ca2+](i). 6. ADP inhibited isoproterenol-induced cyclic AMP accumulation. PTX blocked this effect, but PPADS did not. 7. RT - PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y(1) and P2Y(2). 8. It is concluded that both P2Y(1) and P2Y(2) receptors co-exist in glioma C6 cells. ADP acts as agonist of the first, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).
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PMID:Two subtypes of G protein-coupled nucleotide receptors, P2Y(1) and P2Y(2) are involved in calcium signalling in glioma C6 cells. 1115 87

We used the patch-clamp technique to study the effects of extracellular ATP on the activity of ion channels recorded in rat pancreatic beta-cells. In cell-attached membrane patches, action currents induced by 8.3 mM glucose were inhibited by 0.1 mM ATP, 0.1 mM ADP or 15 microM ADPbetaS but not by 0.1 mM AMP or 0.1 mM adenosine. In perforated membrane patches, action potentials were measured in current clamp, induced by 8.3 mM glucose, and were also inhibited by 0.1 mM ATP with a modest hyperpolarization to -43 mV. In whole-cell clamp experiments, ATP dose-dependently decreased the amplitudes of L-type Ca2+ channel currents (ICa) to 56.7+/-4.0% (p<0.001) of the control, but did not influence ATP-sensitive K+ channel currents observed in the presence of 0.1 mM ATP and 0.1 mM ADP in the pipette. Agonists of P2Y purinoceptors, 2-methylthio ATP (0.1 mM) or ADPbetaS (15 microM) mimicked the inhibitory effect of ATP on ICa, but PPADS (0.1 mM) and suramin (0.2 mM), antagonists of P2 purinoceptors, counteracted this effect. When we used 0.1 mM GTPgammaS in the pipette solution, ATP irreversibly reduced ICa to 58.4+/-6.6% of the control (p<0.001). In contrast, no inhibitory effect of ATP was observed when 0.2 mM GDPbetaS was used in the pipette solution. The use of either 20 mM BAPTA instead of 10 mM EGTA, or 0.1 mM compound 48/80, a blocker of phospholipase C (PLC), in the pipette solution abolished the inhibitory effect of ATP on ICa, but 1 microM staurosporine, a blocker of protein kinase C (PKC), did not. When the beta-cells were pretreated with 0.4 microM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump, ATP lost the inhibitory effect on ICa. These results suggest that extracellular ATP inhibits action potentials by Ca2+-induced ICa inhibition in which an increase in cytosolic Ca2+ released from thapsigargin-sensitive store sites was brought about by a P2Y purinoceptor-coupled G-protein, PI-PLC and IP3 pathway.
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PMID:P2Y-purinoceptor mediated inhibition of L-type Ca2+ channels in rat pancreatic beta-cells. 1123 96

1. Tetanic stimulation of parallel fibres (PFs) produces a slow EPSP (sEPSP) or slow EPSC (sEPSC) in Purkinje neurones (PNs), mediated by type 1 metabotropic glutamate receptors (mGluR1). The conductance change underlying the sEPSP was investigated with rapid photolytic release of L-glutamate from nitroindolinyl (NI)-caged glutamate with ionotropic glutamate receptors blocked, and showed a slow mGluR1-activated cation channel. 2. In cerebellar slices rapid photolytic release (t (1/2) < 0.7 ms) of 7--70 microM L-glutamate on PNs voltage clamped at -65 mV activated first a transient inward current, peaking in 8 ms, followed by a slow inward current with time course similar to the PF sEPSP, peaking at -1 nA in 700 ms. 3. The initial current was inhibited by 300 microM threo-hydroxyaspartate (THA) and did not reverse as the potential was made positive up to +50 mV, suggesting activation of electrogenic glutamate uptake. 4. The slow current was inhibited reversibly by 1 mM (R,S)-MCPG or the non-competitive mGluR1 antagonist CPCCOEt (20 microM), indicating activation of metabotropic type 1 glutamate receptors. The mGluR current was associated with increases of input conductance and membrane current noise, and reversed close to 0 mV, indicating activation of channels permeant to Na(+) and K(+). 5. The sEPSC was not blocked by Cd(2+), Co(2+), Mg(2+) or Gd(3+) ions, by the inhibitor of hyperpolarisation-activated current (I(H)) ZD7288, or by the purinoceptor inhibitor PPADS. Activation was not affected by inhibitors of phospholipase C (PLC) or protein kinase C (PKC), nor mimicked by photorelease of InsP(3) or Ca(2+). The results show that mGluR1 in PNs produces a slow activation of cation-permeable ion channels which is not mediated by PLC activation, Ca(2+) release from stores, or via the activation of PKC.
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PMID:The conductance underlying the parallel fibre slow EPSP in rat cerebellar Purkinje neurones studied with photolytic release of L-glutamate. 1141 Jun 33

1. The role of ATP in the regulatory volume decrease (RVD) after hypotonic cell swelling was examined in cultured endothelial cells isolated from the rat caudal artery. 2. Hypotonic stress increased [Ca2+]i in addition to increasing the overflow of ATP and cell volume. The hypotonicity induced increase in [Ca2+]i was prevented by pyridoxalphosphate-6-azophenyl-1-2',4'-disulphonic acid (PPADS; a P2 purinoceptor antagonist), U-73122 (a phospholipase C inhibitor) and thapsigargin (a Ca2+ pump inhibitor). However, the hypotonicity induced increase in cell volume was potentiated by PPADS, U-73122 and thapsigargin. 3. Similar changes were observed in cells treated with 2-methylthioATP, a P2Y purinoceptor agonist, but not by alpha,beta-methylene ATP, a P2X purinoceptor agonist. Thus, it appears that the responses observed following hypotonic stress are mediated by activation of P2Y purinoceptors. 4. On the basis of these findings, it is suggested that ATP, which is released by hypotonicity, may participate in the RVD as a substantial regulator or initiator via P2 purinoceptor-induced increases in [Ca2+]i.
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PMID:Participation of ATP in cell volume regulation in the endothelium after hypotonic stress. 1155 18


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