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 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

P2 purinoceptor agonists produced whole-cell potassium currents in cerebellar neurons with the order of potency 2-methylthio ATP (2-MeSATP) > ADP > ATP > adenosine > alpha,beta- methylene ATP > AMP > UTP. In the outside-out patch clamp configuration, 2-MeSATP evoked single channel currents with two major classes of slope conductances without latency. The currents were blocked by a G-protein inhibitor, GDP beta S, although they were not affected by a phospholipase C inhibitor, a selective protein kinase C or A inhibitor. In contrast, a potent G-protein activator, GTP gamma S, produced single channel currents with same conductances as those of the currents induced by 2-MeSATP. These provide an indication that the P2 purinoceptor-operated potassium channel is regulated by the beta gamma subunits of a G-protein.
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PMID:P2 purinoceptor-operated potassium channel in rat cerebellar neurons. 857 78

1. The release of prostacyclin (PGI2) from vascular endothelial cells is stimulated by ATP acting at G protein-coupled P2-purinoceptors. Here we investigate the hypothesis that tyrosine protein phosphorylations are involved in this response. 2. The use of Western blots with anti-phosphotyrosine antibodies showed that 30 microM 2MeSATP (selective for P2Y-purinoceptors), 300 microM UTP (selective for P2U-purinoceptors) and 300 microM ATP (effective at both these purinoceptors), each stimulate the tyrosine phosphorylation of proteins in bovine cultured aortic endothelial cells. Each of these agonists also stimulates 6-keto PGF1 alpha accumulation in the medium (an index of PGI2 release) in these cells in the same period. 3. The tyrosine kinase inhibitor, genistein, inhibits the 6-keto PGF1 alpha response with the same concentration-dependency (1-100 microM) as the tyrosine phosphorylation response. 4. Tyrphostin, a structurally and functionally distinct tyrosine kinase inhibitor, is also a potent inhibitor (0.1-10 microM) of the 6-keto PGF1 alpha response. 5. Neither tyrphostin nor genistein inhibit the phospholipase C response to P2-purinoceptor stimulation. Furthermore, these inhibitors do not affect the 6-keto PGF1 alpha response to ionomycin. 6. These results show that the regulation of vascular endothelial cells by ATP acting at both P2Y- and P2U-purinoceptors involves the stimulation of tyrosine phosphorylation, and suggest that this is a necessary event for the purinoceptor-mediated stimulation of PGI2 production.
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PMID:Evidence for requirement of tyrosine phosphorylation in endothelial P2Y- and P2U- purinoceptor stimulation of prostacyclin release. 859 Sep 71

Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 mM), glutamate (100 microM), norepinephrine (10 microM), and substance P (1 microM) did not increase the intracellular calcium concentration ([Ca2+]i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 microM), bradykinin (11%; 10 microM), and histamine (31%; 100 microM), whereas 100% of glia responded to ATP (100 microM). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca2+]i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca2+]i evoked, ATP = UTP > ADP > beta, gamma-methyleneadenosine 5'-triphosphate >> 2-methylthioadenosine 5'-triphosphate = alpha,beta-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P2U receptor. Depletion of D-myo-inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 microM) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 microM), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 nM), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca2+]i. Our data suggest that glial responses to ATP are mediated by a P2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.
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PMID:Enteric glia exhibit P2U receptors that increase cytosolic calcium by a phospholipase C-dependent mechanism. 859 30

ADP evoked outwardly rectifying potassium currents with a latency of 0.6 s in cultured rat medullar neurons. Purinoceptor agonists, such as 2-methylthio ATP (2-MeSATP), ATP, AMP, alpha,beta-methylene ATP (alpha,beta-MeATP), and UTP, produced similar outward currents with the order of their potencies for current amplitudes: 2-MeSATP > ADP > ATP > or = alpha,beta-MeATP > or = AMP > UTP. This order corresponds to that for a subtype of P2Y purinoceptors. ADP-evoked currents were fully blocked by a broad G-protein inhibitor, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), whereas a G(i)/G(o)-protein inhibitor, pertussis toxin (PTX) had no effect. The currents were not affected by a phospholipase C (PLC) inhibitor, neomycin. Furthermore, a selective protein kinase C inhibitor, GF109203X or a selective cAMP-dependent protein kinase inhibitor, H-89 showed no effect on the currents. These results suggest that ADP activates the potassium channel via a P2Y purinoceptor linked to a PTX-insensitive G-protein and its channel regulation may be due to a direct action of the G-protein beta gamma subunits regardless of second messenger signaling cascades. Additionally, ADP enhanced intracellular free Ca2+ concentration ([Ca2+]i) both in the presence and absence of extracellular calcium, and this [Ca2+]i increase was not inhibited by neomycin. This provides an additional evidence that ADP binds to a subtype of P2Y purinoceptors, which is not involved in PLC stimulation.
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PMID:A P2 purinoceptor activated by ADP in rat medullar neurons. 859 44

Human neutrophils or HL60 cells express P2U receptors and respond to micromolar concentrations of ATP, adenosine 5'-O-(thiotriphosphate) (ATPgammaS), or UTP with immediate increases in intracellular Ca2+ through activation of phosphoinositide phospholipase C (Cowen, D. S., Lazarus, H. M., Shurin, S. B., Stoll, S. E., and Dubyak, G. R. (1989) J. Clin. Invest. 83, 1651-1660). P2U agonists reportedly induce limited enzyme secretion and enhance the respiratory burst in response to chemotactic factors. We demonstrate here that P2U agonists are chemotactic for neutrophils or differentiated HL60 cells. Rhodamine phalloidin staining indicates that ATPgammaS treatment induces actin polymerization and shape changes similar to those seen when these cells are treated with chemotactic peptide fMet-Leu-Phe. Although undifferentiated HL60 cells fail to mount a rise in Ca2+ when challenged with fMet-Leu-Phe, they increase Ca2+ in response to P2U agonists. However, functional expression of phospholipase C-coupled receptors is not sufficient for chemotaxis since HL60 cell migration in response to these agonists or to fMet-Leu-Phe occurs only after exposure to differentiating agents such as BT2cAMP. In addition to the well known G protein-linked receptors for lipid or peptide chemotactic factors, neutrophils apparently also can utilize G protein-linked purino/pyrimidino receptors to recognize nucleotides as chemoattractants. High concentrations of ATP and UTP generated at sites of platelet aggregation and tissue injury could thus be important mediators of inflammation.
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PMID:P2U agonists induce chemotaxis and actin polymerization in human neutrophils and differentiated HL60 cells. 866 69

ATP (1 mM) induced a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H- pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca2+. We examined the mechanism of the ATP-elicited sustained [Ca2+]i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 +/- 0.8 mV (n = 10) in Tyrode external solution. The EC50 of ATP was approximately 0.75 mM. The permeability ratio of various cations carrying this current was Na+ (defined as 1) > Li+ (0.92 +/- 0.01; n = 5) > K+ (0.89 +/- 0.03; n = 6) > Rb+ (0.55 +/- 0.02; n = 6) > Cs+ (0.51 +/- 0.01; n = 5) > Ca2+ (0.22 +/- 0.03; n = 3) > N-methyl-D-glucamine (0.13 +/- 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg2+. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5'-thiotriphosphate (ATPgammaS), and adenosine 5'-O-(2-thiodiphosphate), but not adenosine, ADP, alpha,beta-methylene-ATP (AMPCPP), beta,gamma-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPgammaS, but not AMPCPP or AMPPCP, increased the sustained [Ca2+]i increase. We conclude that ATP activates a class of Ca2+-permeable nonselective cation channels via the P2z receptor in NG108-15 cells.
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PMID:ATP-activated nonselective cation current in NG108-15 cells. 866 19

Schwann cells play an important role in both the development and regeneration of peripheral nerves. Proliferation and differentiation of Schwann cells are critically dependent on changes in the levels of cAMP. ATP is a fast excitatory transmitter in the peripheral nervous system, inducing depolarization of the vagus nerve through occupancy of P2-purinergic receptors. In the present study we demonstrate that extracellular ATP stimulates phospholipase C and inhibits adenylate cyclase activities in cultured Schwann cells. Addition of ATP inhibited, in a concentration-dependent manner, forskolin- or isoprenaline-stimulated adenylate cyclase activity. The rank order of potency corresponding to different purinergic receptor agonists was 2-methylthio-ATP > ATP = ADP > or = adenosine 5'-[gamma-thio]triphosphate (ATP[S]) > UTP, consistent with the involvement of a P2y subtype. Adenosine and adenosine 5'-[alpha,beta-methylene]-triphosphate (pp[CH2pA) were ineffective. Preincubation with pertussis toxin completely blocked this inhibitory effect. When Schwann cells were pre-labelled with myo-[3H]inositol and incubated in Hanks' balanced salt solution containing Ca2+ and Mg2+, addition of ATP[S] resulted in a concentration-dependent increase in the release of InsP with a concomitant increase in intracellular free [Ca2+] ([Ca2+]i). Under these conditions, the effects of both ATP and UTP were of lower magnitude. Removal of Ca2+ and Mg2+ from the assay medium resulted in a significant increase in the effects of ATP[S], ATP and UTP. The decreased response observed in the presence of both bivalent cations (1.2 mM Ca2+ and 1 mM Mg2+) could not be explained either by increased degradation of ATP by Ca2+/Mg2+-dependent nucleotidases or by cation influx. The rank order of potency for the effects of agonists on phospholipase C activity was ATP[S] = adenosine 5'[gamma-imido]triphosphate > ATP -UTP > ADP, indicating the involvement of a P(2U) receptor subtype in this response. Adenosine, AMP and pp[CH2]pA were ineffective. These results demonstrate that immortalized Schwann cells express P(2U) and P(2Y) purinoceptors, which are coupled to stimulation of phospholipase C and inhibition of adenylate cyclase, respectively. Our observations unveil signal-transduction pathways that may be used by ATP to regulate proliferation and differentiation of Schwann cells, and ultimately to influence nerve homeostasis.
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PMID:P2-purigenic receptors regulate phospholipase C and adenylate cyclase activities in immortalized Schwann cells. 867 70

Observation that the G protein-coupled P2U receptor (P2Y2 receptor) is activated by UTP as well as ATP provided the first indication that a class of uridine nucleotide-responsive receptors might exist. This hypothesis was confirmed by our identification of a uridine nucleotide-specific receptor on C6-2B rat glioma cells and by the recent cloning of two uridine nucleotide-responsive receptors, the P2Y6 receptor [J. Biol. Chem. 270:26152-26158 (1995)] and the P2Y4 receptor [J. Biol. Chem. 270:30849-30852 (1995) and J. Biol. Chem. 270:30845-30848 (1995)]. The relative nucleotide selectivities of these uridine nucleotide-activated receptors have not been established. Therefore, we cloned and expressed the P2Y6 and P2Y4 receptors in 1321N1 human astrocytoma cells and compared their relative selectivities for UDP, UTP, and other uridine and adenine nucleotides with that of the P2Y2 receptor expressed in the same cells. These comparisons were made by measuring inositol phosphate accumulation under conditions in which the initial purity and stability of agonists were rigidly ensured and quantitatively assessed. The data indicate that the P2Y2 receptor is activated with similar potencies by ATP and UTP but not by ADP or UDP; the P2Y6 receptor is activated most potently by UDP but weakly by UTP, ATP, and ADP; and the P2Y4 receptor is activated most potently by UTP, less potently by ATP, and not at all by nucleotide diphosphates. Furthermore, the P2Y6 receptor, which displays a uridine nucleotide selectivity essentially identical to that of the uridine nucleotide-specific receptor in C6-2B cells, was shown to be natively expressed in C6-2B cells and to account for the uridine nucleotide responses originally identified in these cells. These results define the uridine nucleotide selectivity of three phospholipase C-linked receptors: a receptor that is selectively activated by UDP (P2Y6 receptor), selectively activated by UTP (P2Y4 receptor), and activated by UTP and ATP but not by diphosphate nucleotides (P2Y2 receptor).
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PMID:Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor. 870 Jan 27

Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y7, has 352 amino acids and shares 23-30% amino acid identity with the P2Y1-P2Y6 purinoceptors. The P2Y7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 +/- 6 nM), much less for UTP and ADP (approximately 1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.
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PMID:Molecular cloning of a novel P2 purinoceptor from human erythroleukemia cells. 870 78


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