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Query: UMLS:C0017638 (glioma)
 30,880 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cDNA encoding a novel P2 receptor was isolated from rat aortic smooth muscle cell library and functionally characterized. The cloned P2 receptor exhibits structural features characteristic of the G protein-coupled receptor family and shows 44 and 38% amino acid identity with previously cloned rat P2U and chicken P2Y receptors, respectively. The cloned P2 receptor is functionally coupled to phospholipase C but not to adenylate cyclase in C6 rat glioma cells transfected with the cloned P2 expression vector. The rank order of agonist potency as judged by intracellular Ca2+ mobilization responses is UTP > ADP = 2-methylthioATP > ADP beta S > ATP = ATP gamma S, which is not compatible with any of the previously characterized P2 receptor subtypes. The nonselective P2 antagonists, suramin and reactive blue-2, inhibit nucleotide-induced phospholipase C activation in cells expressing the cloned P2 receptor. The cloned P2 receptor mRNA is abundantly expressed in various rat tissues including lung, stomach, intestine, spleen, mesentery, heart, and, most prominently, aorta. The results indicate that the novel metabotropic P2 receptor has pharmacological characteristics distinct from any of P2 receptor subtypes thus far identified and suggest the existence of a novel regulatory system by extracellular nucleotides of potential significance.
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PMID:Molecular cloning and functional analysis of a novel P2 nucleotide receptor. 759 19

A phospholipase-C-linked nucleotide receptor, sensitive to both uridine and adenosine triphosphate (UTP and ATP) has been cloned from NG108-15 neuroblastoma x glioma hybrid cells. We have tested whether activation of this receptor could inhibit the voltage-dependent K+ current [IK(M) or "M-current"] in NG108-15 cells recorded using whole-cell patch-clamp methods. Both UTP and ATP inhibited IK(M) by 44% and 42%, respectively, at 100 microM. Mean IC50 values were: UTP, 0.77 +/- 0.27 microM; ATP, 1.81 +/- 0.82 microM. The order of nucleotide and nucleoside activity at 100 microM was: UTP = ATP > ATP [gamma S] = ITP > 2-MeSATP > ADP = GTP >> AMP-CPP, adenosine, where ATP[gamma S] is adenosine 5'-O-(3-thiotriphosphate), ITP is inosine 5'-triphosphate, 2-MeSATP is 2-methylthio ATP and AMP-CPP is alpha, beta methylene ATP. This rank order accords with their activities at the cloned P2U receptor. Effects were not inhibited by suramin (up to 500 microM) or by pre-incubation for 12 h in 500 ng.ml-1 Pertussis toxin. Inhibition of IK(M) was frequently preceded by a transient outward current, probably a Ca(2+)-activated K+ current, responding to Ca2+ mobilization. No effect on the delayed rectifier K+ current was observed. These observations match those expected from stimulating other phospholipase-C-linked receptors in NG108-15 cells.
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PMID:Activation of nucleotide receptors inhibits M-type K current [IK(M)] in neuroblastoma x glioma hybrid cells. 789 8

Incubation of C6-2B rat glioma cells with UDP or UTP resulted in a time- and concentration-dependent increase in the accumulation of inositol phosphates. In contrast, ATP, ADP, and analogs of these nucleotides known to be effective agonists at P2U-, P2X-, P2Y-, P2T-, and P2Z-purinergic receptors all had no effect on inositol phosphate levels in C6-2B cells. Pyrimidine nucleotides stimulated inositol phosphate accumulation with an order of potency of UDP > 5-BrUTP > UTP > dTDP > UDP glucose. K0.5 values for UDP, 5-BrUTP, and UTP were 2.3 +/- 0.5, 9 +/- 3, and 57 +/- 10 microM, respectively. A similar uridine nucleotide selectivity was observed for arachidonic acid release presumably occurring as a consequence of activation of phospholipase A2. Cross-desensitization and additivity experiments indicated that UDP and UTP interact with the same population of receptors. The effect of uridine nucleotides on inositol phosphate accumulation was inhibited markedly by pretreatment of cells with pertussis toxin. UDP also caused a guanine nucleotide-dependent increase in inositol lipid hydrolysis in streptolysin-O-permeabilized cells. Taken together these results describe the existence of a novel uridine nucleotide receptor that is not activated by adenine nucleotides. This receptor is pharmacologically distinct from the previously described P2U- and other P2-purinergic receptors, and likely is a member of a new class of receptors for extracellular nucleotides.
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PMID:Identification of a uridine nucleotide-selective G-protein-linked receptor that activates phospholipase C. 816 81

The activation of P2-purinergic receptors on C6-2B rat glioma cells caused a transient increase in cytosolic-free Ca2+ concentration ([Ca2+]i) as detected by Fura 2 fluorescence ratio imaging of single cells. These purinergic receptors are of the P2U subtype because UTP and ATP were equipotent and substantially more potent than the P2X- and P2Y-selective agonists alpha,beta-methylene ATP and 2-methylthio ATP, respectively. There was homologous desensitization of the Ca2+ responses between UTP and ATP but no heterologous desensitization between these nucleotides and another Ca(2+)-mobilizing receptor agonist, alpha-thrombin. The UTP-induced peak [Ca2+]i rise was insensitive to chelation of extracellular Ca2+ with EGTA. However, the response was abolished after either depletion of intracellular Ca2+ stores with the microsomal Ca(2+)-ATPase inhibitor thapsigargin or blockade of Ca2+ release from intracellular stores with the muscle relaxant dantrolene. The activation of P2U-purinergic receptors and thrombin receptors increased the formation of total inositol phosphates (IPs) and inhibited cAMP accumulation elicited with either the beta-adrenergic receptor agonist (-)-isoproterenol, or forskolin, a direct activator of adenylyl cyclase. UTP- and alpha-thrombin-induced changes in the levels of IPs, cytosolic Ca2+, and agonist-elicited cAMP accumulation were dramatically inhibited (> 80%) by acute treatment of the cells with the protein kinase C activator 4 beta-phorbol 12-myristate 13-acetate but not with the inactive ester 4 alpha-phorbol 12,13-didecanoate. We conclude that in C6-2B cells, the increase in [Ca2+]i after activation of P2U-purinergic receptors is primarily a result of IPs-mediated release of Ca2+ from intracellular stores with secondary influx of Ca2+ by capacitative mechanisms. Also, the inhibition by UTP and alpha-thrombin of agonist-elicited cAMP accumulation is mediated through an increase in [Ca2+]i.
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PMID:P2U-purinergic receptors on C6-2B rat glioma cells: modulation of cytosolic Ca2+ and cAMP levels by protein kinase C. 826 55

Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P2 purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P2U receptor in a cultured neuroblastoma x glioma hybrid cell line (NG108-15 cells). The addition of > or = 1 microM ATP to NG108-15 cells caused a transient increase in [Ca2+]i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations > or = 500 microM also elicited a sustained increase in [Ca2+]i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca2+]i elicited by ATP occurred concomitantly with the hydrolysis of [32P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca2+]i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP4-) in the medium and not with the concentration of magnesium-ATP (MgATP2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca2+]i. ADP, ITP, TTP, GTP, ATP gamma S, 2-methylthio ATP, beta, gamma-imidoATP or 3'-O-(4-benzoyl)benzoylATP, but not CTP, AMP, beta, gamma-methylene ATP, or adenosine, also caused an increase in [Ca2+]i. In cells labeled with [32P]P(i) or [14C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [14C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P2 nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A2 and D.
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PMID:Signal transduction pathways coupled to a P2U receptor in neuroblastoma x glioma (NG108-15) cells. 838 62

There is increasing evidence that pituitary ATP receptors may play a novel role in modulating pituitary function. This work reports the isolation and expression of a pituitary ATP receptor gene clone from a rat pituitary complementary DNA library. The isolated clone (rpP2U) has a 1125-bp coding sequence flanked by 483 bp of 5' - and 422 bp of 3'-untranslated sequences. The deduced 374-amino acid product shows structural features common to other G protein-coupled receptors, and when stably transfected into a glioma cell line lacking endogenous ATP receptors, is functionally characterized as a P2U purinoceptor. Specifically, the ATP-induced intracellular Ca2+ mobilization in the transfected cells was inhibited by suramin, 2-methylthio-ATP had a modest stimulatory effect on intracellular Ca2+ mobilization, and beta, gamma-methylene ATP and alpha, beta-methylene ATP had no effect. The cloned receptor exhibited the agonist potency and efficacy profile of ATP approximately equal to uridine triphosphate > ADP approximately equal to uridine diphosphate > GTP. Such characteristics very closely mimic the pharmacologically defined P2U purinoceptor of primary rat gonadotropes and mixed sheep pituitary cells, and Southern blot analysis further indicates that there is only one allele in rat genome for the P2U purinoceptor. These findings suggest that the P2U purinoceptor is the predominant G protein-linked ATP receptor found in the pituitary.
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PMID:Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor. 861 22

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

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

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. 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


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