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

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

Rat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII-related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen-coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three-dimensional structures. The activity of the blood-brain barrier-associated enzyme, gamma-glutamyl transpeptidase (gamma GTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel-related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three-dimensional structures, but every cell inside the aggregates still expressed gamma GTP and ALP activities. Such structures, associated with high levels of gamma GTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood-brain barrier-related gamma GTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood-brain barrier.
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PMID:Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells. 790 23

Acute ethanol treatment of NG108-15 neuroblastoma x glioma hybrid cells results in inhibition of adenosine uptake with consequent increases in extracellular adenosine and intracellular cAMP concentrations. Chronic exposure to ethanol, however, causes heterologous desensitization of receptors coupled to adenylyl cyclase via stimulatory guanine nucleotide regulatory protein. This heterologous desensitization is correlated with a decrease in the amount of protein and mRNA for the GTP-binding subunit of stimulatory guanine nucleotide regulatory protein. In addition, after chronic exposure to ethanol, the adenosine transporter becomes tolerant to acute ethanol inhibition of adenosine uptake, and there is no longer an increase in extracellular adenosine. We have previously shown that extracellular adenosine is required for the development of ethanol-induced heterologous desensitization. To examine the role of adenosine receptors in mediating these responses to ethanol, we used BW A1434U, an adenosine receptor antagonist that does not inhibit nucleoside transport. BW A1434U caused a concentration-dependent inhibition of (-)-N6-(R-phenyl-isopropyl)-adenosine-stimulated cAMP production in NG108-15 cells. BW A1434U also completely blocked acute ethanol-induced increases in intracellular cAMP levels and prevented the development of ethanol-induced heterologous desensitization and the reduction in the GTP-binding subunit of stimulatory guanine nucleotide regulatory protein. In addition, BW A1434U prevented the development of tolerance to ethanol-induced inhibition of adenosine transport. Our results indicate that in NG108-15 cells, adenosine receptors mediate ethanol-induced changed in cAMP signal transduction and adenosine transport and that an adenosine receptor antagonist can block both these acute and chronic affects of ethanol.
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PMID:Adenosine receptors mediate cellular adaptation to ethanol in NG108-15 cells. 796 54

The molecular interfaces between Gs and the beta-adrenergic receptor were investigated using synthetic peptides corresponding to various regions of its alpha subunit, alpha s. These experiments were carried out on saponin-permeable C6 glioma cells in which the beta-adrenergic receptor appears tightly coupled to Gs. Synthetic site-specific peptides from alpha s (corresponding to amino acids 15-29, 354-372, and 384-394) and alpha i (8-22, 315-324, and 345-455) were tested for their ability to interfere with coupling between the beta-adrenergic receptor and Gs. The two carboxyl-terminal peptides from alpha s blocked beta-adrenergic stimulation of adenylyl cyclase in permeable cells. However, only alpha s-354-372 had this effect in C6 membranes. It is suggested that the partial uncoupling of Gs, which occurs subsequent to cell disruption, may be related to a change in the interaction of the alpha s carboxyl terminus with the beta-adrenoreceptor. Two carboxyl-terminal peptides, 354-372 and 384-394, could also mimic the effect of Gs to increase agonist affinity for the beta-adrenergic receptor. In combination, alpha s-354-372 and alpha s-384-394 increased the ability of isoproterenol to compete with 125I-pindolol binding in a partially additive manner. Synthetic peptides from alpha i and amino-terminal peptides from alpha s had no effect on beta-agonist binding, suggesting a high specificity of peptide effects. Two findings suggest that these peptides bind directly to the beta-adrenergic receptor and stabilize its high agonist affinity conformation. First, GTP and hydrolysis-resistant GTP analogs did not alter the high affinity binding in the presence of high concentrations of the peptides. Second, in S49 lymphoma cyc- cells, which lack Gs, these peptides evoked the high affinity agonist binding state of the beta-receptor. Neither peptide had an effect on antagonist binding affinity, as measured by propranolol displacement of 125I-pindolol. These data suggest that at least two regions on the alpha subunit of Gs participate in high affinity Gs binding to the beta-adrenergic receptor. The fact that these small peptides could mimic the holo-Gs effect on the receptor is rather surprising, and the specificity of the effect suggests that the primary and secondary structure of small regions of alpha s contain much of the information for specific interaction with beta-adrenergic receptors.
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PMID:Synthetic peptides as probes for G protein function. Carboxyl-terminal G alpha s peptides mimic Gs and evoke high affinity agonist binding to beta-adrenergic receptors. 806 88

The purpose of the present investigation was to determine whether the coupling of delta-opioid receptors to multiple G proteins in NG108-15 neuroblastoma x glioma cells is a characteristic limited to only this cell line (because of the high density of delta-opioid receptors) and to ascertain whether there is any correlation between delta-opioid agonist potency to inhibit adenylyl cyclase and to activate G proteins. Interactions between receptors and G proteins were investigated using agonist-stimulated incorporation of the photoreactive GTP analog azidoanilido[alpha-32P]GTP ([alpha-32P]AA-GTP) into G protein alpha subunits, with subsequent separation by urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In NG108-15, NS20Y, and N1E115 cell membranes, four alpha subunits (Gi2 alpha, one isoform of Gi3 alpha, and both isoforms of Go alpha) in the 39-41-kDa region were labeled with [alpha-32P]AA-GTP. The delta-opioid agonist [D-Ala2,D-Leu5]-enkephalin (DADLE) produced a dose-dependent, naloxone-reversible increase of [alpha-32P]AA-GTP incorporation into all four alpha subunit subtypes, in all cell lines tested. In addition, with the single exception of Gi3 alpha in NG108-15 cells, the maximal increases in incorporation of the photoaffinity label into all G alpha subunits induced by DADLE were similar. The Bmax values determined for delta-opioid receptors in NG108-15, NS20Y, and N1E115 cell membranes were 570, 370, and 120 fmol/mg of protein, respectively. Finally, although the IC50 values to inhibit intracellular cAMP production and affinity for DADLE were similar across the three cell lines, the EC50 values to produce labeling of the G alpha subunits between cell lines differed by > 100-fold. In fact, only in NS20Y cells were the IC50 and ED50 values comparable. Firstly, these results suggest that simultaneous coupling of the delta-opioid receptor to multiple G protein alpha subunits occurs in a variety of cell lines that express a range of receptor densities. Secondly, the magnitudes with which delta-opioid receptors interact with available G alpha subunits in response to agonist are approximately the same. Finally, there appears to be no relationship between the potency of agonists to inhibit adenylyl cyclase and that required for activation of G proteins.
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PMID:Interaction of delta-opioid receptors with multiple G proteins: a non-relationship between agonist potency to inhibit adenylyl cyclase and to activate G proteins. 819 Jan 15

1. Acetylcholine (ACh) produces two membrane current changes when applied to NG108-15 mouse neuroblastoma x rat glioma hybrid cells transformed (by DNA transfection) to express m1 muscarinic receptors: it activates a Ca(2+)-dependent K+ conductance, producing an outward current, and it inhibits a voltage-dependent K+ conductance (the M conductance), thus diminishing the M-type voltage-dependent K+ current (IK(M)) and producing an inward current. The present experiments were undertaken to find out how far inhibition of IK(M) might be secondary to stimulation of phospholipase C, by recording membrane currents and intracellular Ca2+ changes with indo-1 using whole-cell patch-clamp methods. 2. Bath application of 100 microM ACh reversibly inhibited IK(M) by 47.3 +/- 3.2% (n = 23). Following pressure-application of 1 mM ACh, the mean latency to inhibition was 420 ms at 35 degrees C and 1.79 s at 23 degrees C. Latencies to inhibition by Ba2+ ions were 148 ms at 35 degrees C and 92 ms at 23 degrees C. 3. The involvement of a G-protein was tested by adding 0.5 mM GTP-gamma-S or 10 mM potassium fluoride to the pipette solution. These slowly reduced IK(M), with half-times of about 30 and 20 min respectively, and rendered the effect of superimposed ACh irreversible. Effects of ACh were not significantly changed after pretreatment for 24 h with 500 ng ml-1 pertussis toxin or on adding up to 10 mM GDP-beta-S to the pipette solution. 4. The role of phospholipase C and its products was tested using neomycin (to inhibit phospholipase C), inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4), heparin, and phorbol dibutyrate (PDBu) and staurosporin (to activate and inhibit protein kinase C respectively). Both neomycin (1 mM external) and InsP3 (100 microM intrapipette) inhibited the ACh-induced outward current and/or intracellular Ca2+ transient but did not block ACh-induced inhibition of IK(M). Intrapipette heparin (1 mM) blocked activation of IK(Ca) and reduced Ach-induced inhibitions of IK(M), but also reduced inhibition of ICa via endogeneous m4 receptors. PDBu (with or without intrapipette ATP) and staurosporin had no significant effects.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:On the mechanism of M-current inhibition by muscarinic m1 receptors in DNA-transfected rodent neuroblastoma x glioma cells. 827 Nov 96

We have compared the characteristics of receptors for nucleotide analogues and the involvement of phospholipase C (PLC) in the effector mechanism in NG108-15 neuroblastoma and C6 glioma cells. The relative potency of these analogues to stimulate inositol phosphate (IP) formation is UTP > UDP >> 2-methylthio-ATP (2-MeSATP), GTP > ATP, CTP > ADP > UMP in NG108-15 cells and ATP > UTP > ADP > GTP > UDP >> 2Me-SATP, CTP, UMP, in C6 glioma cells. alpha, beta-Methylene-ATP, beta, gamma-methylene-ATP, AMP, and adenosine had little or no effect in both types of cells. The EC50 values were 3 and 106 microM for UTP in NG108-15 and C6 glioma cells, respectively. The EC50 value for ATP in C6 glioma cells was 43 microM. 2-MeSATP was threefold more potent than ATP in NG108-15 cells but had little effect in C6 glioma cells at 1 mM. In NCB-20 cells, a similar rank order of potency to that found in NG108-15 cells, i.e., UTP >> GTP > ATP > CTP, was observed. In both NG108-15 and C6 glioma cells, preincubation with ATP or UTP caused a pronounced cross-desensitization of subsequent nucleotide-stimulated IP production. ATP and UTP displayed no additivity in terms of IP formation at maximally effective concentrations. In contrast, endothelin-1, bradykinin, and NaF interacted in an additive manner with either nucleotide in stimulating PI hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Heterogeneity of nucleotide receptors in NG108-15 neuroblastoma and C6 glioma cells for mediating phosphoinositide turnover. 829 16

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

This study investigates the functional state of the stimulatory GTP-binding protein GS in neuroblastoma x glioma NG108-15 hybrid cells chronically exposed to an opioid. For this purpose, a novel in situ reconstitution protocol was established using membranes selectively depleted of GS function by transient exposure to low pH and then reconstituted with purified exogenous stimulatory GTP-binding proteins. With prostaglandin E1 (PGE1) receptor-stimulated adenylate cyclase activity as an indicator, reconstituted membranes of cells previously rendered tolerant to the delta-opioid [D-Ala2,D-Leu5]enkephalin (DADLE) exhibited approximately 3-fold elevated cAMP generation upon stimulation with PGE1, compared with nontolerant reconstituted cell membranes. This effect developed dose-dependently with respect to the opioid concentration used for pretreatment of the cells and was blocked by concomitant exposure to naloxone. In contrast, receptor-independent activation of GS by the stable GTP analogue guanosine-5'-O-(3-thio)triphosphate did not reveal any difference in adenylate cyclase activity between reconstituted membranes of control and chronically DADLE-pretreated cells. Furthermore, the functional activity of endogenous GS displayed no difference between control and DADLE-tolerant cells, as assessed in S49 cyc- reconstitution assays using sodium cholate extracts derived from NG108-15 membranes. The data presented suggest that the increase in PGE1 receptor-mediated adenylate cyclase activity in opioid-tolerant/dependent NG108-15 hybrid cells most likely relates to enhanced coupling efficiency between the PGE1 binding site (receptor) and GS. Moreover, our results support the concept that supersensitivity to excitatory drugs reflects an adaptive mechanism of cells chronically exposed to an opioid.
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PMID:Coupling of prostaglandin E1 receptors to the stimulatory GTP-binding protein Gs is enhanced in neuroblastoma x glioma (NG108-15) hybrid cells chronically exposed to an opioid. 838 8

We previously reported that transfection of antisense OBCAM (opioid-binding cell adhesion molecule) cDNA into NG108-15 neuroblastoma x glioma hybrid cells, which contain delta-opioid receptors, results in greatly reduced opioid binding (Ann, D. K., Hasegawa, J., Ko, J. L., Chen, S. T., Lee, N. M., and Loh, H. H. (1992) J. Biol. Chem. 267, 7921-7926. Here we report that these cells show altered coupling between opioid receptors and G-proteins. G-proteins were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for Gi2 and Go alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into a 39-41-kDa protein with the same electrophoretic mobility as Gi2 and Go alpha subunits. This band, which was also a pertussis toxin (PTX) substrate, exhibited decreased CTX-induced ADP-ribosylation in membranes of cells treated chronically with D-Ala2-D-Leu5-enkephalin (DADLE). In cells transfected with antisense cDNA for OBCAM, labeling of this band was also decreased, compared with either sense-transfected or untransfected cells. DADLE inhibition of adenylyl cyclase and DADLE stimulation of GTPase were also greatly impaired in antisense cells, as well as GTP and GppNHp inhibition of basal and forskolin-stimulated adenylyl cyclase. These results provide further evidence for a role of OBCAM in opioid receptor function.
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PMID:Transfection of NG108-15 cells with antisense opioid-binding cell adhesion molecule cDNA alters opioid receptor-G-protein interaction. 839 63


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