Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0017638 (glioma)
 30,880 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Opioids elicit an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells, which, depending upon growth conditions, results from either Ca2+ influx in differentiated cells or Ca2+ release from internal stores in undifferentiated cells (Jin et al., 1992). In this report we describe fura-2-based digital imaging studies that demonstrate that opioid-evoked Ca2+ release in these cells results from the activation of phospholipase C (PLC) and subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive store. D-Ala2-D-Leu5-enkephalin (DA-DLE) evoked concentration-dependent increases in [Ca2+]i (EC50 approximately equal to 4 nM). The response was blocked by naloxone (1 microM). In single cells, sequential application of selective opioid agonists (10 nM) evoked responses of the rank order DADLE = D-Pen2, D-Pen5-enkephalin (DPDPE) > trans-(+/-) 3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488) > D-ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO), consistent with activation of a delta-opioid receptor. Forty percent (n = 198) of the cells responded to 100 nM DADLE with a net [Ca2+]i increase of 483 +/- 40 nM. Bradykinin (100 nM) elicited a response in 91% of the cells with a mean net amplitude of 707 +/- 36 nM. The DADLE-evoked responses were not blocked by removal of extracellular Ca2+; instead, they were abolished by treatment with 10 nM thapsigargin, an agent that depletes and prevents refilling of IP3-sensitive Ca2+ stores. A 1 microM concentration of U73122, an aminosteroid inhibitor of PLC, completely blocked the DADLE-evoked [Ca2+]i increase, while an inactive analog, U73433, was without effect. To explore the possible role of G-proteins in mediating opioid-induced [Ca2+]i increases in NG108-15 cells, we pretreated cells with pertussis or cholera toxin; pertussis toxin blocked the opioid-induced response while cholera toxin was without effect, consistent with a Gi- or Go-mediated effect. Activation of the opioid inhibitory pathway previously described for these cells appears to stimulate the phosphoinositide (PI) cascade as well. Including the PI cascade among the multiple second messenger systems modulated by opioids may be key to understanding the biochemical events that underlie acute and chronic opioid action.
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PMID:Opioids mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells. 815 47

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

Long-term treatment with ethanol increases delta-opioid receptor (DOR) expression in the NG108-15 neuroblastoma x glioma hybrid cell line. To determine the underlying mechanism, we studied the effects of ethanol on [3H]diprenorphine binding to intact cells and DOR gene expression in four related clonal neural cell lines. Incubation with 200 mM ethanol for 48 hr increased [3H]diprenorphine binding by 1.4- (N18TG2), 1.8- (NG108-15), 1.9- (N4TG1), and 3.0-fold (N1E-115). Treatment with 25, 50, or 100 mM ethanol for 1 week caused a dose-dependent increase in receptor expression. Receptor up-regulation was associated with an increase in the potency of etorphine for inhibiting prostaglandin E1-stimulated cAMP accumulation. Constitutive DOR expression differed more than 3-fold among the different cell lines and correlated positively with basal cAMP levels. Long-term ethanol treatment increased basal cAMP levels in three of the four cell lines, but did not induce cellular differentiation. Northern blot analysis demonstrated an identical pattern of multiple transcripts in the four cell lines. Ethanol increased the abundance of DOR mRNA by approximately 3-fold in N18TG2 cells and by approximately 5-fold in the remaining cell lines. These findings indicate that clinically relevant concentrations of ethanol regulate DOR expression by increasing the abundance of DOR mRNA. The disparity between the increase in gene expression and ligand binding suggests that ethanol may also modify mRNA translation or receptor processing.
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PMID:Ethanol increases delta-opioid receptor gene expression in neuronal cell lines. 826 48

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

Using delta opioid receptor as a model system, acute desensitization of neuronal opioid receptor was studied in detail in neuroblastoma x glioma NG108-15 cells and primarily-cultured mouse cortical cells. The opioid desensitization could occur in as short as 3 minutes of agonist treatment and the half-life of the desensitized state was about 90 minutes. This acute opioid desensitization was homologous in nature in both neuronal cells. The acute desensitization was almost abolished by treatment of the neuronal cells with staurosporine, a nonspecific protein kinase inhibitor. Treatment with the protein kinase C-selective inhibitor, calphostin C, however, caused partial block. In conclusion, neuronal opioid receptor undergoes acute, agonist-dependent, and homologous desensitization, during which protein kinases appear to play an important role.
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PMID:delta Opioid receptor in neuronal cells undergoes acute and homologous desensitization. 860 89

In C6 glioma cells stably expressing a homogeneous population of the cloned rat mu opioid receptor, the binding affinities of opioid agonists and subsequent activation of G protein were examined. Opioid receptor number in membranes of these cells was high (10-30 pmol/mg protein [3H]diprenorphine binding sites). Opioids were found to bind to the receptor with high affinity [Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) 0.23 nM; sufentanil 0.034 nM; morphine 0.16 nM]. Activation of G protein by opioid agonists was examined by measuring the stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding. Sufentanil increased [35S]GTP gamma S binding by 326% with an EC50 value of 2.39 nM. Agonist stimulation of [35S]GTP gamma S binding was stereoselective, naltrexone-reversible, and pertussis toxin-sensitive. The "intrinsic activity" of opioids at the mu receptor was reflected by the magnitude of agonist-mediated activation of G protein. The rank order of the stimulation of [35S]GTP gamma S binding was etonitazene = sufentanil = DAMGO = PLO17 = fentanyl > morphine > profadol > meperidine > butorphanol = nalbuphine = pentazocine > cyclazocine = nalorphine > levallorphan > naltrexone. High affinity binding of ligands to the mu opioid receptor was reduced by the addition of sodium and guanosine diphosphate at concentrations used in the [35S]GTP gamma S binding assay. Ligand affinity was reduced in a manner correlating with "intrinsic activity". DAMGO, 1229-fold, nalbuphine 35-fold, naltrexone, 3-fold. The results presented show that the stable expression of the rat mu opioid receptor in C6 cells provides an effective tool to examine opioid receptor signal transduction mechanisms and evaluate the activity of novel opioids at the mu receptor.
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PMID:Characterization of opioid agonist efficacy in a C6 glioma cell line expressing the mu opioid receptor. 881 94

A large body of evidence implicates the second and third intracellular loops and the carboxyl-terminal portion of many G protein-coupled receptors as sites responsible for the interaction to G proteins. We synthesized a number of peptides from selected sites of the murine delta-opioid receptor and measured their ability to modify ligand-stimulated G protein activation and 3H agonist binding to the receptor. In membranes from Rat-1 fibroblasts transfected to express the murine delta-opioid receptor stably (clone D2 cells), the delta-opioid agonist [D-Ser2-Leu5-Thr6]enkephalin (DSLET) stimulated high affinity GTPase activity, which was inhibited by peptides that are derived from the proximal (i3.1) and the distal portions (i3.3) of the third intracellular loop with IC50 values of 15 +/- 5 and 50 +/- 4 microM, respectively. Peptides i3.1 and i3.3 inhibited DSLET-stimulated [35S]guanosine 5'-O-thiotriphosphate binding in the same membranes. However, a peptide designated i4, which was derived from a juxtamembranous region of the carboxyl-terminal tail of the delta-opioid receptor, failed to alter agonist-mediated high affinity GTPase activity or agonist-driven [35S]guanosine 5'-O-thiotriphosphate binding. Specific binding of [3H]DSLET to membrane preparations from clone D2 was reduced by peptides i3.1 and i4. Combinations of these peptides abolished detectable [3H]DSLET binding in the same membranes. Peptides i3.1 and i3.3 also destabilized the high affinity state of the receptor as assessed in 3H agonist binding on membranes from neuroblastoma X glioma (NG108-15) hybrid cells, which express the delta-opioid receptor endogenously; furthermore, delta-opioid receptor-stimulated GTPase activity in the same membranes was inhibited by peptides i3.1 and i3.3 but i4 was inactive. In contrast, peptides derived from the second intracellular loop (i2.1 and i2.2), an intermediate portion of the third intracellular loop (i3.2), and the extreme amino-terminal region of the receptor were without effect in these assays. These observations indicate that although peptides i3.1, i3.3, and i4 act via different mechanisms, they provide evidence that at least two sites of the third intracellular loop and part of the carboxyl-terminal tail of the delta-opioid receptor are important in the interaction between this receptor and cellular G proteins. Collectively, these results provide novel information about regions of the delta-opioid receptor that are involved in G protein coupling and high affinity agonist binding.
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PMID:Identification of the critical domains of the delta-opioid receptor involved in G protein coupling using site-specific synthetic peptides. 886 45

The effects of activation of the adenylyl cyclase-protein kinase A pathway on the expression of delta-opioid receptor mRNA in the NG108-15 neuroblastoma x glioma cell line has been investigated. Activation of prostaglandin E1 (PGE1) receptors, which are positively coupled to adenylyl cyclase, resulted in a reduction in delta-receptor messenger RNA levels. Direct stimulation of adenylyl cyclase by forskolin or treatment of cells with the cyclic AMP analogue dibutyryl cyclic AMP (db-cAMP) mimicked the effect of PGE1. Down-regulation in receptor protein levels, as measured by loss of radioligand binding sites, was also observed and its extent correlated well with the decrease in the amount of delta-opioid receptor transcripts. D-Ser2-Leu-enkephalin-Thr6 (DSLET) inhibition of adenylyl cyclase activity was also diminished after db-cAMP treatment. Inhibitors of protein kinase A (PKA) partially reversed the PGE1- and db-cAMP-mediated repression of the delta-opioid receptor mRNA levels. The rate of degradation of delta-opioid receptor mRNA in the presence of actinomycin D was not altered in response to db-cAMP, suggesting that mRNA stability is not reduced by PKA action. The regulation of delta-opioid receptor mRNA levels by db-cAMP was not sensitive to the protein synthesis inhibitor cycloheximide, suggesting that de novo protein synthesis is not required in this process.
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PMID:Regulation of delta-opioid receptor mRNA levels by receptor-mediated and direct activation of the adenylyl cyclase-protein kinase A pathway. 900 47

The delta-opioid receptors in mouse neuroblastoma x rat glioma NG108-15 cells were characterized by receptor binding and cAMP assays. Saturation binding assays using [3H][D-Pen5]enkephalin (DPDPE) or [3H][D-Ser2, Leu5, Thr6]enkephalin (DSLET) gave similar binding capacities (Bmax). Competition binding assays showed that DPDPE and DSLET have similar affinity for the [3H]DPDPE or 3[H]DSLET binding sites. The rank order of potency of competition with [3H]DPDPE and [3H]DSLET was similar: naltriben approximately DSLET > or = DPDPE > 7-benzylidenenaltrexone (BNTX). Both DPDPE and DSLET were found to decrease cAMP formation. The action of DSLET was antagonized by naltriben but not BNTX, while the action of DPDPE was reversed by both antagonists. Therefore, the delta-opioid receptor in NG108-15 cells has similar affinity for the agonists DPDPE and DSLET, and a higher affinity for the antagonist naltriben than BNTX.
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PMID:Determination of delta-opioid in NG108-15 cells. 903 Sep 5

Neuroblastoma x glioma NG108-15 hybrid cells have been examined for the expression of opioid receptor-like receptor (ORL1). [3H]Nociceptin/orphanin FQ (OFQ) bound to the cell membrane specifically (Kd = 3.6 +/- 0.6 nM) and inhibited forskolin-stimulated cAMP accumulation (EC50 = 0.72 +/- 0.3 nM). The responsiveness of NG108-15 cells to nociceptin/OFQ was blocked by pertussis toxin but not by naltrindole. The inhibitory activity of nociceptin/OFQ was significantly reduced after a prechallenge with the same peptide but was not influenced by DPDPE pretreatment, indicating acute and homologous desensitization of ORL1 receptors. Naltrindole caused the overshoot of cAMP in DPDPE-pretreated cells but not in nociceptin/OFQ-pretreated cells. The results indicate that ORL1 is functionally expressed and does not cross-interact with specific ligands of the delta opioid receptor in NG108-15 cells.
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PMID:Functional expression, activation and desensitization of opioid receptor-like receptor ORL1 in neuroblastoma x glioma NG108-15 hybrid cells. 903 67


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