guanosine-5--o-(3-thiotriphosphate) and Glioma

Regulators of G protein signaling (RGS) proteins act as GTPase-accelerating protein to negatively modulate G protein signaling and are defined by a conserved RGS domain with considerable amino acid diversity. To determine the effects of specific, purified RGS proteins on mu-opioid signaling, C6 cells stably expressing a mu-opioid receptor were rendered permeable to proteins by treatment with digitonin. Mu-opioid inhibition of forskolin-stimulated adenylyl cyclase by [D-Ala(2),N-Me-Phe(4),Gly-ol]-enkephalin (DAMGO), a mu-specific opioid peptide, remained fully intact in permeabilized cells. Purified RGS domain of RGS4 added to permeabilized cells resulted in a twofold loss in DAMGO potency but had no effect in cells expressing RGS-insensitive G proteins. The inhibitory effect of DAMGO was reduced to the same extent by purified RGS4 and RGS8. In contrast, the RGS domain of RGS7 had no effect and inhibited the action of RGS8 as a result of weak physical association with Galphai2 and minimal GTPase-accelerating protein activity in C6 cell membranes. These data suggest that differences in conserved RGS domains of specific RGS proteins contribute to differential regulation of opioid signaling to adenylyl cyclase and that a permeabilized cell model is useful for studying the effects of specific RGS proteins on aspects of G protein-coupled receptor signaling.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Cell Line, Tumor; Colforsin; Cyclic AMP; Digitonin; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Protein Binding; Rats; Receptors, Opioid, mu; RGS Proteins; Signal Transduction

Chronic exposure of cells to mu-opioid agonists leads to tolerance which can be measured by a reduced ability to activate signaling pathways in the cell. Cell signaling through inhibitory G proteins is negatively regulated by RGS (regulator of G protein signaling) proteins. Here we examine the hypothesis that the GTPase accelerating activity of RGS proteins, by altering the lifetime of Galpha and Gbetagamma, plays a role in the development of cellular tolerance to mu-opioids. C6 glioma cells were stably transfected with mu-opioid receptor and pertussis toxin (PTX)-insensitive Galpha(o) that was either sensitive or insensitive to endogenous RGS proteins. Cells were treated with PTX to uncouple endogenous Galpha proteins followed by exposure to the mu-opioid agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) or morphine. Receptor desensitization as measured by agonist-stimulated [(35)S]GTPgammaS binding and receptor down-regulation as measured by [(3)H]diprenorphine binding were increased in cells expressing RGS-insensitive Galpha(o). Exposure to high concentrations of morphine or the peptidic mu-opioid agonist DAMGO led to a tolerance to inhibit adenylyl cyclase activity in both cell types with a rapid (30 min) and a slower component. Using a submaximal concentration of DAMGO to induce a reduced level of tolerance, a shift in the concentration-effect curve for DAMGO to inhibit adenylyl cyclase activity was seen in the cells expressing RGS-insensitive Galpha(o), but not in the cells expressing RGS-sensitive Galpha(o), which can be partly explained by an increased supersensitization of the adenylyl cyclase response. The results show that RGS proteins endogenously expressed in C6 cells reduce agonist-induced mu-opioid receptor desensitization, down-regulation, and sensitivity to tolerance to inhibit adenylyl cyclase activity.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Cell Line, Tumor; Cells, Cultured; Cyclic AMP; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glioma; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Pertussis Toxin; Receptors, Opioid, mu; RGS Proteins

Several G protein-coupled receptors function within lipid rafts plasma membrane microdomains, which may be important in limiting signal transduction. Here we show that treatment of rat C6 glioma cells with the raft disruptor methyl-beta-cyclodextrin (MCD) doubles the binding efficiency (i.e. the ratio between maximum binding and dissociation constant) of type-1 cannabinoid receptors (CB1R), which belong to the rhodopsin family of G protein-coupled receptors. In parallel, activation of CB1R by the endogenous agonist anandamide (AEA) leads to approximately 3-fold higher [35S]GTPgammaS binding in MCD-treated cells than in controls, and CB1R-dependent signaling via adenylate cyclase, and p42/p44 MAPK is almost doubled by MCD. Unlike CB1R, the other AEA-binding receptor TRPV1, the AEA synthetase NAPE-PLD, and the AEA hydrolase FAAH are not modulated by MCD, whereas the activity of the AEA membrane transporter (AMT) is reduced to approximately 50% of the controls. We also show that MCD reduces dose-dependently AEA-induced apoptosis in C6 cells but not in human CHP100 neuroblastoma cells, which mirror the endocannabinoid system of C6 cells but are devoid of CB1R. MCD reduces also cytochrome c release from mitochondria of C6 cells, and this effect is CB1R-dependent and partly mediated by activation of p42/p44 MAPK. Altogether, the present data suggest that lipid rafts control CB1R binding and signaling, and that CB1R activation underlies the protective effect of MCD against apoptosis.

Topics: Animals; Apoptosis; Arachidonic Acids; beta-Cyclodextrins; Biological Transport; Cannabinoid Receptor Modulators; Cell Line, Tumor; Cell Membrane; Cell Separation; Cholesterol; Cyclic AMP; Dose-Response Relationship, Drug; Endocannabinoids; Flow Cytometry; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hydrolysis; Kinetics; Lipid Metabolism; Lipids; Membrane Microdomains; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroblastoma; Neurons; Polyunsaturated Alkamides; Protein Binding; Rats; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Rhodopsin; Signal Transduction; Time Factors

Acute mu opioid application has been shown to activate extracellular signal-related kinases (ERKs) in various non-neural cell lines. However, ERK activation in neuronal cells following acute morphine treatment is more questionable. Moreover, the ERK activation phenomenon observed in vivo after withdrawal of chronic opioids has never been demonstrated in vitro. The goal of this study was to determine if mu agonist treatment induced ERK activation acutely or after withdrawal of chronic opioids in one glial and three neuronal cell lines. We found that acute application of opioids was not able to activate ERK in neuronal cell lines but was able to activate ERK in a glial cell line. In another set of experiments, cells were chronically treated with escalating doses of a mu opioid agonist. After 8 days, the agonist was removed from the media and naloxone applied. Acute ERK activation was not seen in any tested cell line after agonist removal. These findings suggest that opioids may acutely activate ERK in non-neuronal cells, and that the acute ERK activation observed in some brain regions during opioid withdrawal in vivo might be mediated by indirect effects on neuronal cells.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Diprenorphine; Drug Administration Schedule; Enzyme Activation; Fentanyl; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Mitogen-Activated Protein Kinases; Naloxone; Narcotic Antagonists; Narcotics; Neuroblastoma; Neuroglia; Neurons; Radioligand Assay; Rats; Time Factors

A ligand-independent activator of heterotrimeric brain G-protein was partially purified from detergent-solubilized extracts of the neuroblastoma-glioma cell hybrid NG108-15. The G-protein activator (NG108-15 G-protein activator (NG-GPA)) increased [(35)S]guanosine 5'-O-(thiotriphosphate) ([(35)S]GTPgammaS) to purified brain G-protein in a magnesium-dependent manner and promoted GDP dissociation from Galpha(o). The NG-GPA also increased GTPgammaS binding to purified, recombinant Galpha(i2), Galpha(i3), and Galpha(o), but minimally altered nucleotide binding to purified transducin. The NG-GPA increased GTPgammaS binding to membrane-bound G-proteins and inhibited basal, forskolin- and hormone-stimulated adenylyl cyclase activity in DDT(1)-MF-2 cell membranes. In contrast to G-protein coupled receptor-mediated activation of heterotrimeric G-proteins in DDT(1)-MF-2 cell membrane preparations, the action of the NG-GPA was not altered by treatment of the cells with pertussis toxin. ADP-ribosylation of purified brain G-protein also failed to alter the increase in GTPgammaS binding elicited by the NG-GPA. Thus, the NG-GPA acts in a manner distinct from that of a G-protein coupled receptor and other recently described receptor-independent activators of G-protein signaling. These data indicate the presence of unexpected regulatory domains on G(i)/G(o) proteins and suggest the existence of pertussis toxin-insensitive modes of signal input to G(i)/G(o) signaling systems.

Topics: Animals; Brain; Carrier Proteins; Cattle; Cell Line; Cell Membrane; Colforsin; Glioma; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Isoproterenol; NAD; Neuroblastoma; Pertussis Toxin; Phosphorus Radioisotopes; Tumor Cells, Cultured

Opioid agonists acting at their receptors alter intracellular events by initiating activation of various types of Gi/Go proteins. This can be measured by the binding of the stable GTP analog [(35)S]guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS). In this study agonist efficacy is defined by the degree to which an opioid stimulates the binding of [(35)S]GTPgammaS. This allows for a definition of full and partial agonists; a full agonist causing a greater stimulation of [(35)S]GTPgammaS binding than a partial agonist. The hypothesis that the rate of agonist-stimulated [(35)S]GTPgammaS binding is dependent upon agonist efficacy was tested using membranes from C6 glioma cells expressing mu- or delta-opioid receptors. At maximal concentrations the rate of agonist-stimulated [(35)S]GTPgammaS binding followed the efficacy of mu-agonists in stimulating [(35)S]GTPgammaS binding, i.e., [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin > morphine > meperidine > butorphanol > nalbuphine. At submaximal concentrations of mu- or delta-full agonists the [(35)S]GTPgammaS association rate was also reduced, such that the rate of [(35)S]GTPgammaS binding correlated with the extent of [(35)S]GTPgammaS bound, whether this binding was stimulated by a full agonist or a partial agonist. Agonists also stimulated [(35)S]GTPgammaS dissociation, showing that binding of this stable nucleotide was reversible. Comparison of the delta-agonists [D-Ser(2),Leu(5)]-enkephalin-Thr and (+/-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxylbenzyl)-N,N-diethylbenzamide, a compound with slow dissociation kinetics, showed the measured rate of G protein activation was not influenced by the agonist switching between receptors. The results are consistent with the idea that the active state(s) of the receptor induced by full or partial agonists is the same, but the number of activated receptors determines the rate of G protein activation.

Topics: Biotransformation; Cell Membrane; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Half-Life; Humans; Kinetics; Molecular Conformation; Nalbuphine; Narcotic Antagonists; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sulfur Radioisotopes; Tumor Cells, Cultured

No G(i)-linked P2Y receptors have been cloned to date but the presence of such receptors is thought to be restricted to platelets and certain clonal cell lines. Using the functional approach of [(35)S]guanosine 5'-[gamma-thio]-triphosphate autoradiography, we uncovered the widespread presence of such receptors in the CNS. Under conditions in which the prominent signal due to tonic adenosine receptor activity is masked, ADP and ATP stimulated G-protein activity in multiple grey and white matter regions. Localization in the grey matter suggests inhibitory auto-/heteroreceptor function. In the white matter, activated G proteins appeared as 'hot spots' (presumed oligodendrocyte progenitors) with scattered distribution along the main fibre tracts. Responses to ATP were diminished under conditions that inhibited degradation, suggesting that prior conversion to ADP explained agonist action. Uracil nucleotides were ineffective but 2-methylthio-ADP activated G proteins approximately 500-fold more potently than ADP, although both were similarly degraded. Throughout the brain, ADP-dependent G-protein activity was reversed by 2-hexylthio-AdoOC(O)Asp(2), a non-phosphate ATP analogue, whereas selective P2Y(1) receptor antagonists proved ineffective. A similar receptor was also disclosed from the adrenal medulla. These data witness a hitherto unrecognized abundance of G(i/o)-linked ADP receptors in the nervous system. Biochemical and pharmacological behaviour suggests striking similarities to the elusive platelet P2Y(ADP) receptor.

Topics: Adenine Nucleotides; Adenosine; Adenosine Diphosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Adrenal Medulla; Animals; Autoradiography; Blood Platelets; Brain; Brain Chemistry; Capillaries; Cell Line; Dithiothreitol; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Glioma; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); Heterotrimeric GTP-Binding Proteins; Macromolecular Substances; Magnetic Resonance Spectroscopy; Male; Membrane Proteins; Nerve Tissue Proteins; Neuroglia; Organ Specificity; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2Y12; Signal Transduction; Sulfhydryl Reagents; Sulfur Radioisotopes; Thionucleotides; Tumor Cells, Cultured; Uracil Nucleotides; Xanthines

The guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding assay for the determination of relative opioid efficacy has been adapted to measure G protein activation in digitonin-permeabilized C6 rat glioma cells expressing a cloned mu-opioid receptor. The mu-agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) caused a 3-fold increase in [35S]GTPgammaS binding over basal in a naloxone-sensitive manner. Relative mu-agonist efficacy was DAMGO > fentanyl > or = morphine > buprenorphine. Nalbuphine showed no efficacy. G protein activation by receptors has been predicted to occur by random encounter. In this model a reduction in the number of receptors will decrease the rate of G protein activation but not the maximum number of G proteins activated. To test this model C6 mu cells were treated with the irreversible mu-antagonist beta-funaltrexamine (10 nM) prior to permeabilization. This reduced the number of mu-opioid receptors determined with [3H]diprenorphine to 23 +/- 3% of control with no change in affinity. A commensurate reduction (to 29 +/- 10% of control) in the level of [35S]GTPgammaS binding stimulated by DAMGO was observed, but the t(1/2) for [35S]GTPgammaS binding remained unchanged. Thus, random encounters of receptor and G protein failed to occur in this permeabilized cell preparation. A model that assumes an organized association of G proteins with receptors better describes the activation of G proteins by opioid mu-receptors.

Topics: Analgesics, Opioid; Animals; Digitonin; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Indicators and Reagents; Naloxone; Naltrexone; Narcotic Antagonists; Rats; Receptors, Opioid, mu; Tumor Cells, Cultured

1. Caveolins are integral proteins of glycolipid/cholesterol-rich plasmalemmal caveolae domains, where, they may function as a plasma membrane scaffold onto which many classes of signalling molecules, including receptors and heterotrimeric G proteins, can assemble. To ascertain whether caveolins influence G protein-mediated signal transduction, we stably expressed caveolin-1 and -3 isoforms in the neuroblastoma x glioma NG108-15 hybrid cell line, lacking endogenous caveolins. Subsequently, using whole-cell voltage clamp methods, we examined whether the modulation of N-type voltage-gated Ca2+ channels by G(o) protein-coupled, delta-type opioid receptors might be affected by recombinant caveolin expression. 2. In transfected NG108-15 cells, caveolins localized at the plasma membrane and, upon subcellular fractionation on sucrose density gradients, they co-localized in Triton-resistant, low buoyancy fractions, with endogenous G(o) protein alpha-subunits. 3. The voltage-dependent inhibition of omega-conotoxin GVIA-sensitive Ba2+ currents following either activation of delta-opioid receptors by the agonist [o-pen2,o-pen5]-enkephalin (DPDPE), or direct stimulation of G proteins with guanosine 5'-O-(thiotriphosphate) (GTPgammaS) was significantly attenuated in caveolin-expressing cells. The kinetics of Ca2+ channel inhibition were also modified by caveolins. 4. Overall, these results suggest that caveolins may negatively affect G protein-dependent regulation of voltage-gated N-type Ca2+ channels, presumably by causing a reduction of the available pool of activated G proteins.

Topics: Analgesics, Opioid; Animals; Calcium Channels, N-Type; Caveolin 1; Caveolin 3; Caveolins; Electrophysiology; Enkephalin, D-Penicillamine (2,5)-; Gene Expression; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Hybrid Cells; Membrane Potentials; Neuroblastoma; Neurons; Rats; Recombinant Proteins

Chronic opioid regulation of stimulatory receptor activity was investigated in neuroblastoma x glioma (NG108-15) hybrid cells stably transfected to express the human beta(2)-adrenoceptor (beta(2)-AR). Expressed beta(2)-ARs are functionally coupled to G proteins and display ligand-independent signalling activity, as demonstrated by the ability of an inverse agonist to attenuate basal adenylyl cyclase (AC) activity. Despite the relative increase in basal AC activity due to the development of tolerance/dependence, chronic morphine treatment was found to completely abolish spontaneous beta(2)-AR activity by reducing basal receptor/G protein precoupling. A similar chronic opioid effect was observed in transiently transfected COS-7 cells. These results indicate that during the state of opioid tolerance/dependence basal levels of AC activity are no longer under the control of spontaneously active stimulatory receptors.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Binding, Competitive; COS Cells; Drug Tolerance; Enzyme Inhibitors; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hybrid Cells; Iodine Radioisotopes; Morphine; Narcotics; Neuroblastoma; Pindolol; Propanolamines; Receptors, Adrenergic, beta-2; Signal Transduction; Transfection

A C6 glioma cell line stably transfected with the human kappa opioid receptor (kappaOR) was used to characterize receptor binding and G protein activation via the kappaOR by a comprehensive series of opioid ligands. The ligand-binding affinity for [3H]5alpha,7alpha, 8beta(-)-N-methyl-N-(7-Cl-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl)benzene acetamide (U69593) was similar to that observed in monkey brain membranes and was 10-fold lower in the presence of sodium and GDP. Both peptide and nonpeptide agonists maximally stimulated [35S]GTPgammaS binding. The stimulation of [35S]GTPgammaS binding was blocked by pretreatment of cells with pertussis toxin. Partial stimulation of [35S]GTPgammaS binding via the kappaOR was observed for several ligands that are antagonists at the mu opioid receptor, suggesting an additional mechanism of drug action. The ability of isomers of tifluadom and levallorphan to stimulate [35S]GTPgammaS binding indicates that the chiral carbon of levallorphan, a benzomorphan derivative, imparts a greater degree of stereoselectivity than does the chiral carbon in the benzodiazepine derivative tifluadom. In addition, (-)tifluadom, the less potent isomer of tifluadom, which is also a gamma-aminobutyric acidA receptor agonist, stimulated [35S]GTPgammaS binding. In contrast, d-pentazocine, (+)SKF10047, (+)cyclazocine, and d-ethylketocyclazocine displayed no agonist activity. kappaOR-selective antagonist norbinaltorphimine competitively inhibited the stimulation of [35S]GTPgammaS binding by the active isomers of ethylketocyclazocine, cyclazocine, and nalorphine to the same degree, indicating that all three ligands are eliciting an effect via the kappaOR. The results suggest that these cells express a homogeneous population of kappaOR, and that their [35S]GTPgammaS-binding properties make them an excellent means to assess kappaOR efficacy.

Topics: Analgesics, Opioid; Animals; Benzeneacetamides; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Naloxone; Naltrexone; Narcotic Antagonists; Pyrrolidines; Rats; Receptors, Opioid, kappa; Stereoisomerism; Transfection; Tritium; Tumor Cells, Cultured

Phosducin (Phd) regulates the function of G proteins by its ability to tightly bind Gbetagamma subunits. Because the internalization of opioid receptors as well as the activity of adenylyl cyclase (AC) activity depends on G proteins, we tested Phd on these parameters. NG 108-15 hybrid cells stably expressing the phosphoprotein were challenged with [D-penicillamine2,D-penicillamine5]enkephalin to inhibit cAMP generation, demonstrating an increased efficacy of the opioid on AC. Studying the binding of [35S]guanosine-5'-O-(gamma-thio)-triphosphate to membranes from Phd overexpressing cells, we found that [D-penicillamine2, D-penicillamine5 ]enkephalin failed, in the presence of Phd (0.1 nM), to elevate incorporation of the nucleotide. Phd also strongly inhibited opioid-stimulated GTPase activity. NG 108-15 cells were also employed to investigate the effect of Phd on opioid receptor internalization. Control cells and cells overexpressing Phd were transiently transfected to express mu-opioid receptors fused to green fluorescence protein. In controls and in Phd overexpressing cells confocal microscopy identified fluorescence associated with the membrane. Time-lapse series microscopy of living control cells challenged with etorphine (1 microM) revealed receptor internalization within 30 min. In contrast, Phd overexpressing cells largely failed to respond to the opioid. Thus, in Phd overexpressing cells, opioids exhibit an increased efficacy despite the inhibitory action of the phosphoprotein on opioid-stimulated incorporation of [35S]guanosine-5'-O-(gamma-thio)-triphosphate. We suggest that inhibition of GTPase stabilizes the opioid-induced G protein Gi-GTP complex, which is believed to enhance AC inhibition. Finally, scavenging of Gbetagamma by Phd attenuates internalization of opioid receptors, which may contribute to the efficacy of opioids.

Topics: Cyclic AMP; DNA; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Eye Proteins; Flow Cytometry; Glioma; Green Fluorescent Proteins; GTP Phosphohydrolases; GTP-Binding Protein Regulators; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hybrid Cells; Luminescent Proteins; Microscopy, Confocal; Nerve Tissue Proteins; Neuroblastoma; Phosphoproteins; Radioligand Assay; Receptors, Opioid; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured

The present study reports on G-protein activation by recombinant 5-HT receptors and by native 5-HT1A and 5-HT1B receptors in guinea-pig and rat brain using agonist-stimulated [35S]GTPgammaS binding responses mediated by a new 5-HT ligand, a dimer of sumatriptan. Dimerization of sumatriptan increased the binding affinity for h 5-HT1B (pKi: 9.22 vs. 7.79 for sumatriptan), h 5-HT1D (9.07 vs. 8.08) and also h 5-HT1A receptors (7.80 vs. 6.40), while the binding affinity for h 5-ht1E (6.67 vs. 6.19) and h 5-ht1F (7.37 vs. 7.78) receptors was not affected. Sumatriptan dimer (10 microM) stimulated [35S]GTPgammaS binding mainly in the superficial gray layer of the superior colliculi, hippocampus and substantia nigra of guinea-pig and rat coronal brain sections. This fits with the labelling by the 5-HT1B/1D receptor antagonist [3H] GR 125743. The observed [35S]GTPgammaS binding responses in the substantia nigra are likely to be mediated by stimulation of the 5-HT1B receptor subtype, since they were antagonized by the 5-HT1B inverse agonist SB 224289 (10 microM), and not by the 5-HT2A/1D antagonist ketanserin (10 microM). Quantitative assessment of the [35S]GTPgammaS binding responses in the substantia nigra of rat showed highly efficacious responses for both sumatriptan dimer and its monomer. In contrast, less efficacious agonist responses (51+/-10% and 35+/-13%, respectively) were measured in the guinea-pig substantia nigra. This may suggest that the G-protein coupling efficacy of 5-HT1B receptors is different between the substantia nigra of both species. In addition, the sumatriptan dimer also activated guinea-pig and rat hippocampal 5-HT1A receptors with high efficacy in contrast to sumatriptan. Therefore, dimerization of sumatriptan can be considered as a new approach to transform a partial 5-HT1A agonist into a more efficacious agonist. In conclusion, the sumatriptan dimer stimulates G-protein activation via 5-HT1B receptors besides 5-HT1A receptors in guinea-pig and rat brain. The magnitude of the 5-HT1B receptor responses is superior for sumatriptan and its dimer in rat compared to guinea-pig substantia nigra.

Topics: Animals; Autoradiography; Binding, Competitive; Brain Chemistry; Dimerization; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guinea Pigs; HeLa Cells; Humans; Male; Membrane Proteins; Oxadiazoles; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT1B; Receptors, Serotonin; Receptors, Serotonin, 5-HT1; Serotonin Receptor Agonists; Sulfur Radioisotopes; Sumatriptan; Tryptamines

The influence of membrane microviscosity on mu-opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH-SY5Y cells and from a C6 glioma cell line stably expressing the rat mu-opioid receptor (C6mu). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) for the mu-opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS. The effect of CHS on agonist binding was reversed by subsequent addition of cis-vaccenic acid, suggesting that the effect of CHS is the result of increased membrane microviscosity and not a specific sterol-receptor interaction. CHS addition increased the potency of DAMGO to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate binding by fourfold, whereas the potency of nalbuphine was unaffected. However, nalbuphine efficacy relative to that of the full agonist DAMGO was strongly increased in CHS-treated membranes compared with that in control membranes. Membrane rigidification also resulted in an increased efficacy for the partial agonists meperidine, profadol, and butorphanol relative to that of DAMGO as measured by agonist-stimulated GTPase activity in control and CHS-modified membranes. These findings support a regulatory role for membrane microviscosity in receptor-mediated G protein activation.

Topics: Animals; Cell Line; Cell Membrane; Cholesterol Esters; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Membrane Fluidity; Nalbuphine; Naltrexone; Narcotic Antagonists; Narcotics; Neuroblastoma; Protein Conformation; Rats; Receptors, Opioid, mu; Sodium Chloride; Sufentanil; Tumor Cells, Cultured; Viscosity

Chronic exposure of neuroblastoma x glioma (NG108-15) hybrid cells and rat mu-receptor-transfected Chinese hamster ovary (CHO) cells to 10 microM morphine resulted in a compensatory and antagonist-precipitated increase in cAMP accumulation. However, incubation of these cells with 10 microM methadone during chronic exposure to morphine substantially prevented the actions of morphine. Chronic methadone treatment caused a pronounced reduction in agonist-stimulated binding of [35S]GTPgammaS to G proteins, but it did not produce significant down-regulation of delta-opioid receptors, whereas chronic morphine treatment failed to induce either uncoupling of delta-opioid receptors from G proteins or down-regulation of delta-opioid receptors. In contrast to chronic treatment with morphine alone, treatment of cells with morphine and methadone simultaneously resulted in a significant decrease in agonist-stimulated binding of [35S]GTPgammaS to G proteins. The action of methadone-mediated uncoupling of the receptor from the G protein was blocked by the nonselective protein kinase inhibitor [1-(5-isoqinolinesulfony)-2-methylpiprazine](H7), but not by the specific protein kinase C inhibitor, chelerythrine. The data demonstrate that methadone desensitizes the delta-opioid receptor by uncoupling the receptor from the G protein. In this way, methadone antagonizes the morphine-mediated adaptive sensitization and overshoot of adenylate cyclase. The functional desensitization of opioid receptors by methadone may explain why methadone is effective in the treatment of morphine dependence.

Topics: Animals; CHO Cells; Cricetinae; Cyclic AMP; Down-Regulation; Drug Interactions; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Methadone; Morphine; Neuroblastoma; Protein Binding; Rats; Receptors, Opioid, delta; Time Factors; Tumor Cells, Cultured

To observe attenuative effects of agmatine on opiate desensitization and substance dependence.. Guanosine 5'-O-(3-[35S] thiotriphosphate) ([35S]GTTP) binding and cellular cyclic AMP (cAMP) level were determined by radioligand binding assay and radioimmunoassay in NG108-15 cells, respectively.. Agmatine increased stimulative action of opioids on [35S]GTTP binding by about 35% and inhibitory effects of opioids on cellular cAMP concentration by about 114.3% in NG108-15 cells pretreated with opioids. On the other hand, it also inhibited cAMP over-shooting by 214.9% of morphine substance dependent cells precipitated by naloxone compared with that of control. These effects of agmatine were antagonized by idazoxan in a concentration-dependent manner.. Agmatine reversed the formative process of adaptation in cAMP signal transduction cascade.

Topics: Adaptation, Physiological; Adrenergic alpha-Antagonists; Agmatine; Analgesics; Animals; Cyclic AMP; Enkephalin, D-Penicillamine (2,5)-; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Hybrid Cells; Idazoxan; Imidazoline Receptors; Mice; Morphine; Neuroblastoma; Rats; Receptors, Drug; Signal Transduction; Tumor Cells, Cultured

Acute incubation of NMDA with neuroblastoma x glioma hybrid (NG108-15) cells or neuroblastoma SK-N-SH cells produced significant attenuation of nociceptin/orphanin FQ (N/OFQ)-induced activation of G protein and inhibition of adenylyl cyclase. The attenuation of N/OFQ signaling by NMDA was dose-dependent, blockable by NMDA antagonists, and not observed in cells lacking NMDA receptors, indicating that the effect of NMDA is mediated by the NMDA receptor. Furthermore, NMDA antagonist pretreatment greatly attenuated N/OFQ-induced acute homologous desensitization of ORL1. Interestingly, the signaling induced by etorphine, an opioid agonist of wide spectrum, was sensitive to NMDA treatment in NG108-15 but insensitive in SK-N-SH cells, suggesting differential modulation of opioid signaling by NMDA. The attenuation effects of NMDA on mu opioid receptor-mediated signaling were also observed.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hybrid Cells; Kinetics; Mice; N-Methylaspartate; Narcotic Antagonists; Neuroblastoma; Neurons; Nociceptin; Nociceptin Receptor; Opioid Peptides; Rats; Receptors, Opioid; Signal Transduction

Chemokine receptor CCR5 is not only essential for chemotaxis of leukocytes but also has been shown to be a key coreceptor for HIV-1 infection. In the present study, hemagglutinin epitope-tagged human CCR5 receptor was stably expressed in Chinese hamster ovary cells or transiently expressed in NG108-15 cells to investigate CCR5-mediated signaling events. The surface expression of CCR5 was confirmed by flow cytometry analysis. The CCR5 agonist RANTES stimulated [35S]GTPgammaS binding to the cell membranes and induced inhibition on adenylyl cyclase activity in cells expressing CCR5. The effects of RANTES were CCR5 dependent and could be blocked by pertussis toxin. Furthermore, overexpression of Gialpha2 strongly increased both RANTES-dependent G-protein activation and inhibition on adenylyl cyclase in cells cotransfected with CCR5. These data demonstrated directly that activation of CCR5 stimulated membrane-associated inhibitory G proteins and indicated that CCR5 could functionally couple to G-protein subtype Gialpha2. The abilities of CCR5 to activate G protein and to inhibit cellular cAMP accumulation were significantly diminished after a brief prechallenge with RANTES, showing rapid desensitization of the receptor-mediated responsiveness. Prolonged exposure of the cells to RANTES caused significant reduction of surface CCR5 as measured by flow cytometry, indicative of agonist-dependent receptor internalization. Our data thus demonstrated that CCR5 functionally couples to membrane-associated inhibitory G proteins and undergoes agonist-dependent desensitization and internalization.

Topics: Adenylate Cyclase Toxin; Animals; Chemokine CCL5; CHO Cells; Cricetinae; Glioma; GTP-Binding Protein alpha Subunit, Gi2; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Hemagglutinin Glycoproteins, Influenza Virus; Humans; Hybrid Cells; Neuroblastoma; Pertussis Toxin; Proto-Oncogene Proteins; Receptors, CCR5; Recombinant Proteins; Transfection; Virulence Factors, Bordetella

To compare activation of G proteins by opioid receptors, opioid agonist-stimulated guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP gamma S) binding in the presence of excess GDP was assayed in membranes from NG108-15 (delta) and SK-N-SH (primarily mu) cells. Basal [35S]GTP gamma S binding consisted of a single class of low-affinity sites (KD 400-500 nM). Addition of agonists produced a high-affinity site 100-300-fold higher in affinity than the basal site. The receptor/transducer amplification factor (ratio of activated G protein Bmax to opioid receptor Bmax) was 10-fold higher for SK-N-SH mu receptors than for NG108-15 delta receptors. Chronic delta agonist ([D-Ser2]-Leu-enkephalin-Thr; DSLET) treatment of NG108-15 cells resulted in an 80% loss of DSLET-stimulated [35S]-GTP gamma S binding within 1 h. Morphine treatment of SK-N-SH cells decreased mu agonist ([D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin; DAMGO)-stimulated [35S]GTP gamma S binding by 45% after 16 h, with no effect after 1 h. Loss of agonist response was due to a decrease in the Bmax of activated G proteins with no change in the KD. These results provide a quantitative description of G protein activation occurring on acute and chronic exposure to opioid agonists.

Topics: Analgesics; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hybrid Cells; Membrane Proteins; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Narcotics; Neuroblastoma; Protein Binding; Rats; Receptors, Opioid, delta; Receptors, Opioid, mu; Signal Transduction; Sulfur Radioisotopes; Time Factors

In this study we examined the biochemical properties and subcellular localization of Rab3A, Rab3B and Rab3C in bovine adrenal chromaffin cells. The Kd for guanosine 5'-[gamma-thio]triphosphate (GTP[S]) of the three Rab3 proteins was 15, 2700 and 204 nM for Rab3A, Rab3B and Rab3C respectively. The intrinsic GTPase activity of the three Rab3 proteins seemed similar and was increased approx. 3-fold by bovine chromaffin cell lysate. Truncation of the C-terminal 31 amino acid residues decreased the binding affinity for GTP[S] of the three Rab3 proteins. When the C-terminus of Rab3C was replaced with that of Rab3A, the binding affinity of Rab3C for GTP[S] was decreased, but the replacement did not affect the affinity of Rab3B for GTP[S]. Immunostaining experiments showed that Rab3A, Rab3B and Rab3C are localized separately within chromaffin cells. Anti-Rab3A and anti-Rab3C antibodies stained vesicle-like structures, whereas anti-Rab3B antibody distinctly stained the plasma membrane. In summary, bovine chromaffin cells express the three Rab3 proteins but the subcellular localization and biochemical properties of the three Rab3 proteins are distinct.

Topics: Adrenal Medulla; Amino Acid Sequence; Animals; Base Sequence; Cattle; Chromaffin Cells; DNA Primers; Fluorescent Antibody Technique; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Hybrid Cells; Kinetics; Molecular Sequence Data; Neuroblastoma; Polymerase Chain Reaction; Proto-Oncogene Proteins; rab3 GTP-Binding Proteins; Recombinant Proteins; Sequence Deletion

N-Acetylsphingosine (C2-ceramide), a membrane-permeable analogue, induced apoptosis in C6 glial cells. Phase-contrast micrographs showed that the round cells appeared 3 h after exposure to 25 microM C2-ceramide and the number of floating cells increased time-dependently. Staining with Hoechst 33258 dye showed condensed or fragmented nuclei in round cells at 12 h. DNA fragmentation was also observed by agarose gel electrophoresis at 12 h. To understand the mechanism underlying glial cell death induced by C2-ceramide treatment, changes in phospholipase D (PLD) activity in response to guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and expression of mRNA levels of PLD isozymes were examined. In cell lysate, GTPgammaS-dependent PLD activity was down-regulated after ceramide treatment in a time-dependent manner. In the in vitro PLD assay, membrane-associated PLD activation in response to recombinant ADP-ribosylation factor 1 was greatly suppressed. Furthermore, levels of rPLD1a and rPLD1b mRNAs were found to be down-regulated, whereas the level of rPLD2 mRNA increased gradually, peaking at 3 h, followed by a slow decrease, as inferred by reverse transcription-polymerase chain reaction. Decreases in GTPgammaS-dependent PLD activity were well correlated with those in rPLD1a and rPLD1b mRNAs levels. Taken together, these data suggest that levels of PLD enzymes might be decreased by ceramide treatment.

Topics: ADP-Ribosylation Factors; Animals; Apoptosis; Bisbenzimidazole; Cell Survival; Ceramides; DNA Fragmentation; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Isoenzymes; Neuroglia; Phospholipase D; Rats; Recombinant Proteins; RNA, Messenger; Tumor Cells, Cultured

A C6 glioma cell line stably transfected with the rat delta opioid receptor (C6delta) was used to characterize receptor binding and G protein activation by both peptide and nonpeptide delta opioid ligands. The ligand binding affinities for [3H]naltrindole and [3H]pCl-[D-Pen2,D-Pen5]enkephalin (DPDPE) were similar to those observed in monkey brain membranes. The nonpeptide agonists, BW373U86 and SNC80, as well as peptide agonist [D-Ser2, L-Leu5]enkephalyl-Thr maximally stimulated [35S]GTPgammaS binding by 640, 654 and 576%, respectively, over basal. The peptide agonists, DPDPE and deltorphin II, both stimulated [35S]GTPgammaS binding by 375%. Etorphine, diprenorphine, oxymorphindole and 7-spiroindanyloxymorphone were also partial agonists in this assay, although they were less efficacious than deltorphin II. Stimulation of [35S]GTPgammaS binding by agonists was blocked completely by pertussis toxin pretreatment. Both delta-1 and delta-2 selective antagonists 7-benzylidenenaltrexone and a benzofuran analog of naltrindole displayed high affinity for the cloned receptor (0.04 and 0.08 nM) and antagonized the stimulation of [35S]GTPgammaS binding by BW373U86 and DPDPE with similar potencies. Other evidence suggesting the lack of receptor subtypes includes the finding that stimulation of [35S]GTPgammaS binding by receptor subtype selective ligands DPDPE and deltorphin II was not additive. BW373U86, SNC80 and DPDPE maximally inhibited forskolin-stimulated adenylyl cyclase. These cells highly express a homogeneous population of delta opioid receptor that couple to inhibitory Go/Gi proteins. Ligand affinity for the delta opioid receptor correlates with ligand EC50 values for stimulation of [35S]GTPgammaS binding.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Animals; Cholera Toxin; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Narcotics; Pertussis Toxin; Rats; Receptors, Opioid, delta; Tumor Cells, Cultured; Virulence Factors, Bordetella

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma x glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharmacological characteristics of [3H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes. 2. Although the KD of specified [3H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the Bmax was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [3H]WIN 55212-2 binding. However, IC50 values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the Ki values for CP55,940 were 1.2 nM in brain and > 5000nM in NG108-15 membranes. Moreover, sodium and GTP-gamma-S decreased [3H]WIN 55212-2 binding in brain but not in NG108-15 membranes. 3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindole binding sites are not coupled to G proteins.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Calcium Channel Blockers; Cannabinoids; Cerebellum; Cyclohexanols; Endocannabinoids; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Hybrid Cells; Male; Membrane Proteins; Morpholines; Naphthalenes; Neuroblastoma; Polyunsaturated Alkamides; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Sensitivity and Specificity; Tritium

GTP gamma S-dependent phospholipase D (PLD) activity time-dependently increased during differentiation of rat C6 glioma cells to astrocytic phenotypes induced by dibutyryl cyclic AMP (dbcAMP)/theophylline. The changes in PLD mRNA level were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) method using the degenerate primers designed based on two conserved amino acid sequences in PLDs of human and yeast. The amplified three DNA fragments (tentatively termed as rPLDa, b, and c) contained the conserved regions present in PLDs of various organisms. RT-PCR using non-degenerate primers showed that rPLDa mRNA increased within 12h following treatment with dbcAMP, reaching a broad plateau and then returned to the initial level at 48h. In contrast, the level of rPLDb mRNA showed a concurrent decrease. rPLDc decreased in a time-dependent manner. These results suggest that the expression of PLD mRNAs are differentially regulated during differentiation in C6 glioma cells.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bucladesine; Cell Differentiation; DNA Primers; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Isoenzymes; Molecular Sequence Data; Phospholipase D; Rats; RNA, Messenger; Sequence Homology, Amino Acid; Theophylline

Prolonged exposure of neuroblastoma x glioma (NG 108-15) hybrid cells to inhibitory acting drugs results in sensitization of adenylate cyclase. We now report that chronic activation (3 days) of either inhibitory delta-opioid receptors, alpha 2B-adrenoceptors, or muscarinic M4 receptors significantly decreases the number of stimulatory, adenylate cyclase-coupled prostaglandin E1 receptors. Pharmacological characterization further revealed that the loss of [3H]prostaglandin E1-binding sites most likely corresponds to a reduction of the number of high-affinity, G protein-coupled prostaglandin E1 receptors. The decline in functionally active prostaglandin E1 receptors developed in a time- and dose-dependent manner and could be prevented by pretreatment of the cells with pertussis toxin. Heterologous prostaglandin E1 receptor regulation was blocked by concomitant exposure of the cells to antagonists for inhibitory receptors and was rapidly reversed (t 1/2 < 30 min) upon termination of chronic inhibitory drug treatment. The decrease in high-affinity prostaglandin E1 receptors developed regardless of whether full or partial agonists were used for pretreatment. In addition, the concentrations of inhibitory drugs required to maximally affect prostaglandin E1 receptor number closely resembled those mediating maximal adenylate cyclase inhibition. The data demonstrate that chronic inhibitory drug treatment of NG 108-15 hybrid cells reduces the number of functionally active, excitatory prostaglandin E1 receptors. Thus, it is proposed that adaptations at the level of stimulatory receptor systems contribute to the regulatory mechanisms associated with drug dependence.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Adrenergic alpha-Agonists; Alprostadil; Animals; Binding, Competitive; Cattle; Clonidine; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hybrid Cells; Muscarinic Agonists; Naloxone; Narcotic Antagonists; Neuroblastoma; Oxotremorine; Pertussis Toxin; Receptors, Opioid, delta; Receptors, Prostaglandin E; Virulence Factors, Bordetella

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.

Topics: Animals; Binding, Competitive; Cell Membrane; Enzyme Activation; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Radioligand Assay; Rats; Receptors, Opioid, mu; Recombinant Proteins; Signal Transduction; Sufentanil; Tumor Cells, Cultured

Purified bovine brain G-protein was used in a solution phase assay to identify membrane-associated proteins that influenced the activation of heterotrimeric G-proteins. Detergent-solubilized membrane extracts from the neuroblastoma-glioma cell hybrid NG108-15, but not the parent C6B4 glioma cell line, increased [35S]GTPgammaS binding to purified G-protein by approximately 460%. The G-protein activator was heat-sensitive, and the magnitude of its action was related to the amount of extract protein. The biophysical and biochemical properties of the G-protein activator were determined using DEAE ion exchange chromatography, gel filtration, and a lectin affinity matrix. In the presence of added GDP (1 microM), the enriched G-protein activator increased the initial rate of [35S]GTPgammaS binding to brain G-protein by up to 4-fold. In the absence of added GDP, the G-protein activator elicited an initial burst in [35S]GTPgammaS binding to brain G-protein within the first 30 s, after which the rate of nucleotide binding to G-protein was similar in the absence or presence of the G-protein activator. The stimulation of nucleotide binding to brain G-protein by the activator was also observed after resolution of Galpha from Gbetagamma. The G-protein activator was distinct from other proteins (neuromodulin, tubulin, and beta-amyloid precursor protein) that influence nucleotide binding to G-protein, indicating the existence of a novel signal accelerator.

Topics: Animals; Brain; Cattle; Chromatography, Gel; Chromatography, Ion Exchange; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Hybrid Cells; Neuroblastoma; Tumor Cells, Cultured

Heterotrimeric Go proteins have recently been described as regulators of vesicular traffic. The Goalpha gene encodes, by alternative splicing, two Goalpha polypeptides, Go1alpha and Go2alpha. By immunofluorescence and electron microscopy, we detected Go1alpha on the membrane of small intracellular vesicles in C6 glioma cells. After stable transfection of these cells, overexpression of Go1alpha but not Go2alpha was followed by a rise in the secretion of a serine protease inhibitor, protease nexin-1 (PN-1). This secretion was enhanced as a function of the amount of expressed Go1alpha. Metabolic cell labeling indicated that this increase in PN-1 secretion was not the result of an enhancement in PN-1 biosynthesis or a decrease in its uptake, but revealed a potential role of Go1alpha in the regulation of vesicular PN-1 trafficking. Furthermore, activators of Go proteins, mastoparan and a peptide derived from the amino terminus of the growth cone-associated protein GAP43, increased PN-1 secretion in parental and Go1alpha-overexpressing cells. Brefeldin A, an inhibitor of vesicular traffic, inhibited both basal and mastoparan-stimulated PN-1 secretions. These results indicate, that in C6 glioma cells, PN-1 secretion could be regulated by both Go1alpha expression and activation.

Topics: Amyloid beta-Protein Precursor; Carrier Proteins; Glioma; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Kinetics; Plasminogen Inactivators; Protease Nexins; Receptors, Cell Surface; Serine Proteinase Inhibitors; Serpin E2; Transfection; Tumor Cells, Cultured

C6 glioma cells possess endothelin ETA receptor and P2 purinoceptor coupled to two signaling pathways, i.e. phosphoinositide turnover and inhibition of adenylyl cyclase. In this study, the effects of raising cyclic AMP levels on the inositol phospholipid hydrolysis and adenylyl cyclase inhibition caused by endothelin-1 and ATP in C6 glioma cells were examined. Pretreatment with cAMP generating agents (forskolin, isoproterenol and cholera toxin) or dibutyryl cAMP for 10 min-3 h did not affect the inositol phosphate accumulation caused by endothelin and ATP. Long-term (8-24 h) pretreatment with isoproterenol, forskolin, cholera toxin or dibutyryl cAMP resulted in a 40-50% inhibition of endothelin- and ATP-stimulated inositol phosphate accumulation, whereas the EC50 values of endothelin and ATP were not affected. Consistent with the effects on endothelin and ATP, NaF-induced inositol phosphate formation was also inhibited by cAMP generating agents to a similar extent. Permeabilized cells from 24 h isoproterenol-or forskolin-pretreated C6 cells also showed a diminished Ca(2+)-sensitivity of phosphoinositide-specific phospholipase C and also attenuated the potentiation response caused by GTP gamma S. The inhibitory effects on adenylyl cyclase by endothelin, ATP and 2-methylthio-ATP were unaffected by 24 h pretreatment with isoproterenol or forskolin. Long-term treatment with dibutyryl cGMP did not affect the two signaling pathways caused by ATP and endothelin. It is concluded that the phosphoinositide turnover, but not the adenylyl cyclase inhibition caused by endothelin and ATP in C6 cells, was inhibited by protein kinase A-dependent pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Adenylyl Cyclase Inhibitors; Animals; Brain Neoplasms; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Endothelins; Enzyme Activation; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Phosphatidylinositols; Rats; Signal Transduction; Stimulation, Chemical; Tumor Cells, Cultured

To investigate the role of calmodulin (CaM)-dependent pathways in agonist-induced phosphoinositide (PI) turnover, the influence of several CaM antagonists on PI-phospholipase C (PLC) activation in intact and permeabilized C6 glioma cells was examined. The extent of PI turnover was assessed by measuring the accumulation of inositol phosphates (IPs) in the presence of LiCl in C6 glioma cells prelabelled with myo-[3H]inositol. Trifluoperazine, N-(6-aminohexyl)-5-chloro-1- naphthalenesulphonamide (W-7), fendiline and calmidazolium themselves had no effect on basal IP formation, but concentration-dependently (1-30 microM) potentiated ATP-, NaF- and A23187-stimulated IP formation. The maximal response to ATP (1 mM) was increased by up to 50%, while the concentration for half-maximal effect (EC50, 60 microM) was unaffected by trifluoperazine. In digitonin-permeabilized C6 glioma cells, the concentration-dependent increase of PI-PLC activation elicited by free Ca2+ was potentiated by the GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), with an EC50 of 6 microM. Trifluoperazine (1-30 microM) enhanced the Ca(2+)-stimulated IP formation concentration dependently and this potentiation was counteracted by the addition of CaM. In the combined presence of each CaM antagonist studied and GTP gamma S, an additive increase in IP formation was observed. The results indicate that CaM antagonists enhance stimulus-induced IP formation in C6 glioma cells primarily by increasing the Ca(2+)-dependent activation of PI-PLC.

Topics: Adenosine Triphosphate; Calcimycin; Calmodulin; Digitonin; Dose-Response Relationship, Drug; Enzyme Activation; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Hydrolysis; Phosphatidylinositols; Signal Transduction; Sodium Fluoride; Trifluoperazine; Tumor Cells, Cultured; Type C Phospholipases

Topics: Adenosine Diphosphate Ribose; Animals; Calcium Channels; Carbachol; Cholera Toxin; Electrophysiology; Epinephrine; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanylyl Imidodiphosphate; Homeostasis; Hybrid Cells; Ion Channels; Macromolecular Substances; Neuroblastoma; Oligonucleotides, Antisense; Patch-Clamp Techniques; PC12 Cells; Potassium Channels; Rats; RNA, Messenger; Virulence Factors, Bordetella

Transfection of a human dopamine D3 receptor cDNA in a neuroblastoma-glioma hybrid cell line (NG 108-15) provided clonal cell lines stably expressing up to 600 fmol per mg protein of [125I]iodosulpiride binding sites. Dopamine and several agonists distinguished two receptor-affinity states in membranes. In the case of dopamine, the high-affinity state (Ki = 0.9 nM, 30% of total binding) was completely converted into a low-affinity state (Ki = 57 nM) in the presence of 10 microM guanosine-5'-O-(3-thiotriphosphate). In addition to these two sites, a site with a very low affinity for dopamine was evidenced in whole cells. The dopamine D3 receptor mediated two responses: c-fos activation, as measured by the appearance of Fos-like immunoreactivity, and increased mitogenesis, as measured by incorporation of [3H]thymidine. The Fos-like immunoreactivity appeared within 30 min, lasted 2 h and was blocked by the partially selective dopamine D3 receptor compound (+)-UH 232 (cis-(+)-5-methoxy-1-methyl-2-(di-n-propylamino)tetralin). The mitogenic effect, which occurred after a lag time (over 2 h stimulation), was produced with subnanomolar potency and full intrinsic activity by several compounds previously identified as dopamine D2 receptor agonists, e.g. quinpirole, (+)-7-OH-DPAT ((+)-7-hydroxy-2-(di-n-propylamino)tetralin) and RU 24926 (N-n-propyl-di-beta(3-hydroxyphenyl)-ethylamine), and was reversibly blocked by (+)-UH 232 (Ki = 9 nM). Talipexole (B-HT 920, 5-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin) was identified as a partial agonist at the dopamine D3 receptor. Dopamine D3 receptor-mediated mitogenesis was potentiated by a phorbol ester and was abolished by pretreatment with pertussis toxin. A mitogenic effect of same amplitude was elicited by bradykinin or carbachol, both acting through constitutive receptors. Bradykinin markedly activated inositol phosphate turnover, and had no effect on forskolin-stimulated cyclic AMP accumulation. Carbachol inhibited forskolin-stimulated cyclic AMP accumulation and had no effect on inositol-phosphate turnover. Quinpirole had no effect on any of these second messenger pathways. Thus, in transfected NG 108-15 cells, the dopamine D3 receptor is coupled to a pertussis toxin-sensitive G protein and mediates two possibly unrelated biological effects, through initial biochemical events that remain to be identified.

Topics: Animals; Cyclic AMP; Ergolines; Genes, fos; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hybrid Cells; Mice; Neuroblastoma; Quinpirole; Rats; Receptors, Dopamine; Receptors, Dopamine D2; Receptors, Dopamine D3; Sulpiride; Thymidine; Transfection; Tumor Cells, Cultured

In NG108-15 cells, bradykinin (BK) activates a potassium current (IK,BK) and inhibits the voltage-dependent calcium current (ICa,V). BK also stimulates a phosphatidylinositol-specific phospholipase C (PI-PLC). The subsequent release of inositol 1,4,5-trisphosphate and increase in intracellular calcium contribute to IK,BK, through activation of a calcium-dependent potassium current. In membranes from these cells, stimulation of PI-PLC by BK is mediated by Gq and/or G11, two homologous, pertussis toxin-insensitive G proteins. Here, we have investigated the role of Gq/11 in the electrical responses to BK. GTP gamma S mimicked and occluded both actions of BK, and both effects were insensitive to pertussis toxin. Perfusion of an anti-Gq/11 alpha antibody into the pipette suppressed IK,BK, but not the inhibition of ICa,V by BK. Thus, BK couples to IK,BK via Gq/11, but coupling to ICa,V is most likely via a different, pertussis toxin-insensitive G protein.

Topics: Animals; Bradykinin; Calcium Channels; Electrophysiology; Enkephalin, Leucine; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Hybrid Cells; Kinetics; Models, Biological; Neuroblastoma; Pertussis Toxin; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Potassium Channels; Tetrodotoxin; Tumor Cells, Cultured; Virulence Factors, Bordetella

In permeabilized C6 glioma cells and NIH 3T3 cells, the peptide endothelin 1 (ET-1) in combination with GTP gamma S stimulates the formation of inositol phosphates. In the presence of 10 microM GTP gamma S, ET-1 induces the formation of inositol phosphates with an EC50 value of 2.5 nM for C6 glioma cells and 1.6 nM for NIH 3T3 cells. The analogous peptide endothelin 3 (ET-3) is less potent than ET-1 in such action. In NIH 3T3 cells, ET-1+GTP gamma S-induced formation of inositol phosphates could be detected after 1 min of stimulation, and it increased for up to 30 min. ET-1-induced effects were partially reduced by pretreatment of the cells with pertussis toxin (1 microgram/ml) in C6 glioma cells, but were unaffected in NIH 3T3 cells. In binding studies in whole C6 cells and NIH 3T3 cells, specific binding for [125I]ET-1 was detected. Cross-linking of [125I]ET-1 in whole C6 cells revealed the presence of two binding proteins for ET-1 of 74 kDa and 55 kDa. ET-1 at 100 nM inhibited the labeling of both proteins by [125I]ET-1. However, ET-3 inhibited the labeling of the 55 kDa protein only. The results provide direct evidence for endothelin receptor coupling to phospholipase C through guanine nucleotide binding (G) proteins. In addition, in C6 cells, endothelin-mediated phospholipase C activation is partially inhibited by pertussis toxin pretreatment. The endothelin receptor involved in phospholipase C stimulation in C6 cells seems to correspond to a 74 kDa protein which binds ET-1 but not ET-3.

Topics: 3T3 Cells; Animals; Cross-Linking Reagents; Endothelins; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Inositol Phosphates; Mice; Pertussis Toxin; Receptors, Cell Surface; Receptors, Endothelin; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

In rat glioma C6 cells, extracellular ATP stimulated phosphoinositide (PI) hydrolysis in concentration- and time-dependent manners with a median effective dose value of 60 microM. The maximal response was attained at 300 microM ATP. Of adenine nucleotides, ATP and adenosine 5'-O-(3-thiotriphosphate) were most effective, while adenosine, AMP and beta,gamma-methylene ATP were ineffective. Similar results were obtained in cultured rat astrocytes. The stimulatory effects of ATP and ADP were negated by removal of external Ca++ in C6 cells. ATP at 300 microM induced an elevation of intracellular Ca++ concentration in 1-[2-(5-carboxyoxazol-2-yl)-6-amino-benzofuran-5-oxy]-2-(2'-amino- 5'- methylphenoxy)-ethane-N,N,N',N' acid-loaded C6 cells. This response was not blocked by nifedipine (10 microM) and verapamil (10 microM). A Ca++ ionophore A23187 (10 microM) stimulated PI hydrolysis in C6 cells. The responses to ATP (300 microM) and A23187 (10 microM) were additive. In digitonin-permeabilized C6 cells, Ca++ at the concentration of 100 microM evoked PI hydrolysis, and ATP alone did not affect the Ca++ dependence. GTP gamma S (100 microM) stimulated the PI hydrolysis at a range of 0.1 to 10 microM Ca++, and ATP enhanced the GTP gamma S response in the permeabilized cells. These results suggest that activation of P2-purinergic receptors by ATP causes phospholipase C to be activated by subthreshold concentrations of Ca++ via GTP-binding proteins, resulting in an activation of the enzyme in response to stimulated Ca++ influx.

Topics: Adenosine Triphosphate; Animals; Astrocytes; Calcimycin; Calcium; Cells, Cultured; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Hydrolysis; Phosphatidylinositols; Rats; Tumor Cells, Cultured

In synaptosomal membranes from rat brain cortex, the mu selective agonist [3H]dihydromorphine in the absence of sodium, and the nonselective antagonist [3H]naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with Kd values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 microM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP[gamma-35S] binding sites by 90% and low Km, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP[gamma S] was diminished. High-affinity (Kd, 0.72 nM), guanine nucleotide-sensitive agonist binding was reconstituted by polyethylene glycol-induced fusion of the alkali-treated membranes with (opioid receptor devoid) C6 glioma cell membranes. Also restored was opioid agonist-stimulated, naltrexone-inhibited GTPase activity. In contrast, antagonist binding in the fused membranes was unaltered. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low Km GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor-guanine nucleotide binding protein complex.

Topics: Animals; Binding, Competitive; Cell Line; Cell Membrane; Cells, Cultured; Cerebral Cortex; Dihydromorphine; Glioma; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Male; Membrane Fusion; Naloxone; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, mu; Synaptosomes

Accumulation of inositol phosphates in NG108-15 neuroblastoma x glioma hybrid cells, pre-labeled for 24h to equilibrium, was stimulated by bradykinin, guanosine 5'-O-(3-thiotriphosphate) and the diacylglycerol kinase inhibitor R59022. Only the stimulation by bradykinin was inhibited by the bradykinin receptor antagonist [D-Arg0, Hyp3, Phe7, Thi5,8] bradykinin. Neither bradykinin nor R059022 increased the labeling of the inositol phospholipids. The sulfhydryl-alkylating reagent N-ethylmaleimide at 100 microM essentially abolished the stimulation caused by all three agents, possibly by preventing the binding of GTP to a guanine nucleotide-binding regulatory protein of as yet unknown size.

Topics: Animals; Bradykinin; Cell Line; Drug Interactions; Ethylmaleimide; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hybrid Cells; Inositol; Inositol Phosphates; Kinetics; Mice; Neuroblastoma; Platelet Activating Factor; Pyrimidinones; Rats; Thiazoles; Thionucleotides

In neuroblastoma x glioma hybrid NG108-15 cells, bradykinin (BK) receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates and mobilization of intracellular calcium. Treatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) suppressed the spike phase of increases in intracellular calcium concentration. In radioligand binding studies, TPA treatment did not interfere with [3H]BK specific binding to intact cells or to cell membranes. The ability of guanyl-5'-yl-imidodiphosphate to promote the conversion of the high affinity sites of the BK receptors into a low affinity sites was unaffected by TPA. TPA treatment showed the dose-dependent, noncompetitive inhibition of BK-stimulated formation of inositol trisphosphate. In the membrane preparations from TPA-treated cells, guanosine 5'-(3-O-thio)triphosphate-stimulated inositol trisphosphate formation was inhibited by 50%. These data indicate that TPA exerts its inhibitory action on BK responses at the sites of guanine nucleotide-binding protein or phospholipase C or both.

Topics: Bradykinin; Calcium; Cell Line; Diglycerides; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Neuroblastoma; Protein Kinase C; Receptors, Bradykinin; Receptors, Neurotransmitter; Sugar Phosphates; Tetradecanoylphorbol Acetate; Thionucleotides; Type C Phospholipases

The addition of bradykinin to NG108-15 cells resulted in an increase in the intracellular Ca2+ concentration [( Ca2+]i) and the formation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in these cells. The bradykinin-stimulated formation of inositol polyphosphates in plasma membrane preparations was dependent on the presence of GTP or guanosine-5'-O-thiotriphosphate (GTP gamma S) but not of GDP. GTP gamma S, unlike GTP, increased the basal formation of inositol polyphosphate in NG108-15 membranes. Iontophoretic injection of GTP gamma S into single cells induced increases in [Ca2+]i. These effects of bradykinin and GTP gamma S on [Ca2+]i and the formation of inositol phosphates in the intact cells and membranes were not affected by treatment of the cells with pertussis toxin or cholera toxin. Data on binding of bradykinin to membrane preparations indicated the presence of two classes of binding sites with Kd values of 0.80 +/- 0.26 and 9.63 +/- 0.13 nM. Approximately 74% of the receptors were in the high affinity state. In the presence of guanyl-5'-yl-imidodiphosphate [Gpp(NH)p], the high affinity sites in the membrane preparations were converted to low affinity sites with no change in the total receptor number. These toxin treatments had no effect on binding of bradykinin to its receptors. Thus, these results indicate that a guanine nucleotide regulatory protein, which is not a substrate of pertussis toxin or cholera toxin, is involved in mediating the effects of bradykinin on membrane-bound phosphoinositide-specific phospholipase C to induce the increase of cytosolic calcium.

Topics: Animals; Bradykinin; Calcium; Cells, Cultured; Cholera Toxin; Glioma; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Mice; Neuroblastoma; Pertussis Toxin; Phosphatidylinositols; Rats; Receptors, Bradykinin; Receptors, Neurotransmitter; Thionucleotides; Virulence Factors, Bordetella

We have compared the regulation of adenylate cyclase activity in membrane fractions from C6 glioma cells and in monolayer cultures of C6 cells that had been permeabilized with saponin. Guanine nucleotides (GTP and GTP gamma S) and isoproterenol increase adenylate cyclase activity in C6 membranes and in permeabilized C6 cells. In C6 membranes, guanine nucleotides activate adenylate cyclase in the presence or absence of isoproterenol; in permeabilized cells, however, guanine nucleotides increase adenylate cyclase activity only in the presence of isoproterenol. We suggest that the properties of the permeabilized cells more closely resemble those of intact cells, and that some component which is present in permeabilized cells but is lost following cell disruption may be important for the normal regulation of adenylate cyclase activity.

Topics: Adenylyl Cyclases; Cell Line; Cell Membrane Permeability; Enzyme Activation; Glioma; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Isoproterenol; Saponins; Thionucleotides