guanylyl-imidodiphosphate and Glioma

Topics: Adenylyl Cyclase Inhibitors; Adipose Tissue; Adrenergic alpha-Agonists; Animals; Blood Platelets; Calmodulin; Cholera Toxin; Glioma; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Macromolecular Substances; Models, Biological; Molecular Weight; Neuroblastoma; Receptors, Cell Surface; Receptors, Cholinergic; Receptors, Opioid; Sodium; Surface Properties

While the cytoskeleton is known to play several roles in the biology of the cell, one role, which has been revealed only recently, is that of a participant in the signal transduction process. Tubulin binds specifically to the alpha subunits of Gs (stimulatory GTP-binding regulatory protein of adenylyl cyclase), Gi1 (inhibitory protein of adenylyl cyclase), and Gq and transactivates those molecules through direct transfer of GTP. The relevance of this transactivation process to G proteins which are normally activated by a neurotransmitter-occupied receptor is the subject of this study. C6 glioma cells, made permeable with saponin, retained tight coupling between Gs and the beta-adrenergic receptor. Although 5-guanylylimidodiphosphate (GppNHp) was incapable of activating Gs (and subsequently, adenylyl cyclase) in the absence of agonist, tubulin with GppNHp bound (tubulin-GppNHp) activated adenylyl cyclase with an EC(50) of 30 nM. Desensitization of beta-adrenergic receptors by isoproterenol exposure had no effect on the ability of tubulin-GppNHp to activate Gs and adenylyl cyclase. When the photoaffinity GTP analog, azidoanilido GTP (AAGTP; P3(4-azidoanilido)-P1-5'-GTP), was added to C6 membranes or permeable C6 cells, it was only weakly incorporated by G alpha s in the absence of isoproterenol. When the same concentration of dimeric tubulin with AAGTP bound was introduced, AAGTP was transferred from tubulin to G alpha s, activating the latter species. Similar 'preferential' activation of G alpha s by tubulin-AAGTP versus the free nucleotide was seen using purified components. Thus, membrane-associated tubulin may serve to activate G alpha s, independent of signals not normally coupled to that protein. Tubulin may act as an agent to link a variety of membrane-associated signalling systems.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Animals; Azides; Cell Membrane; Cell Membrane Permeability; Enzyme Activation; Glioma; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Precipitin Tests; Rats; Receptors, Adrenergic, beta; Saponins; Signal Transduction; Tubulin; Tumor Cells, Cultured

The mechanism of ethanol inhibition of glucose uptake was investigated using C6 glioma cells. Basal [3H]2-deoxy-D-glucose (2DG) uptake by C6 cells was inhibited by ethanol in a concentration-dependent manner. Fifty, 75 and 100 mM ethanol significantly inhibited basal 2DG uptake by 12, 20 and 23%, respectively (p < 0.05). Carbachol (an agonist acting via G protein-coupled receptors) stimulated the uptake by 26% (p < 0.05). In the presence of 100 mM ethanol, the ability of carbachol to stimulate 2DG uptake was abolished. In contrast, ethanol did not inhibit the ability of insulin to stimulate 2DG uptake. These results suggest that ethanol inhibits 2DG uptake by selectively interfering with G protein-mediated signal transduction pathway.

Topics: Animals; Carbachol; Cell Count; Cell Survival; Culture Media; Deoxyglucose; Ethanol; Glioma; Glucose; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Insulin; Muscarinic Agonists; Rats; Tumor Cells, Cultured

The role of G proteins in glucose uptake was investigated using C6 glioma cells. Carbachol (an agonist acting via G protein coupled receptors) and 5'-guanylylimidodiphosphate (Gpp(NH)p; a nonhydrolysable guanine nucleotide analog which bypasses the receptors and directly activates G proteins) stimulated [3H]2-deoxy-D-glucose (2DG) uptake by C6 cells, suggesting that hexose uptake is a G protein-mediated process. To identify the G protein involved in glucose uptake by C6 cells, the effect of carbachol on 2DG uptake was examined in the presence of pertussis toxin. Pertussis toxin treatment did not alter the ability of C6 cells to respond to carbachol, ruling out the involvement of G(i alpha) in 2DG uptake. C6 cells were transfected with G(q alpha) or GLUT1 cDNA for 48 h, exposed to 1 mM carbachol for 2 h, and processed for 2DG uptake. Carbachol stimulated 2DG uptake in both G(q alpha) and GLUT1-transfected cells. Gpp(NH)p, also stimulated 2DG uptake in G(q alpha) and GLUT1-transfected cells. These results suggest that muscarinic receptor coupling to G(q alpha) regulates hexose uptake in C6 cells.

Topics: Animals; Carbachol; Deoxyglucose; Glioma; Glucose; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Pertussis Toxin; Rats; Stimulation, Chemical; Tumor Cells, Cultured; Virulence Factors, Bordetella

Metabotropic glutamate receptors in rat C6 glioma cells have been characterized by pharmacological and kinetic binding experiments, using both L-[3H]glutamate and [3H(+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid ([3H](+/-)-trans-ACPD) radioligands. Saturation experiments revealed a single binding site with a Kd = 1250 +/- 101 nM and Bmax = 12.1 +/- 1.8 pmol/mg protein when the assays were performed with L-[3H]glutamate as radioligand in the presence of AMPA, kainate, NMDA and DL-threo-beta-hydroxyaspartic acid. When [3H](+/-)-trans-ACPD was used as radioligand, the kinetic parameters obtained were Kd = 2605 +/- 1042 nM and Bmax = 13.66 +/- 5.01 pmol/mg protein. Pharmacological characterization indicated that specific binding of L-[3H]glutamate was sensitive to different agonists of mGlu receptors, showing a rank order of affinity L-glutamate > L-quisqualic acid > (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) > ibotenic acid >>> (2S, 'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Specific binding of L-[3H]glutamate to mGlu receptors is regulated by guanine nucleotides. Guanylyl imidodiphosphate (Gpp(NH)p) causes an affinity shift on the L-glutamate dose-response curve, increasing the IC50 value. These results support the evidence that metabotropic glutamate receptors are present in rat C6 glioma cells and they are coupled to a G-protein.

Topics: Animals; Brain Neoplasms; Cell Membrane; Cycloleucine; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glioma; Glutamic Acid; Guanylyl Imidodiphosphate; Kinetics; Neuroprotective Agents; Radioligand Assay; Rats; Receptors, Metabotropic Glutamate; Tumor Cells, Cultured

The G proteins G S and Gi1 appear to be capable of binding to tubulin specifically, and it has been suggested that such binding results in G protein activation via direct transfer of GTP. This study was undertaken to demonstrate that consequences of G protein activation by tubulin, i.e., stimulation or inhibition of adenyl cyclase, were dependent on the G proteins expressed as well as unique aspects of the membrane or cytoskeleton in a given cell type. Membranes from rat C6 glioma cells, which express G s alpha but not G i alpha 1, responded to the addition of tubulin with a stable activation of adenyl cyclase. Conversely, membranes from rat cerebral cortex, which contain both G s and G i 1, responded to exogenous tubulin with a stable inhibition of adenyl cyclase. Unlike C6 membranes, cerebral cortex membranes are richly endowed with tubulin, and antitubulin antibodies immunoprecipitated complexes of tubulin and G i 1 and G s from detergent extracts of these membranes. Nearly 90% of the G s alpha from Triton X-114 extracts coimmunoprecipitated with tubulin, suggesting that these proteins exist as a complex in the synaptic membrane. Such complexes may provide the framework for a G protein-cytoskeleton link that participates in the modulation of cellular signal transduction.

Topics: Adenylyl Cyclase Inhibitors; Affinity Labels; Animals; Azides; Cerebral Cortex; Chickens; Glioma; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Rats; Sheep; Synaptic Membranes; Synaptosomes; Tubulin; Tumor Cells, Cultured

We studied the activity and the ultracytochemical localization of membrane-bound guanylate cyclase (GC) after stimulation with rat atrial natriuretic peptide (rANP), porcine brain natriuretic peptide (pBNP), rat brain natriuretic peptide (rBNP), or porcine C-type natriuretic peptide (CNP) in rat C6 glioma cells during proliferation or following exposure of confluent cells to dibutyryl cyclic AMP (db-cAMP) or retinoic acid (RA). Under our experimental conditions all peptides were activators of GC as demonstrated by the accumulation of cGMP within cells. During proliferation of C6 cells, the amounts of cGMP remained approximately constant. However, at subconfluency, confluency and postconfluency, the GC reaction product was located at different sites in C6 cells. At subconfluency, GC reaction product was on membranes of protoplasmic extensions, at postconfluency, GC reaction product was in association with membranes of cell bodies, and at confluency, both localizations of GC reaction product were detected. Incubation of confluent cells in culture medium containing db-cAMP or RA induced the appearance of long and slender protoplasmic extensions. Under these conditions, the GC reaction product was localized exclusively to these processes. These data suggest that GC is differentially located depending on the state of growth of glial cells, and that in differentiating glial cells GC is preferentially located in cell processes.

Topics: Animals; Atrial Natriuretic Factor; Bucladesine; Cell Division; Culture Media; Cyclic GMP; Glioma; Guanylate Cyclase; Guanylyl Imidodiphosphate; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Nerve Tissue Proteins; Proteins; Rats; Swine; Tretinoin; Tumor Cells, Cultured

It has been reported that antidepressant treatment in rats results in a significant increase of Gs-mediated stimulation of adenylyl cyclase and this effect correlates well with the clinical therapeutic response. This increased activity occurs despite a down-regulation of several receptors linked normally to the stimulation of that enzyme. To distinguish between these effects and to determine whether presynaptic components of the cell are required, C6 glioma cells were treated with antidepressants. Tricyclic (amitriptyline and desipramine) or atypical (iprindole) antidepressant exposure to C6 cells for 5 days significantly increased guanylyl-5'-imidodiphosphate [Gpp(NH)p]-stimulated adenylyl cyclase activity in membrane preparations in a manner similar to that seen for rat brain membranes after 21-day treatment. This effect was drug dose and exposure time dependent. Nevertheless, stimulation of adenylyl cyclase by isoproterenol was decreased after antidepressant treatment. By comparison, the antidepressant-induced beta-receptor desensitization occurred earlier than the enhancement of Gpp(NH)p-activated adenylyl cyclase, and extensive desensitization of beta receptors by isoproterenol treatment did not enhance the Gpp(NH)p-stimulated adenylyl cyclase activity. These results indicated that the antidepressant has a direct effect on cell signaling and this enhanced Gpp(NH)p-stimulated adenylyl cyclase activity is not correlated with desensitization of beta-adrenergic receptor stimulated adenylyl cyclase. These data contribute to the suggestion that G proteins (especially Gs) are the target of antidepressant actions. Immunoblotting showed that neither the number of G protein subunits (alpha s, alpha i, alpha o, and beta) nor their association with the plasma membrane was changed after antidepressant treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Animals; Antidepressive Agents; Cell Membrane; Cytosol; Desipramine; Dose-Response Relationship, Drug; Enzyme Activation; Glioma; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Isoproterenol; Rats; Receptors, Adrenergic, beta; Time Factors; Tumor Cells, Cultured

NG108-15 mouse neuroblastoma x rat glioma cells were treated with the prostanoid IP receptor agonist iloprost (1 microM) and the time course of changes in the levels of prostanoid IP receptors, adenylyl cyclase activity, and the alpha-subunit of the stimulatory guanine nucleotide binding regulatory protein, Gs, were measured. Incubation of cells with iloprost produced a biphasic time course of desensitisation of prostanoid IP receptor-activated adenylyl cyclase. Parallel analysis of iloprost-induced loss of membrane Gs alpha, NaF-stimulated adenylyl cyclase and [3H]iloprost binding suggested only monophasic curves, with t0.5 values similar to the initial phase of desensitisation of iloprost-stimulated adenylyl cyclase activity. This suggests that the loss of receptor and Gs alpha occur at the same time and account for the initial period of desensitisation due to iloprost pretreatment. Pretreatment of NG108-15 cells with cholera toxin produced a near complete loss of membrane-associated Gs alpha, but the loss of [3H]iloprost binding due to iloprost treatment was not affected by pretreatment with cholera toxin, suggesting that prostanoid IP receptors can be down-regulated in the absence of any coupling to Gs. The second phase of desensitisation of iloprost-stimulated adenylyl cyclase activity, during which there was no further change in NaF-stimulated adenylyl cyclase or in the membrane levels of Gs alpha, was not due to protein kinase A activation, since elevating intracellular cyclic AMP levels with forskolin did not subsequently decrease iloprost-stimulated adenylyl cyclase activity or [3H]iloprost binding. These results demonstrate that iloprost pretreatment of NG108-15 cells induces two distinct phases of desensitisation; an initial desensitisation due to concurrent loss of prostanoid IP receptors and Gs alpha, and then a further desensitisation by an as yet uncharacterized mechanism during which there is no further loss of Gs alpha.

Topics: Adenylyl Cyclases; Animals; Cholera Toxin; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Glioma; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hybrid Cells; Iloprost; Mice; Neuroblastoma; Rats; Receptors, Prostaglandin; Tumor Cells, Cultured

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

To understand the details of regulation of guanine-nucleotide-binding-protein-linked transmembrane cellular-signalling cascades, it is important to know the absolute levels of each polypeptide component and the stoichiometry of their interactions. Amounts of the IP prostanoid receptor, the stimulatory G protein of the adenylyl cyclase cascade (Gs alpha) and the functional complex of Gs alpha with adenylyl cyclase, which acts as the cyclic AMP generator, were measured in membranes of neuroblastoma x glioma hybrid, NG108-15, cells. As measured by the specific binding of [3H]prostaglandin E1, the IP prostanoid receptor was present in some 100,000 copies/cell. Gs alpha assessed by quantitative immunoblotting with recombinantly expressed protein, was present in considerably higher levels (1,250,000 copies/cell). However, the maximal formation of a complex of Gs alpha and adenylyl cyclase represented only some 17,500 copies/cell. The previously established 8:1 stoichiometry of concurrent downregulation of Gs alpha and the IP prostanoid receptor in these cells [Adie, E. J., Mullaney, I., McKenzie, F. R. & Milligan, G. (1992) Biochem. J. 285, 529-536] indicates that full-agonist occupation of the receptor should be able to activate some 65% of the expressed Gs. Despite the potential 70-fold excess of Gs alpha over the Gs alpha/adenylyl cyclase complex, IP prostanoid-receptor-agonist-mediated reduction of Gs alpha levels by some 35% resulted in a 25% reduction in the maximal formation of the Gs alpha/adenylyl cyclase complex. Such results demonstrate that adenylyl cyclase is quantitatively the least highly expressed component of this signalling cascade and suggests that much of the cellular Gs alpha may not have access to adenylyl cyclase.

Topics: Adenylyl Cyclases; Alprostadil; Animals; Colforsin; Cyclic AMP; Glioma; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Mice; Neuroblastoma; Rats; Receptors, Prostaglandin; Recombinant Proteins; Sodium Fluoride; Tumor Cells, Cultured

We developed a newly devised method for receptor binding assays in permeable C6 cell suspensions, which permits investigation of beta-adrenergic receptor behavior under conditions where the receptor appears tightly coupled to Gs protein to activate adenylyl cyclase. Tubulin, the cytoskeletal element, is a G protein with similarities to other G proteins. Although dimeric tubulins have been implicated as modulators of the adenylyl cyclase system, the total mechanism of this regulation has not been clear. Guanine nucleotides have been known to modulate agonist binding affinity for G protein-linked receptors. We demonstrate that tubulin alters the coupling between receptors and G proteins involved in the stimulation or inhibition of adenylyl cyclase system via direct transfer of GTP from tubulin to Gs alpha and Gi 1 alpha. In this study, the effects of tubulin-GppNHp have been compared to those of GppNHp in changing beta-adrenergic agonist affinity in both permeable C6 cells and C6 membranes. Competitive binding studies, including time-course assays, were performed with isoproterenol and the beta-adrenergic antagonist, [125I]pindolol. These results show that tubulin-GppNHp is more effective than GppNHp in decreasing receptor affinity in both permeable C6 cells and C6 membranes. These results imply that tubulin plays a dynamic role in neuronal signal transduction system at multiple loci.

Topics: Adenylyl Cyclases; Animals; Cell Membrane Permeability; Cells, Cultured; Glioma; GTP-Binding Proteins; Guanine Nucleotides; Guanylyl Imidodiphosphate; Isoproterenol; Radioligand Assay; Rats; Receptors, Adrenergic, beta; Signal Transduction; Tubulin

Nuclear opioid binding sites have been discovered in NG108-15 neurohybrid cells. Marker enzyme analyses as well as electron and fluorescence microscopy studies attested to the high degree of purity of the nuclear preparations. Immunohistochemical studies on cryostat sections of NG108-15 cells with an antibody to the opioid receptor corroborated a nuclear localization. 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE), 3H-[D-Ala2,D-Leu5]enkephalin (3H-DADLE), and 3H-diprenorphine binding parameters, Kd and Bmax values, and heterologous competition binding and stereospecificity data satisfied criteria for the presence of delta-opioid sites in purified nuclear preparations. Neither mu-([D-Ala2,mephe4,gly-ol5] enkephalin), dihydromorphine, nor kappa-(U69593) specific binding was detectable in purified nuclear preparations. Rates of association and dissociation of 3H-[D-Ser2,L-Leu5]enkephalyl-Thr were comparable to values obtained previously for opioid receptors. Opioid binding was also shown in subnuclear preparations from NG108-15 cell cultures. Agonists, 3H-DADLE and 3H-DPDPE, bind with high affinity to nuclear membranes and with lower affinity to chromatin. In contrast, partial agonist 3H-diprenorphine high-affinity binding sites were predominant in chromatin, while low-affinity binding was found in the nuclear membrane. Accordingly, 5'-guanylylimidodiphosphate sensitivity of 3H-DADLE binding was detected in nuclear membranes but not in chromatin. Both agonist and partial agonist opioid binding to nuclear membrane and chromatin were abolished upon cycloheximide treatment of NG108-15 cells. Taken together, the results suggest that NG108-15 cells contain newly synthesized GTP binding regulatory protein (G-protein)-coupled delta-opioid receptors in nuclear membranes and uncoupled opioid binding sites in chromatin.

Topics: Binding Sites; Cell Nucleus; Glioma; Guanylyl Imidodiphosphate; Hybrid Cells; Immunohistochemistry; Kinetics; Narcotics; Neuroblastoma; Tissue Distribution; Tumor Cells, Cultured

Electrophysiological evidence shows that voltage-dependent calcium channel (VDCC) activity can be regulated by a large number of neurotransmitters. In particular, guanine nucleotide binding regulatory protein (G protein)-mediated inhibitory modulation of the channel activity has been deduced from evidence that GTP analogues and purified G proteins are able to mimic this effect. The G proteins involved are pertussis toxin (PTx) sensitive. The purpose of the present study was to investigate, using biochemical techniques, whether G protein activation modulates the recognition site for omega-conotoxin GVIA (CgTx), a peptide neurotoxin that selectively labels a population of high-threshold VDCC. Undifferentiated and differentiated (1 mM dibutyryl cyclic AMP, 4 days) NG 108-15 cells were used. In both crude cellular extracts specific binding of 125I-CgTx was characterized. Differentiation induced a sixfold increase in the number of binding sites and doubled the KD value. The in vitro addition of guanylylimidodiphosphate (GMP-PNP; a nonhydrolyzable analogue of GTP) to extracts prepared from differentiated cells reduced the 125I-CgTx binding by 48%. This effect, observed in undifferentiated cells as well, was also caused by other triphosphate guanine nucleotides, such as GTP, but not by guanosine 5'-O-(2-thiodiphosphate) or adenine nucleotides. Treatment of the cells with PTx prevented the GMP-PNP effect. Moreover, the results obtained after preincubation with specific antisera raised against the alpha subunits of Gi1-2 and Go suggest that Go is the G protein responsible for the observed effect.

Topics: Animals; Binding Sites; Brain Neoplasms; Calcium Channels; Glioma; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hybrid Cells; Immunoblotting; Iodine Radioisotopes; Neuroblastoma; omega-Conotoxin GVIA; Peptides, Cyclic; Pertussis Toxin; Precipitin Tests; Protein Binding; Receptors, Neurotransmitter; Tumor Cells, Cultured; Virulence Factors, Bordetella

Neuroblastoma x glioma NG 108-15 hybrid cells contain a homogeneous population of delta-opioid receptors. NG 108-15 membranes were labelled either with the opiate agonist, [3H]etorphine or the opiate antagonist [3H]diprenorphine under various conditions: absence or presence of Na+ and/or 5'-guanylylimidophosphate (GppNHp). Ultracentrifugation in linear sucrose gradients after digitonin solubilization of prelabeled receptor was performed. In the soluble extracts from NG 108-15 hybrid cell membranes, bound [3H]etorphine and bound [3H]diprenorphine sedimented in the same position, even in the presence of NaCl and/or GppNHp. These data were analyzed in terms of relative agonist potency of diprenorphine on this specific model, using equilibrium binding studies and inhibition of adenylate cyclase activity. Diprenorphine, at the concentrations used for sedimentation studies, behaving as an opiate antagonist, it is concluded that the delta-opioid receptor could be strongly precoupled to the G-protein in the NG 108-15 cell.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cell Membrane; Centrifugation, Density Gradient; Glioma; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hybrid Cells; Mice; Narcotics; Nervous System Neoplasms; Neuroblastoma; Rats; Receptors, Opioid; Receptors, Opioid, delta; Tumor Cells, Cultured

The beta-adrenoceptor-coupled adenylate cyclase system in rat C6 glioma cells displays many characteristics observed in brain tissue: using nonlinear regression analysis of agonist competition binding curves, we demonstrated that the bulk of beta-adrenoceptors show high nanomolar affinity for isoproterenol; like in brain tissue, Gpp(NH)p does not shift agonist competition binding curves to the right; and the agonist isoproterenol rapidly downregulates the number of beta-adrenoceptors and deamplifies the norepinephrine signal. However, unlike in brain tissue, where (-)-oxaprotiline fails to decrease the number of beta-adrenoceptors and to desensitize the cyclic adenosine monophosphate generating system, it desensitizes the beta-adrenoceptor-coupled adenylate cyclase system in C6 glioma cells. Determination of the relative steady-state levels of beta-adrenoceptor messenger ribonucleic acid (mRNA) by Northern blot analysis showed a twofold increase in the steady-state levels of the mRNA at 30 minutes following exposure to (-)-isoproterenol or (-)-oxaprotiline. At 48 hours, basal values of mRNA were observed at a time when beta-adrenoceptors were maximally decreased. Further experiments on transcriptional activation, and mRNA stability and translation will be required to unravel the complexity of agonist-dependent and agonist-independent regulation of beta-adrenoceptor density and function.

Topics: Adenylyl Cyclases; Animals; Antidepressive Agents; Autoradiography; Blotting, Northern; Brain Neoplasms; Cells, Cultured; DNA Probes; Glioma; Guanylyl Imidodiphosphate; Isoproterenol; Maprotiline; Rats; Receptors, Adrenergic, beta; Regression Analysis; RNA; Tumor Cells, Cultured

The binding characteristics of [125I]angiotensin II (ANG II) to membranes prepared from undifferentiated and differentiated neuroblastoma x glioma hybrid cells (NG108-15) were investigated. Scatchard analysis revealed the existence of high and low affinity sites in differentiated cells, but only a low affinity site in undifferentiated cells. Similarly, self-displacement studies revealed competition to a single low affinity site in undifferentiated cells, and to high and low affinity sites in differentiated cells. Angiotensin III (ANG III) displaced high affinity binding in differentiated cells but did not displace low affinity binding in either differentiated or undifferentiated cells. Furthermore, 5-guanyl imidodiphosphate (GPP(NH)P) inhibited [125I]ANG II binding to differentiated cells, in a dose-dependent fashion, but had no effect on binding to indifferentiated cells. These findings suggest that the high affinity site represents a G-protein linked receptor with approximately equal affinities for ANG II and ANG III. We hypothesize that the low affinity site represents a non-specific membrane-bound aminopeptidase.

Topics: Angiotensin II; Animals; Binding, Competitive; Cell Differentiation; Cell Membrane; Glioma; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Mice; Neuroblastoma; Rats; Receptors, Angiotensin

Peptides derived from various regions of the alpha 2A-adrenergic receptor (alpha 2A-AR) were used to study receptor-G protein interactions. Binding of the partial agonist [125I]-p-iodoclonidine and the full agonist [3H]bromoxidine (UK14,304) to membrane preparations from human platelet was potently reduced by peptides (12-14 amino acids) from the second cytoplasmic loop (A) and the C-terminal side of the third cytoplasmic loop (Q). Binding of the antagonist [3H]yohimbine was significantly less affected. Five other peptides had no significant effects on ligand binding at concentrations less than 100 microM. The IC50 values for peptides A and Q were 7 and 27 microM for [125I]-p-iodoclonidine binding at the platelet alpha 2A receptor, 15 and 71 microM for the neuroblastoma-glioma (NG108-15) alpha 2B receptor, and greater than 300 microM for yohimbine binding at both alpha 2A and alpha 2B receptors. Competition studies demonstrate that at concentrations of 100 microM, peptides A and Q reduce the affinity of bromoxidine for the platelet alpha 2A-AR and this effect was abolished in the presence of guanine nucleotide. Alpha 2A-AR-stimulated GTPase activity in platelet membranes was inhibited by peptide Q with an IC50 of 16 microM but A was inactive. These data suggest that both the second cytoplasmic loop and the C-terminal part of the third cytoplasmic loop of the alpha 2A-AR are important in the interaction between the alpha 2-AR and Gi protein. Peptide Q appears to destabilize the high affinity state of the alpha 2-AR by binding directly to Gi thus preventing it from coupling to the receptor under both binding and GTPase assay conditions. The peptide from the second cytoplasmic loop (A) also reduces high affinity agonist binding in a G protein-dependent manner but its interaction with receptor and G protein is distinct in that it does not prevent activation of the G protein. These results provide new information about regions of the alpha 2-adrenergic receptor involved in G protein coupling and high affinity agonist binding.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Blood Platelets; Cell Line; Cell Membrane; Clonidine; Glioma; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Humans; Hybrid Cells; Kinetics; Molecular Sequence Data; Neuroblastoma; Peptides; Protein Conformation; Receptors, Adrenergic, alpha; Yohimbine

A synthetic peptide corresponding to the second extracellular loop of the beta 1-adrenergic receptor was used as an antigen for antibody production in three rabbits. Antibodies of high titers were obtained in all rabbits. Only one rabbit yielded antibodies which decreased radioligand binding on the receptor in a similar way to that described for autoantibodies in patients with dilated cardiomyopathy. These antibodies recognized the receptor protein in immunoblots. Epitope mapping indicated that the N-terminal sequence of the loop used as antigen was the target of the major antigen fraction. Incubation of antibodies with C6 glioma cell membranes or inner membranes of E. coli, which express the human beta 1-adrenergic receptor, resulted in a decrease in number of radioligand binding sites. This decrease was dependent on the concentration of antibody and of Mg++ ions. It was not affected by the GTP analog GppNHp or the beta 1 subtype-specific antagonist metoprolol. The agonist, isoproterenol, also induced a decrease but the effects of antibody and agonist were not additive. These results suggest that the antibodies induce a Mg(++)-dependent, 'active', labile conformation of the receptor, independent from coupling to the GTP regulatory protein, but similar to that induced by the agonist isoproterenol. This interpretation was corroborated by the beta 1-adrenergic receptor agonist-like effect of the antibodies on cardiomyocytes in culture.

Topics: Amino Acid Sequence; Animals; Autoantibodies; Binding Sites; Binding, Competitive; Cardiomyopathy, Hypertrophic; Dose-Response Relationship, Immunologic; Glioma; Guanylyl Imidodiphosphate; Humans; Iodocyanopindolol; Isoproterenol; Magnesium; Metoprolol; Molecular Sequence Data; Pindolol; Rabbits; Radioligand Assay; Receptors, Adrenergic, beta

Cholera toxin treatment (up to 1 microgram/ml, 16 h) of neuroblastoma x glioma hybrid NG108-15 cells produced a decrease of some 35% in both delta opioid receptor-mediated stimulation of high-affinity GTPase activity and inhibition of forskolin-amplified adenylate cyclase. Coincident with these decreases was a down-regulation of some 35% in the delta opioid receptor population. A similar pattern of a decrease in signalling capacity was noted for the alpha 2B-adrenergic receptor in these cells after cholera toxin treatment. Half-maximal effects of cholera toxin on all of the parameters assayed were noted at concentrations between 2 and 5 ng/ml. Neither levels of Gi2, as assessed by immunoblotting with specific antisera, nor the intrinsic activity of the alpha subunit of the guanine-nucleotide-binding protein which acts as the inhibitory G-protein of the adenylate cyclase in these cells, as assessed by guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p)-mediated inhibition of adenylate cyclase, was lowered by cholera toxin treatment. Furthermore, levels of another pertussis toxin-sensitive G-protein (Go) expressed by these cells was also not lowered by cholera toxin treatment. However, as previously noted in other cells [Milligan, Unson & Wakelam (1989) Biochem. J. 262, 643-649], marked down-regulation of the alpha subunit of the stimulatory G-protein (Gs) of the adenylate cyclase cascade was observed in response to cholera toxin treatment. Previous studies [Klee, Milligan, Simonds & Tocque (1985) Mol. Aspects Cell Regul. 4, 117-129] have shown that cholera toxin treatment can result in a decrease in the maximal effectiveness of agonists which function to inhibit adenylate cyclase. These data have been used as evidence to suggest a functional interaction between Gs and 'Gi'. The results provided herein demonstrate that such effects of the toxin can be explained adequately by a decrease in the number of receptors that function to produce inhibition of adenylate cyclase.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adrenergic alpha-Agonists; Brimonidine Tartrate; Cholera Toxin; Colforsin; Enkephalin, Leucine-2-Alanine; Glioma; GTP Phosphohydrolases; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hybrid Cells; Immunoblotting; Neuroblastoma; Pertussis Toxin; Quinoxalines; Receptors, Opioid; Receptors, Opioid, delta; Signal Transduction; Tumor Cells, Cultured; Virulence Factors, Bordetella

Chronic treatment of neuroblastoma x glioma NG108-15 hybrid cells with the opioid agonist D-Ala,2 D-Leu5-enkephalin (DADLE) induces a homologous desensitization of the delta opioid receptors present in these cells. Since the Kd value of the delta opioid receptor's high-affinity state reflects the potency of the agonist, we examined the effect of receptor desensitization in NG108-15 cells on the percentage of receptor in the high-affinity state. When NG108-15 hybrid cells were treated with 10 or 100 nM DADLE for 4 hr at 24 degrees C, loss of DADLE's ability to inhibit adenylate cyclase was observed. However, when competition binding experiments were carried out with P2P3 membranes isolated from the delta opioid-desensitized hybrid cells, it was determined that 41.7 +/- 3.4% of the total binding sites remained in the high-affinity state, with no apparent alteration in the Kd value of either high- or low-affinity states. Similarly, when NG108-15 cells were treated with 100 ng/ml of pertussis toxin for 3 hr at 37 degrees C, 39.9 +/- 3.6% of the binding sites remained in the high-affinity state. This reduction in the percentage of receptor in high-affinity state was agonist specific, for chronic treatment of hybrid cells with levorphanol, a partial agonist, or the antagonist naloxone did not alter the percentage of opioid receptors in the high-affinity state. Furthermore, the delta opioid receptors remaining in the high-affinity state after chronic DADLE treatment were still sensitive to both Na+ and guanyldylimidodiphosphate, indicating that opioid ligand binding remained coupled to the G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylate Cyclase Toxin; Binding, Competitive; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Guanosine Diphosphate; Guanylyl Imidodiphosphate; Humans; Hybrid Cells; Magnesium; Neuroblastoma; Pertussis Toxin; Receptors, Opioid; Receptors, Opioid, delta; Sodium; Virulence Factors, Bordetella

Topics: Adenylyl Cyclases; Animals; Azides; Calmodulin; Cerebral Cortex; Cytoskeleton; Enzyme Activation; Glioma; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Microtubules; Neurotransmitter Agents; Rats; Signal Transduction; Tubulin

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Amino Acid Sequence; Animals; Base Sequence; Cell Line; DNA, Neoplasm; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; GTP Phosphohydrolases; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hybrid Cells; Immune Sera; Kinetics; Mice; Molecular Sequence Data; NAD; Neuroblastoma; Oligonucleotide Probes; Pertussis Toxin; Rats; Receptors, Opioid; Receptors, Opioid, delta; RNA, Neoplasm; Signal Transduction; Virulence Factors, Bordetella

In membranes of undifferentiated neuroblastoma x glioma hybrid cell line NG108-15, the apparent specific binding of [3H]yohimbine measured in the presence of 1 microM noradrenaline, was increased substantially by the presence of the poorly hydrolysed analogue of GTP, guanylyl-imidodiphosphate (Gpp[NH]p) or by preincubation of membranes with antibodies against the C-terminal decapeptide of the alpha subunit of the G-protein Gi2. Such an effect was not produced by antibodies against the equivalent region of Go alpha Gi3 alpha or Gs alpha or from non-immune serum. By contrast, total specific binding of [3H]yohimbine was not modified by co-incubation with Gpp[NH]p or by preincubation with the antibodies from any of the anti-G protein antisera. These results demonstrate a direct interaction of the alpha 2B adrenergic receptor of NG108-15 cells with Gi2.

Topics: Amino Acid Sequence; Animals; Cell Line; Glioma; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hybrid Cells; Immune Sera; Immunoglobulin G; Kinetics; Mice; Molecular Sequence Data; Neuroblastoma; Norepinephrine; Peptides; Protein Binding; Rats; Receptors, Adrenergic, beta; Transducin; Virulence Factors, Bordetella; Yohimbine

We have carried out an extensive pharmacological characterization of muscarinic binding sites in rabbit lung and chicken heart in parallel with M1, M2, and M3 sites, [3H]Pirenzepine, a selective antagonist at M1 receptors, bound saturably and reversibly to membranes from chicken heart and rabbit lung. These binding sites were not M1 receptors, however, because the cardioselective antagonist himbacine had 10-fold higher affinity at these sites than at [3H]pirenzepine sites in rat and rabbit cortex (true M1 sites). We measured the inhibitory potency of 28 antagonists at [3H]N-methylscopolamine-labeled sites in chicken heart, rabbit lung, rat heart (M2 sites), and rat submandibular gland (M3 sites) and at M1 sites in rat cortex. The sites in rabbit lung were different from M1, M2, and M3 sites, because they had moderate to high affinity for M1-selective compounds (pirenzepine and telenzepine), M2-selective compounds (himbacine and methoctramine), and M3-selective compounds (hexahydrosiladifenidol and 4-diphenylacetoxy-N-methylpiperidine methiodide). The sites in chicken heart resembled most those in rabbit lung, with similar high affinity for secoverine, but they were not the same because tropicamide, diphenylacetoxybutynyl dimethylamine, and [3H]-N-methylscopolamine were more potent in rabbit lung. In a further series of experiments, we compared the affinity of six of the most discriminating antagonists in membranes from rabbit lung and NG108-15 cells, a neuroblastoma-glioma cell line reported to express the muscarinic m4 receptor gene. The antagonists had very similar affinities in the two tissues, the largest discrepancy being that pirenzepine was twice as potent in rabbit lung as in NG108-15 cells. Northern blots using probes designed to discriminate between five species of muscarinic receptor RNA detected only m4 mRNA in rabbit lung. We conclude that rabbit lung contains a muscarinic M4 binding site with a quite distinctive pharmacology and that chicken heart contains a receptor with similarities to the M4 sites. This is the first report to characterize native M4 binding sites in a nonneuronal mammalian tissue.

Topics: Animals; Chickens; Glioma; Guanylyl Imidodiphosphate; In Vitro Techniques; Kinetics; Lung; Male; Myocardium; N-Methylscopolamine; Neuroblastoma; Pirenzepine; Rabbits; Rats; Receptors, Muscarinic; RNA, Messenger; Scopolamine Derivatives; Species Specificity; Tumor Cells, Cultured

Regulation of [125I]beta h-endorphin binding by guanine nucleotides was investigated in membrane preparations from two opioid receptor-containing cell lines: NG108-15, which contains only delta opioid receptors, and SK-N-SH, which contains predominantly mu opioid receptors. In contrast to the binding of the delta-selective agonist [3H][D-penicillamine2,D-penicillamine5]enkephalin to NG108-15 cell membranes, and of the mu-selective agonist [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin to SK-N-SH cell membranes, [125I]beta h-endorphin binding to NG108-15 and SK-N-SH cell membranes was not altered by guanosine triphosphate (GTP) or guanylyl-5'-imidodiphosphate (Gpp(NH)p) in the absence of cations. However, in the presence of NaCl, [125I]beta h-endorphin binding to both cell lines was inhibited by GTP and Gpp(NH)p in a concentration-dependent manner. In SK-N-SH cell membranes, the ability of sodium to promote regulation of [125I]beta h-endorphin binding by GTP was mimicked by the monovalent cations lithium and potassium, but not by the divalent cations magnesium, calcium, or manganese. In NG108-15 cell membranes, only sodium was effective in promoting inhibition of [125I]beta h-endorphin binding by GTP. The effect of GTP or Gpp(NH)p in the presence of sodium was also observed with guanosine diphosphate, but not guanosine monophosphate or any of the non-guanine nucleotides tested. These results indicate that the presence of monovalent cations is required for regulation of [125I]beta h-endorphin binding by guanine nucleotides, and that the specificity of this cation requirement differs between the mu and delta receptor-containing cell lines.

Topics: beta-Endorphin; Cations; Cell Membrane; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Glioma; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Hybrid Cells; Neuroblastoma; Oligopeptides; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sodium Chloride; Tumor Cells, Cultured

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Animals; Calcium; Cell Line; Central Nervous System; Cyclic AMP; Glioma; Guanylyl Imidodiphosphate; Inositol Phosphates; Models, Biological; Neurotransmitter Agents; Phenylisopropyladenosine; Signal Transduction; Vasoactive Intestinal Peptide

Incubation of membranes of neuroblastoma x glioma hybrid, NG108-15 cells with GDP beta S followed by immunoblotting of resolved membrane and supernatant fractions with specific anti-peptide antisera showed essentially all of the alpha subunit of Go to be associated with the membrane. Similar experiments with poorly hydrolyzed analogues of GTP caused release of a significant fraction (some 50% within 60 minutes) of Go alpha into the supernatant. This was not mimicked by analogues of ATP. Antisera directed against peptides corresponding to the extreme N and C-termini of GO alpha demonstrated that the released polypeptide was not proteolytically clipped. These experiments show that the alpha subunit of GO need not be invariably bound to the plasma membrane and that guanine nucleotide activation can release the alpha subunit of GO from its site of membrane attachment.

Topics: Animals; Cell Line; Cell Membrane; Glioma; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Macromolecular Substances; Neuroblastoma; Thionucleotides

Using neuroblastoma cells as a model of developing neurons, we have tested the hypothesis that thyroid hormones alter cAMP metabolism. Neuroblastoma cells were grown in serum-free defined medium for 48 h with or without thyroid hormones. Treatment with 20-200 nM 3,5,3'-triiodo-L-thyronine (T3) increased the accumulation of cAMP by intact cells without altering growth, gross morphology, or DNA or protein content. The increase in cAMP accumulation could be detected 5 h after the addition of T3 and was abolished by the addition of cycloheximide. The maximum stimulation produced by prostaglandin E1 was increased in T3 cells without a significant alteration of the half-maximal concentration. T4 and D-T3 in concentrations up to 20 microM did not increase cAMP accumulation. Adenylate cyclase activity in response to forskolin, guanine nucleotides, and stimulatory hormones was increased in purified membranes from cells grown in T3, suggesting that increased adenylate cyclase is probably the major mechanism of the observed response to thyroid hormone.

Topics: Adenylyl Cyclases; Alprostadil; Animals; Astrocytoma; Cell Line; Colforsin; Cyclic AMP; Glioma; Guanylyl Imidodiphosphate; Kinetics; Neuroblastoma; Thyroid Hormones; Thyroxine; Triiodothyronine

Desensitization of the responsiveness to hormones or drugs is often mediated by down-regulation of receptors. The stimulatory coupling protein (Ns) of adenylate cyclase has been shown to be involved in the down-regulation of stimulatory beta-adrenergic receptors. Whether the inhibitory coupling protein (Ni) is involved in the down-regulation of receptors that inhibit adenylate cyclase is not known. We wished to determine whether down-regulation of inhibitory muscarinic cholinergic and alpha 2-adrenergic receptors occurs in neuroblastoma X glioma hybrid cells after the ability of Ni to inhibit adenylate cyclase is inactivated by pertussis toxin. After treatment of cells with pertussis toxin, the ability of carbachol or epinephrine to inhibit prostaglandin E1-stimulated cAMP accumulation in intact cells was either completely prevented or markedly attenuated, respectively, indicating functional inactivation of Ni. Furthermore, pertussis toxin treatment of membrane fragments from these cells did not result in labeling of the 41,000-dalton alpha-subunit of Ni with ADP ribose from [32P] NAD, indicating maximal ADP ribosylation of Ni by prior treatment of cells with pertussis toxin. Carbachol treatment of cells resulted in down-regulation of muscarinic cholinergic receptors to 45.7 +/- 12.5% and 52.5 +/- 13.5% of control values for toxin-untreated and toxin-treated cells, respectively. Epinephrine treatment of cells caused homologous desensitization of alpha 2-receptor-mediated inhibition of cAMP accumulation and down-regulation of alpha 2-adrenergic receptors to 42.9 +/- 11.4% and 53.2 +/- 5.3% of control values for toxin-untreated and toxin-treated cells, respectively. Down-regulation of muscarinic cholinergic receptors by carbachol and of alpha 2-adrenergic receptors by epinephrine was not due to the effect of retained agonist and was agonist specific, since it could be prevented by the antagonists atropine and yohimbine, respectively. We conclude that agonist-mediated down-regulation of both the muscarinic cholinergic receptor and the alpha 2-adrenergic receptor does not require functional inhibitory coupling.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Alprostadil; Animals; ATPase Inhibitory Protein; Carbachol; Cell Line; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Epinephrine; Glioma; Guanylyl Imidodiphosphate; Molecular Weight; NAD; Neuroblastoma; Pertussis Toxin; Proteins; Receptors, Adrenergic, alpha; Receptors, Cholinergic; Receptors, Muscarinic; Scopolamine; Virulence Factors, Bordetella; Yohimbine

The existence of multiple affinity states for the opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells has been demonstrated by competition binding studies with tritiated diprenorphine and [D-Ala2, D-Leu5]enkephalin (DADLE). In the presence of 10 mM Mg2+, all receptors exist in a high affinity state with Kd = 1.88 +/- 0.16 nM. Addition of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) decreased the affinity of DADLE to Kd = 8.08 +/- 0.93 nM. However, in the presence of 100 mM Na+, which is required for opiate inhibition of adenylate cyclase activity, analysis of competition binding data revealed three sites: the first, consisting of 17.5% of total receptor population has a Kd = 0.38 +/- 0.18 nM; the second, 50.6% of the population, has a Kd = 6.8 +/- 2.2 nM; and the third, 31.9% of the population, has a Kd of 410 +/- 110 nM. Thus, in the presence of sodium, a high affinity complex between receptor (R), GTP binding component (Ni), and ligand (L) was formed which was different from that formed in the absence of sodium. These multiple affinity states of receptor in the hybrid cells are agonist-specific, and the percentage of total opiate receptor in high affinity state is relatively constant in various concentrations of Na+. Multiple affinity states of opiate receptor can be demonstrated further by Scatchard analysis of saturation binding studies with [3H]DADLE. In the presence of Mg2+, or Gpp(NH)p, analysis of [3H]DADLE binding demonstrates that opiate receptor can exist in a single affinity state, with apparent Kd values of [3H]DADLE in 10 mM Mg2+ = 1.75 +/- 0.28 nM and in 10 microM Gpp(NH)p = 0.85 +/- 0.12 nM. There is a reduction of Bmax value from 0.19 +/- 0.02 nM in the presence of Mg2+ to 0.14 +/- 0.03 nM in the presence of Gpp(NH)p. In the presence of 100 mM Na+, Scatchard analysis of saturation binding of [3H]DADLE reveals nonlinear plots; two-site analysis of the curves yields Kd = 0.43 +/- 0.09 and 7.9 +/- 3.2 nM. These Kd values are analogous to that obtained with competition binding studies. Again, this conversion of single site binding Scatchard plots to multiple sites binding plots in the presence of Na+ is restricted to 3H-agonist binding only.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Binding, Competitive; Cell Line; Diprenorphine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Magnesium; Neuroblastoma; Receptors, Opioid; Sodium

Neuroblastoma-glioma NG108-15 cells that were cultured for 48 h with the opiate antagonist, naloxone, respond to the guanosine 5'-triphosphate (GTP) analogue guanosine 5'-[beta, gamma-imido]-triphosphate (GMP-PNP) in the binding assay as the control, non-treated, cells. This was observed when the guanyl nucleotide was tested in the presence or absence of sodium chloride and also after subcellular fractionation of the membranes on a sucrose gradient which separated between two receptor-containing fractions. The findings suggest that the increase in delta type enkephalin receptors in naloxone-treated NG108-15 cells does not reflect an alteration in the interaction between the receptor and the adenylate cyclase-GTP-binding protein system.

Topics: Animals; Cells, Cultured; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Naloxone; Neuroblastoma; Rats; Receptors, Opioid; Receptors, Opioid, delta

Topics: Adaptation, Physiological; Adenylyl Cyclases; Animals; Cell Line; Clone Cells; Cytosol; Endorphins; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Etorphine; Glioma; Guanylyl Imidodiphosphate; Lanthanum; Mice; Naloxone; Narcotics; Neuroblastoma; Rats; Receptors, Opioid; Sodium; Time Factors

Exposure of NG108-15 hybrid cells to islet-activating protein (IAP), pertussis toxin, caused strong ADP-ribosylation of one of the membrane proteins with a molecular weight of 41,000. This ADP-ribosylation was paralleled by decreases in the inhibition of cAMP accumulation in intact cells or associated with reversal of the inhibition of GTP-dependent membrane adenylate cyclase, via alpha-adrenergic, cholinergic muscarinic, or opiate receptors. The affinity of these receptors for agonists was lowered by guanyl-5'-yl beta-gamma-imidodiphosphate (Gpp(NH)p) reflecting their coupling to the guanine nucleotide regulatory protein in this cell line. This effect of Gpp(NH)p was lost in membranes of IAP-treated cells; in the absence of Gpp(NH)p, the affinity for agonist was lower in treated than in nontreated cells. In contrast, the function of these receptors to bind antagonists remained unaltered in IAP-treated cells. Thus, IAP treatment of NG108-15 cells caused specific uncoupling of negative signal transduction from inhibitory receptors to the adenylate cyclase catalytic unit via the guanine nucleotide regulatory protein, as a result of ADP-ribosylation of one of the subunits of the regulatory protein.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Animals; Bacterial Proteins; Cyclic AMP; Glioma; Guanylyl Imidodiphosphate; Hybrid Cells; Membrane Proteins; Mice; Neuroblastoma; Pertussis Toxin; Rats; Receptors, Adrenergic; Receptors, Adrenergic, alpha; Receptors, Cholinergic; Receptors, Opioid; Virulence Factors, Bordetella

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Animals; Cell Line; Dihydroalprenolol; Enzyme Activation; Epinephrine; Glioma; Guanylyl Imidodiphosphate; Isoproterenol; Kinetics; Propranolol; Rats; Receptors, Adrenergic; Receptors, Adrenergic, beta

Prostaglandin E1 receptor sites were measured in homogenates of NG108-15 neuroblastoma-glioma hybrid cells after exposure of intact cells to PGE1. Scatchard analysis of competitive binding studies showed that incubation of NG108-15 cells in the presence of 2.5 microM PGE1 for 16 h resulted in a loss of PGE1 receptors and an increase in the dissociation constant of the remaining receptors. Thus, cells challenged with PGE1 not only lose adenylate cyclase activity, but also lose PGE1 receptors and decreased the affinity of the remaining receptors for PGE1.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Alprostadil; Animals; Cyclic AMP; Cytidine Triphosphate; Glioma; Guanylyl Imidodiphosphate; Hybridomas; Neuroblastoma; Phosphocreatine; Prostaglandins E; Receptors, Cell Surface; Receptors, Prostaglandin; Receptors, Prostaglandin E; Time Factors

The cAMP content of intact cells as well as adenylate cyclase of the membrane-rich particulate fractions was studied with C6 glioma cells that had been exposed to the culture medium supplemented with islet-activating protein (IAP), one of the pertussis toxins. Both the increase in the cellular cAMP content in response to a beta-adrenergic agonist and the stimulation of membrane adenylate cyclase by the beta-agonist and/or GTP were markedly enhanced by the IAP treatment of C6 cells, but no change was induced in affinities of the agonist (or an antagonist) or GTP for their respective sites of action (or binding). The concentration of IAP required for the half-maximal enhancement was as low as 1 pg/ml, when the time of cell exposure to the toxin was prolonged to 18 h. No enhancement was observed for the basal cAMP content or basal enzyme activity, nor was activation of adenylate cyclase by Gpp(NH)p (or NaF) affected by IAP treatment. The Vmax value of a specific and low Km GTPase was significantly smaller in the membranes of IAP-treated cells than in those of control cells. Cholera toxin treatment of cells activated adenylate cyclase without exerting any influence on these IAP actions. Thus, IAP would appear to enhance beta-receptor-coupled stimulation of adenylate cyclase, in a manner distinct from cholera toxin, by rendering more GTP available to the GTP sites on the regulatory subunit of the receptor-enzyme system.

Topics: Adenine Nucleotides; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Bacterial Proteins; Calcium; Cell Line; Cell Membrane; Cyclic AMP; Glioma; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hypoglycemic Agents; Isoproterenol; Pertussis Toxin; Rats; Virulence Factors, Bordetella

The guanine nucleotides GDP, GTP, and guanosine-5'-(beta, gamma-imido)triphosphate inhibit binding of opiates and opioid peptides to receptors solubilized from membranes of neuroblastoma X glioma NG108-15 hybrid cells. The inhibition reflects decreased affinity of receptors for opioid ligands. Whereas in membranes, only opioid agonist binding is sensitive to guanine nucleotide inhibition, both agonist and antagonist binding is reduced in the case of soluble receptors. Furthermore, soluble receptors are more sensitive to the effects of guanine nucleotides than are membrane-bound receptors. These observations are consistent with the suggestion that solubilized receptors may be complexes of an opiate binding protein and a guanine nucleotide-sensitive regulatory component.

Topics: Animals; Cell Line; Cell Membrane; Diprenorphine; Etorphine; Glioma; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Mice; Morphinans; Neuroblastoma; Rats; Receptors, Opioid

D-Ala2-Met5-enkephalin, morphine, and noradrenaline inhibit the adenylate cyclase in homogenates of neuroblastoma x glioma hybrid cells in a dose-dependent manner even after the enzyme has been preactivated by cholera toxin. Half-maximal inhibition and extent of inhibition are the same with native or cholera toxin-activated enzyme. The inhibition caused by opioids or noradrenaline are antagonized by naloxone or phentolamine, respectively. The effect of D-Ala2-Met5-enkephalin on cholera toxin-activated enzyme is immediate in onset and rapidly reversed by the addition of naloxone. Guanyl-5'-yl-imidodiphosphate stimulates basal activity but inhibits the enzyme activated by cholera toxin or prostaglandin E1. Stimulation occurs at a concentration of 100 microM or above, inhibition even at 0.1 microM. The inhibitory effect of the non-hydrolysable GTP analog is antagonized by GTP. Guanyl-5'-yl-methylenediphosphonate, another nonhydrolysable GTP analog, inhibits basal as well as cholera toxin-stimulated or prostaglandin E1-stimulated adenylate cyclase. Other guanine derivatives such as GDP, GMP, cyclic GMP, guanyl-5'-yl-phosphoric acid amide and guanosine have no effect under the same conditions. The results may be taken as a piece of evidence for two separate guanyl nucleotide-binding sites accompanying the adenylate cyclase in the hybrid cells and mediating, respectively, stimulation and inhibition of the enzyme by hormones.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cholera Toxin; Clone Cells; Diphosphonates; Endorphins; Enkephalin, Methionine; Enkephalins; Glioma; Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Mice; Morphine; Neuroblastoma; Norepinephrine; Rats

Topics: Adenylyl Cyclase Inhibitors; Cell Line; Glioma; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Neuroblastoma; Prostaglandins E; Receptors, Adrenergic, alpha; Receptors, Cholinergic; Receptors, Muscarinic; Receptors, Opioid; Sodium

Topics: Adenosine; Adenylyl Cyclases; Animals; Caffeine; Cell Line; Cell Membrane; Deoxyadenosines; Enzyme Activation; Glioma; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Macromolecular Substances; Mice; Neuroblastoma

Topics: Adenylyl Cyclase Inhibitors; Adrenergic alpha-Antagonists; Animals; Cells, Cultured; Cyclic AMP; Cycloheximide; Glioma; Guanylyl Imidodiphosphate; Hybrid Cells; Mice; Neoplasms, Experimental; Neuroblastoma; Norepinephrine; Rats; Receptors, Adrenergic; Receptors, Adrenergic, alpha; Sodium Fluoride

Topics: Animals; Binding, Competitive; Cell Line; Enkephalins; Glioma; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Neuroblastoma; Receptors, Opioid; Sodium

Topics: Cell Membrane; Cells, Cultured; Enkephalins; Etorphine; Glioma; Guanylyl Imidodiphosphate; Hybrid Cells; Magnesium; Manganese; Naloxone; Neuroblastoma; Nucleotides; Receptors, Opioid; Sodium

Reactions mediated by the opiate receptors that inhibit adenylate cyclase (EC 4.6.1.1) are closely coupled to subsequent reactions that gradually increase adenylate cyclase activity of neuroblastoma X glioma NG108-15 hybrid cells. Opiate-treated cells have higher basal-, prostaglandin E1-, and 2-chloroadenosine-stimulated activities than do control cells. However, NaF or guanosine 5'-(beta, gamma-imido)triphosphate abolishes most of the differences in adenylate cyclase activity observed with homogenates from control and opiate-treated cells. Cycloheximide blocked some, but not all, of the opiate-dependent increase in adenylate cyclase activity. These results suggest that the opiate-dependent increase in adenylate cyclase is due to conversion of adenylate cyclase to a form with altered activity. Protein synthesis also is required for part of the opiate effect. We propose that activity of adenylate cyclase determines the rate of conversion of the enzyme from one form to the other and that opiates, by inhibiting adenylate cyclase, alter the relative abundance of low- and high-activity forms of the enzyme.

Topics: Adenosine; Adenylyl Cyclases; Cell Line; Cycloheximide; Enzyme Activation; Etorphine; Fluorides; Glioma; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Morphinans; Morphine; Naloxone; Neuroblastoma; Prostaglandins E