guanosine-triphosphate and Glioma

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder whose major feature is the occurrence of multiple neurofibromas, which are benign tumors of the nerve sheath. It affects an estimated one in 3000 to 4000 individuals. In addition to neurofibromas, there are many other clinical manifestations, including malignant tumors such as gliomas or malignant peripheral nerve sheath tumors, and nontumor effects such as skeletal dysplasia and learning disability. Diagnosis is established on the basis of clinical criteria. Molecular genetic testing is feasible, but the large size of the gene and wide range of pathogenic mutations have so far impeded the development of a clinical diagnostic test. Insights into pathogenesis have followed from identification of the NF1 gene and the development of animal models. The major function of the gene product appears to be regulation of the ras protein. Tumors are believed to arise by the loss of function of the NF1 protein, suggesting that NF1 behaves as a tumor suppressor gene. Heterozygous effects on some cell types are also likely, however. The role of ras in the pathogenesis of tumors in NF1 has suggested an approach to treatment using ras inhibitors, some of which are likely to begin in clinical trials in NF1 patients in the near future.

Topics: Animals; Brain Neoplasms; Cafe-au-Lait Spots; Cell Transformation, Neoplastic; Female; Genes, Dominant; Genes, Neurofibromatosis 1; Glioma; Guanosine Triphosphate; Humans; Hypertension; Learning Disabilities; Leukemia; Male; Mice; Mice, Knockout; Neurofibroma; Neurofibromatosis 1; Neurofibromin 1; Protein Structure, Tertiary; ras Proteins; Rhabdomyosarcoma; Scoliosis

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

Tubulin is known to form high-affinity complexes with certain G proteins. The formation of such complexes allows tubulin to activate Galpha and fosters a system whereby elements of the cytoskeleton can influence G-protein signaling. This article describes the interaction between tubulin and G proteins and discusses methods for examining this interaction.

Topics: Animals; Cell Line; Cell Membrane; Cytoskeleton; Enzyme Activation; Glioma; GTP-Binding Protein alpha Subunits; GTP-Binding Proteins; Guanosine Triphosphate; Isoenzymes; Phospholipase C beta; Protein Binding; Rats; Recombinant Fusion Proteins; Second Messenger Systems; Tubulin; Type C Phospholipases

Cyclic ADP-ribose (cADP-ribose) is a putative second messenger or modulator. However, the role of cADP-ribose in the downstream signals of the metabotropic glutamate receptors (mGluRs) is unclear. Here, we show that glutamate stimulates ADP-ribosyl cyclase activity in rat or mouse crude membranes of retina via group III mGluRs or in superior cervical ganglion via group I mGluRs. The retina of mGluR6-deficient mice showed no increase in the ADP-ribosyl cyclase level in response to glutamate. GTP enhanced the initial rate of basal and glutamate-stimulated cyclase activity. GTP-gamma-S also stimulated basal activity. To determine whether the coupling mode of mGluRs to ADP-ribosyl cyclase is a feature common to individual cloned mGluRs, we expressed each mGluR subtype in NG108-15 neuroblastoma x glioma hybrid cells. The glutamate-induced stimulation of the cyclase occurs preferentially in NG108-15 cells over-expressing mGluRs1, 3, 5, and 6. Cells expressing mGluR2 or mGluRs4 and 7 exhibit inhibition or no coupling, respectively. Glutamate-induced activation or inhibition of the cyclase activity was eliminated after pre-treatment with cholera or pertussis toxin, respectively. Thus, the subtype-specific coupling of mGluRs to ADP-ribosyl cyclase via G proteins suggests that some glutamate-evoked neuronal functions are mediated by cADP-ribose.

Topics: Adenosine Diphosphate Ribose; ADP-ribosyl Cyclase; Animals; Cell Membrane; Cells, Cultured; Cholera Toxin; Enzyme Activation; Glioma; Glutamic Acid; Guanosine Triphosphate; Mice; Mice, Knockout; Neuroblastoma; Neurons; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Retina; Signal Transduction; Superior Cervical Ganglion

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

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event.

Topics: Adenosine Triphosphate; Adrenergic beta-Agonists; Animals; Cell Differentiation; Cell Division; Cells, Cultured; Centrosome; Glioma; Guanosine Triphosphate; Immunohistochemistry; Isoenzymes; Microtubules; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Precipitin Tests; Rats; Recombinant Proteins; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tubulin

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 presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the alpha- and gamma-phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for approximately 50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 microM abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a Km for ATP of 17 +/- 5 microM. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.

Topics: Adenosine Triphosphate; Animals; Astrocytes; Enzyme Inhibitors; Extracellular Space; Glioma; Guanosine Triphosphate; Hydrolysis; Nucleoside-Diphosphate Kinase; Phosphorus Radioisotopes; Phosphorylation; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Pyrophosphatases; Rats; Receptors, Purinergic; Stem Cells; Tumor Cells, Cultured

Preliminary studies have demonstrated that the Ras family and related guanosine triphosphate-dependent proteins are overactivated in malignant gliomas and that inhibition of the activation of such proteins, by blockade of their post-translational processing, reduces tumor cell growth in vitro. The current study evaluates the utility of this therapeutic strategy in vivo, using preclinical glioma model systems.. We examined the efficacy against U-87 human malignant glioma cells, in both subcutaneous and intracranial nude mouse models, of selective peptidomimetic inhibitors of farnesyltransferase (FTI-276) and geranylgeranyltransferase (GGTI-297), which are involved in critical steps in the post-translational processing of Ras and related guanosine triphosphate-dependent proteins. For the subcutaneous model, 2 x 10(5) U-87 cells were implanted; after measurable tumors were detected on Day 7, animals were treated with either FTI-276, GGTI-297, or vehicle, administered by continuous infusion for 7 days. Differences in tumor volumes among the treatment groups were examined for significance using a Student's t test. For the intracranial model, 2 x 10(5) U-87 cells were implanted in the right frontal lobe and treatment was initiated on Day 7. In initial studies, animals received a 7-day course of either FTI-276, GGTI-297, or vehicle. In subsequent studies, a 28-day treatment period was used. Comparisons of survival times among treatment groups were performed using a rank-sum test.. Although the two agents exhibited comparable antiproliferative activities in previous in vitro studies, an obvious difference in efficacy was apparent in this study. Whereas the geranylgeranyltransferase inhibitor failed to improve survival rates, compared with those observed for control animals, in either the subcutaneous or intracranial model, the farnesyltransferase inhibitor produced objective regression of tumor growth in the subcutaneous model and significant prolongation of survival times in the intracranial model, without apparent toxicity. In the subcutaneous model, tumor volumes for the control, GGTI-297-treated, and FTI-276-treated animals on Day 28 after implantation were 621+/-420, 107+/-104, and 18.5+/-12.7 mm3, respectively (P < 0.05). In the 7-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 27.7+/-2.9, 29.8+/-2.1, and 43.6+/-2.7 days, respectively (P < 0.001). In the 28-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 29.2+/-3.7, 28.3+/-3.9, and 58.7+/-6.2 days, respectively, with five of six animals in the latter group surviving more than 55 days after tumor implantation (P < 0.001).. These studies demonstrate that farnesyltransferase inhibition is effective in diminishing the growth of human glioma cells in vivo. Evaluation of this treatment approach in clinical trials is warranted.

Topics: Alkyl and Aryl Transferases; Animals; Benzamides; Brain Neoplasms; Cell Division; Drug Evaluation, Preclinical; Enzyme Inhibitors; Farnesyltranstransferase; Geranyltranstransferase; Glioma; Guanosine Triphosphate; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Proto-Oncogene Proteins p21(ras); Treatment Outcome; 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

The coupling efficiencies of beta-1 and beta-2-adrenergic receptors (ARs) in rat C6 glioma cells and the effect of changes in subtype density and ratio caused by dexamethasone (DEX) treatment were studied. Radioligand binding studies in membranes suggested that beta-2-ARs showed slightly larger GTP-induced decreases in agonist affinity than beta-1-ARs, which suggests more ternary complex formation with Gs. Treatment with DEX increased the proportion of beta-2-ARs from 20% to 60% without affecting GTP-induced decreases in agonist affinity. Coupling efficiency was determined directly by progressive inactivation of beta-ARs with the irreversible alkylating agent pindobind, which caused similar reductions in beta-1 and beta-2-AR binding sites. Studies on cyclic AMP accumulation showed that DEX-induced decreases in beta-1/beta-2 ratio reduced the receptor reserve for the beta-1-selective agonist norepinephrine but increased the maximal response to the beta-2-selective agonist zinterol without creating a receptor reserve. DEX treatment did not alter occupancy-response curves or maximal responses to the nonselective agonists isoproterenol and epinephrine. It was concluded that changing beta-2-AR density alters the maximal response to beta-2-AR activation; changing beta-1-AR density alters the apparent beta-1-AR reserve. Both subtypes contribute to the responses to nonselective agonists in a nonadditive manner. Beta-1- and beta-2-ARs appear to couple with different efficiencies (beta-1 > beta-2) to cyclic AMP accumulation in C6 glioma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cyclohexane Monoterpenes; Dexamethasone; Glioma; Guanosine Triphosphate; In Vitro Techniques; Pindolol; Rats; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Tumor Cells, Cultured

The effects of dibutyryl cyclic AMP (dbcAMP) treatment on Ca2+ channel activities, Ca2+ accumulation by intracellular Ca2+ pools, and sizes of IP3- and GTP-releasable pools in neuroblastoma x glioma hybrid NG108-15 cells were studied. High extracellular K+ induced a greater rise in intracellular calcium concentration ([Ca2+]i) in dbcAMP-treated cells than in control cells. In dbcAMP-treated cells, the initial phase of the high K(+)-induced [Ca2+]i rise displayed a much higher sensitivity to omega-conotoxin than it did in control cells, whereas the plateau phase of the [Ca2+]i rise was sensitive only to nifedipine. These results indicate that predominantly L-type Ca2+ channels exist in control cells, and that N-type channels develop only after dbcAMP treatment. In dbcAMP-treated cells, mitochondria showed an increased Ca2+ uptake capacity (5.3 nmol Ca2+/mg protein) compared with that in control cells (4.2 nmol Ca2+/mg protein). However, dbcAMP treatment did not cause significant change in the affinity for Ca2+. Dibutyryl cAMP treatment enhanced the Ca2+ accumulation activity by nonmitochondrial pools (from 0.84 to 0.97 nmol Ca2+/mg protein) and increased the affinity for Ca2+ (EC50 for Ca2+ decreased from 0.146 microM to 0.063 microM). Our data also indicate that the pool that is sensitive to both IP3 and GTP was enlarged. The affinities for IP3 and GTP in causing Ca2+ release remained the same before or after dbcAMP treatment.

Topics: Bucladesine; Calcium; Calcium Channels; Electrophysiology; Glioma; Guanosine Triphosphate; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Mitochondria; Neuroblastoma; Signal Transduction; Tumor Cells, Cultured

Topics: Affinity Labels; Animals; Autoradiography; Azides; Electrophoresis, Polyacrylamide Gel; Glioma; GTP-Binding Proteins; Guanosine Triphosphate; Indicators and Reagents; Macromolecular Substances; Molecular Structure; Phosphorus Radioisotopes; Recombinant Proteins; Synaptic Membranes; Tubulin; Tumor Cells, Cultured

We used 31P-NMR spectroscopy to investigate the response of living C6 glioma cells to stimulation by a beta-adrenergic agonist, isoproterenol. In the presence of 3-isobutyl-1-methylxanthine, stimulation induced an accumulation of cAMP, making possible the NMR detection of the second messenger in living cells grown on microcarrier beads and perfused in the NMR tube. The cAMP signal rose to a maximum level within 20-25 min of stimulation; thereafter it decreased to the detection threshold within 60 min. At the same time, 40% increases of phosphomonoester and diphosphodiester signals were observed, whereas no significant change in phosphocreatine and nucleotide signals was detected. The kinetics of changes of the cellular content in phosphorylated metabolites were analyzed after recording 31P-NMR spectra of cell perchloric acid extracts as a function of time of stimulation. cAMP accumulation in stimulated cells was evidenced by a near linear increase of its NMR signal as a function of incubation time (from 0 to 60 min). Concomitantly with the production of cAMP, the data showed 30% decreases of phosphocreatine and ATP levels within 60 min of stimulation, and an unexpected redistribution of pyrimidine and purine nucleoside triphosphates. At the same time, levels of phosphomonoesters (phosphorylcholine and phosphorylethanolamine) and phosphodiesters (glycerophosphorylcholine and glycerophosphorylethanolamine) rose (50% increase). 13C-NMR spectra of cell perchloric acid extracts prepared after isoproterenol stimulation of cells incubated in the presence of [1-13C]glucose indicated a higher glucose content in stimulated cells, whereas the resonance of ribose C1 was diminished. Moreover, the resonances of C1 of ethanolamine and choline (and their derivatives) were increased in spectra of stimulated cells, whereas that of C3 of serine was decreased. In addition, the 13C-NMR data indicated that neither the pattern of glutamate carbon enrichment nor the glutamate/glutamine ratio was modified in stimulated cells. On the other hand, the heteronuclear coupling pattern of the lactate (methyl group) resonance in 1H-NMR spectra of cell incubation media indicated that no change occurred in the carbon flux through the pentose-phosphate shunt under stimulation. The results of this multinuclear NMR approach are discussed in terms of metabolic responses of C6 cells to beta-adrenergic stimulation and cAMP overproduction.

Topics: Adenosine Triphosphate; Animals; Carbon Isotopes; Cyclic AMP; Glioma; Glucose; Glutamates; Glutamine; Guanosine Triphosphate; Isoproterenol; Kinetics; Lactates; Magnetic Resonance Spectroscopy; Phosphorus; Phosphorylation; Receptors, Adrenergic, beta; Tumor Cells, Cultured

Interactions between beta-adrenergic and ADP purinergic receptors in C6 glioma cell membrane preparations were investigated under steady state and then pre-steady state conditions of adenylyl cyclase (EC 4.6.1.1) activity, in order to determine how fast the second receptor antagonizes the transduction mechanism of the first. Cell membranes were washed to deplete them as thoroughly as possible of low molecular weight compounds, especially ATP and ADP, and to ensure better control of both substrate and agonist nucleotide concentrations. ATP concentrations were kept constant with the use of an ATP-regenerating system; the C6 cell line exhibited very active ectonucleotidases. The purinergic agonist ADP was replaced by its nonhydrolyzable congener adenosine 5'-O-(2-thio)diphosphate (ADP beta S), which was demonstrated, like ADP, to inhibit isoproterenol-stimulated adenylyl cyclase activity in intact cells (IC50 for ADP, 0.5 +/- 0.1 microM; IC50 for ADP beta S, 25 +/- 2 microM) and in membrane preparations (IC50 for ADP beta S, 79 +/- 20 microM). In the case of membrane preparations, ADP beta S did not compete with ATP, the substrate of the cyclase-catalyzed reaction, and behaved apparently as a non-competitive inhibitor of the enzyme. The pre-steady state kinetics of isoproterenol-stimulated adenylyl cyclase activity measured with a pulsed quenched-flow apparatus have previously been shown to include two steps, the first very rapid (taking place within 1-2 sec) and giving rise to a burst of cAMP synthesis and the second much slower and corresponding to the steady state reaction. ADP beta S inhibited the occurrence of both steps with comparable IC50 values (mean value, 55 +/- 20 microM). In the presence of increasing concentrations of the purinergic receptor agonist, the time constant of the exponential burst reaction was not affected, but its amplitude progressively decreased to zero. These results showed that the extinction of the beta receptor cAMP response by the purinergic ADP receptor occurred within the dead-time of the pulsed quenched-flow apparatus, which was 50 msec. Such a rapid inhibition of cAMP production excluded modulation of isoproterenol-stimulated adenylyl cyclase activity by the ADP receptor by a pathway other than its direct negative coupling to the cyclase via a Gi protein. In this respect, the P2 purinergic ADP receptor of the C6 glioma cell line appears comparable to the P2t receptor of platelets.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adenylate Cyclase Toxin; Adenylyl Cyclases; Cell Membrane; Cyclic AMP; Enzyme Activation; Glioma; Guanosine Triphosphate; Isoproterenol; Kinetics; Pertussis Toxin; Receptors, Adrenergic, beta; Receptors, Purinergic; Thionucleotides; Tumor Cells, Cultured; Virulence Factors, Bordetella

Incubation of the human glioma cell line HS 683 in the presence of IFN-gamma or retinoic acid strongly stimulates the cell-surface expression of the intercellular adhesion molecule ICAM-1. We have investigated the role of the cAMP-mediated signal transduction pathway in this process and report that pharmacological agents which increased the intracellular levels of cAMP exhibited a biphasic action on ICAM-1 expression in human glioma cell line HS 683. Treatment for 1 hr with 25 microM forskolin or 1 mM isobutylmethylxanthine, or for 12 hr with 100 ng/ml pertussis toxin or 50 micrograms/ml cholera toxin transiently stimulated ICAM-1 expression with a maximal level of expression 8 hr post treatment, after which time ICAM-1 expression returned to the basal level. On the other hand, such pretreatments inhibited the inducing effects of either retinoic acid or IFN-gamma. Indeed, 24 hr after treatment with cAMP-elevating agents, both the retinoic-acid- and the IFN-gamma-induced ICAM-1 expression were inhibited by 60 to 80%, with a maximal 90 to 100% inhibition 72 hr post treatment. This inhibition of the cell-surface expression of ICAM-1 was confirmed at the mRNA level. The intracytoplasmic levels of cAMP were also quantified following treatments with forskolin, retinoic acid or IFN-gamma. In response to forskolin, cAMP levels increased 30-fold within 5 min, whereas a 10-fold increase occurred 60 min following treatment with 10 microM retinoic acid. Interferon gamma, in contrast, did not induce cAMP accumulation. These results were also correlated with an in vitro activation of adenylyl cyclase activity by retinoic acid and inhibition of this activity by IFN-gamma, in a dose-dependent and a GTP-dependent manner. Our results suggest that the suppression of IFN-gamma-induced ICAM-1 expression, obtained upon pre-treatment with cAMP-elevating agents, is due to direct antagonism with IFN-gamma action on adenylyl cyclase. However, the inhibition of retinoic-acid-induced ICAM-1 expression cannot be explained by the same mechanisms. The timing of adenylyl cyclase stimulation and cAMP accumulation, as well as the levels of cAMP accumulation, are probably involved in this inhibition. Our results also emphasize the fact that the induction of ICAM-1 expression is a multi-step process implicating different transductional signals among which cAMP might be involved as a second messenger.

Topics: 1-Methyl-3-isobutylxanthine; Adenylate Cyclase Toxin; Adenylyl Cyclases; Cell Adhesion Molecules; Cholera Toxin; Colforsin; Cyclic AMP; Cytosol; Gene Expression Regulation, Neoplastic; Glioma; Guanosine Triphosphate; Humans; Intercellular Adhesion Molecule-1; Interferon-gamma; Kinetics; Pertussis Toxin; Recombinant Proteins; Stimulation, Chemical; Time Factors; Tretinoin; Tumor Cells, Cultured; 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

The intracellular nonmitochondrial calcium pools of saponin-permeabilized NG108-15 cells were characterized using inositol 1,4,5-trisphosphate (IP3) and GTP. IP3 or GTP alone induced release of 47 and 68%, respectively, of the calcium that was releasable by A23187. GTP induced release of a further 24% of the calcium after IP3 treatment, whereas IP3 induced release of a further 11% of the calcium after GTP treatment. Guanosine 5'-O-(3-thio)triphosphate had little effect on IP3-induced calcium release but completely inhibited GTP-induced calcium release. In contrast, heparin inhibited the action of IP3 but not that of GTP. The results imply the existence of at least three nonmitochondrial pools: (a) 31% is releasable by IP3 and GTP, (b) 11% is releasable by IP3 alone, and (c) 24% is releasable by GTP alone. GTP enhanced calcium uptake in the presence of oxalate with an EC50 of 0.6 microM and stimulated calcium release in the absence of oxalate with an EC50 of 0.32 microM. The similar EC50 values for these dual effects of GTP on calcium movement suggest that GTP exerts its dual action by the same mechanism.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Calcimycin; Calcium; Calcium-Transporting ATPases; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Cell Membrane Permeability; Glioma; Guanosine Triphosphate; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Kinetics; Mice; Neuroblastoma; Oligomycins; Rats

As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5'-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.

Topics: Animals; Cell Membrane; Glioma; GTP-Binding Proteins; Guanosine Triphosphate; Rats; Tumor Cells, Cultured

The effect of GTP on Ca2+ uptake and release was studied in a microsomal fraction isolated from neuroblastoma x glioma hybrid NG108-15 cells. GTP did not alter the ATP-dependent initial uptake of Ca2+ but markedly enhanced the efflux of Ca2+ from microsomes. GTP-dependent Ca2+ release requires the presence of millimolar concentration of Mg2+. The effect of GTP was not mimicked by other nucleotides and was competitively blocked by the thiophosphate analogue of GTP, GTP gamma S but not by the non-hydrolyzable nucleotide GMP-PNP. Addition of an inhibiting concentration of GTP gamma S after completion of GTP-induced calcium release did not result in a re-uptake of Ca2+, showing the irreversibility of the releasing effect of GTP. Our data are consistent with the hypothesis of Ca2+-dependent GTP-induced opening of a channel responsible for vectorial transport of Ca2+ ions from one intracellular compartment to another. A model is proposed suggesting that the GTP-binding protein is a GTP-specific diacylglycerol kinase.

Topics: Animals; Calcimycin; Calcium; Cell Line; Glioma; Guanosine Triphosphate; Hybrid Cells; Intracellular Membranes; Kinetics; Magnesium; Mice; Microsomes; Neuroblastoma; Oxalates; Rats

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

The activity of beta-adrenergic receptors at the plasma membrane level was investigated in viable, electropermeabilized C6 glioma cells. Electric field pulses were applied directly to the plated cells without any previous proteinase treatment. The affinity for isoproterenol and the density of the beta-adrenergic receptors, as judged from the number of [3H]CGP-12177 binding sites, were not affected by the electropermeabilization whereas the isoproterenol-stimulated cAMP accumulation was transiently impaired. This decrease in activity is due to an electropermeabilization-induced GTP leak. Normal activity could be obtained either by treating the cells by the electric field in a GTP-containing buffer, or by spontaneous recovery of the cells after the resealing of the plasma membrane, with a delay depending on the temperature. The activity of the receptors was not affected by the structural organization of the membrane associated to its electropermeabilization.

Topics: Adenosine Triphosphate; Adrenergic beta-Antagonists; Animals; Cell Line; Cell Membrane; Cell Membrane Permeability; Cell Nucleus; Cyclic AMP; Electric Stimulation; Glioma; Guanosine Triphosphate; Isoproterenol; Kinetics; Propanolamines; Receptors, Adrenergic, beta; Signal Transduction

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

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

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

In membranes of neuroblastoma x glioma hybrid (NG108-15) cells, bradykinin (EC50 approximately equal to 5 nM) stimulates GTP hydrolysis by a high-affinity GTPase (Km approximately equal to 0.2 microM). The octapeptide, des-Arg9-bradykinin, was inactive. Stimulation of GTP hydrolysis by bradykinin and an opioid agonist was partially additive. Treatment of NG108-15 cells with pertussis toxin, which inactivates Ni, eliminated GTPase stimulation by the opioid agonist but not by bradykinin. The data suggest that bradykinin activates in NG108-15 membranes a guanine nucleotide-binding protein which is not sensitive to pertussis toxin and which may be involved in bradykinin-induced stimulation of phosphoinositide metabolism in these cells.

Topics: Bradykinin; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Hybrid Cells; Hydrolysis; Membranes; Neuroblastoma; Pertussis Toxin; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Virulence Factors, Bordetella

Target size analysis of opioid receptor is complicated by the presence of multi-exponential inactivation curves. Irradiation of intact frozen tissue proved essential to eliminate such artifacts, due to indirect irradiation effects. Upon irradiation condition, opioid binding activity was inactivated in a single mono-exponential manner. Identical inactivation curves were obtained for mu, delta and kappa binding activities in brain membranes from rat, guinea-pig and frog and in NG 108-15 cells: the molecular mass obtained was 98 +/- 2 kDa. However, when opioid binding was assayed in the presence of Na+, Mg2+ and GTP, the molecular mass was found to be only 56 +/- 4.4 kDa. We suggest that the opioid recognition site comprises a unit of 56 kDa and that in the absence of Na+, Mg2+ and GTP an additional membrane component of 40-44 kDa is necessary for high-affinity opioid binding.

Topics: Animals; Brain Chemistry; Cell Line; Freeze Drying; Freezing; Glioma; Guanosine Triphosphate; Guinea Pigs; Magnesium; Male; Molecular Weight; Neuroblastoma; Rana esculenta; Rats; Receptors, Opioid; Sodium; Swine; Synaptic Membranes

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

The interactions of the atypical agonists pindolol and celiprolol with beta adrenergic receptors were compared with those of the full agonist, isoproterenol. Studies were carried out using intact cells as well as membranes prepared from C6 glioma cells. Computer-assisted analysis of dose-response curves resulting from the inhibition of the binding of [125I]iodopindolol by the beta-1 and beta-2 selective compounds ICI 89,406 and ICI 118,551 revealed that approximately one-third of the beta adrenergic receptors on these cells were beta-1 receptors. Addition of GTP to the binding assay simplified the dose-response curve for inhibition of the binding of [125I]iodopindolol by isoproterenol and diminished the potency of the agonist. GTP had no effect on the binding of pindolol or celiprolol, suggesting that these drugs do not induce the formation of a ternary complex with the receptor and the guanine nucleotide-binding protein for stimulation of adenylate cyclase activity. When added to the growth medium of intact C6 cells, isoproterenol induced a 40-fold increase in cyclic AMP accumulation. Pindolol and celiprolol, however, caused no elevation of enzyme activity. Addition of isoproterenol to the growth medium of intact cells resulted in an 80% decrease in the density of both beta-1 and beta-2 adrenergic receptors within 8 hr. Growing cells in the presence of pindolol or celiprolol induced a 50% decrease in the density of beta-2 receptors, which was inhibited by beta adrenergic antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Animals; Celiprolol; Cells, Cultured; Glioma; Guanosine Triphosphate; Iodine Radioisotopes; Isoproterenol; Pindolol; Propanolamines; Rats; Receptors, Adrenergic, beta

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

When C6-rat glioma cells were incubated for 20 min with beta-adrenoceptor agonists, a part of the beta-adrenoceptors was localized in light density vesicles. These receptors have the same affinity for dihydroalprenolol as plasma membrane receptors but have a lowered affinity for agonists as well as for the hydrophilic beta-adrenoceptor antagonist CGP-12177. The affinity of these vesicular receptors for a variety of hydrophobic beta-blockers as well as for the hydrophilic beta-blocker timolol is compared with that of plasma membrane receptors. None of the ligands investigated showed any difference in affinity but CGP-12177. Both, introducing a lipophilic side chain into CGP-12177 or altering the benzimidazol-2-one ring system of CGP-12177 led to an increase in the affinity of the vesicular receptors. Since in the presence of the pore-forming agent alamethicin the same affinity was determined for vesicular receptors as for those located on the plasma membrane, it is concluded that the apparent low affinity of the vesicular receptors in the absence of alamethicin is caused by a membrane barrier. The lower affinity of the vesicular receptors for agonists was only slightly increased by alamethicin.

Topics: Animals; Cell Line; Cell Membrane; Glioma; Guanosine Triphosphate; Isoproterenol; Rats; Receptors, Adrenergic, beta; Solubility; Subcellular Fractions

It has been repeatedly demonstrated that the neuroblastoma-glioma (NG 108-15) cell line has opiate receptors that inhibit adenylate cyclase and it has been proposed that this inhibition is mediated by a naloxone reversible stimulation of a low Km GTPase (Koski and Klee, Proc. Natl. Acad. Sci. 78:4185, 1981). The guanine nucleotides of NG cells were labeled with [3H]guanine followed by incubation with 10(-6)M guanine. Etorphine (10(-6)M) or vehicle were added and the incubations continued for 1-4 min. The reaction was stopped with 5 percent TCA containing nucleotides as carriers and markers for the HPLC. Marker nucleotides were detected at 254 nm and the labeled nucleotides by liquid scintillation spectrometry. In several experiments, etorphine failed to produce any measurable change in the labeled nucleotides or in the GTP/GDP ratios. To verify that the opiate receptors were functional we measured its capacity to inhibit the formation of cAMP induced by PGE1. We also studied the effects of naloxone and PGE1 on the formation of cAMP in opiate tolerant cells. Tolerant cells responded to naloxone with a 50 percent increase in cAMP, indicating again that the opiate receptors were functional. Our results are consistent with the idea that in intact NG108-15 cells the opiate-mediated hydrolysis of GTP observed in cell membrane preparations is of very small magnitude.

Topics: Alprostadil; Animals; Cell Line; Cyclic AMP; Etorphine; Glioma; Guanine Nucleotides; Guanosine Triphosphate; Mice; Naloxone; Neuroblastoma; Prostaglandins E; Tritium

Opiates and opioid peptides inhibit adenylate cyclase and stimulate specific low Km GTPase activity in membranes from neuroblastoma x glioma NG108-15 hybrid cells. The effects of opiate agonists on both enzymes are mediated by high affinity stereospecific receptors and require Mg2+, GTP, and Na+. In the presence of Mg2+, Na+ inhibits basal GTPase activity; opiates stimulate GTP hydrolysis by antagonizing the Na+-induced inhibition. Activation of GTPase leads, in turn, to inactivation of GTP-stimulated adenylate cyclase activity. The intrinsic activities (or efficacies) of a series of opiates are identical for stimulation of GTPase and inhibition of adenylate cyclase. These results provide a mechanism for the dual requirement for Na+ and GTP in the inhibitory coupling of opiate receptors to the adenylate cyclase system in these cells and may be of general significance to the action of other inhibitory hormones.

Topics: Adenylyl Cyclase Inhibitors; Animals; Cations, Monovalent; Cell Line; Glioma; GTP Phosphohydrolases; Guanosine Triphosphate; Hybrid Cells; Kinetics; Magnesium; Magnesium Chloride; Mice; Narcotics; Neuroblastoma; Phosphoric Monoester Hydrolases; Rats; Sodium

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

Topics: Adenylyl Cyclase Inhibitors; Animals; Cell Line; Cyclic AMP; Enkephalins; Glioma; Guanosine Triphosphate; Hybrid Cells; Kinetics; Mice; Narcotics; Neoplasms, Experimental; Neuroblastoma; Rats; Receptors, Opioid

Topics: Animals; Binding, Competitive; Cell Line; Enkephalins; Glioma; Guanosine Triphosphate; Hybrid Cells; Kinetics; Macromolecular Substances; Manganese; Mice; Neuroblastoma; Rats; Receptors, Opioid; Receptors, Opioid, delta; Sodium

Cholera toxin catalyzed the transfer of radioactive label from [adenine-2,8-3H2]NAD+ or ((32P]NAD+ to rat C6 glioma cell membrane and cytosolic proteins. Labeled proteins were resolved by polyacrylamide-NaDodSO4 gel or two-dimensional gel electrophoresis and stained with Coomassie blue, and the gels were subjected to fluorography or autoradiography. Autoradiograms of gels revealed labeled Mr 42000 and 46000-48000 membrane proteins that are putative subunits of the regulatory component (G/F) of the C6 cell hormone-sensitive adenylate cyclase. Cholera toxin also catalyzed the labeling of several cytosolic proteins including a Mr 54000 protein that was observed in autoradiograms of two-dimensional gels to migrate as an acidic satellite relative to Coomassie-stained C6 cell tubulin. Tubulin modified by ADP-ribosylation would undergo an acid shift relative to the stained unmodified tubulin in two-dimensional gels. The data led us to postulate that tubulin undergoes cholera toxin catalyzed ADP-ribosylation. Bovine brain tubulin prepared by three cycles of warm/cold polymerization/depolymerization was incubated with [32P]NAD+, GTP, and cholera toxin and then subjected to two-dimensional gel electrophoresis. Autoradiograms of the gels revealed the presence of [32P]ADP-ribosylated proteins that migrated as acidic satellites relative to the Coomassie-stained brain alpha and beta tubulin. Peptide maps of bovine brain tubulin and the associated [32P]ADP-ribosylated proteins showed a correspondence between the autoradiographic images and the stained peptide fragments. The data demonstrate that cholera toxin catalyzes the ADP-ribosylation of tubulin.

Topics: Adenosine Diphosphate Ribose; Animals; Brain Chemistry; Cattle; Cell Line; Cell Membrane; Cholera Toxin; Cytosol; Glioma; Guanosine Triphosphate; NAD; Nucleoside Diphosphate Sugars; Rats; Tubulin

[3H]Dihydroergocryptine ([3H]DHE) was shown to bind to sites in membranes from neuroblastoma X glioma hybrid cells (NG 108-15) that had the characteristics expected of alpha-adrenergic receptors. The binding was saturable with 0.3 pmol [3H]DHE bound per mg of protein and of high affinity, with an apparent dissociation constant (KD) of 1.8 nM. The specificity of the binding site for various ligands was more similar to that of alpha 2 receptors than to that of alpha 1. No specific binding of [3H]WB-4101 was found in the membranes derived from NG 108 cells. This finding also indicated that the [3H]DHE binding site in the cell is the alpha 2 receptor. GTP lowered the affinity of agonists for the [3H]DHE binding site, although the nucleotide hardly affected the affinity of antagonists including [3H]DHE.

Topics: Adrenergic alpha-Antagonists; Animals; Brain; Cell Line; Dihydroergotoxine; Dioxanes; Glioma; Guanosine Triphosphate; Hybrid Cells; Kinetics; Neoplasms, Experimental; Neuroblastoma; Rats; Receptors, Adrenergic; Receptors, Adrenergic, alpha; Synaptic Membranes

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

The adenylate cyclase of C6 glioma cell cultures was characterized for sensitivity to the beta-adrenergic agonist isoproterenol, as well as fluoride, and GTP as a function of the cell cycle. The mitotic phase of the cell cycle was emphasized because both the basal cellular cyclic AMP level and the intact C6 cell's capacity to accumulate cyclic AMP in response to isoproterenol decreased during mitosis. Basal and stimulated adenylate cyclase activities in mitotic cells were decreased relative to the enzyme activities in the G1, S, and G2 phases of the cell cycle. Analysis of the beta-adrenergic receptor using the radioligand(-)[3H]dihydroalprenolol showed that neither ligand affinity nor receptor density changed during the cell cycle, indicating that the reduced adenylate cyclase activity of the mitotic C6 cell was not caused by alterations in this hormone receptor. The reduction in the mitotic cell's basal adenylate cyclase activity was more prominent than the decrease in isoproterenol-, fluoride, or GTP-stimulated activities suggesting that the effectiveness of these enzymes activators (i.e., the efficiency of the coupling mechanism) was not attenuated during mitosis. These studies indicate that the intrinsic catalytic capacity (not the beta-adrenergic receptor or the coupling mechanism) of the C6 adenylate cyclase complex is reduced during mitosis and contributes to the mitotic cell's inability to accumulate and maintain the cyclic AMP concentration at the interphase level.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cyclic AMP; Dihydroalprenolol; Glioma; Guanosine Triphosphate; Interphase; Mitosis; Rats; Receptors, Adrenergic, beta; Sodium Fluoride

Topics: Adenylyl Cyclases; Animals; Binding, Competitive; Blood Platelets; Brain; Cattle; Cell Line; Cell Membrane; Clonidine; Erythrocytes; Glioma; Guanosine Triphosphate; Humans; Muscle, Smooth, Vascular; Neuroblastoma; Norepinephrine; Rats; Receptors, Adrenergic; Receptors, Adrenergic, alpha; Synaptic Membranes

An elevation of the intracellular cAMP concentration in C6 cells by cholera toxin or the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine causes a densitization of the beta-adrenergic-receptor-dependent synthesis of adenosine 3',5'-monophosphate. The specific binding of [3H]dihydroalprenolol to the beta-adrenergic receptors and the activation of the adenylate cyclase in vitro by fluoride anions and guanyl imidotriphosphate remain unchanged. It is likely that the desensitization is caused by an inhibition of beta-receptor coupling to the GTP-binding coupling protein in the adenylate cyclase complex. Furthermore, these experiments provide evidence that the loss of beta-receptor binding observed after incubation of cells with catecholamines is not a necessary consequence of the densensitization of receptor coupling dependent on adenosine 3',5'-monophosphate.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Animals; Carrier Proteins; Cholera Toxin; Cyclic AMP; Dihydroalprenolol; Fluorides; Glioma; Guanosine Triphosphate; Isoproterenol; Rats; Receptors, Adrenergic; Receptors, Adrenergic, beta

Adenylate cyclase of homogenates of NG108-15 neuroblastoma x glioma hybrid cells is activated by low concentrations of Ca2+ ions and is inhibited by higher (greater than 0.1 mM) concentrations of Ca2+ ions. Activation of either opiate receptors by 10 microM morphine or alpha-adrenergic receptors by 10 microM norepinephrine inhibits adenylate cyclase by 55% in the absence of Ca2+ ions, and inhibits the Ca2+-dependent activation of adenylate cyclase by more than 90%. Concentrations of Ca2+ ions greater than 0.1 mM inhibit adenylate cyclase and also reduce the extent inhibition of adenylate cyclase by morphine but not by norepinephrine. Guanosine-5'-triphosphate (0.1-1 microM) is required for inhibition of adenylate cyclase by morphine. The results show that morphine inhibits adenylate cyclase by a guanosine-5-triphosphate-dependent process and that the extent of inhibition of adenylate cyclase by morphine or norepinephrine is a function of the Ca2+ ion concentration and the proportion of adenylate cyclase molecules that are activated or inhibited by Ca2+ ions.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Calcium; Cell Line; Dose-Response Relationship, Drug; Glioma; Guanosine Triphosphate; Morphine; Neuroblastoma; Norepinephrine; Receptors, Opioid

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

Cholinergic agonists inhibit the basal and PGE1-activated adenylate cyclase activity in membranes isolated from the mouse neuroblastoma x glioma hybrid cell NG108-15. Inhibition is observed with acetylcholine, acetyl-beta-methylcholine and carbachol and is blocked by two specific muscarinic antagonists, atropine and quinuclydinylbenzilate. Inhibition of basal and PGE1-activated activity is only partial. Carbachol-directed inhibition has an apparent Km of 6 microM in the presence or absence of PGE1. Both the guanine nucleotide GTP and the monovalent cation Na+ are required for this muscarinic inhibition of basal and PGE1-activated NG108-15 adenylate cyclase. The selectivity observed for monovalent cations (all chloride salts) in this process is Na+ congruent to Li+ greater than K+ greater than Choline+ with the ED50 for Na+ congruent 40 microM. Of the nucleotides tested, only IT (and not ATP, UTP or CTP) replaces GTP in this process. GTP at 10 microM represents a saturating nucleotide concentration. Opiate-directed inhibition of NG108-15 adenylate cyclase has recently been shown to exhibit a similar requirement for GTP and Na+ [Blume, A. J., Lichtshtein, D. and Boone, G. (1979) Proc. National Academy of Sciences, USA, in press]. The data presented here therefore support the hypothesis that the general transfer of inhibitory information from membrane receptors to adenylate cyclase involves both a Na+ and GTP-sensitive process.

Topics: 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Acetylcholine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Atropine; Carbachol; Cations, Monovalent; Cell Line; Decamethonium Compounds; Glioma; Guanosine Triphosphate; Hexamethonium Compounds; Hybrid Cells; Mice; Neuroblastoma; Nucleotides; Prostaglandins E; Quinuclidinyl Benzilate; Receptors, Cholinergic; Receptors, Muscarinic; Sodium

Topics: Adenylyl Cyclases; Alprenolol; Binding Sites; Clone Cells; Drug Interactions; Enzyme Activation; Glioma; Guanosine Triphosphate; Isoproterenol; Kinetics; Receptors, Adrenergic; Receptors, Adrenergic, beta; Structure-Activity Relationship; Time Factors

1. When C6 glioma cells were incubated with mycophenolic acid, a potent and specific inhibitor of IMP:NAD oxidoreductase (EC 1.2.1.14) there was a marked depletion of the cellular content of GTP. The viability of the cells was unaffected. 2. The adenosine 3':5'-monophosphate (cyclic AMP) response of C6 glioma cells to the beta-adrenergic stimulant, (+/-)isoprenaline, was considerably reduced after treatment with mycophenolic acid. The diminished response to (+/-)isoprenaline was prevented by the inclusion of guanine in the culture medium along with mycophenolic acid. 3. The adenylate cyclase response to (+/-)isoprenaline of whole homogenates from C6 cells treated with mycophenolic acid was also depressed; the response was restored to normal by the addition of GTP. 4. The adenylate cyclase response to (+/-)isoprenaline of a membrane fraction prepared from homogenates of C6 cells was almost totally dependent on the presence of added GTP. Membrane fractions from control and mycophenolic-acid-treated C6 cells gave similar adenylate cyclase responses to (+/-)isoprenaline in the presence of GTP. 5. It is concluded that mycophenolic acid may depress the beta-adrenergic sensitivity of C6 cells by depleting the cellular content of GTP.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Cell Line; Cell Membrane; Cyclic AMP; Enzyme Activation; Glioma; Guanosine Triphosphate; Isoproterenol; Kinetics; Mycophenolic Acid; Time Factors

Nuclear RNA polymerase activity was studied in homotransplanted rat glial tumors where the primary tumor was produced by transplacental injection of ethylnitrosourea. Alpha amanitin, cycloheximide, and rifampicin were tested as inhibitors of this activity. Alpha amanitin significantly inhibited RNA polymerase activity in all tumors. This indicated that the major nuclear RNA polymerase activity seen in vitro in the tumor nuclei was RNA polymerase II. This is similar to the activity seen in normal glial nuclei. Cycloheximide and rifampicin which have no effect on RNA polymerase activity in normal glial nuclei inhibited about 20% of the polymerase activity in three of the tumors. The size and multiplicity of the nucleoli in these tumor cells suggests that RNA polymerase I could account for the activity which is inhibited by cycloheximide.

Topics: Amanitins; Animals; Autoradiography; Brain Neoplasms; Cell Nucleolus; Cell Nucleus; Cycloheximide; DNA-Directed RNA Polymerases; DNA, Neoplasm; Endoplasmic Reticulum; Ethylnitrosourea; Fibrosarcoma; Glioma; Guanosine Triphosphate; Neoplasm Transplantation; Neoplasms, Experimental; Neurofibroma; Rats; Rifampin; Transplantation, Homologous