guanosine-triphosphate and Neuroblastoma

In this article the broad spectrum of biological properties of 6-keto-Prostaglandin E1 will be reviewed. This includes assay procedures, biosynthesis and metabolism, various aspects of pharmacological activities, disease states, therapeutic potential and analogues of 6-keto-Prostaglandin E1.

Topics: Alprostadil; Animals; Blood Platelets; Cholesterol; Cholesterol Esters; Cyclic AMP; Epoprostenol; Guanosine Triphosphate; Humans; Muscle, Smooth; Neuroblastoma; Platelet Aggregation; Prostaglandins E; Renin; Shock; Synaptic Transmission; Vasodilation

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

We have demonstrated previously that the Myc oncoprotein blocks cancer cell differentiation by forming a novel transcriptional repressor complex with histone deacetylase and inhibiting gene transcription of tissue transglutaminase (TG2). Moreover, induction of TG2 gene transcription and transamidase activity is essential for the differentiating effects of retinoids in cancer cells. Here, we show that two structurally distinct TG2 protein isoforms, the full-length (TG2-L) and the short form (TG2-S), exert opposing effects on cell differentiation. Repression of TG2-L with small interfering RNA, which did not affect TG2-S expression, induced dramatic neuritic differentiation in neuroblastoma cells. In contrast, overexpression of TG2-S or a GTP-binding-deficient mutant of TG2-L (R580A), both of which lack the GTP-binding Arg-580 residue, induced neuroblastoma cell differentiation, which was blocked by an inhibitor of transamidase activity. Whereas N-Myc repressed and retinoid activated both TG2 isoforms, repression of TG2-L, but not simultaneous repression of TG2-L and TG2-S, enhanced neuroblastoma cell differentiation due to N-Myc small interfering RNA or retinoid. Moreover, suppression of vasoactive intestinal peptide (VIP) expression alone induced neuroblastoma cell differentiation, and VIP was up-regulated by TG2-L, but not TG2-S. Taken together, our data indicate that TG2-L and TG2-S exert opposite effects on cell differentiation due to differences in GTP binding and modulation of VIP gene transcription. Our findings highlight the potential importance of repressing the GTP binding activity of TG2-L or activating the transamidase activity of TG2-L or TG2-S for the treatment of neuroblastoma, and possibly also other Myc-induced malignancies, and for enhancing retinoid anticancer effects.

Topics: Arginine; Binding Sites; Cell Differentiation; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Enzymologic; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Immunoblotting; Isoenzymes; Neurites; Neuroblastoma; Protein Binding; Protein Glutamine gamma Glutamyltransferase 2; Proto-Oncogene Proteins c-myc; Retinoids; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transfection; Transglutaminases; Vasoactive Intestinal Peptide

Defect of the purine salvage enzyme, hypoxanthine phosphoribosyl transferase (HPRT), results in Lesch-Nyhan disease (LND). It is unknown how the metabolic defect translates into the severe neuropsychiatric phenotype characterized by self-injurious behavior, dystonia and mental retardation. There are abnormalities in GTP, UTP and CTP concentrations in HPRT-deficient cells. Moreover, GTP, ITP, XTP, UTP and CTP differentially support Gs-protein-mediated adenylyl cyclase (AC) activation. Based on these findings we hypothesized that abnormal AC regulation may constitute the missing link between HPRT deficiency and the neuropsychiatric symptoms in LND. To test this hypothesis, we studied AC activity in membranes from primary human skin and immortalized mouse skin fibroblasts, mouse Neuro-2a neuroblastoma cells and rat B103 neuroblastoma cells. In B103 control membranes, GTP, ITP, XTP and UTP exhibited profound stimulatory effects on basal AC activity that approached the effects of hydrolysis-resistant nucleotide analogs. In HPRT- membranes, the stimulatory effects of GTP, ITP, XTP and UTP were strongly reduced. Similarly, in human and mouse skin fibroblast membranes we also observed a decrease in GTP-stimulated AC activity in HPRT-deficient cells compared with the respective controls. In mouse Neuro-2a neuroblastoma membranes, AC activity in the presence of GTP was below the detection limit of the assay. We discuss several possibilities to explain the abnormalities in AC regulation in HPRT deficiency that encompass various species and cell types.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Fibroblasts; Guanosine Triphosphate; Humans; Hypoxanthine Phosphoribosyltransferase; Lesch-Nyhan Syndrome; Mice; Mice, Knockout; Neuroblastoma; Rats

Mutations in the the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson disease and some cases of sporadic Parkinson disease. Here we found that LRRK2 kinase activity was regulated by GTP via the intrinsic GTPase Roc domain, and alterations of LRRK2 protein that reduced kinase activity of mutant LRRK2 correspondingly reduced neuronal toxicity. These data elucidate the pathogenesis of LRRK2-linked Parkinson disease, potentially illuminate mechanisms of sporadic Parkinson disease and suggest therapeutic targets.

Topics: Analysis of Variance; Blotting, Western; Cell Survival; Cells, Cultured; Cerebral Cortex; Green Fluorescent Proteins; Guanosine Triphosphate; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mutagenesis; Mutation; Neuroblastoma; Neurons; Neurotoxicity Syndromes; Phosphotransferases; Protein Serine-Threonine Kinases; Transfection

The best studied oncogenic mechanisms are inactivating defects in both alleles of tumor suppressor genes and activating mutations in oncogenes. Chromosomal gains and losses are frequent in human tumors, but for many regions, like 1p36 and 17q in neuroblastoma, no mutated tumor suppressor genes or oncogenes were identified. Amplification of N-myc in neuroblastoma is strongly correlated with loss of 1p36 and gain of 17q. Here we report that N-myc down-regulates the mRNA expression of many genes with a role in cell architecture. One of them is the 1p36 gene Cdc42. Restoring the Cdc42 expression in neuroblastoma cells strongly induced differentiation. N-myc also inhibited Cdc42 functioning at the protein level. This was mediated by nm23-H1 and nm23-H2, which are located in the amplified 17q region. Nm23-H1 and nm23-H2 are strongly up-regulated downstream targets of N-myc. Nm23-H1 was shown to bind Cdc42 and prevented the induction of differentiation. Overexpression of Nm23 due to gain of 17q and induction by N-myc combined with weak expression of Cdc42 due to loss of 1p36 and down-regulation by N-myc can thus block differentiation. Although this marks Cdc42 as a candidate tumor suppressor gene, no mutations were found. Further silencing of Cdc42 by small interfering RNA induced massive apoptosis, indicating that tumor cell survival requires a minimal Cdc42 activity. Three regions of chromosomal gain and loss thus affect genes functioning in one pathway in neuroblastoma. They converge to bring the pathway out of balance and prevent Cdc42 mediated differentiation.

Topics: cdc42 GTP-Binding Protein; Cell Differentiation; Cell Division; Cell Line, Tumor; Down-Regulation; Gene Dosage; Gene Expression Regulation, Neoplastic; Genes, myc; Genes, Tumor Suppressor; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Neuroblastoma; Neurons; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; RNA Interference; RNA, Messenger; Transfection

Hereditary spastic paraplegias (HSPs) are neurodegenerative diseases caused by mutations in more than 20 genes, which lead to progressive spasticity and weakness of the lower limbs. The most frequently mutated gene causing autosomal dominant HSP is SPG4, which encodes spastin, a protein that belongs to the family of ATPases associated with various cellular activities (AAAs). A number of studies have suggested that spastin regulates microtubule dynamics. We have studied the ATPase activity of recombinant human spastin and examined the effect of taxol-stabilized microtubules on this activity. We used spastin translated from the second ATG and provide evidence that this is the physiologically relevant form. We showed that microtubules enhance the ATPase activity of the protein, a property also described for katanin, an AAA of the same spastin subgroup. Furthermore, we demonstrated that human spastin has a microtubule-destabilizing activity and can bundle microtubules in vitro, providing new insights into the molecular pathogenesis of HSP.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Cell Line; DNA Mutational Analysis; Dose-Response Relationship, Drug; Drug Interactions; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression; Green Fluorescent Proteins; Guanosine Triphosphate; Histidine; Humans; Katanin; Microscopy, Electron, Scanning; Microtubules; Models, Biological; Molecular Biology; Mutagenesis; Neuroblastoma; Paclitaxel; Protein Binding; Spastin; Time Factors; Transfection; Tubulin

Mycophenolic acid (MPA) specifically inhibits inosine-5'-monophosphate dehydrogenase, the first committed step toward GMP biosynthesis. In its morpholinoethyl ester pro-drug form it is one of the most promising immunosuppressive drugs recently developed. The aim of the present study was to investigate the in vitro effects of MPA, at concentrations readily attainable during immunosuppressive therapy, on 3 human neuroblastoma cell lines (LAN5, SHEP and IMR32). Mycophenolic acid (0.1-10 microM) caused a decrease of intracellular levels of guanine nucleotides, a G(1) arrest and a time- and dose-dependent death by apoptosis. These effects, associated with an up-regulation of p53, p21 and bax, a shuttling of p53 protein into the nucleus and a down-regulation of bcl-2, survivin and p27 protein, were reversed by the simultaneous addition of guanine or guanosine and were more evident using nondialysed serum containing hypoxanthine. These results suggest that in neuroblastoma cell lines clinically attainable concentrations of mycophenolic acid deplete guanine nucleotide pools triggering G(1) arrest and apoptosis through p53-mediated pathways, indicating a potential role of its morpholinoethyl ester pro-drug in the management of patients with neuroectodermal tumors.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Nucleus; Dose-Response Relationship, Drug; Down-Regulation; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Hypoxanthine; Immunohistochemistry; Immunosuppressive Agents; Inhibitor of Apoptosis Proteins; Microfilament Proteins; Microtubule-Associated Proteins; Muscle Proteins; Mycophenolic Acid; Neoplasm Proteins; Neuroblastoma; Prodrugs; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); Reverse Transcriptase Polymerase Chain Reaction; Survivin; Time Factors; Tumor Suppressor Protein p53

The pseudopeptide [Nphe(1)]N/OFQ(1-13)NH(2) (Nphe) has been shown to act as a pure, selective and competitive antagonist of nociceptin/orphanin FQ (N/OFQ) receptors in different tissues. However, Nphe displayed a highly variable potency, with pA(2) values ranging from 5.96 to 8.45. In the present study, we show that sodium ions and GTP markedly affect the potency of Nphe in blocking N/OFQ receptors coupled to cyclic AMP inhibition in different cellular systems. In intact N1E-115 neuroblastoma cells, the pA(2) value of Nphe increased from 7.13 to 8.02 when the extracellular sodium concentration was reduced from 138 to 2.5 mM. When N/OFQ inhibition of adenylyl cyclase activity was assayed in cell membranes, 100 mM NaCl decreased the pK(i) value of Nphe from 8.38 to 7.32, but increased that of the nonpeptide N/OFQ receptor antagonist CompB from 8.61 to 8.92. Similar effects of sodium ions on the potencies of Nphe and CompB were observed when the compounds were used to antagonize the N/OFQ inhibition of adenylyl cyclase activity in membranes of the external plexiform layer of the rat olfactory bulb. In the same assay, the increase of GTP concentration from 0.1 to 200 micro M decreased Nphe potency by 8-fold. These data demonstrate that sodium ions and GTP affect the potency of Nphe in a manner similar to that of agonists but not of pure antagonists and suggest that these factors may contribute to the reported variability of Nphe affinity constant.

Topics: Adenylyl Cyclases; Animals; Brain Neoplasms; Cell Membrane; Cyclic AMP; Guanosine Triphosphate; Male; Mice; Narcotic Antagonists; Neuroblastoma; Nociceptin Receptor; Olfactory Bulb; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Sodium; Somatostatin; Tumor Cells, Cultured

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

Prion diseases are fatal and transmissible neurodegenerative disorders characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrP(C)) denoted PrP(Sc). To identify intracellular organelles involved in PrP(Sc) formation, we studied the role of the Ras-related GTP-binding proteins Rab4 and Rab6a in intracellular trafficking of the prion protein and production of PrP(Sc). When a dominant-negative Rab4 mutant or a constitutively active GTP-bound Rab6a protein was overexpressed in prion-infected neuroblastoma N2a cells, there was a marked increase of PrP(Sc) formation. By immunofluorescence and cell fractionation studies, we have shown that expression of Rab6a-GTP delocalizes PrP within intracellular compartments, leading to an accumulation in the endoplasmic reticulum. These results suggest that prion protein can be subjected to retrograde transport toward the endoplasmic reticulum and that this compartment may play a significant role in PrP(Sc) conversion.

Topics: Animals; Biological Transport; Cell Fractionation; Endoplasmic Reticulum; Guanosine Triphosphate; Mice; Mutation; Neuroblastoma; Proto-Oncogene Proteins c-myc; PrPC Proteins; rab GTP-Binding Proteins; rab4 GTP-Binding Proteins; Tumor Cells, Cultured

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

A series of CCK(2) receptor ligands were analysed with respect to their interaction with binding sites in the membranes of COS-7 cells and SK-N-MC cells transiently expressing the human CCK(2) receptor (short isoform). The ligands were YF476, YM022, AG041R, L-740,093, JB93182, PD134308, and PD136450. Their binding was analysed by radioligand competition using [3H]L-365,260 as the labelled ligand. Saturation binding analysis indicated that [3H]L-365,260 interacted with a single class of binding sites. In competition binding experiments using COS-7-cell membranes, all seven ligands were incubated together with 2 nM [3H]L-365,260. The data for four of the compounds fitted a one-site model (pK(i) values: YM022: 9.2+/-0.02; YF476: 9.6+/-0.04; L-740,093: 9.2+/-0.01; and AG041R: 8.3+/-0.06), while the data for the three others fitted a two-site model (pK(i) values: JB93182: 8.8+/-0.04 and 6.0+/-0.15; PD134308: 9.0+/-0.04 and 6.1+/-0.15; and PD136450: 9.0+/-0.02 and 5.4+/-0.41). SK-N-MC cell membranes and 2 nM [3H]L-365,260 were incubated together with YM022, YF476, JB93182, and PD134308. The data for YM022 and YF476 fitted a one-site model (pK(i) values: YM022: 9.3+/-0.06; YF476: 9.4+/-0.02), while the data for JB93182 and PD134308 fitted a two-site model (pK(i) values: JB93182: 8.7+/-0.06 and 6.2+/-0.06; PD134308: 9.1+/-0.06 and 7.0+/-0.17). Competition binding experiments in the presence of the GTP-analogue guanylylimidodiphosphate, using either of the two cell types, produced similar binding data for PD134308 and JB93182 as in the absence of GTP-analogue. The human receptor seems to exist in a low and/or high affinity state. The shift from low to high affinity does not seem to reflect the degree of G protein coupling.

Topics: Animals; Binding Sites; Binding, Competitive; Cell Line; Cell Membrane; COS Cells; Gene Expression; Guanosine Triphosphate; Humans; Kinetics; Ligands; Models, Theoretical; Neuroblastoma; Radioligand Assay; Receptor, Cholecystokinin B; Receptors, Cell Surface; Receptors, Cholecystokinin; Structure-Activity Relationship; Transfection; Tumor Cells, Cultured

Guanine nucleotide exchange factors from the Dbl family are proto-oncogenic proteins that activate small GTPases of the Rho family. Here we report the characterization of GEF720, a novel Dbl-like protein related to p115Rho-GEF. GEF720 activated RhoA both in our recently developed Yeast Exchange Assay and in biochemical in vitro exchange assays. GEF720 induced RhoA dependent assembly of actin stress fibers in REF52 fibroblastic cells. In NIH3T3 cells this Dbl-like protein elicited formation of transformation foci with a morphology similar to RhoA-V14 induced foci. In the PC12 neuron-like cell line, expression of GEF720, whose mRNA is brain specific, inhibited NGF-induced neurite outgrowth. Finally, GEF720 gene is located on human chromosome 1 on band 1p36, between Tumor Protein 73 and Tumor Necrosis Factor Receptor 12, two genes rearranged in many neuroblastoma cell lines. Together, these results show that this new Dbl related protein, GEF720, is an exchange factor that can directly activate RhoA in vivo and is potentially involved in the control of neuronal cell differentiation. GEF720 is also a new candidate gene involved in the progression of neuroblastoma and developmental abnormalities associated with rearrangements in the 1p36 chromosomal region.

Topics: 3T3 Cells; Actins; Amino Acid Sequence; Animals; Base Sequence; Brain; Brain Chemistry; Cell Differentiation; Cell Line, Transformed; Cell Transformation, Neoplastic; Chromosome Mapping; Chromosomes, Human, Pair 1; Disease Progression; Enzyme Activation; Exons; Fibroblasts; Genes; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Mice; Molecular Sequence Data; Multigene Family; Nerve Tissue Proteins; Neurites; Neuroblastoma; PC12 Cells; Protein Binding; Protein Structure, Tertiary; Rats; Recombinant Fusion Proteins; rhoA GTP-Binding Protein; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Stress Fibers; Transfection; Tumor Cells, Cultured

Tissue transglutaminase (tTG) is a transamidating enzyme that is elevated in Huntington's disease (HD) brain and may be involved in the etiology of the disease. Further, there is evidence of impaired mitochondrial function in HD. Therefore, in this study, we examined the effects of mitochondrial dysfunction on the transamidating activity of tTG. Neuroblastoma SH-SY5Y cells stably overexpressing human tTG or mutated inactive tTG were treated with 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. 3-NP treatment of tTG-expressing cells resulted in a significant increase of TG activity in situ. In vitro measurements demonstrated that 3-NP had no direct effect on tTG activity. However, 3-NP treatment resulted in a significant decrease of the levels of GTP and ATP, two potent inhibitors of the transamidating activity of tTG. No significant changes in the intracellular levels of calcium were observed in 3-NP-treated cells. Treatment with 3-NP in combination with antioxidants significantly reduced the 3-NP-induced increase in in situ TG activity, demonstrating that oxidative stress is a contributing factor to the increase of TG activity. This study demonstrates for the first time that impairment of mitochondrial function significantly increases TG activity in situ, a finding that may have important relevance to the etiology of HD.

Topics: Adenosine Triphosphate; Antioxidants; Calcium; Dose-Response Relationship, Drug; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Huntington Disease; Marine Toxins; Mitochondria; Neuroblastoma; Nitro Compounds; Oxidative Stress; Oxocins; Propionates; Protein Glutamine gamma Glutamyltransferase 2; Succinate Dehydrogenase; Transfection; Transglutaminases; Tumor Cells, Cultured

Differences in the specificity of coupling of delta-opioid receptor with G-protein have been reported in the literature. We have observed a differential desensitization of delta-opioid receptors, endogenously expressed in the neuroblastoma cell line SK-N-BE, induced by peptide and alkaloid agonists. By combining photoaffinity labelling of receptor-activated G-proteins with [alpha-(32)P]azidoanilide-GTP and an anti-sense oligodeoxynucleotide strategy, we examined whether the chemical nature of opioid agonists, alkaloid or peptide, has a critical role in determining a G(i)alpha/G(o)alpha-protein-selective activation by the human delta-opioid receptors. Etorphine, a non-selective alkaloid agonist, was shown to stimulate the incorporation of [alpha-(32)P]azidoanilide-GTP into G(i)alpha1, G(i)alpha2, G(i)alpha3 and pertussis-toxin-insensitive Galpha subunits. In contrast, [d-Pen(2),d-Pen(5)]enkephalin (DPDPE; Pen is penicillamine) and Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH(2) (deltorphin I), selective peptide agonists, mainly activated G(i)alpha2 and G(o)alpha2 subunits. The 'knock-down' of G(o)alpha2 subunits by anti-sense oligodeoxynucleotides selectively decreased the inhibition of adenylate cyclase induced by DPDPE and deltorphin I, whereas anti-sense oligodeoxynucleotides directed against G(i)alpha2 subunits only decreased the potency of etorphine in inhibiting cAMP accumulation. These results suggest that the nature of the agonist, peptide or alkaloid is critical in determining the interaction between human delta-opioid receptors and Galpha subunits.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Alkaloids; Animals; Azides; Cell Membrane; Colforsin; Cyclic AMP; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Etorphine; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Isoelectric Point; Neuroblastoma; Neurons; Oligodeoxyribonucleotides, Antisense; Oligopeptides; Peptides; Pertussis Toxin; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Tumor Cells, Cultured; Virulence Factors, Bordetella

Topics: Biopterins; Cell Differentiation; Guanosine Triphosphate; Humans; Mycophenolic Acid; Neuroblastoma; Tumor Cells, Cultured

A previous analyzer of adenine compounds by high-performance liquid chromatography was converted for the determination of guanine, its nucleoside and nucleotides by a post-column fluorescence derivatization with phenylglyoxal (PGO) in place of bromoacetoaldehyde. The gel filtration column (Asahipak GS-320H) was used for separation by a mobile phase consisting of 25 mM sodium citrate buffered (pH 4.0)-150 mM NaCl solution and CH3CN (85:15, v/v) containing 15 mM PGO. The separated analytes reacted with flow through PGO in a reaction coil at 90 degrees C into fluorescent derivatives. Those derivatives were detected fluorimetrically, highly selective and quantitatively. The activity of soluble guanylate cyclase (sGC) in the neuroblastoma N1E-115 cell was measured by tracing the peak height of cGMP synthesized from substrate GTP using this guanine analyzer. The sensitivity of the present method was lower than the radioisotope method. However, our modified method was simpler, safer and quicker than the radioisotope method. Furthermore, this method could trace other guanine compounds simultaneously, allowing measurement of guanine metabolizing enzymatic activity. Therefore, it will be useful for screening of effectors on sGC.

Topics: Chromatography, Gel; Chromatography, High Pressure Liquid; Cyclic GMP; Fluorescent Dyes; Guanine; Guanosine Triphosphate; Guanylate Cyclase; Indicators and Reagents; Neuroblastoma; Phenylglyoxal; Sensitivity and Specificity; Tumor Cells, Cultured

1. We have used somatostatin (SRIF) receptor subtype-selective ligands to determine some of the operational characteristics of somatostatin receptors in Neuro2A mouse neuroblastoma cells. The potent SRIF1-receptor selective ligand, BIM-23027, was able to displace completely the specific binding of radioiodinated somatostatin, [125I]-Tyr11-SRIF-14, with a pIC50 of 10.3, suggesting that Neuro2A cells contain predominantly receptors of the SRIF1 receptor group. The rank order of affinities for several somatostatin analogues tested in competition studies, together with the high affinity of BIM-23027, indicate that the majority of receptors in Neuro2A cells are of the sst2 subtype. 2. The stable radioligand, [125I]-BIM-23027, bound with high affinity (Kd = 13 pM, Bmax = 0.2 pmol mg-1 protein) to Neuro2A cell membranes, but its binding was only partially reversible at room temperature and below. Thus at 4 degrees C, only 36% of the bound ligand dissociated within 2 h. In contrast, 60% of the ligand dissociated at 15 degrees C and 89% of the ligand dissociated at 37 degrees C. 3. Equilibrium binding of [125I]-BIM-23027 was partially (25%) inhibited by 10 microM GTP, and by 120 mM NaCl (42% inhibition) but this inhibition was increased to 75% when sodium chloride and GTP were added together. This effect of GTP and sodium chloride was also seen in dissociation experiments. After incubation to equilibrium with [125I]-BIM-23027, dissociation was initiated with excess unlabelled ligand in the presence of GTP (10 microM) and sodium chloride (120 mM). Under these conditions 67% of the ligand dissociated at 4 degrees C, 81% at 15 degrees C and 93% at 37 degrees C. Binding was totally inhibited by pretreatment of cells with pertussis toxin. 4. Functionally, BIM-23027 inhibited forskolin-stimulated cyclic AMP accumulation in a concentration-dependent manner with an IC50 of 1.0 nM and a maximal inhibition of 37%. This effect was abolished by pretreatment of the cells with pertussis toxin. However, unlike in studies reported with the recombinant sst2 receptor, no rise in intracellular calcium concentration was observed with SRIF-14. 5. We conclude that Neuro2A cells provide a stable neuronal cell line for the study of functionally coupled endogenous somatostatin receptors of the sst2 type. In addition, we have found that activation of the receptor is associated with ligand-receptor internalisation.

Topics: Adenosine Triphosphate; Animals; Brain Neoplasms; Calcium; Cell Membrane; Cyclic AMP; Guanosine Triphosphate; Kinetics; Ligands; Mice; Neuroblastoma; Peptides, Cyclic; Receptors, Somatostatin; Second Messenger Systems; Somatostatin; Tumor Cells, Cultured

The amyloid beta protein (25-35) stimulated appearance of 3H-inositol phosphates from [3H]inositol-prelabeled LA-N-2 cells was investigated. This stimulation was unaltered by extra- and intracellular calcium chelators in a calcium-free medium or by several protein kinase inhibitors. This phospholipase C stimulation by amyloid beta protein appeared to be pertussis toxin sensitive. It is possible that this phospholipase C stimulation by amyloid beta protein is a receptor-mediated process. This possibility is based on two related observations. The stimulation is ablated by the presence of conventional antagonists for metabotropic, adrenergic, and bombesin agonists. The IC50 values were 12 microM for propranolol, 15 microM for AP-3, and 25 nM for [Tyr4,D-Phe12]bombesin. Additional support comes from results of desensitization and resensitization experiments. Amyloid beta protein stimulation of phospholipase C was absent from LA-N-2 cells previously treated with norepinephrine, trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), bombesin, or amyloid beta peptide. In a similar manner, LA-N-2 cells previously treated with amyloid beta protein were no longer responsive to norepinephrine, t-ACPD, or bombesin. The responsiveness to amyloid beta protein returned, subsequent to a period of resensitization for the individual agonists. It is suggested that this observed amyloid beta protein stimulation of phospholipase C may be responsible for the elevated quantity of inositol seen in the brains of Alzheimer's disease patients.

Topics: Adrenergic Agonists; Adrenergic alpha-Agonists; Amyloid beta-Peptides; Bombesin; Calcium; Chelating Agents; Cholera Toxin; Cycloleucine; Egtazic Acid; Enzyme Inhibitors; Epinephrine; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; Neuroblastoma; Neuroprotective Agents; Norepinephrine; Peptide Fragments; Pertussis Toxin; Protein Kinase Inhibitors; Protein Kinases; Sensitivity and Specificity; Thionucleotides; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

The wasp venom, mastoparan (MP), is a direct activator of reconstituted pertussis toxin-sensitive G-proteins and of purified nucleoside diphosphate kinase (NDPK) [E.C. 2.6.4.6.]. In HL-60 membranes, MP activates high-affinity GTPase [E.C. 3.6.1.-] and NDPK-catalyzed GTP formation, but not photolabeling of G-protein alpha-subunits with GTP azidoanilide; this suggests that the venom activates G-proteins in this system indirectly via stimulation of NDPK. Moreover, the MP analogue, mastoparan 7 (MP 7), is a much more effective activator of reconstituted G-proteins than MP, whereas with regard to NDPK and GTPase in HL-60 membranes, the two peptides are similarly effective. In our present study, we investigated NDPK- and G-protein activation by MP in membranes of the human neuroblastoma cell line, SH-SY5Y, the human erythroleukemia cell line, HEL, the rat basophilic leukemia cell line, RBL 2H3, and the hamster ductus deferens smooth muscle cell line, DDT1MF-2. All these membranes exhibited high NDPK activities that were increased by MP. Compared to basal GTP formation rates, basal rates of high-affinity GTP hydrolysis in cell membranes were low. MP activated high-affinity GTP hydrolysis in cell membranes but did not enhance incorporation of GTP azidoanilide into G-protein alpha-subunits. As with HL-60 membranes, MP and MP 7 were similarly effective activators of NDPK and GTPase in SH-SY5Y membranes. Pertussis toxin inhibited MP-stimulated GTP hydrolyses in SH-SY5Y- and HEL membranes, whereas NDPK activations by MP were pertussis toxin-insensitive. Our data suggest that indirect G-protein activation via NDPK is not restricted to HL-60 membranes but is a more general mechanism of MP action in cell membranes. Pertussis toxin-catalyzed ADP-ribosylation of alpha-subunits may inhibit the transfer of GTP from NDPK to G-proteins. NDPK may play a much more important role in transmembrane signal transduction than was previously appreciated and, moreover, the GTPase of G-protein alpha-subunits may serve as GDP-synthase for NDPK.

Topics: Animals; Cell Line; Cell Membrane; Cricetinae; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Intercellular Signaling Peptides and Proteins; Leukemia, Erythroblastic, Acute; Neuroblastoma; Nucleoside-Diphosphate Kinase; Peptides; Pertussis Toxin; Rats; Tumor Cells, Cultured; Virulence Factors, Bordetella; Wasp Venoms

Effects of ethanol on signal transduction in neuronal membranes are supposed to occur by the interaction with heterotrimeric guanine nucleotide-binding proteins (G-proteins). Several substances affect signal transduction by activation of G-proteins directly independent of receptors. We show that similar to those substances, ethanol stimulates high-affinity guanosine triphosphate (GTP)-hydrolysis in SH-SY5Y membranes at concentrations of 50 mM and higher in a pertussis toxin-sensitive manner. Compared with ethanol at a concentration of 170 mM, other alcohols were without or with respect to methanol with a slight effect on high-affinity GTP-hydrolysis in SH-SY5Y membranes. Ethanol also stimulates the GTPase of the purified G-protein transducin. The findings suggest that ethanol affects signal transduction in neuronal membranes by direct activation of pertussis toxin-sensitive G-proteins.

Topics: Animals; Cattle; Cell Membrane; Central Nervous System Depressants; Enkephalin, Leucine-2-Alanine; Ethanol; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Hydrolysis; Neuroblastoma; Pertussis Toxin; Receptors, Opioid; Retinal Rod Photoreceptor Cells; Sensitivity and Specificity; Time Factors; Transducin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Platelet-activating factor (PAF) may be a neuromodulator involved in neural cell differentiation, cerebral inflammation, and ischemia. The PAF receptor is a member of the G protein-coupled receptor superfamily. In the present study, we sought to define the specific G protein(s) that mediate PAF-stimulated phosphoinositide (PI) metabolism in an immortalized hippocampal cell line, HN33.11. PAF increased the production of 3H-labeled inositol phosphates (IPs) with EC50 values of 1.2-1.5 nM. The effect of PAF on 3H-IPs formation was completely blocked by the PAF antagonist BN 50739 at a concentration of 300 nM. Pertussis toxin pretreatment attenuated PAF-stimulated 3H-IPs production by 20-30% (p < 0.05). Consistent with a role for Gi1/2 in this response, antiserum against G alpha i1/2 blocked the response to a similar degree. Pretreatment of permeabilized cells with G alpha q/11 antiserum attenuated the response by 70% (p < 0.05), suggesting a role for Gq/11 in mediating the PAF response in this cell line. Stimulation with PAF increased [alpha-32P]-GTP binding to both G alpha q and G alpha i1/2 proteins. Moreover, specific [3H]PAF binding sites coprecipitated with G alpha q and G alpha i1/2 proteins. The results suggest that PAF-stimulated PI metabolism in HN33.11 cells is mediated by both Gq and Gi1/2 proteins.

Topics: Animals; Azepines; Cell Line, Transformed; Cell Membrane; GTP-Binding Proteins; Guanosine Triphosphate; Hippocampus; Hybrid Cells; Immune Sera; Mice; Mice, Inbred C57BL; Neuroblastoma; Neurons; Pertussis Toxin; Phosphatidylinositols; Platelet Activating Factor; Triazoles; Virulence Factors, Bordetella

Incubation of SH-SY5Y neural cells with mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (the key enzyme in purine nucleotide biosynthesis), reduced the cellular content of GTP by 94% relative to its concentration in control cells (43 nmol/mg protein) without altering the level of GDP. Although in GTP-depleted intact cells the receptor binding parameters (Kd and Bmax) of the opioid antagonist [3H]naltrexone were unchanged from those in untreated cells, the binding affinity of the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly-(Me)- Phe-Gly-ol ([3H]DAMGO) was enhanced 2-fold. Furthermore, the kinetics of ligand/receptor interaction revealed that in the nucleotide-depleted cells, the dissociation rate constant for [3H]DAMGO was reduced by 44%. Initial exposure of SH-SY5Y cells to pertussis toxin reduced high-affinity ligand binding by 95% and abolished the effect of MPA treatment. Renewed incubation of the GTP-depleted cells with guanosine restored the original GTP levels and agonist binding. Neither MPA nor guanosine treatment changed the Bmax of [3H]DAMGO binding. Forskolin- and prostaglandin E1-stimulated adenylyl cyclase activities were decreased significantly in GTP-depleted cells. DAMGO and [D-Pen2,D-Pen5]enkephalin inhibitions of adenylyl cyclase were also affected with MPA treatment. Maximal inhibition of forskolin-stimulated adenylyl cyclase activity by both of the agonists was reduced, whereas MPA caused a 2-fold reduction in potency for DAMGO. The results show that reduction in endogenous GTP levels leads to noticeable changes in agonist, receptor, and G protein interactions, as measured by agonist binding, and to subsequent diminution of the signal transduction, as reflected by the cAMP levels.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Analgesics; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Kinetics; Molecular Sequence Data; Mycophenolic Acid; Naltrexone; Narcotic Antagonists; Neuroblastoma; Neurons; Receptors, Opioid, mu; Tritium; Tumor Cells, Cultured

Murine N1E-115 neuroblastoma cells are shown to express a single class of angiotensin II (Ang II) receptors that display all the pharmacological properties defining the Ang II receptor subtype 2 (AT2): high affinity for 125I-labelled AT2-selective agonist CGP 42112 (Kd 91 +/- 19 pM); expected rank order of potency (CGP 42112 = (Sar1,Ile8)Ang II > or = Ang II > PD 123319 >> DUP 753) for several Ang II analogues; increased binding in the presence of the reducing reagent dithiothreitol (DTT); and insensitivity to analogues of GTP. Molecular cloning of cDNA encoding AT2 receptors from N1E-115 cells reveals nucleotide sequence identity with the AT2 subtype expressed in fetal tissue. Murine AT2 receptors transiently expressed in COS cells display the same pharmacological profile as endogenous Ang II receptors of N1E-115 cells. Taken together, these data reveal the exclusive presence of the AT2 receptor subtype in N1E-115 cells. Incubation of N1E-115 cells with Ang II leads to a marked decrease in the level of tyrosine phosphorylation of several proteins with apparent molecular masses of 80, 97, 120, 150 and 180 kDa respectively. Tyrosine dephosphorylation of the same set of proteins is observed after treatment with the AT2-specific agonist CGP 42112. The response to both effectors is rapid and transient, showing a maximum between 5 and 10 min, and returning to basal levels after 20-30 min. In both cases, tyrosine dephosphorylation can be prevented by co-incubation with an excess of the antagonist Sarile. These data thus establish that AT2 receptor activation leads to protein tyrosine dephosphorylation in N1E-115 cells, and support a possible role for AT2 receptors in the negative regulation of cell proliferation.

Topics: Animals; Base Sequence; Cloning, Molecular; Dithiothreitol; DNA, Complementary; Gene Expression; Guanosine Triphosphate; Mice; Molecular Sequence Data; Neuroblastoma; Oligopeptides; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Receptors, Angiotensin; Transfection; Tumor Cells, Cultured; Tyrosine

Nerve growth factor (NGF) is essential for the differentiation and survival of sympathetic and sensory neurones and is thought to play a role in the differentiation of neuroblastoma. In this study we have shown NGF decreased the mRNA level of the two GTPase activating proteins neurofibromin (containing the NF1-GRD) and type 1 GAP120 in two neuroblastoma cell lines, IMR-32 and SK-N-SH. This effect was seen within 15 min exposure to NGF and was maintained up to 2 h after the addition of NGF. Treatment with NGF increased the amount of GTP bound p21ras 3-fold, within 20 min exposure. Western blot analysis showed SK-N-SH and IMR-32 cells to contain equal amounts of p21ras protein and these levels were unchanged by NGF treatment. However, NGF induced an increase in the level of neurofilament L protein, which was accompanied by an increase in neurite extension. These effects of NGF occurred in the absence of growth inhibition. In conclusion, our results demonstrate a decrease in GTPase activating proteins and activation of p21ras by NGF in IMR-32 and SK-N-SH cells, thus implicating p21ras in NGF signal transduction in neuroblastoma.

Topics: Base Sequence; Cell Differentiation; Cell Division; GTPase-Activating Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Molecular Sequence Data; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; Neurofibromin 1; Neurons; Proteins; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; RNA, Messenger; 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: Alcohol Oxidoreductases; Animals; Biopterins; Cell Line; Dihydropteridine Reductase; GTP Cyclohydrolase; Guanosine Triphosphate; Kinetics; Mice; Mycophenolic Acid; Neuroblastoma; Phosphorus-Oxygen Lyases; Tumor Cells, Cultured

Opioids are regarded to act via receptors interacting with heterotrimeric pertussis toxin (PTX)-sensitive G proteins. In membranes of SH-SY5Y cells, the mu-selective agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO) and the delta-selective agonist [D-Pen2,Pen5]-enkephalin (DPDPE) stimulated incorporation of the photoreactive GTP analog [alpha-32P]GTP azidoanilide into proteins comigrating with the alpha subunits of G(i1), G(i2), G(i3), G(o1), and another form of G(o), presumably G(o2). In membranes of PTX-treated cells, both agonists were ineffective. Subtype-specific immunoprecipitation of G protein alpha subunits photolabeled in the absence or presence of agonists revealed profound differences between mu and delta opioid receptors in coupling to PTX-sensitive G proteins. Whereas activated delta opioid receptors preferentially coupled to G(i1), activated mu opioid receptors more effectively coupled to G(i3). Additionally, we provide evidence that G(o) subtypes are also differentially activated by the two receptors. Thus, mu and delta opioid receptors appear to discriminate between PTX-sensitive G proteins and lead to activation of distinct G protein subtypes.

Topics: Affinity Labels; Azides; Cell Membrane; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Immunosorbent Techniques; Neuroblastoma; Pertussis Toxin; Photochemistry; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured; Virulence Factors, Bordetella

Evidence is presented for linkage of opioid receptors directly to the stimulatory G protein (guanine nucleotide-binding protein), Gs, in addition to the generally accepted linkage to the inhibitory and "other" G proteins, gi and Go, in F-11 (neuroblastoma-dorsal root ganglion neuron) hybrid cells. Treatment of intact F-11 cells with cholera toxin decreased specific binding of the opioid agonist [D-Ala2,D-Leu5]enkephalin to F-11 cell membranes by 35%, with the remaining binding retaining high affinity for agonist. Under these conditions cholera toxin influenced the alpha subunit of Gs (Gs alpha) but had no effect on the alpha subunit of Gi/o (Gi/o alpha), based on ADP-ribosylation studies. Pertussis toxin treatment decreased high-affinity opioid agonist binding by about 50%; remaining binding was also of high affinity, even though pertussis toxin had inactivated Gi/o alpha selectively and essentially completely. Simultaneous treatment with both toxins had an additive effect, reducing specific binding by about 80%. While opioid agonists inhibited forskolin-stimulated adenylate cyclase activity of F-11 cells as expected, opioids also stimulated basal adenylate cyclase activity, indicative of interaction with Gs as well as Gi. Cholera toxin treatment attenuated opioid-stimulation of basal adenylate cyclase, whereas pertussis toxin treatment enhanced stimulation. In contrast, inhibition by opioid of forskolin-stimulated activity was attenuated by pertussis toxin but not by cholera toxin. It is concluded that a subset of opioid receptors may be linked directly to Gs and thereby mediate stimulation of adenylate cyclase. This Gs-adenylate cyclase interaction is postulated to be responsible for the novel excitatory electrophysiologic responses to opioids found in our previous studies of sensory neurons and F-11 cells.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Binding, Competitive; Cell Line; Cell Membrane; Cholera Toxin; Colforsin; Cricetinae; Cricetulus; Enkephalin, Leucine-2-Alanine; Ganglia, Spinal; GTP-Binding Proteins; Guanosine Triphosphate; Hybrid Cells; Kinetics; Levorphanol; Mice; Neuroblastoma; Neurons, Afferent; Pertussis Toxin; Receptors, Opioid; Sodium; Tumor Cells, Cultured; Virulence Factors, Bordetella

The introduction of human chromosome 17 suppresses the tumourigenicity of a neuroblastoma cell line in the absence of any effects on in vitro growth and the neurofibromatosis type 1 (NF1) gene may be responsible. Here we report that 4 out of 10 human neuroblastoma lines express little or no neurofibromin and that two of these lines show evidence of NF1 mutations, providing further proof that NF1 mutations occur in tumours that are not commonly found in NF1 patients. We also show that NF1 deficient neuroblastomas show only moderately elevated ras-GTP levels, in contrast to NF1 tumour cells, indicating that neurofibromin contributes differently to the negative regulation of ras in different cell types.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Chromosomes, Human, Pair 17; Genes, Neurofibromatosis 1; Guanosine Triphosphate; Humans; Mice; Molecular Sequence Data; Mutation; Neuroblastoma; Neurofibromin 1; Precipitin Tests; Proteins; Proto-Oncogene Proteins p21(ras); Tumor Cells, Cultured

GTP cyclohydrolase I exhibits a positive homotropic cooperative binding to GTP, which raises the possibility of a role for GTP in regulating the enzyme reaction (Hatakeyama, K., Harada, T., Suzuki, S., Watanabe, Y., and Kagamiyama, H. (1989) J. Biol. Chem. 264, 21660-21664). We examined whether or not the intracellular GTP level is within the range of affecting GTP cyclohydrolase I activity, using PC-12 rat pheochromocytoma and IMR-32 human neuroblastoma cells. Since GTP cyclohydrolase I was the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in these cell lines, the intracellular activities of this enzyme were reflected in the tetrahydrobiopterin contents. We found that the addition of guanine or guanosine increased GTP but not tetrahydrobiopterin in these cells. On the other hand, three IMP dehydrogenase inhibitors, tiazofurin, 2-amino-1,3,4-thiadiazole, and mycophenolic acid, decreased both GTP and tetrahydrobiopterin in a parallel and dose-dependent manner, and these effects were reversed by the simultaneous addition of guanine or guanosine. There was no evidence suggesting that these inhibitors inhibited other enzymes involved in the biosynthesis and regeneration of tetrahydrobiopterin. Comparing intracellular activities of GTP cyclohydrolase I in the inhibitor-treated cells with its substrate-velocity curve, we estimated that the intracellular concentration of free GTP is 150 microM at which point the activity of GTP cyclohydrolase I is elicited at its maximum velocity. Below this GTP concentration, GTP cyclohydrolase I activity is rapidly decreased. Therefore GTP can be a regulator for tetrahydrobiopterin biosynthesis.

Topics: Animals; Antineoplastic Agents; Biopterins; GTP Cyclohydrolase; Guanine; Guanosine; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Kinetics; Mycophenolic Acid; Neuroblastoma; PC12 Cells; Ribavirin; Thiadiazoles; Tumor Cells, Cultured

1. Dimethylsulfoxide-induced differentiated neuroblastoma express high levels of membrane 21 to 23-kDa carboxyl methylated proteins. Relationships among methylation, isoprenylation, and GTP binding in these proteins were investigated. Protein carboxyl methylation, protein isoprenylation, and [alpha-32P]GTP binding were determined in the electrophoretically separated proteins of cells labeled with the methylation precursor [methyl-3H]methionine or with an isoprenoid precursor [3H]mevalonate. 2. A broad band of GTP-binding proteins, which overlaps with the methylated 21 to 23-kDa proteins, was detected in [alpha-32P]GTP blot overlay assays. This band of proteins was separated in two-dimensional gels into nine methylated proteins, of which four bound GTP. 3. The carboxyl-methylated 21 to 23-kDa proteins incorporated [3H]mevalonate metabolites with characteristics of protein isoprenylation. The label was not removed by organic solvents or destroyed by hydroxylamine. Incorporation of radioactivity from [3H]mevalonate was enhanced when endogenous levels of mevalonate were reduced by lovastatin, an inhibitor of mevalonate synthesis. Lovastatin blocked methylation of the 21 to 23-kDa proteins as well (greater than 70%). 4. Methylthioadenosine, a methylation inhibitor, inhibited methylation of these proteins (greater than 80%) but did not affect their labeling by [3H]mevalonate. The results suggest that methylation of the 21 to 23-kDa proteins depends on, and is subsequent to, isoprenylation. The sequence of events may be similar to that known in ras proteins, i.e., carboxyl methylation of a C-terminal cysteine that is isoprenylated. 5. Lovastatin reduced the level of small GTP-binding proteins in the membranes and increased GTP binding in the cytosol. Methylthioadensoine blocked methylation without affecting GTP binding. 6. Thus, isoprenylation appears to precede methylation and to be important for membrane association, while methylation is not required for GTP binding or membrane association. The role of methylation remains to be determined but might be related to specific interactions of the small GTP-binding proteins with other proteins.

Topics: Adenosine; Animals; Deoxyadenosines; Dimethyl Sulfoxide; GTP-Binding Proteins; Guanosine Triphosphate; Lovastatin; Membrane Proteins; Methylation; Mevalonic Acid; Mice; Molecular Weight; Neoplasm Proteins; Neuroblastoma; Protein Processing, Post-Translational; Thionucleosides; Tumor Cells, Cultured

We have previously shown that bradykinin-induced production of second messengers such as inositol trisphosphate and diacylglycerol in neurotumor cells is inhibited by raising cellular cyclic AMP levels, which in turn inhibit phospholipase C. A monoclonal antibody to phospholipase C-II immunoprecipitated the 140-kDa form of phospholipase C-II from [35S]methionine/[3H]eucine-labeled cells, but not [32P]orthophosphate-labeled phospholipase C-II, following treatment with either forskolin or dibutyryl cyclic AMP. This suggested that phospholipase C is not the target for cyclic AMP-dependent protein kinase-mediated phosphorylation. In vitro studies confirmed that phospholipase C activity was inhibited by raising cellular cAMP levels, and partial sensitivity to Bordetella pertussis toxin suggested the involvement of a GTP-binding protein which could be the target for protein kinase A. The involvement of a GTP-binding protein in coupling the bradykinin receptor to phospholipase C was further suggested by the ability of both guanosine 5'-O-(thio-triphosphate) and fluoride (NaF) to release inositol phosphates from NCB-20 cell membranes previously labeled with [3H]inositol. Both effects were blocked by pretreatment of the cells with protein kinase A activators, further suggesting a GTP-binding protein as the target for protein kinase A-mediated phosphorylation. When whole NCB-20 cell extracts were blotted onto nitrocellulose and incubated with [alpha- 32P]GTP, a major 24-kDa band plus minor bands at 22 and 20 kDa were revealed by autoradiography. A pH 3.0/6.0 soluble (basic protein) NCB-20 cell extract revealed the major 24-kDa band plus the 20-kDa band, and similar basic proteins were shown to be heavily phosphorylated following [32P]orthophosphate labeling and pretreatment with forskolin. The size and ability to bind GTP on Western blots are characteristic of the ras, rho, smg, etc. family of GTP-binding proteins recently suggested to be the much sought after GPLC (Lapetina, E.G., Lacal, J. C., Reep, B. R., and Molina y Vedia, L. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3131-3134; Wang, P., Nishihata, J., Takabori, E., Yamamoto, K., Toyoshima, S., and Osawa, T. (1989) J. Biochem. (Tokyo) 105, 461-466; Nagata, K.-I., Nagao, S., and Nozawa, Y. (1989) Biochem. Biophys. Res. Commun. 160, 235-242). We propose that GPLC is uniquely sensitive to protein kinase A-mediated phosphorylation and that phosphorylation inhibits stimulus-secretion coupling in these cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Arachidonic Acid; Arachidonic Acids; Bradykinin; Brain; Cell Line; Cholera Toxin; Colforsin; Cricetinae; Embryo, Mammalian; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hybrid Cells; Inositol Phosphates; Kinetics; Molecular Weight; Neuroblastoma; Phosphorylation; Protein Kinases; Thionucleotides; Type C Phospholipases

1. NaF (10 mM) produced a 2-3 fold increase in adenylate cyclase activity in homogenates of NG108-15 cells incubated in the presence of 1 microM GTP. Higher concentrations of NaF suppressed adenylate cyclase activity. 2. In the presence of the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA; 100 microM) or the prostacyclin receptor agonist iloprost (10 nM), NaF produced a much smaller increase in adenylate cyclase activity, whereas in the presence of a saturating concentration of iloprost (1 microM), NaF only inhibited adenylate cyclase activity. 3. Similarly, Gpp(NH)p activated basal adenylate cyclase activity, and inhibited 1 microM iloprost-activated enzyme activity. In the presence of 10 microM forskolin, NaF or Gpp(NH)p increased adenylate cyclase activity synergistically. Analysis of concentration-effect curves indicated that NaF (2 mM) or Gpp(NH)p (100 microM) increased the potency with which forskolin activated adenylate cyclase, whilst reducing the maximum activation of adenylate cyclase by iloprost. 4. Opiate receptors mediate inhibition of adenylate cyclase, and the opiate agonist morphine (100 microM) reduced the capacity of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. Unexpectedly, pertussis toxin treatment enhanced the ability of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. 5. In the absence of GTP, NaF and Gpp(NH)p remained able both to activate basal adenylate cyclase and to be synergistic with forskolin in activating the enzyme. In contrast the ability of NaF and Gpp(NH)p to inhibit iloprost-activated adenylate cyclase was substantially lost in the absence of added GTP. These results suggest that NaF modulates adenylate cyclase activity in NG108-15 cell membranes by interacting with the alpha subunits of both G0 and Gi regulatory proteins. The effects of NaF and Gpp(NH)p are critically dependent on the prior mode and extent of activation or inhibition of this transmembrane signalling pathway. This simple system may be of use in assessing alterations in GSO-O interaction following manipulations such as hormone receptor desensitization.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Animals; Cell Membrane; Colforsin; Enzyme Activation; Epoprostenol; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Iloprost; Mice; Morphine; Nervous System Neoplasms; Neuroblastoma; Pertussis Toxin; Sodium Fluoride; Tumor Cells, Cultured; Virulence Factors, Bordetella

The effects of L-arginine (Arg) derivatives on soluble guanylate cyclase from neuroblastoma N1E 115 cells were examined. The Arg derivatives were modified at the -NH2, -COOH, C alpha-proton or guanidino group of Arg. Among the synthesized derivatives, eight compounds, i.e. the 5-(dimethylamino)-1-naphthalenesulfonyl (DNS) ones, especially N-cyclohexyl-2-(N-DNSamino)-5-guanidino-2-methylvaleramide and 1-[2-(N-DNSamino)-2-(2-imino-1,2,3,4,5,6-hexahydropyrimidin- 4-yl)acetyl]- piperidine, were found to inhibit the activity of crude guanylate cyclase in the 105,000 g supernatant fraction of the cell homogenate. The enzyme, partially purified by a column of Chelex 100 Na+, was also inhibited by these eight compounds. The mode of the inhibition was competitive. The Ki values were in the range of 2-8 microM for the enzyme in the 105,000 g supernatant fraction and 3-16 microM for the partially purified enzyme, in the presence of Mg2+ as a metal cofactor. In contrast, a new derivative, methyl 2-amino-5-guanidinovalerate (M Arg ME), as well as the Arg methyl ester (Arg ME) and Arg; were found to enhance the activity of the partially purified guanylate cyclase; KA values of M Arg ME, Arg ME and Arg were approximately 9, 4 and 3 microM respectively. From these results, the free guanidino group including 2-imino-1,2,3,4,5,6-hexahydropyrimidin-4-yl or 2-imino-1,2,3,4,5,6-hexahydropyrimidin-5-yl and modification of the --NH2 residue with a hydrophobic group such as DNS seemed to be essential for inhibition of the guanylate cyclase; however, the guanidino and --NH2 residue of Arg should be free for activation by these Arg derivatives.

Topics: Arginine; Binding, Competitive; Dansyl Compounds; Guanosine Triphosphate; Guanylate Cyclase; Magnesium; Molecular Structure; Neuroblastoma; Structure-Activity Relationship; Tumor Cells, Cultured

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

The actions of tumor promoters on the coupling of muscarinic receptors to the hydrolysis of inositol lipids and the generation of Ca2+ signals were examined in the human neuroblastoma SH-SY5Y cell line. Pretreatment of SH-SY5Y cells with 50 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 5 days resulted in neuronal differentiation, a 28% decrease in both N-[3H]methylscopolamine and [3H]-scopolamine binding, and a significantly larger reduction (48%) in agonist-stimulated 3H-inositol phosphate generation. Whereas mezerein could mimic the effects produced by TPA, the biologically inactive 4 alpha-phorbol 12,13-didecanoate was without effect on both antagonist binding and agonist-stimulated phosphoinositide (PPI) turnover. A decline (approximately 50%) in the agonist-mediated rise in cytoplasmic Ca2+ and a substantial loss of protein kinase C activity also were observed following pretreatment with TPA or mezerein. The ability of fluoride, an agent capable of direct activation of guanine nucleotide binding proteins, to stimulate 3H-inositol phosphate release was significantly reduced in SH-SY5Y cells treated with these agents. Furthermore, pretreatment of SH-SY5Y neuroblastoma cells with TPA or mezerein impaired 3H-inositol phosphate formation induced by the addition of either guanosine 5'-O-(3-thiotriphosphate) or carbamylcholine to digitonin-permeabilized cells, but not that elicited by the addition of 2 mM CaCl2. Although cells cultured in the presence of serum-free media also exhibited neuronal differentiation, no significant alteration in either muscarinic receptor number or agonist-stimulated PPI hydrolysis was observed. The results suggest that TPA and mezerein decrease agonist-stimulated PPI hydrolysis and Ca2+ signaling in SH-SY5Y cells not only by a reduction in muscarinic receptor number but also through an inhibition of guanine nucleotide-stimulated PPI turnover.

Topics: Calcium; Culture Media; Cytoplasm; Diterpenes; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydrolysis; Inositol Phosphates; Neuroblastoma; Osmolar Concentration; Phosphatidylinositols; Protein Kinase C; Receptors, Muscarinic; Sodium Fluoride; Terpenes; Tetradecanoylphorbol Acetate; Thionucleotides; Time Factors; Tumor Cells, Cultured

The effect of triiodothyronine (T3) on the differentiation of cultured neuroblastoma (NB) cells was studied after 9 days of treatment with a dose of 10(-4) M/10(6) cells per day. Using phase contrast microscopy, 30-50% of NB cells showed formation of neurites as a morphological sign of cellular differentiation. The initial rise of the mitosis rate was followed by a plateau. Changes in cyclic nucleotide content, in the triphosphates and in the activity of the enzyme ornithine decarboxylase (ODC) were assessed in 2 human and 2 murine cell lines to serve as biochemical parameters of the cell differentiation induced by T3. Whereas the cAMP level increased significantly (3 to 7 fold compared with its initial value), the cGMP value dropped to 30 to 50% of that of the control group. ATP and GTP increased about 200%, the ODC showed a decrease of about 50%. The present studies show a biphasic effect of T3 on neuroblastoma cells: the initial rise of mitotic activity is followed by increased cell differentiation starting from day 4 of the treatment.

Topics: Adenosine Triphosphate; Cell Differentiation; Cell Line; Cyclic AMP; Cyclic GMP; Guanosine Triphosphate; Humans; In Vitro Techniques; Microscopy, Phase-Contrast; Mitosis; Neuroblastoma; Ornithine Decarboxylase; Triiodothyronine; Tumor Cells, Cultured

Both ethanol and neurotensin produce sedation and hypothermia. When administered in combination the behavioral effects of these two substances are potentiated. In order to better understand the biochemical nature of this interaction, the direct effects of ethanol on neurotensin receptors and an associated signal transduction process were determined in NIE-115 neuroblastoma cells. Ethanol in physiologically relevant concentrations (50mM) significantly reduced neurotensin stimulated [3H]inositol phosphate production while having no effect on the specific binding of [3H]neurotensin. In addition, ethanol up to 200 mM had no effect on GTPYS mediated [3H]inositol phosphate production. The results indicate that acute exposure to ethanol partially disrupts the normal coupling of activated neurotensin receptors to the guanine nucleotide binding protein associated with phospholipase C.

Topics: Animals; Dose-Response Relationship, Drug; Ethanol; Guanine Nucleotides; Guanosine Triphosphate; Inositol Phosphates; Mice; Neuroblastoma; Neurons; Neurotensin; Receptors, Neurotensin; Receptors, Neurotransmitter; Signal Transduction; Tritium; Tumor Cells, Cultured; Type C Phospholipases

Serotonin (5-hydroxytryptamine; 5-HT) and its analogs activate adenylate cyclase in membrane particles from neuroblastoma NCB.20 cells. Low concentrations of GTP (EC50 = 60 nM) were required for activation by serotonin. Guanosine 5'-O-(2-thiodiphosphate) inhibited serotonin-activated cyclase in these cells. The nonhydrolyzable GTP analogs guanosine 5'-O-(3-thiotriphosphate) (EC50 = 3 nM) and guanylyl-imidodiphosphate (EC50 = 100 nM) substituted for GTP in potentiating serotonin activation. Pretreatment of the cells with cholera toxin potentiated enzyme activation by serotonin, whereas pertussis toxin was found to have little effect, indicating the involvement of the alpha subunit of a stimulatory GTP-binding protein in enzyme activation. Homologous desensitization of the serotonin-stimulated adenylate cyclase was demonstrated in membranes prepared from intact cells pretreated with serotonin. Cell membrane particles that were desensitized to serotonin were still responsive to beta-adrenergic agonists and to prostaglandin E1. Evidence is presented indicating that serotonin stimulation of adenylate cyclase is mediated by receptors that are distinct from other positively coupled receptors (beta-adrenergic, histamine, and prostacyclin). Equilibrium binding analysis with [3H]serotonin, [3H]lysergic acid diethylamide, and [3H]dihydroergotamine suggested that the site density was below the level of detection of binding of these radioligands. The pharmacological characteristics of the serotonin-activated cyclases were analyzed in order to compare these serotonin receptors with the family of different receptor subtypes. Correlation analysis between the potencies of different agonists and antagonists at the cyclase in these cells and their reported relative potencies for different serotonin receptor subtypes showed no correlation with the 5-HT1A, 5HT1B, 5HT1D, 5-HT2, and 5-HT3 receptors. On the other hand, the analysis showed that the NCB.20 serotonin receptors are similar but not identical to the rat and pig brain 5-HT1C receptors and to the serotonin receptors coupled to adenylate cyclase in the trematodes Schistosoma mansoni and Fasciola hepatica. The results point to a novel serotonin receptor which has a low density in these cells.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Cholera Toxin; Enzyme Activation; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; NAD; Neuroblastoma; Receptors, Serotonin; Serotonin; Serotonin Antagonists; Thionucleotides; 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

Opioid receptors of NG 108-15 cell membranes are distributed in two membrane fractions sedimenting at 20,000 g (P2) and 200,000 g(P3). The number of receptors is identical in P2 and P3, but in P2 all sites are present in one high-affinity state (2 nM), whereas in P3 60% of these receptors display lower affinity (150 nM). Upon addition of GTP or pretreatment with pertussis toxin, 80% of the sites exist in low affinity in both P2 and P3. Therefore, the effect of GTP and pertussis toxin on agonist binding appears to be smaller in P2 than in P3. In contrast, sodium inhibits agonist binding in P2 and P3 to the same extent and with identical potency. Opioid-mediated stimulation of GTPase is much greater in P2 than in P3, whereas inhibition of adenylate cyclase does not differ in the two fractions. Using site-specific antibodies and pertussis toxin-catalyzed ADP-ribosylation, we found that the amount of G proteins in P3 is only 30-50% of that in P2. Treatment of intact cells with the hydrophilic protein-modifying agent sulfosuccinimido-biotin results in biotinylation of proteins from both fractions and in a similar reduction of opioid binding in P2 and P3. Likewise, exposure of intact cells to the alkylating opioid antagonist, chlornaltrexamine, produces identical degrees of receptor inactivation in P2 and P3. The rate of in vivo pertussis toxin-mediated modification of G proteins is not different in the two fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Cell Fractionation; Cell Membrane; Centrifugation; Diprenorphine; Electrophoresis, Polyacrylamide Gel; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Neuroblastoma; Pertussis Toxin; Receptors, Opioid; Tumor Cells, Cultured; Virulence Factors, Bordetella

The relationship between muscarinic receptor-mediated inositol lipid hydrolysis and the generation of Ca2+ signals has been examined in human SK-N-SH neuroblastoma cells. The resting cytoplasmic calcium concentration [( Ca2+]i) as determined by fura-2 fluorescence measurements was 59 +/- 2 nM. Upon the addition of oxotremorine-M, there was a 4-fold increase in [Ca2+]i (293 +/- 18 nM), with half-maximal stimulation obtained at an agonist concentration of 8 microM, a value similar to that previously observed for the enhancement of phosphoinositide hydrolysis. Addition of partial muscarinic agonists for phosphoinositide turnover (bethanechol, oxo-2, and arecoline) elicited correspondingly smaller increases in [Ca2+]i than did oxotremorine-M. Inclusion of EGTA lowered the basal [Ca2+]i within 2 min and markedly reduced (greater than 60%) the magnitude of the agonist-induced rise in [Ca2+]i. Addition of muscarinic agonists to SK-N-SH cells that had been prelabeled with [3H]inositol led to the rapid (5-15 sec) release of inositol mono-, bis-, and triphosphates. When assayed under conditions similar to those employed for the fluorescence measurements, EGTA also inhibited both the basal and oxotremorine-M-stimulated release of inositol phosphates by 45-61%. Conversely, ionomycin both elevated [Ca2+]i and stimulated the release of inositol phosphates. The addition of Ca2+ (10 nM-2 microM) to digitonin-permeabilized cells directly stimulated the release of labeled inositol mono-, bis-, and trisphosphates by 3-4-fold with a half-maximal effect (EC50) observed at 145 nM free Ca2+ (Ca2+f). A further (6-fold) calcium-dependent increase in inositol phosphate release was obtained by inclusion of either guanosine-5-O-(3-thio)-trisphosphate (GTP gamma S) or oxotremorine-M. In the combined presence of agonist and GTP gamma S, a synergistic release of all three inositol phosphates occurred, with half-maximal stimulation observed at 35-40 nM Ca2+f, a value similar to the [Ca2+]i in quiescent cells. These results indicate (i) that the magnitude of the initial rise in [Ca2+]i is directly related to the production of phosphoinositide-derived second messenger molecules and (ii) that the phospholipase C-mediated breakdown of inositol lipids in SK-N-SH cells is particularly sensitive to regulation by physiologically relevant Ca2+ concentrations. It is concluded that, in SK-N-SH cells, either an elevation above or reduction below basal [Ca2+]i can modulate the extent of hydrolysis o

Topics: Calcium; Cytoplasm; Egtazic Acid; Enzyme Activation; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Neuroblastoma; Phosphatidylinositols; Receptors, Muscarinic; Thionucleotides; Tumor Cells, Cultured; Type C Phospholipases

The effects of mastoparan on phospholipase C-catalysed phosphoinositide hydrolysis were examined in [3H]inositol-labelled human neuroblastoma SH-SY5Y cells. [3H]Inositol phosphate formation in intact cells was not altered by 20 microM mastoparan. In contrast, [3H]inositol phosphate formation in electrically permeabilized cells stimulated with guanosine 5'-[gamma-thio]triphosphate and/or carbachol was inhibited by mastoparan with half-maximal effects at approx. 3 microM. The peptide was much less effective in inhibiting stimulatory effects of Ca2+. Similar but less potent inhibitory effects were observed with the cations, neomycin and spermine, indicating that direct interaction of mastoparan with polyphosphoinositides might account for its inhibitory effects on inositol phosphate formation.

Topics: Bee Venoms; Calcium; Carbachol; Cell Membrane Permeability; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydrolysis; Inositol Phosphates; Intercellular Signaling Peptides and Proteins; Neuroblastoma; Peptides; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Receptors, Muscarinic; Thionucleotides; Tumor Cells, Cultured; Type C Phospholipases; Wasp Venoms

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

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

It has been found that neuroblastoma contains a relatively high immunoreactivity for the alpha subunit of guanine nucleotide-binding protein G(o) (G(o) alpha) (Kato et al., Cancer Res. 47, 5800-5805, 1987), which is predominantly localized in the nervous tissues and neuroendocrine cells. To make clear whether neuroblastoma indeed produces G(o) alpha, we purified a guanine nucleotide-binding protein from human neuroblastoma and compared it with G(o) alpha from human brain. A guanine nucleotide-binding protein serving as the specific substrate of islet-activating protein, pertussis toxin, was purified from a human neuroblastoma tissue obtained at surgical resection. The protein had a molecular mass of 39 kDa and reacted with rabbit antibodies to bovine G(o) alpha. Heat stability and kinetics of guanosine 5'-(3-O-thio)triphosphate binding of this protein were very similar to those of G(o) alpha purified from human brain. The results of peptide mapping analysis with a limited proteolysis and amino acid analysis indicated that there was no difference between these proteins. Therefore, it was concluded that this guanine nucleotide-binding protein isolated from neuroblastoma was very similar, if not identical, to brain G(o) alpha.

Topics: Amino Acids; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Neuroblastoma; Thionucleotides

The rates of phosphodiesterase-promoted hydrolysis of cGMP and cAMP have been measured in intact neuroblastoma N1E-115 cells by determining rates of 18O incorporation from 18O-water into the alpha-phosphoryls of guanine and adenine nucleotides. The basal rate of guanine nucleotide alpha-phosphoryl labeling ranged from 180 to 244 pmol X mg protein-1 X min-1. Sodium nitroprusside (SNP) caused a sustained 3.4-fold increase in this 18O-labeling rate in conjunction with 28- and 50-fold increases in cellular cGMP concentration at 3 and 6 min, respectively. This 18O-labeling rate (795 pmol X mg protein-1 X min-1) corresponded with the sum of the low (1.7 microM) and high (34 microM) Km phosphodiesterase activities assayable in cell lysates which exhibited a combined maximum velocity of 808 pmol X mg protein-1 X min-1 to which the high Km species contributed 84%. This information and the characteristics of the profile of 18O-labeled molecular species indicate that cGMP metabolism was restricted to a very discrete cellular compartment(s) of approximately 12% of the cell volume. Carbachol (1 mM) produced a transient increase (6-fold) in cellular cGMP concentration and a transient increase (90%) in the rate of 18O labeling of alpha-GTP during the first minute of treatment which translates into 30 additional cellular pools of cGMP hydrolyzed in this period. IBMX (1 mM) produced a relatively rapid increase in cellular cGMP (3- to 5-fold) and cAMP (2-fold) concentrations and a delayed inhibition of 18O labeling of guanine and adenine nucleotide alpha-phosphoryls without further elevation of cyclic nucleotide levels. These results indicate that besides inhibiting cyclic nucleotide hydrolysis, IBMX also imparts a time-dependent inhibitory influence on the generation of cyclic nucleotides. The data obtained show that measurement of 18O labeling of guanine and adenine nucleotide alpha-phosphoryls combined with measurements of cyclic nucleotide steady state levels provides a means to assess the rates of cyclic nucleotide synthesis and hydrolysis within intact cells and to identify the site(s) of action of agents that alter cellular cyclic nucleotide metabolism.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Adenine Nucleotides; Animals; Carbachol; Cell Line; Cyclic AMP; Cyclic GMP; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Isotope Labeling; Kinetics; Neuroblastoma; Nitroprusside; Oxygen Radioisotopes

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

Membranes prepared from mammalian brain or intestine contain two types of specific binding sites for neurotensin that differ by their affinity and by their sensitivity to sodium ions, GTP, and the antihistamine drug levocabastine. Only the high affinity sites are present in cell cultures and in soluble extracts of CHAPS-treated membranes. These sites represent functional neurotensin receptors coupled to GTP-binding proteins that regulate intracellular levels of cAMP, cGMP and inositol phosphates in neuroblastoma N1E115 cells. The molecular weight of neurotensin receptors in cells and membrane preparations of various origin is about 110,000.

Topics: Animals; Brain Chemistry; Cell Membrane; Cells, Cultured; Cholic Acids; Cyclic AMP; Guanosine Triphosphate; Intestines; Kinetics; Neuroblastoma; Neurotensin; Receptors, Neurotensin; Receptors, Neurotransmitter

Recent evidence has revealed that a highly sensitive and specific guanine nucleotide regulatory process controls intracellular Ca2+ release within N1E-115 neuroblastoma cells (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464). The present report documents GTP-induced Ca2+ release within quite distinct cell types, including the DDT1MF-2 smooth muscle cell line. GTP-induced Ca2+ release has similar GTP sensitivity and specificity among cells and rapidly mobilizes up to 70% of Ca2+ specifically accumulated within a nonmitochondrial Ca2+-pumping organelle within permeabilized DDT2MF-2 cells. Maximal GTP-induced release of Ca2+ is observed to be greater than inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (the latter being approximately 30% of total releasable Ca2+). After maximal IP3-induced release, further IP3 addition is ineffective, whereas subsequent addition of GTP further releases Ca2+ to equal exactly the extent of Ca2+ release observed by addition of GTP in the absence of IP3. This suggests that IP3 releases Ca2+ from the same pool as GTP, whereas GTP also releases from an additional pool. The effects of GTP appear to be reversible since simple washing of GTP-treated cells restores their previous Ca2+ uptake properties. Electron microscopic analysis of GTP-treated membrane vesicles reveals their morphology to be unchanged, whereas treatment of vesicles with 3% polyethylene glycol, known to enhance GTP-mediated Ca2+ release, clearly induces close coalescence of membranes. In the presence of 4 mM oxalate, GTP induces a rapid and profound uptake, as opposed to release, of Ca2+. The findings suggest that GTP-activated Ca2+ movement is a widespread phenomenon among cells, which can function on the same Ca2+ pool mobilized by IP3, and although activating Ca2+ movement by a mechanism distinct from IP3, does so via a process that does not appear to involve fusion between membranes.

Topics: Animals; Calcium; Cell Line; Cell Membrane Permeability; Guanosine Triphosphate; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Intracellular Membranes; Kinetics; Microscopy, Electron; Microsomes; Muscle, Smooth; Neuroblastoma; Neurons; Subcellular Fractions; Sugar Phosphates

The GTP-activated Ca2+ release process we recently described (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464) was revealed in the preceding report to operate via a mechanism likely to be induced by close membrane association but which appears not to involve membrane fusion (Chueh, S. H., Mullaney, J. M., Ghosh, T. K., Zachary, A. L., and Gill, D. L. (1987) J. Biol. Chem. 262, 13857-13864). To determine more about the GTP-activated Ca2+ translocation process, effects of GTP on cells loaded with Ca-oxalate were investigated. Using permeabilized cells of both the N1E-115 neuroblastoma and DDT1MF-2 smooth muscle cell lines, 10 microM GTP activates a profound uptake of Ca2+ in the presence of oxalate, as opposed to release observed without oxalate. GTP stimulation of Ca2+ uptake was observed at oxalate concentrations (2 mM) only slightly augmenting Ca2+ uptake without GTP; with 8 mM oxalate (which alone induces linear Ca2+ accumulation) GTP still increases the rate of uptake. GTP-activated uptake in the presence of oxalate is completely reversed by 1 mM vanadate. 3% polyethylene glycol enhances the effect of GTP although GTP-activated uptake is still observed without polyethylene glycol. The Km for GTP for activation of Ca2+ uptake is 0.9 microM. Uptake is not activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp); however, GTP gamma S (but not GppNHp) completely blocks the action of GTP. GDP gives a delayed uptake response which is blocked by ADP, indicating its action arises from conversion to GTP. In the presence of ADP, GDP blocks the action of GTP; guanosine 5'-O-(2-thio)diphosphate, which does not activate uptake, also blocks the action of GTP. These data reveal almost exact correlation between parameters affecting GTP-activated uptake and release, strongly suggesting the same process mediates both events. To explain the opposite effects of GTP in the absence and presence of oxalate, it is proposed that GTP activates a transmembrane conveyance of Ca2+ between oxalate-permeable and -impermeable compartments.

Topics: Animals; Biological Transport; Calcium; Cell Line; Cell Membrane; Guanosine Triphosphate; Kinetics; Models, Biological; Muscle, Smooth; Neuroblastoma; Neurons; Oxalates; Polyethylene Glycols

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

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Cell Line; Cell Membrane Permeability; Guanine Nucleotides; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Mice; Microsomes; Neuroblastoma; Neurons; Polyethylene Glycols; Saponins; Sugar Phosphates

A sensitive and specific guanine nucleotide regulatory process has recently been shown to rapidly mediate a substantial release of Ca2+ from endoplasmic reticulum within the N1E-115 neuronal cell line (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464). The relationship between this mechanism and Ca2+ efflux mediated by the intracellular regulator inositol 1,4,5-trisphosphate (IP3) has been investigated. Using saponin-permeabilized N1E-115 cells, studies reveal a number of distinctions between the activation of Ca2+ release mediated by GTP and IP3. Thus, the GTP-mediated Ca2+ release process is specifically activated by polyethylene glycol which increases both GTP sensitivity and the extent of GTP-activated Ca2+ release; in contrast, IP3-dependent Ca2+ release is unaffected by polyethylene glycol. The non-hydrolyzable GTP analogue guanosine 5'-O-(3-thio)triphosphate, which completely inhibits GTP-mediated Ca2+ release, does not alter release mediated by IP3. Decreasing the release temperature from 37 to 4 degrees C decreases IP3-activated Ca2+ release by only 20%, whereas the action of GTP on Ca2+ release is abolished at 4 degrees C. Activation of Ca2+ release by IP3 is completely inhibited by increasing free Ca2+ from 0.1 to 10 microM, whereas the fraction of GTP-dependent Ca2+ release (approximately 50% of ionophore-releasable Ca2+) remains unaltered with increasing free Ca2+. These distinctions between IP3- and GTP-mediated Ca2+ release indicate that the two effectors function via distinct mechanisms to activate Ca2+ release; however, they do not preclude the possibility that coupling between the two mechanisms can occur or that a common Ca2+-translocating pathway activated by both effectors exists.

Topics: Animals; Calcium; Cell Line; Endoplasmic Reticulum; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Neuroblastoma; Permeability; Polyethylene Glycols; Sugar Phosphates; Temperature; Thionucleotides; Time Factors

The inhibition of adenylate cyclase activity by cannabimimetic compounds in a membrane fraction from cultured neuroblastoma cells has been examined. The inhibition was shown to be concentration-dependent over a nanomolar range for both delta 9-tetrahydrocannabinol and its synthetic analog, desacetyllevonantradol. Inhibition was rapid and reversible. The cannabimimetic compounds caused a decrease in Vmax of the enzyme, with no alteration in the Km for substrate. The effects of these compounds were related to the ability of the enzyme to be regulated by divalent cations and guanine nucleotides. The inhibition was greatest at micromolar Mg2+ or Mn2+ concentrations and was abolished at less than 1 mM MnCl2. In the hormone-stimulated state, the enzyme appeared to be regulated by one Mg2+ site. The addition of cannabimimetic or muscarinic cholinergic agents transformed the enzyme into one in which more complex regulation by divalent cations was observed. Half-maximal inhibition of adenylate cyclase was observed at 800 nM GTP for both cannabimimetic and muscarinic cholinergic agents. The substitution for GTP of a nonhydrolyzable analog resulted in activation of the enzyme and failure to respond to either class of inhibitory agents. If the Mg2+ concentration was reduced and exposure to the GTP analog was of short duration, inhibition by both cannabimimetic and muscarinic agents could be observed in the presence of forskolin. This study points to the similarities between the enzyme inhibition by cannabimimetic compounds and by muscarinic cholinergic compounds. It is inferred that the cannabimimetic compounds must act via regulatory mechanisms similar to those operating for receptor-mediated inhibition of adenylate cyclase.

Topics: Adenosine Triphosphate; Adenylyl Cyclase Inhibitors; Animals; Cannabinoids; Cell Line; Cell Membrane; Chlorides; Dronabinol; Guanosine Triphosphate; Kinetics; Magnesium; Magnesium Chloride; Manganese; Manganese Compounds; Mice; Neuroblastoma; Phenanthridines; Secretin; Substrate Specificity

eIF-2 purified from neuroblastoma cells consists of three subunits, which appear to be of molecular weight identical to those of the subunits of rabbit reticulocyte eIF-2. A protein fraction has been isolated from neuroblastoma cells with characteristics similar to eRF from reticulocytes: stimulation of amino acid incorporation in a hemin-deprived reticulocyte lysate, the removal of GDP from eIF-2-GDP complexes, a 4-5-fold stimulatory effect in a two-step reaction measuring 40 S preinitiation complex formation and a 3-3.5-fold stimulation in the methionyl-puromycin synthesis. In the methionyl-puromycin-synthesizing system phosphorylated eIF-2 is not responsive to the addition of this fraction from neuroblastoma cells. The protein fraction contains eRF which seems to be similar to the eRF isolated from Ehrlich ascites tumor cells and somewhat distinct from the reticulocyte factor. Incubation of neuroblastoma cell lysate in the presence of [gamma-32P]ATP results in the phosphorylation of a protein of Mr 36 000, migrating on SDS-polyacrylamide gels to the position of eIF-2 alpha. This protein is also phosphorylated in vitro by HRI from reticulocytes. These results may reflect a common underlying principle for the quantitative regulation of protein synthesis in eukaryotic cells.

Topics: Animals; Eukaryotic Initiation Factor-2; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Neuroblastoma; Peptide Initiation Factors; Protein Biosynthesis; Proteins

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

Secretin, a gut-brain peptide, elicited cyclic AMP production in a clone of neuroblastoma cells derived from the C1300 mouse tumor. Adenylate cyclase (EC 4.6.1.1) in plasma membranes from these cells was stimulated by secretin greater than vasoactive intestinal peptide greater than peptide histidine isoleucine amide, but not by the related peptides glucagon, gastric inhibitory polypeptide, or human growth hormone releasing factor. Hill coefficients for stimulation approximated one and the response to submaximal peptide concentrations was additive, as expected for hormones competing for a single receptor associated with the enzyme. Binding of 125I-labeled secretin to the neuroblastoma plasma membranes was saturable, time-dependent, and reversible. The KD determined from kinetic and equilibrium binding studies approximated 1 nM. The binding site displayed marked ligand specificity that paralleled that for stimulation of adenylate cyclase. The secretin receptor was regulated by guanine nucleotides, with guanosine 5'-(beta, gamma-imino)-triphosphate being the most potent to accelerate the rate of dissociation of bound secretin. These findings demonstrate the functional association of the secretin receptor with adenylate cyclase in neuronally derived cells.

Topics: Adenylyl Cyclases; Animals; Binding, Competitive; Cell Line; Cell Membrane; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Neuroblastoma; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; Secretin; Vasoactive Intestinal Peptide

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

Since there is no nutritional requirement for the biopterin cofactor, we attempted to create a drug-induced deficiency in rats in order to study the role of tetrahydrobiopterin in regulating the biosynthesis of dopamine and serotonin. The hypothesis that dihydrofolate reductase (EC 1.5.1.3) mediates the final step in the de novo synthesis of tetrahydrobiopterin was tested by treating rats with methotrexate along with leucovorin as a protective agent; there was no reduction in total biopterin or in the fraction present as tetrahydrobiopterin in adrenal medulla, adrenal cortex, pituitary, brain, or pineal glands. Similar results were obtained with metoprine, a lipid-soluble inhibitor of dihydrofolate reductase which readily enters the central nervous system. Treatment with loading doses of phenylalanine along with methotrexate reduced the level of tetrahydrobiopterin in liver. Neuroblastoma N115 cells growing in medium supplemented with thymidine and hypoxanthine continued to form normal amounts of tetrahydrobiopterin in the presence of concentrations of methotrexate which completely inhibited dihydrofolate reductase; higher concentrations of methotrexate increased the tetrahydrobiopterin content of the cells 2-fold and the total biopterin in the medium 3-fold. Although attempts to create a drug-induced deficiency were unsuccessful, the evidence indicates that the de novo synthesis of tetrahydrobiopterin proceeds by a pathway independent of dihydrofolate reductase and that folate antagonists, such as methotrexate are unlikely to impair the hydroxylation of tyrosine and tryptophan, which is dependent upon the availability of the biopterin cofactor.

Topics: Animals; Biopterins; Cell Line; Clone Cells; Folic Acid Antagonists; GTP Cyclohydrolase; Guanosine Triphosphate; Male; Methotrexate; Neuroblastoma; Pteridines; Rats; Rats, Inbred Strains

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

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

Na+ has been implicated as a requirement for the inhibition of adenylate cyclase by hormones and neurotransmitters. This study examines effects of salt concentration on neuroblastoma plasma membranes that occur in the absence of an inhibitory hormone. The adenylate cyclase response to stimulatory agonists (GTP plus PGE1 (3), PGI2 or PGE2) was influenced by NaCl. As the [NaCl] increased to 150 mM, an increase in maximal activity and a decrease in apparent affinity was observed. At concentrations above 150 mM, NaCl decreased prostaglandin affinity and progressively decreased maximal activation. The GTP requirement was not altered by 30 or 150 mM NaCl in the presence of PGE1 or PGI2. The rate of Gpp(NH)p stimulated activity increased as the [NaCl] was increased in the assay. This increased rate was conserved when membranes activated in the presence of Gpp(NH)p and NaCl were reassayed in the absence of guanine nucleotide or salt. The salt evoked rate increase was proportionally greater at submaximal MgCl2 concentrations. The concentration requirement for Mg2+ was reduced by salt for adenylate cyclase in the presence of GTP or Gpp(NH)p. However, the enzyme stimulated by hormone exhibited a Mg2+ requirement that was low in the absence of salt and could not be further reduced by increased [NaCl]. Alternative monovalent cations (150 mM Li+, K+, Cs+, but not choline or tetramethylammonium) and anions (SO4=) substituted for NaCl. The observed effects were reversible upon washing the membranes and neither ouabain nor tetrodotoxin altered the response. These effects may result from a conformational alteration of a protein particularly sensitive to neutral salts in the assay.

Topics: Adenylyl Cyclases; Alprostadil; Animals; Cell Line; Dinoprostone; Epoprostenol; Guanine Nucleotides; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Mice; Neuroblastoma; Prostaglandins; Prostaglandins E; Sodium Chloride

[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

Topics: Animals; Brain; Cells, Cultured; Diprenorphine; Endorphins; Enkephalins; Guanosine Triphosphate; In Vitro Techniques; Magnesium; Naloxone; Neoplasms, Experimental; Neuroblastoma; Rats; Receptors, Opioid; Sodium

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

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

The binding of many opiates and enkephalins to enkephalin (delta) and morphine (mu) receptors was compared by using three different binding assays: (i) 125I-labeled[D-Ala2, D-Leu5]enkephalin or 125I-labeled[D-Ala2,N-Me-Phe4,Met(O)5ol]-enkephalin to brain membranes; (ii) [3H]ethylketocyclazocine to brain membranes; and (iii) [3H]diprenorphine and [3H]naloxone to neuroblastoma cell and brain membranes, respectively. According to their relative binding potencies and the effects of Na+ and GTP on the binding to these two receptors, opiates and enkephalins can be classified into seven classes: (i) morphine-type mu agonists; (ii) enkephalin-type delta agonists; (iii) mixed agonists-antagonists; (iv) putative kappa agonists; (v) putative sigma agonists; (vi) nalorphine-type antagonists; and (vii) opiate antagonists. Studies with [3H]ethylketocyclazocine do not reveal specific kappa receptors distinct from those already described that bind morphine and enkephalins. The benzomorphan analogs ketocyclazocine and ethylketocyclazocine (putative kappa agonists) and N-allylnormetazocine (putative sigma agonist) bind to morphine (mu) and enkephalin (delta) receptors with similarly high affinities. The potency of putative kappa agonists, measured by competition with binding of the 3H-labeled antagonist, is greatly reduced by the presence of Na+ and GTP; the "Na+ and GTP ratios" are similar to those of morphine and enkephalins. However, Na+ and GTP greatly decrease the potency of binding of putative sigma agonists to enkephalin receptors but only slightly decrease the binding to morphine receptors. These data suggest that putative kappa agonists have agonistic activity toward both receptors, whereas putative sigma agonists behave as agonists for enkephalin receptors but have antagonist activity for morphine receptors. Mixed agonist-antagonists also show smaller difference in affinity to both receptors. These findings may have important implications for understanding the differences in the pharmacological effects of these drugs.

Topics: Animals; Benzomorphans; Brain; Cyclazocine; Diprenorphine; Endorphins; Enkephalins; Ethylketocyclazocine; Guanosine Triphosphate; Morphinans; Morphine; Naloxone; Neuroblastoma; Rats; Receptors, Opioid; Sodium; Synaptic Membranes

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

Topics: Adenylyl Cyclases; Animals; Brain; Cell Line; Cricetinae; Enzyme Activation; Guanosine Triphosphate; Hybrid Cells; Kinetics; Mice; Neuroblastoma; Serotonin

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: Antipsychotic Agents; Carbachol; Cell Line; Cyclic GMP; Dose-Response Relationship, Drug; Guanosine Triphosphate; Kinetics; Neuroblastoma; Phenothiazines; Receptors, Cholinergic; Receptors, Muscarinic

Mycophenolic acid, an inhibitor of inosinate dehydrogenase, had cytostatic and cytotoxic effects on cultured neuroblastoma cells. Proliferation was inhibited by 50% when cells were incubated with 0.07 micrometerM mycophenolic acid, and cell viability was reduced by 83% when cells were treated with 10 micrometerM mycophenolic acid for 24 hr. Treatment of monolayer cultures with mycophenolic acid reduced intracellular concentrations of guanosine triphosphate by 70% within 3 hr, whereas cytidine triphosphate and uridine triphosphate concentrations were significantly elevated, and adenosine triphosphate concentrations were increased only slightly. Reduction of cellular guanine nucleotides had differential effects on rates of macromolecular synthesis: incorporation of radioactive thymidine into acid-insoluble material was inhibited by mycophenolic acid to a much greater extent than was that of adenosine and leucine. Although proliferation of neuroblastoma cells was inhibited, differentiation, as judged by formation of neuronlike processes in serum-free medium, was unaffected by decreased intracellular concentrations of guanosine triphosphate.

Topics: Adenosine Triphosphate; Cell Differentiation; Cell Division; Cell Survival; Cells, Cultured; Cytosine Nucleotides; DNA, Neoplasm; Guanine Nucleotides; Guanosine Monophosphate; Guanosine Triphosphate; IMP Dehydrogenase; Mycophenolic Acid; Neoplasm Proteins; Neoplasms, Experimental; Neuroblastoma; RNA, Neoplasm; Uracil Nucleotides

The effect of acetylcholine, 3,4-dihydroxyphenylethylamine, prostaglandin (PGE1), guanosine triphosphate (GTP), and divalent ions on adenylate cyclase activity in homogenates of ""differentiated" and malignant mouse neuroblastoma cells was studied. The sensitivity of adenylate cyclase to acetylcholine and 3,4-dihydroxyphenylethylamine markedly increased in adenosine cyclic 3:5-monophosphate-induced differentiated neuroblastoma cells. Although 3,4-dihydroxyphenylethylamine stimulated adenylate cyclase activity in malignant neuroblastoma cells, it failed to do so in X-irradiation induced differentiated cells. PGE1 and GTP stimulated adenylate cyclase activity in malignant and adenosine cyclic 3:5-monophosphate induced differentiated neuroblastoma cells to about the same level. GTP protentiated the PGE1 effect in differentiated concentrations of magnesium and manganese inhibited adenylate cyclase activity; this effect was more pronounced in differentiated cells than in malignant cells. Calcium stimulated adenylate cyclase activity in malignant and differentiated cells to about the same level. There was no significant difference in the values of Km and Vmax of neuroblastoma cells. This study shows that the sensitivity of adenylate cyclase to neurotransmitters and divalent ions (magnesium and manganese) and the sensitivity of PGE1 stimulated enzyme activity to GTP increase in adenosine cyclic 3:5-monophosphate-induced differentiated neuroblastoma cells. Therefore, we suggest that the reverse may be true during malignant transformation of nerve cells.

Topics: Acetylcholine; Adenosine Triphosphate; Adenylyl Cyclases; Animals; Calcium; Cell Differentiation; Cells, Cultured; Cyclic AMP; Depression, Chemical; Dopamine; Drug Synergism; Guanosine Triphosphate; Kinetics; Magnesium; Manganese; Mice; Neuroblastoma; Prostaglandins; Stimulation, Chemical

Topics: Animals; Centrifugation, Density Gradient; Colchicine; Dithiothreitol; Glycerol; Guanosine Triphosphate; Half-Life; In Vitro Techniques; Mice; Neuroblastoma; Protein Binding; Protein Conformation; Sucrose; Tritium; Vinblastine

Topics: Adenosine Triphosphate; Animals; Axons; Cells, Cultured; Culture Media; Cyclic AMP; Guanosine Triphosphate; Mice; Mice, Inbred A; Microscopy, Electron; Microtubules; Neoplasms, Experimental; Neuroblastoma; Temperature

Topics: Adenosine Triphosphate; Animals; Brain; Calcium; Cattle; Cobalt; Cyclic AMP; Cyclic GMP; Guanosine Triphosphate; Kinetics; Magnesium; Manganese; Muscles; Nephropidae; Nerve Tissue Proteins; Neuroblastoma; Phosphotransferases; Rabbits; Rats; Thermodynamics