guanosine-monophosphate and Neuroblastoma

To observe the role of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels.. Using whole-cell patch-clamp techniques applied to NG108-15 cells, investigate the effect of opioid receptor agonist on the delayed rectified potassium channels by administration of Guanosine-5'-0'-2-thiociphosphate (GDP beta S), Pertusis Toxin (PTX), Tetroacetic acid nueleoside diphosphate kinase (NDPK) and Adenosine-3' 5' cyclic monophosphate cAMP in the pipette solution.. (1) GDP beta S could block the changes induced by both high and low concentration of (D-Pen2.5)-enkephalin (DPDPE) (P < 0.05). (2) PTX could inhibit the excitative regulation on K+ channel by high concentration of DPDPE (P < 0.05). But CTX had no effect on K+ channel caused by DPDPE. (3) UDP could block the excitative effect of K+ channel by high concentration of NDPK, while have no changes on the inhibitory effect caused by low concentration of opioid agonists. (4) cAMP took part in the regulation in high concentration of agonist administration (P < 0.05), while no changes for low concentration of agonists.. Dual changes were observed on delayed rectifier potassium channel by agonist treatment on NG108-15 cells. The excitative effect was Gi/o coupled in high concentration of agonist incubation, related to cAMP. While the inhibitory effect was possibly induced by G protein beta gamma subunit directly.

Topics: Animals; Enkephalin, D-Penicillamine (2,5)-; Glioma; GTP-Binding Proteins; Guanosine Monophosphate; Hybrid Cells; Mice; Neuroblastoma; Patch-Clamp Techniques; Pertussis Toxin; Potassium Channels, Inwardly Rectifying; Rats; Receptors, Opioid; Thionucleotides

Fura-2 fluorescence imaging was used to measure changes in intracellular Ca2+ concentration in individual N1E-115 neuroblastoma cells during repeated activation of M1 muscarinic receptors with carbachol. Ca2+ transients could be elicited repeatedly at 4 min intervals with little decrement as long as external Ca2+ was present. When the cells were bathed in Ca(2+)-free saline, however, the response amplitude decreased rapidly in a use-dependent fashion, indicating that external Ca2+, and presumably Ca2+ influx, is required for refilling Ca2+ stores during the interval between trials. The response amplitude also decreased during repeated stimulation in cells treated with the NO-synthase inhibitor L-NMMA or with the guanylyl cyclase inhibitor LY-83583 even when Ca2+ was present. Application of the membrane permeable cGMP analog 8-Br-cGMP reversed the effect of L-NMMA and promoted refilling in the continued presence of NO-synthase inhibitor. These results indicate that activation of the NO/cGMP pathway is necessary for refilling Ca2+ stores during muscarinic signaling. Evidence is also presented suggesting that the NO/cGMP pathway is involved in long term modulation of the content of Ca2+ stores.

Topics: Animals; Calcium; Enzyme Inhibitors; Guanosine Monophosphate; Mice; Neuroblastoma; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Signal Transduction; Sympathetic Nervous System; Tumor Cells, Cultured

The activity of IMP dehydrogenase (EC 1.2.1.14), the key enzyme of de novo guanylate biosynthesis, was shown to be increased in tumor cells. Tiazofurin (TR), a potent and specific inhibitor of this enzyme, proved to be effective in the treatment of refractory granulocytic leukemia in blast crisis. We examined the effects of tiazofurin as a single agent and in combination with hypoxanthine and allopurinol in six different neuroectodermal tumor cell lines, the STA-BT-3 and 146-18 human glioblastoma cell lines, the SK-N-SH, LA-N-1 and LA-N-5 human neuroblastoma cell lines, and the STA-ET-1 Ewing tumor cell line. Tiazofurin inhibited tumor cell growth with IC50 values between 2.2 microM (LA-N-1 cell line) and 550 microM (LA-N-5 cells) and caused a significant decrease of intracellular GTP pools (GTP concentrations decreased to 39-79% of control). Incorporation of [8-14C]guanine into GTP pools was determined as a measure of guanylate salvage activity; incubation with 100 microM hypoxanthine caused a 62-96% inhibition of the salvage pathway. Incubation with tiazofurin (100 microM) and hypoxanthine (100 microM) synergistically inhibited tumor cell growth, and the addition of allopurinol (100 microM) strengthened these effects. Therefore, this drug combination, inhibiting guanylate de novo and salvage pathways, may prove useful in the treatment of human neuroectodermal tumors.

Topics: Allopurinol; Cell Division; Dose-Response Relationship, Drug; Drug Synergism; Guanosine Monophosphate; Humans; Hypoxanthine; Hypoxanthines; IMP Dehydrogenase; Neuroblastoma; Ribavirin; Tumor Cells, Cultured

Acute desensitization of M1 muscarinic receptor-mediated responses (cyclic GMP formation and inositol phosphate release) was studied in murine neuroblastoma cells (N1E-115 clone). After a 45-min incubation at 37 degrees of N1E-115 cells either in monolayer or in suspension, with the muscarinic agonist carbachol (1 mM), the receptor-mediated cyclic GMP response to carbachol was nearly completely lost. This loss was associated with greater than 80% loss of carbachol-mediated inositol phosphate release. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) inhibited both responses with similar potencies. Carbachol or PMA reduced by 30-40% the number of muscarinic receptor sites for antagonist and agonist on intact cells (determined in binding assays using [3H]N-methylscopolamine) only for cells in monolayer and not for those in suspension. PMA but not carbachol pretreatment of cells in monolayer or in suspension caused a translocation of [3H]phorbol 12,13-dibutyrate binding and protein kinase C activity. In addition, desensitization to carbachol occurred in cells largely depleted of protein kinase C by chronic exposure to PMA. Thus, agonist-mediated down-regulation is not needed for muscarinic M1 receptor desensitization, which may be a result of the activation of a receptor-activated kinase different from protein kinase C.

Topics: Animals; Carbachol; Down-Regulation; Guanosine Monophosphate; Inositol Phosphates; Mice; N-Methylscopolamine; Neuroblastoma; Parasympatholytics; Protein Kinase C; Receptors, Muscarinic; Scopolamine Derivatives; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

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: Adenosine Monophosphate; Animals; Astrocytes; Brain; Clone Cells; Culture Techniques; Cytidine Monophosphate; Glioma; Guanosine Monophosphate; Haplorhini; Mice; Neoplasms, Experimental; Neuroblastoma; Nucleotides; Rats; RNA; Uridine Monophosphate

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