iloprost and Glioma

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Down-Regulation; Glioma; GTP-Binding Proteins; Hybrid Cells; Iloprost; Isoproterenol; Neuroblastoma; Transfection; Tumor Cells, Cultured

Exposure of NG108-15 cells to 50 mM nicotinamide [an inhibitor of mono(ADP-ribosyl)transferase] for 18 hr led to an increase in membrane associated Gs alpha measured either as cholera toxin substrate or by immunoblotting with a specific antiserum. Prolonged exposure of NG108-15 cells to iloprost is followed by homologous loss of iloprost sensitivity, and heterologous loss of fluoride-dependent activation of adenylate cyclase. Nicotinamide reversed the loss of fluoride sensitivity, but failed to restore iloprost-dependent activation of adenylate cyclase. These results with nicotinamide in NG108-15 cells contrasted with those from platelets, which also exhibit heterologous desensitization of fluoride sensitivity following prolonged exposure to iloprost. Treatment of platelets with 50 mM nicotinamide for 18 hr led to an increase of 75.0 +/- 19.4% in the amount of membrane associated cholera toxin substrate. However, there was no associated increase in the abundance of Gs alpha as determined by immunoblotting. Furthermore, in platelets there was no restoration by nicotinamide of the iloprost-dependent loss of fluoride-sensitive adenylate cyclase activity. It follows that heterologous desensitization in platelets is accompanied by inactivation of Gs alpha, which is retained within the plasma membrane in its inactive state. The nicotinamide-dependent increase in the abundance of membrane associated cholera toxin substrate and immunoreactive Gs alpha in NG108-15 cells is associated with an increase of 72.0 +/- 20.3% in the levels of mRNA encoding Gs alpha. The capacity of nicotinamide to increase the abundance of membrane associated Gs alpha was reversed when the cells were cultured in the presence of 20 micrograms/mL cycloheximide. These results suggest that the ability of nicotinamide to increase the abundance of Gs alpha in NG108-15 cells is mediated by de novo protein synthesis.

Topics: Adenylyl Cyclase Inhibitors; ADP Ribose Transferases; Blood Platelets; Fluorides; Glioma; GTP-Binding Proteins; Hybrid Cells; Iloprost; Neuroblastoma; Niacinamide; Poly(ADP-ribose) Polymerase Inhibitors; Tumor Cells, Cultured

Topics: Animals; Cell Line; Colchicine; Colforsin; Cyclic AMP; Glioma; GTP-Binding Proteins; Hybrid Cells; Iloprost; Kinetics; Mice; Neuroblastoma; Rats; Receptors, Prostaglandin; Tumor Cells, Cultured

Topics: Adenylyl Cyclases; Animals; Down-Regulation; Epitopes; Glioma; GTP-Binding Proteins; Hemagglutinin Glycoproteins, Influenza Virus; Hemagglutinins, Viral; Humans; Hybrid Cells; Iloprost; Kinetics; Mice; Neuroblastoma; Rats; Receptors, Adrenergic, beta; Receptors, Prostaglandin; Recombinant Proteins; Transfection; Tumor Cells, Cultured; Viral Envelope Proteins

Topics: Adenylyl Cyclases; Animals; Binding Sites; Binding, Competitive; Cell Membrane; Colforsin; Glioma; GTP-Binding Proteins; Homeostasis; Hybrid Cells; Iloprost; Kinetics; Mice; Neuroblastoma; Rats; Signal Transduction; Tumor Cells, Cultured

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

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

We studied the effect of intracarotid administration of prostacyclin analogue iloprost on the regional cerebral blood flow in transplanted rat C6 glioma by the hydrogen clearance method. Iloprost at doses of 0.1 and 0.5 micrograms/kg/min produced a selective increase of the regional cerebral blood flow in the tumour (17.8 +/- 5.6%, p < 0.05 and 27.3 +/- 10.3%, p < 0.05, respectively) without significant change of the regional cerebral blood flow in the ipsilateral hemisphere and the systemic arterial pressure. At a dose of 1 microgram/kg/min, iloprost produced a significant reduction of a systemic blood pressure, but did not change the regional cerebral blood flow significantly both in the tumour and the ipsilateral hemisphere. These results indicated that brain tumour vessels could respond to iloprost in a different fashion from the normal brain capillaries. The selective action of iloprost to the tumour vessels might contribute to the drug-delivery in malignant brain tumour.

Topics: Animals; Brain Neoplasms; Carotid Arteries; Cerebrovascular Circulation; Glioma; Iloprost; Infusions, Intra-Arterial; Male; Neoplasm Transplantation; Rats; Rats, Wistar; Reference Values

NG108-15 neuroblastoma x glioma somatic hybrid cells were permeabilized in the presence of [32P]NAD+ and then cultured for 18 h. Resolution of the cell proteins on polyacrylamide gels revealed [32P]ADP-ribosylation of five major protein species with molecular mass values of 52 kDa, 44 kDa, 35 kDa, 30 kDa and 25 kDa. A similar pattern of labelling was also seen when NG108-15 cell membranes were incubated with [32P]NAD+ and hydrolysis of the product revealed mono(ADP-ribosyl)ation. Immunoprecipitation of these products with anti-Gs alpha antiserum revealed a single band identical to cholera toxin substrate. Culture of [32P]NAD(+)-loaded cells for 18 h in the presence of 50 mM-nicotinamide inhibited the eukaryotic mono(ADP-ribosyl)transferase activity. Inhibition of the eukaryotic enzyme was also accompanied by an increase in the abundance of Gs alpha, whether measured by Western blotting with anti-Gs alpha antibody (two separate antisera) or by cholera toxin-dependent [32P]ADP-ribosylation. There was no accompanying change in the abundance of G beta. The increase in Gs alpha abundance in nicotinamide-treated NG108-15 cells was accompanied by a 2-fold increase in basal adenylate cyclase activity (measured in the presence of GTP), and by a smaller but significant increase in iloprost-dependent activation of adenylate cyclase. Receptor number or affinity was not affected by nicotinamide, since this treatment did not alter the binding parameters of [3H]iloprost to NG108-15 cell membranes. Short-term exposure of cells to nicotinamide for 1 h revealed no significant difference in either basal or agonist-stimulated adenylate cyclase activity. These results reveal that mono(ADP-ribosyl)ation of Gs alpha by eukaryotic ADP-ribosyltransferase modifies the abundance and activity of Gs alpha in NG108-15 cells, and hence may play a role in the hormonal regulation of cell function.

Topics: Adenosine Diphosphate Ribose; ADP Ribose Transferases; Blotting, Western; Cholera Toxin; Glioma; GTP-Binding Proteins; Hybrid Cells; Iloprost; Immunosorbent Techniques; NAD; Neuroblastoma; Niacinamide; Substrate Specificity; Tumor Cells, Cultured