Inhibition of ADP-ribosyltransferase increases synthesis of Gs alpha in neuroblastoma x glioma hybrid cells and reverses iloprost-dependent heterologous loss of fluoride-sensitive adenylate cyclase

Abstract

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.