Guanine nucleotide binding proteins and the regulation of cyclic AMP synthesis in NS20Y neuroblastoma cells: role of D₁ dopamine and muscarinic receptors

D₁ dopamine receptors on NS20Y neuroblastoma cells stimulate adenylate cyclase activity, whereas muscarinic receptors on the same cells negatively regulate adenylate cyclase. To determine the mechanisms which underlie these processes, cyclic AMP accumulation was measured in intact cells following either cholera or pertussis toxin treatment. Pretreatment with pertussis toxin (100 ng/ml), which ribosylated 95% of inhibitory guanine nucleotide binding protein (G_i), caused the complete loss of muscarinic induced inhibition. Conversely, pertussis toxin did not affect the ability of dihydrexidine (1 μM, a full efficacy D₁ agonist), PGE₁ (100 nM), or forskolin (1 μM, a direct activator) to stimulate cAMP accumulation. Both the dihydrexidine-induced stimulation and the carbachol-induced inhibition of cyclic AMP accumulation were unaffected by either removal of extracellular calcium, or increased intracellular calcium caused by the addition of the calcium ionophore A23187. Cholera toxin dose- and time-dependently induced large accumulations of cAMP. At low cholera toxin concentrations, the effects of dihydrexidine (300 nM) were additive with those of cholera toxin. At cholera toxin concentrations >100 ng/ml, dihydrexidine became ineffective in stimulating further cAMP synthesis. Conversely, forskolin (1 μM) still caused marked increases in cAMP accumulation after all cholera toxin treatments. Dihydrexidine-stimulated cAMP accumulation was additive with forskolin-stimulated cAMP accumulation at low forskolin concentrations (10 nM-3 μM), but synergistic at high concentrations (3-100 μM). Additionally, forskolin was much more potent after cholera toxin treatment, suggesting that an activated stimulatory guanine nucleotide binding protein (G_s) may be required for full activation of adenylate cyclase by forskolin in this cell type. Together, these data suggest that adenylate cyclase activity in NS20Y cells is regulated by both stimulatory G-protein(s) linked to D₁ dopamine receptors and inhibitory G-protein(s) linked to muscarinic receptors.