Cyclic AMP accumulation alters calmodulin localization in SK-N-SH human neuroblastoma cells

Abstract

In SK-N-SH human neuroblastoma cells, the muscarinic agonist carbachol promotes polyphosphoinositide (PPI) hydrolysis via M3 receptors and increases cyclic AMP levels through an unidentified mechanism. Activation of PPI hydrolysis by carbachol elicits a robust translocation of CaM from membranes into cytosol which was previously shown to be mimicked by the addition of the calcium ionophore ionomycin and the phorbol ester TPA28. The effect of agonist-stimulated second messenger production on CaM localization was determined by activating receptors that increase and decrease adenylyl cyclase activity on SK-N-SH cells. VIP (10 [mu]M), prostaglandin E1 (30 [mu]M) and forskolin (10 [mu]M) all increased adenylyl cyclase activity 8- to 10-fold above the activity with 1 [mu]M GTP. Carbachol (100 [mu]M) did not stimulate adenylyl cyclase activity. The [alpha]2-adrenergic agonist UK 14,304 (0.1 [mu]M) and the [delta] and [mu] opioid DPDPE (10 [mu]M) and DAMGO (10 [mu]M) inhibited forskolin-stimulated cyclic AMP formation by 27-32%. CaM did not stimulate adenylyl cyclase activity. Incubation of cells with vasoactive intestinal polypeptide (VIP), dibutyryl cyclic AMP and forskolin, resulted in 30% decrease in membrane CaM and an increase in cytosolic CaM of 40-50%. The CaM translocation with the combination of an agent that elevates cyclic AMP levels and a low dose of carbachol was not different from that observed with either agent alone. UK 14,304, DPDPE and DAMGO potentiated carbachol-stimulated increases in cytosolic CaM. Upon the addition of carbachol, a 5-fold increase in intracellular calcium concentration measured with fura-2 fluorescence was observed. VIP and UK 14,304 elevated intracellular calcium concentrations 2 to 3 fold, while forskolin (10 [mu]M) had no effect. Thus, receptor-mediated cyclic AMP synthesis and Ca2+ fluxes alter CaM localization, but with a smaller magnitude than the CaM translocation stimulated by PPI hydrolysis.