Regulation of exocytosis by protein kinases and Ca2+ in pancreatic duct epithelial cells

We asked if the mechanisms of exocytosis and its regulation in epithelial c ells share features with those in excitable cells. Cultured dog pancreatic duct epithelial cells were loaded with an oxidizable neurotransmitter, dopa mine or serotonin, and he subsequent release of these exogenous molecules d uring exocytosis was detected by carbon-fiber amperometry. Loaded cells dis played spontaneous exocytosis that may represent constitutive membrane tran sport. The quantal amperometric events induced by fusion of single vesicles had a rapid onset and decay, resembling those in adrenal chromaffin cells and serotonin-secreting leech neurons. Quantal events were frequently prece ded by a "foot," assumed to be leak of transmitters through a transient fus ion pore, suggesting that those cell types share a common fusion mechanism. As in neurons and endocrine cells, exocytosis in the epithelial cells coul d be evoked by elevating cytoplasmic Ca2+ using ionomycin. Unlike in neuron s, hyperosmotic solutions decreased exocytosis in the epithelial cells, and giant amperometric events composed of many concurrent quantal events were observed occasionally. Agents known to increase intracellular cAMP in the c ells, such as forskolin, epinephrine, vasoactive intestinal peptide, or 8-B r-cAMP, increased the rate of exocytosis. The forskolin effect was inhibite d by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whe reas the Sp-isomer, a specific agonist of PKA, evoked exocytosis. Thus, PKA is a downstream effector of cAMP. Finally, activation of protein kinase C by phorbol-12-myristate-13-acetate also increased exocytosis. The PMA effec t was not mimicked by the inactive analogue, 4 alpha-phorbol-12,13-didecano ate, and it was blocked by the PKC antagonist, bis-indolylmaleimide I. Elev ation of intracellular Ca2+ was not needed for the actions of forskolin or PMA. In summary, exocytosis in epithelial cells can he stimulated directly by Ca2+, PKA, or PKC, and is mediated by physical mechanisms similar to tho se in neurons and endocrine cells.