Pancreatic Duct Epithelial Cells

We asked if the mechanisms of exocytosis and its regulation in epithelial cells share features with those in excitable cells. Cultured dog pancreatic duct epithelial cells were loaded with an oxidizable neurotransmitter, dopamine or serotonin, and the subsequent release of these exogenous molecules during exocytosis was detected by carbon-fiber amperometry. Loaded cells displayed spontaneous exocytosis that may represent constitutive membrane transport. 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 preceded by a “foot,” assumed to be leak of transmitters through a transient fusion pore, suggesting that those cell types share a common fusion mechanism. As in neurons and endocrine cells, exocytosis in the epithelial cells could be evoked by elevating cytoplasmic Ca 2 1 using ionomycin. Unlike in neurons, 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 cells, such as forskolin, epinephrine, vasoactive intestinal peptide, or 8-Br-cAMP, increased the rate of exocytosis. The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas 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 effect was not mimicked by the inactive analogue, 4 a -phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I. Elevation of intracellular Ca 2 1 was not needed for the actions of forskolin or PMA. In summary, exocytosis in epithelial cells can be stimulated directly by Ca 2 1 , PKA, or PKC, and is mediated by physical mechanisms similar to those in neurons and endocrine cells. key words: secretion • secretagogue • cyclic AMP • photometry • amperometry I N T R O D U C T I O N In neurons and some endocrine cells, Ca 2 1 plays a pivotal role as the final signal for rapid stimulus-evoked release of neurotransmitters and hormones. An elevation of intracellular Ca 2 1 will also trigger exocytosis in various nonexcitable cells (Morimoto et al., 1995; Coorssen et al., 1996). Nevertheless, although the machinery of exocytosis uses related universal molecular components, Ca 2 1 may not be the physiological signal for exocytosis in all nonneuronal cells. For example, in neutrophils, eosinophils, and mast cells, the intracellular signal for exocytosis and degranulation seems not to be Ca 2 1 , protein kinase A, or protein kinase C (Neher and Almers, 1986; Almers and Neher, 1987; Scepek et al., 1998). On the other hand, the hormonal regulation of secretion in various gastrointestinal and airway epithelial cells is described as using cAMP or PKC, depending on the stimulating hormone (Forstner et al., 1993; Larivee et al., 1994; Klinkspoor et al., 1996; Oda et al., 1996; Abdullah et al., 1997; Urushidani and Forte, 1997; Brown et al., 1998; Fujita-Yoshigaki, 1998; reviewed in Hille et al., 1999). To compare this kind of exocytosis with that in neurons, we decided to investigate properties of exocytosis in cultured dog pancreatic duct epithelial cells using amperometry. In these cells, roles for cAMP and Ca 2 1 in mucin secretion are known (Oda et al., 1996; Nguyen et al., 1998). The principal questions were whether PKA, PKC, and Ca 2 1 all can trigger secretion in one cell type and whether they act independently.