ROLE OF CALCIUM IN CARBACHOL-INDUCED AND NEUROTENSIN-INDUCED MUCIN EXOCYTOSIS IN A HUMAN COLONIC GOBLET CELL-LINE AND CROSS-TALK WITH THE CYCLIC-AMP PATHWAY

The mechanisms of Ca2+-induced mucin secretion were examined in monolayers of the differentiated epithelial colon cell line Cl.16E by combined measurements of free intracellular Ca2+ ([Ca2+](i)) using a fluorescence indicator and mucous secretion using a specific and sensitive electrophoretic assay. Carbachol, a cholinergic agonist, induced an initial concentration-dependent [Ca2+](i) peak increasing from 129 +/- 3 nM (basal [Ca2+](i)) to 608 +/- 101 nM at 1 x 10(-4) M carbachol with an ED(50) of 7 mu M, and this was followed by a lower-level plateau. These biphasic effects were reversed by the muscarinic-receptor antagonist atropine. In the absence of extracellular Ca2+, the initial [Ca2+](i) peak was maintained while the sustained plateau was abolished. The regulatory peptide neurotensin caused a monophasic transient rise in [Ca2+](i) followed by a very rapid return to baseline. The neurotensin-induced rise in [Ca2+](i) was concentration-dependent with an ED(50) of 4 nM, and was maximal at 1 x 10(-6) M (598 +/- 127 nM). The [Ca2+](i) response to neurotensin was not significantly affected by extracellular Ca2+ depletion. Carbachol-induced mucin exocytosis was concentration-dependent with an ED(50) of 15 mu M, and was inhibited by 35% upon removal of extracellular Ca2+. Neurotensin caused a concentration-dependent rise in mucous secretion with an ED(50) of 36 nM, not significantly affected upon removal of extracellular Ca2+. Together our results suggest that while the mucin secretory response to carbachol depends on both the release of Ca2+ from intracellular stores and a Ca2+ influx from external medium, the secretory response to neurotensin is based solely on intracellular Ca2+ mobilization. Finally, evaluation of the cross-talk between the cyclic AMP pathway stimulated by vasoactive intestinal peptide (VIP) and the Ca2+ pathway stimulated by neurotensin or carbachol led to the conclusion that the potentiated secretory response elicited by the combined action of carbachol and VIP requires extracellular Ca2+.