Role of calcium in carbachol- and neurotensin-induced mucin exocytosis in a human colonic goblet cell line and cross-talk with the cyclic AMP pathway.
The mechanisms of Ca(2+)-induced mucin secretion were examined in monolayers of the differentiated epithelial colon cell line C1.16E 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 ED50 of 7 microM, 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 ED50 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 ED50 of 15 microM, and was inhibited by 35% upon removal of extracellular Ca2+. Neurotensin caused a concentration-dependent rise in mucous secretion with an ED50 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+.
Bou-Hanna
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INSERM U 239, Faculté Xavier Bichat, Paris, France.
Berthon
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Combettes
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Claret
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Laboisse
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D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D002999:Clone Cells; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003110:Colonic Neoplasms; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005089:Exocytosis; D005944:Glucosamine / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis; D006801:Humans; D007700:Kinetics; D009077:Mucins / Q000378:metabolism*; D009496:Neurotensin / Q000494:pharmacology*; D015398:Signal Transduction; D014316:Tritium; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D009077:Mucins; D014316:Tritium; D009496:Neurotensin; D001285:Atropine; D002217:Carbachol; D005944:Glucosamine; D002118:Calcium