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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.

ABSTRACT: 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+.

SUBMITTER: Bou-Hanna C

PROVIDER: S-EPMC1138310 | biostudies-other | 1994 Apr

REPOSITORIES: biostudies-other

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Project description:UnlabelledCryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely, PKC1, BCK1, MKK2, and MPK1 results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions of BCK1, MKK2, and MPK1 compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis.ImportanceCryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall are primarily controlled by the cell wall integrity (CWI) signaling pathway. In this study, we demonstrate that deletion of any of three core kinases in the CWI pathway impacts not only the cell wall but also the amount of surface capsule. Deletion of any of the kinases results in significantly reduced cellular cyclic AMP (cAMP) levels, and addition of exogenous cAMP rescues the capsule defect and some cell wall defects, supporting a direct role for the CWI pathway in regulation of capsule in conjunction with the cAMP/protein kinase A pathway.

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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.

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