cAMP and inositol 1,4,5-trisphosphate increase Ca2+ in HT-29 cells by activating different Ca2+ influx pathways

Ca2+ plays a central role in regulating transepithelial fluid and electrolyte transport in intestinal epithelial cells. To investigate the mechanisms regulating the cytosolic free Ca2+ concentration ([Ca2+]c), we examined the effect of secretory agonists on [Ca2+]c in the intestinal epithelial cell line HT-29 clone 19A cells. We found that [Ca2+]c increased after addition of either adenosine 3',5'-cyclic monophosphate (cAMP)-dependent agonists or a D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]dependent agonist carbachol. Several lines of evidence suggest that cAMP- and Ins(1,4,5)P3-dependent agonists act through separate pathways. First, isoproterenol and forskolin increased cellular levels of cAMP but not Ins(1,4,5)P3, whereas carbachol increased cellular levels of Ins(1,4,5)P3 and stimulated inositol phosphate turnover without increasing cAMP. Second, carbachol increased [Ca2+]c by stimulating the release of Ca2+ from intracellular stores and influx of extracellular Ca2+. In contrast, cAMP agonists increased [Ca2+]c by stimulating Ca2+ influx alone. Third, the responses to maximal concentrations of cAMP agonists and carbachol were approximately additive. Finally, Ins(1,4,5)P3- but not cAMP agonist-dependent Ca2+ influx was inhibited by inorganic Ca2+ channel blockers. Thus, in intestinal epithelial cells, [Ca2+]c is regulated by at least two different second-messenger pathways, involving Ins(1,4,5)P3 or cAMP. In addition, cAMP stimulates influx of extracellular Ca2+ through a pathway distinct from that mediated by Ins(1,4,5)P3.