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 ([Ca^2-](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 [Ca^2-](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)P_3- 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.