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)P-3]-dependent agonist carbachol. Several lines of evidence suggest that cAMP- and Ins(1,4,5)P-3-dependent agonists act through separate pathways. First, isoproterenol and forskolin increased cellular levels of cAMP but not Ins(1,4,5)P-3, whereas carbachol increased cellular levels of Ins(1,4,5)P-3 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)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)P-3 or cAMP. In addition, cAMP stimulates influx of extracellular Ca2+ through a pathway distinct from that mediated by Ins(1,4,5)P-3.