CAMP AND INOSITOL 1,4,5-TRISPHOSPHATE INCREASE CA2-29 CELLS BY ACTIVATING DIFFERENT CA2+ INFLUX PATHWAYS( IN HT)

Ca2+ plays a central role in regulating transepithelial fluid and elec trolyte transport in intestinal epithelial cells. To investigate the m echanisms regulating the cytosolic free Ca2+ concentration ([Ca2+](c)) , we examined the effect of secretory agonists on [Ca2+](c) in the int estinal epithelial cell line HT-29 clone 19A cells. We found that [Ca2 +](c) increased after addition of either adenosine 3',5'-cyclic monoph osphate (cAMP)-dependent agonists or a D-myo-inositol 1,4,5-trisphosph ate [Ins(1,4,5)P-3]-dependent agonist carbachol. Several lines of evid ence suggest that cAMP- and Ins(1,4,5)P-3-dependent agonists act throu gh separate pathways. First, isoproterenol and forskolin increased cel lular levels of cAMP but not Ins(1,4,5)P-3, whereas carbachol increase d cellular levels of Ins(1,4,5)P-3 and stimulated inositol phosphate t urnover without increasing cAMP. Second, carbachol increased [Ca2+](c) by stimulating the release of Ca2+ from intracellular stores and infl ux of extracellular Ca2+. In contrast, cAMP agonists increased [Ca2+]( c) by stimulating Ca2+ influx alone. Third, the responses to maximal c oncentrations of cAMP agonists and carbachol were approximately additi ve. 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 se cond-messenger pathways, involving Ins(1,4,5)P-3 or cAMP. In addition, cAMP stimulates influx of extracellular Ca2+ through a pathway distin ct from that mediated by Ins(1,4,5)P-3.