ROLE OF CALCIUM IN CHLORIDE SECRETION MEDIATED BY CAMP PATHWAY ACTIVATION IN RABBIT DISTAL COLON MUCOSA

Effects of Ca2+ on adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl-secretion were investigated in intact mucosa and isolated crypt cells of rabbit descending colon. Addition of 10 muM prostaglandin (PG)E2 or forskolin to tissues incubated in Ca2+-free medium increased the size of short-circuit current (I(sc)) and Cl-secretion as estimated by unidirectional Cl-36 flux measurements (net flux = -2.31 +/- 0.24 vs. -1.22 +/- 0.10 mueq . h-1 . cm-2, n = 4, P < 0.001). Addition of 10 muM PGE2 to tissues incubated in 1.2 mM Ca2+ Ringer induced a 7-fold increase in mean cAMP level, whereas it produced an 11-fold increase in tissues exposed to Ca2+-free medium. Membrane preparations from whole mucosa incubated in Ca2+-free medium displayed a cyclic nucleotide phosphodiesterase activity significantly lower than controls (18.76 +/- 0.54 vs. 31.20 +/- 0.39 pmol cAMP . mg protein-1 . min-1, means +/- SE, n = 4, P < 0.001). Ca2+ removal also affected adenylate cyclase (AC) responsiveness to agonists; AC activity increased in controls by 54 and 226% after stimulation with 10 muM PGE2 and forskolin, respectively, but it increased more (77 and 325%, respectively) after incubation in Ca2+-free solutions. Simultaneous measurements of intracellular Ca2+ ([Ca2+]i) by fura-2 fluorimetry and cAMP in isolated crypt cells pretreated with 500 muM 3-isobutyl-1-methylxanthine showed that increase in [Ca2+]i by 10 muM ionomycin (from 97 +/- 3.8 to 680 +/- 25 nM, means +/- SE) was associated with lower cAMP levels after stimulation with 10 muM forskolin than controls (25.24 +/- 1.42 vs. 41.94 +/- 2.0 pmol/mg protein, n = 6, P < 0.02). By contrast, crypt cells incubated with 2 muM of the [Ca2+]i chelator (1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester) or in Ca2+-free medium displayed lower [Ca2+]i (29.3 +/- 4.2 and 41.4 +/- 5.6 nM, respectively, means +/- SE) and higher cAMP levels (77.88 +/- 4.69 and 71.22 +/- 3.3 pmol/mg protein, n = 6). Incubation with 5-30 muM calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl (W-7) did not prevent the Ca2+-free-induced cAMP increase. These findings are consistent with a model of Cl- secretion in which [Ca2+]i modulates cAMP regulatory steps and, hence, the size of Cl- secretion.