Involvement of calmodulin in Ca(2+)-activated K+ efflux in human colonic cell line, HT29-19A.

The receptor-mediated agonist, neurotensin (NT) stimulated Ba(2+)- and charybdotoxin-sensitive 86Rb (K+) efflux in the HT29-19A colonic cell line. Efflux was also stimulated by ionomycin and thapsigargin and could be abolished by incubation with the intracellular Ca2+ chelator, BAPTA. Together, these data suggest a rise in [Ca2+]i is prerequisite for activation of K+ efflux in these cells. Comparison of the temporal profiles for NT-induced increases in [Ca2+]i and 86Rb efflux, however, failed to show a direct relationship between these parameters. The NT-stimulated increase in [Ca2+]i was transient, returning to baseline within 4-5 min, while efflux was sustained over a much longer period (> 12 min). Ca(2+)-activated 86Rb efflux was inhibited by pretreatment with calmodulin (CaM) antagonist, W7. W7 had no effect on basal efflux, but reduced both NT- and IM-activated efflux up to 80%, with a Ki of 38 microM. Other CaM antagonist inhibited efflux with an order of potency (TFP approximately W8 > W7 >> W5) consistent with inhibition of a CaM-dependent process. Inhibition by W7 was not abolished by ouabain or bumetanide, indicating its effects are not mediated by action upon K+ uptake processes. W7 did not inhibit NT-stimulated 125I efflux but significantly reduced efflux stimulated by the Ca2+ ionophore, ionomycin. NT-stimulated 86Rb+ efflux was localized to the basolateral membrane of HT29-19A monolayers grown on permeable supports. These data are consistent with the involvement of CaM in mediating Ca(2+)-dependent activation of K+ conductance in HT29-19A colonocytes.