INVOLVEMENT OF CALMODULIN IN CA2+-ACTIVATED K+ EFFLUX IN HUMAN COLONIC CELL-LINE, HT29-19A

The receptor-mediated agonist, neurotensin (NT) stimulated Ba2+- and charybdotoxin-sensitive Rb-86 (K+) ellux 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 Rb-86 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). Ca2+-activated Rb-86 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 K-i of 38 mu M. Other CaM antagonists inhibited efflux with an order of potency (TFP approximate to Wg > W7 much greater than 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 I-125 efflux but significantly reduced efflux stimulated by the Ca2+ ionophore, ionomycin. NT-stimulated Rb-86(+) 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 Ca2+-dependent activation of K+ conductance in HT29-19A colonocytes.