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

Abstract

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 Ca 2+ chelator, BAPTA. Together, these data suggest a rise in [Ca 2+] i is prerequisite for activation of K + efflux in these cells. Comparison of the temporal profiles for NT-induced increases in [Ca 2+] i and 86Rb efflux, however, failed to show a direct relationship between these parameters. The NT-stimulated increase in [Ca 2+] 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 K i of 38 μM. Other CaM antagonists inhibited efflux with an order of potency (TFP ≈ 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 Ca 2+ 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.