title: 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 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.
authors.affiliation: Epithelial Membrane Research Centre, University of Manchester, Hope Hospital, Salford, UK.
authors.forename: K E
authors.initials: KE
authors.lastname: Fogg
authors.forename: N B
authors.initials: NB
authors.lastname: Higgs
authors.forename: G
authors.initials: G
authors.lastname: Warhurst
mesh_terms: D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism; D003106:Colon / Q000187:drug effects / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes; D015759:Ionomycin / Q000494:pharmacology; D009496:Neurotensin / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D012414:Rubidium Radioisotopes; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D018118:Chloride Channels; D007457:Iodine Radioisotopes; D015221:Potassium Channels; D012414:Rubidium Radioisotopes; D013449:Sulfonamides; D009496:Neurotensin; D004533:Egtazic Acid; D015759:Ionomycin; C017967:W 7; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium