Regulation of calcium-activated potassium efflux by neurotensin and other agents in HT-29 cells.
Neurotensin receptors have been shown previously to be coupled to polyphosphoinositide turnover and intracellular Ca2+ ([Ca2+]i) mobilization in HT-29 colonic epithelial cells (Bozou et al. Biochem. J. 264: 871, 1989; Turner et al. J. Pharmacol. Exp. Ther. 253: 1049, 1990). In this study, neurotensin was found to enhance dramatically the Ba2(+)- and tetraethylammonium chloride-sensitive K(+)-efflux rate (measured with 86Rb+) in the presence of ouabain and bumetanide, with basal efflux increasing 4.5 +/- 0.5-fold with 10 nM neurotensin. The K(+)-efflux rate, which was partially dependent on the extracellular Ca2+ concentration, was also increased by carbachol and ATP, two other [Ca2+]i-mobilizing agonists in HT-29 cells, as well as by the Ca2+ ionophores ionomycin and A23187, suggesting that the efflux was through Ca2(+)-activated K+ channels. Pretreatment of cells with neurotensin, carbachol, or ATP desensitized subsequent neurotensin-stimulated efflux by 82, 57, and 63%, respectively, confirming our previous results which indicated homologous and heterologous desensitization of the neurotensin receptor-signal transduction pathway. Pretreatment of cells with the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and mezerein did not affect [Ca2+]i mobilization or K+ efflux directly but desensitized neurotensin-stimulated efflux by greater than 80%. Pretreatment (2 h) with PMA also decreased K+ efflux in response to ionomycin by 59%, although ionomycin-induced [Ca2+]i mobilization was not inhibited. Downregulation of protein kinase C by overnight pretreatment with PMA resulted in recovery of ionomycin-stimulated efflux. These results suggest that agonist-stimulated Ca2(+)-activated K+ channels in HT-29 cells are regulated at multiple steps in the signal transduction pathway.
Wu
H
H
Department of Pharmacology, School of Medicine, University of Missouri-Columbia 65212.
Franklin
C C
CC
Kim
H D
HD
Turner
J T
JT
D000230:Adenocarcinoma; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D003110:Colonic Neoplasms; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D009496:Neurotensin / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D012413:Rubidium / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D015221:Potassium Channels; D013757:Tetraethylammonium Compounds; D001464:Barium; D009496:Neurotensin; D015759:Ionomycin; D019789:Tetraethylammonium; D000255:Adenosine Triphosphate; D002217:Carbachol; D012413:Rubidium; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium; D002118:Calcium