AJP - Cell Physiology, Vol 260, Issue 1 C35-C42, Copyright © 1991 by American Physiological Society
Regulation of calcium-activated potassium efflux by neurotensin and other agents in HT-29 cells
H. Wu, C. C. Franklin, H. D. Kim and J. T. Turner
Department of Pharmacology, School of Medicine, University of Missouri-Columbia 65212.
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.
