Intracellular Ca2+ signalling is modulated by K+ channel blockers in colonic epithelial cells (HT-29/B6)

Abstract

We investigated the inhibitory action of K+ channel blockers on carbachol-stimulated Ca2+ entry into human Cl(-)-secretory colonic epithelial cells (HT-29/B6). Digital imaging of the fluorescent calcium indicator dye fura-2 was performed to monitor effects of K+ channel blockers on cytosolic calcium in resting and carbachol-stimulated HT-29/B6 cells. Stimulation with the muscarinic agonist carbachol (100 microM) caused a clearly biphasic intracellular calcium (Cai) response: Cai was stimulated from resting levels (85 +/- 3 nM, n = 100) to a sudden transient peak (821 +/- 44 nM) followed by a sustained plateau (317 +/- 12 nM). The maintained elevation was dependent on external Ca2+ and represented a new steady state between Ca2+ entry and exit across the plasma membrane. A monophasic Ca2+ response was induced in the absence of external Ca2+ and after the initial peak Cai returned to baseline. The Cai plateau was reduced to resting levels by either the muscarinic antagonist atropine (1 microM) or the inorganic Ca2+ channel blocker lanthanum (effective concentration for 50% inhibition of Cai plateau EC50 = 68 +/- 18 nM), but it was unaffected by the organic Ca2+ channel blockers verapamil and nifedipine. Barium, lidocaine and 4-nitro- 2-(3-phenylpropylamino)benzoate (NPPB), well-known blockers of basolateral K+ channels of HT-29/B6 cells, rapidly and reversibly reduced carbachol-stimulated Ca2+ entry.(ABSTRACT TRUNCATED AT 250 WORDS)