Intracellular Ca2+ signalling is modulated by K+ channel blockers in colonic epithelial cells (HT-29/B6).
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)
Illek
B
B
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Fischer
H
H
Machen
T E
TE
D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002217:Carbachol / Q000037:antagonists & inhibitors / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology*; D007811:Lanthanum / Q000494:pharmacology; D008012:Lidocaine / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D009579:Nitrobenzoates; D015221:Potassium Channels; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D007811:Lanthanum; D001285:Atropine; D002217:Carbachol; D008012:Lidocaine; D002118:Calcium