INTRACELLULAR CA-2+ SIGNALING IS MODULATED BY K+ CHANNEL BLOCKERS IN COLONIC EPITHELIAL-CELLS (HT-29/B6)

被引:13
作者
ILLEK, B
FISCHER, H
MACHEN, TE
机构
[1] Department of Molecular and Cell Biology, Division of Cell and Developmental Biology, 235 Life Science Addition, University of California at Berkeley, Berkeley, 94720, CA
来源
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY | 1992年 / 422卷 / 01期
关键词
CA-2+ ENTRY; CARBACHOL; ATROPINE; LANTHANUM; BARIUM; LIDOCAINE; NPPB;
D O I
10.1007/BF00381512
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
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 muM) caused a clearly biphasic intracellular calcium (Ca(i)) response: Ca(i) 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 Ca(i) returned to baseline. The Ca(i) plateau was reduced to resting levels by either the muscarinic antagonist atropine (1 muM) or the inorganic Ca2+ channel blocker lanthanum (effective concentration for 50% inhibition of Ca(i) 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. The Ca(i) plateau was calculated to be 50% inhibited by barium (96 +/- 2 muM), lidocaine (74 +/- 3 muM) and NPPB (27 +/- 10 muM). The Ca(i) plateau was transiently increased by 1 muM and 10 muM NPPB to 50% and 34%, respectively, probably via hyperpolarization of the membrane potential by blockade of Cl- channels (so that the membrane potential approached V(K)). The resting Ca(i) was transiently increased by 50 muM and 300 muM NPPB to 308 +/- 13 nM and 447 +/- 153 nM, respectively, suggesting that NPPB induced a Ca2+ release from internal Ca stores. We conclude that carbachol-stimulated Ca2+ entry into HT-29/B6 cells (a) requires muscarinic receptor occupation, (b) is highly sensitive to lanthanum and (c) is dependent on membrane potential and therefore inhibited by channel blockers that depolarize the cell potential. Also, the sensitivity of Ca(i) levels to K+ channel blockers indicates that there are feedback relationships among rates of Ca2+ entry, activity of Ca2+-activated K+ and Cl- channels and membrane potential.
引用
收藏
页码:48 / 54
页数:7
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