Intracellular Ca2+ signalling is modulated by K+ channel blockers in colonic epithelial cells (HT-29/B6).
Intracellular Ca2+ signalling is modulated by K+ channel blockers in colonic epithelial cells (HT-29/B6).
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结肠上皮细胞 (HT-29/B6) 中的 K 通道阻滞剂可调节细胞内 Ca2 信号传导。
DOI：
10.1007/bf00381512
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Machen,TE
中科院分区：
文献类型：
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作者：
Illek,B;Fischer,H;Machen,TE
我们研究了K+通道阻滞剂对碳水化合物刺激的Ca2+进入人Cl -分泌结肠上皮细胞（HT-29/B6）的抑制作用。采用荧光钙指示剂fura-2的数字成像技术监测K+通道阻滞剂对静息和碳水化合物刺激HT-29/B6细胞胞质钙的影响。毒蕈碱受体激动剂卡巴醇（100 μM）刺激引起明显的双相细胞内钙（Cairesponse）反应：cai被从静息水平（85±3 nM,n=100）刺激到一个突然的瞬时峰值（821±44 nM），然后是一个持续的平台（317±12 nM）。维持的升高依赖于外部Ca2+，并代表Ca2+通过质膜进入和退出之间的新的稳定状态。在没有外部Ca2+的情况下，在初始峰值Ca2+恢复到基线后，诱导单相Ca2+反应。麝香碱拮抗剂阿托品（1 μM）或无机Ca2+通道阻滞剂镧（50% ca1平台抑制的有效浓度EC50=68±18 nM）可将Caiplateau降低到静息水平，但有机Ca2+通道阻滞剂维拉帕米和硝苯地平对Caiplateau没有影响。钡、利多卡因和4-硝基-2-（3-苯基丙基氨基）苯甲酸盐（NPPB）是众所周知的HT-29/B6细胞基底侧K+通道阻滞剂，可快速可逆地减少碳水化合物刺激的Ca2+进入。计算出钡（96±2 μM）、利多卡因（74±3 μM）和NPPB（27±10 μM）对Caiplateau的抑制作用为50%。Caiplateau瞬时增加了1 μM和10 μM NPPB，分别达到50%和34%，这可能是通过阻断Cl -通道使膜电位超极化（使膜电位接近dvk）。静息Ca2+分别增加50 μM和300 μM NPPB至308±13 nM和447±153 nM，表明NPPB诱导Ca2+从Ca存储中释放。我们得出结论，碳水化合物刺激的Ca2+进入HT-29/B6细胞(a)需要毒毒碱受体占据，(b)对镧高度敏感，(c)依赖于膜电位，因此被通道阻滞剂抑制，使细胞电位去极化。此外，钙离子水平对K+通道阻滞剂的敏感性表明，Ca2+进入率、Ca2+激活的K+和Cl -通道的活性和膜电位之间存在反馈关系。
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 μM) caused a clearly biphasic intracellular calcium (Cairesponse: Caiwas 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 Caireturned to baseline. The Caiplateau was reduced to resting levels by either the muscarinic antagonist atropine (1 μM) or the inorganic Ca2+channel blocker lanthanum (effective concentration for 50% inhibition of Ca1plateau 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 Caiplateau was calculated to be 50% inhibited by barium (96±2 μM), lidocaine (74±3 μM) and NPPB (27±10 μM). The Caiplateau was transiently increased by 1 μM and 10 μM NPPB to 50% and 34%, respectively, probably via hyperpolarization of the membrane potential by blockade of Cl−channels (so that the membrane potential approachedVK). The resting Caiwas transientlyincreasedby 50 μM and 300 μM 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 Cailevels 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.