Carbachol-induced cytosolic free Ca2+ increases in T84 colonic cells seen by microfluorimetry.
Carbachol-induced cytosolic free Ca2+ increases in T84 colonic cells seen by microfluorimetry.
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通过显微荧光测定法观察到 T84 结肠细胞中卡巴胆碱诱导的胞质游离 Ca2+ 增加。
DOI：
10.1152/ajpgi.1989.257.6.g950
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Donowitz,M
中科院分区：
文献类型：
--
作者：
Reinlib,L;Mikkelsen,R;Zahniser,D;Dharmsathaphorn,K;Donowitz,M
在人结肠癌细胞系T84（一种模型Cl-分泌细胞）中，细胞内游离Ca2+ ([Ca2+]i)对分泌物碳醇的反应发生了变化。在本研究中，用荧光指示剂fura-2在单细胞水平上用荧光显微镜成像系统测定[Ca2+]i。Ringer-HCO3溶液中T84细胞的基础[Ca2+]i为76 +/- 4 nM，暴露于无Ca2+溶液或25微米维拉帕米后降低。胆碱能激动剂碳胆醇引起[Ca2+]i浓度依赖性升高，Km为4微米，[Ca2+]i峰值增加约50 nM。[Ca2+]i的升高在3 s内开始，细胞间均匀发生，10-15 s达到峰值。[Ca2+]i的增加在[Ca2+]i保持高于基础水平的时间长度上是异质性的，细胞反应可以在此基础上至少分为两组。用丹trolene阻断细胞内Ca2+的贡献可以抑制[Ca2+]i的增加，而用乙二醇-双（β -氨基乙醚）-N，N，N‘，N’-四乙酸（EGTA），维拉帕米或硝苯地平阻断细胞外贡献可以抑制[Ca2+]i的稍晚增加。总之，最初可检测到的[Ca2+]i的增加是由于钙离子从内部储存的释放，而细胞外Ca2+的贡献发生晚，至少部分涉及硝苯地平和维拉帕米敏感的过程。
Changes in cytosolic free Ca2+ ([Ca2+]i) in response to the secretagogue carbachol have been characterized in the human colon cancer cell line T84, a model Cl- secretory cell. In this study, [Ca2+]i was determined with the fluorescence indicator fura-2 at the single-cell level with a fluorescent microscope-imaging system. Basal [Ca2+]i in T84 cells in Ringer-HCO3 solution was 76 +/- 4 nM and was decreased by exposure to Ca2+ free solution or 25 microM verapamil. The cholinergic agonist carbachol caused a concentration-dependent rise in [Ca2+]i with a Km of 4 microM and a peak increase in [Ca2+]i of approximately 50 nM. The onset of the [Ca2+]i increase was within 3 s, occurred uniformly among cells, and peaked at 10-15 s. The increase in [Ca2+]i was heterogenous in the length of time the [Ca2+]i remained elevated above basal, and cell responses could be divided into at least two groups on that basis. Blocking the contributions of intracellular Ca2+ with dantrolene inhibited the increase in [Ca2+]i as early as could be determined, whereas blocking the extracellular contribution with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), verapamil, or nifedipine inhibited a slightly later increase in [Ca2+]i. In conclusion, the initial detectable increase in [Ca2+]i caused by carbachol is due to the release of Ca2+ from internal stores, whereas the contribution of extracellular Ca2+ occurs later and at least partially involves a nifedipine- and verapamil-sensitive process.