A novel fluorescence chamber for the determination of volume changes in human CaSki cell cultures attached on filters.
A novel fluorescence chamber for the determination of volume changes in human CaSki cell cultures attached on filters.
复制标题
一种新型荧光室，用于测定附着在过滤器上的人类 CaSki 细胞培养物的体积变化。
DOI：
10.1007/bf02737900
复制
发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Whittembury,J
中科院分区：
文献类型：
--
作者：
Gorodeski,GI;Whittembury,J
该研究的目的是检验以下假设：在培养的人宫颈上皮细胞 CaSki 中，钙动员剂对跨上皮电导 (GTE) 的影响是细胞体积减少的结果。附着在滤光片上的 CaSki 细胞装载有 fura-2，并在新设计的荧光室中进行等吸光波长 360 nm 处的荧光测量（激发/发射 [F360/510]）；这种设计还使我们能够确定胞质钙 ([Ca2+]i) 的变化。实验条件与用于测量 Ussing 室中细胞旁通透性变化的实验条件相似，它们使我们能够比较 [Ca2+]i、F360/510 和 GTE 中的变化的时间过程。高渗性增加，低渗性降低F360/510和GTE，但对[Ca2+]i没有影响，并且F360/510和GTE的变化呈线性相关。细胞内fura-2的代谢、漂白和挤出是最小的，表明F360/510的变化反映了染料浓度的变化。高渗性降低，低渗性增加分散的 CaSki 细胞的大小，表明渗透压诱导的 F360/510 变化反映了贴壁细胞大小的变化。离子霉素增加[Ca2+]i、F360/510和GTE，但[Ca2+]的增加先于F360/510和GTE。钙螯合剂 BAPTA 可阻断离子霉素诱导的 [Ca2+]i、F360/510 和 GTE 增加。用 4-acetamido-4'isothiocyanatostilbene-2,2'disulfonic Acid (SITS) 预孵育可增强离子霉素诱导的 [Ca2+]i 增加，但阻止 F360/510 和 GTE 的增加。用高渗溶液预处理细胞消除了离子霉素引起的 F360/510 和 GTE 的增加，但对离子霉素诱导的 [Ca2+]i 的增加几乎没有影响。基于这些结果，我们认为离子霉素诱导的 GTE 增加是由 [Ca2+]i 依赖性氯离子分泌和渗透水损失介导的。
The objective of the study was to test the hypothesis that, in the cultured human cervical epithelium, CaSki, the effect of calcium mobilizing agents on transepithelial electrical conductance (GTE), is the result of cell volume decrease. CaSki cells attached on filters were loaded with fura-2, and measurements of fluorescence at the isosbestic wavelength 360 nm (excitation/emission [F360/510]) were made in a newly designed fluorescence chamber; this design allowed us also to determine changes in cytosolic calcium ([Ca2+]i). The experimental conditions were similar to those used to measure changes in paracellular permeability in the Ussing chamber, and they enabled us to compare the time-course of changes in [Ca2+]i, in F360/510, and in GTE. Hypertonicity increased, and hypotonicity decreased F360/510and GTE, without having an effect on [Ca2+]i, and the changes in F360/510and in GTEcorrelated linearly. Metabolism, bleaching, and extrusion of intracellular fura-2 were minimal, indicating that the changes in F360/510reflect changes in dye concentration. Hypertonicity decreased, and hypotonicity increased the size of dispersed CaSki cells, suggesting that osmolarity-induced changes in F360/510reflect changes in size of the attached cells. Ionomycin increased [Ca2+]i, F360/510, and GTE, but the increases in [Ca2+]ipreceded those in F360/510and GTE. The calcium chelator BAPTA blocked the ionomycin-induced increase in [Ca2+]i, F360/510, and in GTE. Preincubation with 4-acetamido-4′isothiocyanatostilbene-2,2′disulfonic acid (SITS) augmented the ionomycin-induced increase in [Ca2+]i, but blocked the increases in F360/510and in GTE. Pretreatment of cells with hypertonic solution abrogated the increases in F360/510and in GTEin response to ionomycin, but had little effect on the ionomycin-induced increase in [Ca2+]i. On the basis of these results we suggest that the ionomycin-induced increase in GTEis mediated by [Ca2+]i-dependent chloride secretion and osmotic water loss.