CELL-VOLUME MEASURED BY TOTAL INTERNAL-REFLECTION MICROFLUORIMETRY - APPLICATION TO WATER AND SOLUTE TRANSPORT IN CELLS TRANSFECTED WITH WATER CHANNEL HOMOLOGS
CELL-VOLUME MEASURED BY TOTAL INTERNAL-REFLECTION MICROFLUORIMETRY - APPLICATION TO WATER AND SOLUTE TRANSPORT IN CELLS TRANSFECTED WITH WATER CHANNEL HOMOLOGS
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DOI：
10.1016/s0006-3495(95)80335-8
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发表时间：
1995-04-01
影响因子：
3.4
通讯作者：
VERKMAN, AS
中科院分区：
文献类型：
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作者：
FARINAS, J;SIMANEK, V;VERKMAN, AS
建立了全内反射（TIR）微荧光法作为连续测量贴壁细胞体积的方法，并将其应用于检测转染了水通道同源物的细胞的膜透性。细胞质溶胶用不含荧光基团的钙黄蛋白标记。在细胞-底物界面附近的细胞质薄层（约150nm）上，TIR消失场激发荧光。因为每个细胞的胞质荧光团数保持不变，所以TIR荧光信号应该与细胞体积成反比。Sf-9和lc - pk1细胞体积变化较小时，相对TIR荧光几乎等于细胞体积的反比；从理论上模拟了与理想的偏差。通过TIR荧光信号推测渗透诱导细胞体积变化的时间过程，测量质膜渗透性水透性P-f。表达CHIP28水通道的lc - pk1细胞对HgCl2敏感，P-f比未转染的细胞增加了三倍（P-f = 0.0043 cm/s, 10℃）。溶质渗透率是通过TIR荧光时间过程对溶质梯度的响应来测量的。在表达水通道同源GLIP的Sf-9细胞中，甘油通透性为（1.3 +/- 0.2）× 10（-5）。（22℃），大于对照细胞的（0.36 +/- 0.04）× 10(-5) cm/s (n = 4, p < 0.05)，说明GLIP具有功能性表达。在表达glp的细胞和对照细胞中，水和尿素的通透性相似。TIR方法应适用于研究任意形状和大小的细胞中的水和溶质渗透率以及细胞体积调节。
Total internal reflection (TIR) microfluorimetry was established as a method to measure continuously the volume of adherent cells and applied to measure membrane permeabilities in cells transfected with water channel homologs. Cytosol was labeled with the membrane-impermeant fluorophore calcein. Fluorescence was excited by the TIR evanescent field in a thin section of cytosol (similar to 150 nm) adjacent to the cell-substrate interface. Because cytosolic fluorophore number per cell remains constant, the TIR fluorescence signal should be inversely related to cell volume. For small volume changes in Sf-9 and LLC-PK1 cells, relative TIR fluorescence was nearly equal to inverse relative cell volume; deviations from the ideal were modeled theoretically. To measure plasma membrane osmotic water permeability, P-f, the time course of osmotically induced cell volume change was inferred from the TIR fluorescence signal. LLC-PK1 cells expressing the CHIP28 water channel had an HgCl2- sensitive, threefold increase in P-f compared to nontransfected cells (P-f = 0.0043 cm/s at 10 degrees C). Solute permeability was measured from the TIR fluorescence time course in response to solute gradients. Glycerol permeability in Sf-9 cells expressing the water channel homolog GLIP was (1.3 +/- 0.2) x 10(-5).cm/s (22 degrees C), greater than that of (0.36 +/- 0.04) x 10(-5) cm/s (n = 4, p < 0.05) for control cells, indicating functional expression of GLIP. Water and urea permeabilities were similar in GLIP-expressing and control cells. The TIR method should be applicable to the study of water and solute permeabilities and cell volume regulation in cells of arbitrary shape and size.