FLUORESCENCE MEASUREMENT OF CHLORIDE TRANSPORT IN MONOLAYER CULTURED-CELLS - MECHANISMS OF CHLORIDE TRANSPORT IN FIBROBLASTS
FLUORESCENCE MEASUREMENT OF CHLORIDE TRANSPORT IN MONOLAYER CULTURED-CELLS - MECHANISMS OF CHLORIDE TRANSPORT IN FIBROBLASTS
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DOI：
10.1016/s0006-3495(89)82755-9
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发表时间：
1989-12-01
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：
CHAO, AC;DIX, JA;VERKMAN, AS
利用包埋氯敏感的荧光载体6-甲氧基-N-[3-磺丙基]喹啉(SPQ)，建立了测量培养细胞中氯的活度和转运的方法。将该方法应用于肾上皮细胞系LLC-PKI和非上皮细胞系瑞士3T3成纤维细胞。当SPQ在培养液中存在48h时，对细胞无毒。用SPQ(5 Mm)负载细胞，用低渗缓冲液(150mOsm，4min)使细胞瞬时通透。用荧光显微镜连续监测细胞内的荧光，采用306.+-的低照度。激发波长为5 nm，光电倍增管检测波长为410 nm。在37度时超过60分钟。细胞内SPQ荧光均匀，无光漂白现象，细胞外SPQ漏出量为10%。SPQ荧光用含有离子载体黑色和三丁基锡的高K溶液与细胞内的[Cl]进行校准。在无氯条件下，成纤维细胞和LLC-PKI细胞内SPQ被氯猝灭的Stern-Volmer常数为13M-1，SPQ在水溶液中的寿命为26 ns，在水溶液中的寿命为3.7.+-。0.6 ns，表明细胞内的KQ低于游离溶液(KQ=118M-1)是由于细胞内阴离子猝灭SPQ所致。为了研究CI的转运机制，测量了在离子或pH梯度存在的情况下，细胞内[Cl]快速添加和去除的时间过程。在成纤维细胞中，确定了三种不同的氯转运系统：二苯乙烯可抑制的氯/二氧化碳交换系统、速尿敏感的Na/K/2Cl共转运体和钙调节的氯电导。这些结果建立了一种在培养细胞内连续测定细胞内[Cl]的直接光学方法。
The methodology has been developed to measure Cl activity and transport in cultured cells grown on a monolayer using the entrapped Cl-sensitive fluorophore 6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ). The method was applied to a renal epithelial cell line, LLC-PKI, and a nonepithelial cell line, Swiss 3T3 fibroblasts. SPQ was nontoxic to cells when present for > 48 h in the culture media. To load with SPQ (5 mM), cells were made transiently permeable by exposure to hypotonic buffer (150 mOsm, 4 min). Intracellular fluorescence was monitored continuously by epifluorescence microscopy using low illumination intensity at 306 .+-. 5 nm excitation wavelength and photomultiplier detection at > 410 nm. Over 60 min at 37.degree. C, there was no photobleaching and < 10% leakage of SPQ out of cells; intracellular SPQ fluorescence was uniform. SPQ fluorescence was calibrated against intracellular [Cl] using high K solutions containing the ionophores nigericin and tributyltin. The Stern-Volmer constant (Kq) for quenching of intracellular SPQ by Cl was 13 M-1 for fibroblasts and LLC-PKI cells in the absence of Cl, SPQ lifetime ws 26 ns in aqueous solution and 3.7 .+-. 0.6 ns in cells, showing that the lower Kq in cells than in free solution (Kq = 118 M-1) was due to SPQ quenching by intracellular anions. To examine CI transport mechanisms, the time course of intracellular [Cl] was measured in response to rapid Cl addition and removal in the presence of ion or pH gradients. In fibroblasts, three distinct Cl transporting systems were identified: a stilbene-inhibitable Cl/HCO2 exchanger, a furosemide-sensitive Na/K/2Cl cotransporter, and a Ca-regulated Cl conductance. These results establish a direct optical method to mesure intracellular [Cl] continuously in cultured cells.