ESTIMATION OF INTRACELLULAR CHLORIDE ACTIVITY IN ISOLATED PERFUSED RABBIT PROXIMAL CONVOLUTED TUBULES USING A FLUORESCENT INDICATOR
ESTIMATION OF INTRACELLULAR CHLORIDE ACTIVITY IN ISOLATED PERFUSED RABBIT PROXIMAL CONVOLUTED TUBULES USING A FLUORESCENT INDICATOR
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DOI：
10.1016/s0006-3495(88)83176-x
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发表时间：
1988-06-01
影响因子：
3.4
通讯作者：
VERKMAN, AS
中科院分区：
文献类型：
--
作者：
KRAPF, R;BERRY, CA;VERKMAN, AS
已开发的方法来测量细胞的氯离子活性的荧光显微镜使用的氯离子敏感染料，6-甲氧基-1-（3-sulfonatopropyl）quinolinium（SPQ）。通过在38 ℃用20 mM SPQ进行10分钟的管腔灌注，将SPQ加载到体外微灌注的兔近曲小管的细胞中。用宽带激发滤光片（340和380 nm）激发荧光，并用435 nm截止滤光片检测。信号与背景（自发荧光）的比率为4.6 ±。0.6.在38 ℃下SPQ从细胞渗漏的半衰期为8.6 ±。1.1在悬浮小管中，SPQ对O2消耗没有显著影响。使用离子载体尼日利亚菌素和三丁基锡，高外部钾浓度，和不同的细胞外氯离子浓度进行细胞内SPQ校准。细胞荧光与细胞内氯离子的Stern-Volmer关系，猝灭常数为12 M-1。用晚近曲小管特有的溶液灌注的小管中的表观氯化物浓度为27.5 ± 0.01。5 mM（活性20.6 mM）。在加入氯化物浴时，细胞氯化物活性的瞬时半衰期为约3秒（38 ℃）。讨论了这种新的荧光方法在研究细胞氯离子转运中的应用和局限性。
The methodology has been developed to measure cell chloride activity by fluorescence microscopy using the chloride-sensitive dye, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). SPQ was loaded into cells of the in vitro microperfused rabbit proximal convoluted tubule by a 10 min luminal perfusion with 20 mM SPQ at 38.degree.C. Fluorescence was excited with a broad band excitation filter (340 and 380 nm) and detected with a 435 nm cut-on filter. The signal to background (autofluorescence) ratio was 4.6 .+-. 0.6. The halftime for SPQ leakage from cells at 38.degree.C was 8.6 .+-. 1.1 min. In suspended tubules, SPQ did not affect O2 consumption significantly. Intracellular SPQ calibration was performed using the ionophores nigericin and tributyltin, high external potassium concentrations, and varying extracellular chloride concentrations. Cell fluorescence was related to intracellular chloride by a Stern-Volmer relation with a quenching constant of 12 M-1. Apparent chloride concentration in tubules perfused with solutions characteristic for the late proximal convoluted tubule was 27.5 .+-. 5 mM (activity 20.6 mM). The halftime of the transient in cell chloride activity upon bath chloride addition was .apprx.3 s (38.degree.C). Applications and limitations of this new fluorescence method to study cell chloride transport are discussed.