Cutaia M, Davis R, Parks N, Rounds S
Department of Medicine, Providence Veterans Affairs Medical Center, Rhode Island 02908, USA.
J Appl Physiol (1985). 1996 Jul;81(1):509-15. doi: 10.1152/jappl.1996.81.1.509.
The fluoroprobe sodiumbinding benzofuran isophthalate (SBFI) is used to measure intracellular cytosolic sodium concentration ([Na]i). A problem with the use of this probe is the difficulty in loading it into cells. ATP reversibly increases membrane permeability of some cells via activation of receptors of the tetrabasic form of ATP (ATP4-). We investigated the effect of ATP-induced membrane permeabilization on loading of the acetoxymethyl ester (AM) form of SBFI (SBFI-AM) into bovine pulmonary arterial endothelial cells. Monolayers were incubated in a series of solutions that reversibly opened pores, loaded the fluoroprobe, and finally sealed the proes. ATP (1-5 mM) or 3'-O-(4-benzoyl)benzoyl-ATP (0.1-1 mM), an analogue 30-100x more specific for ATP4- receptors, was utilized to permeabilize the cell membrane. The signal-to-background ratio of the intracellular SBFI fluorescent signal was used as an indicator of the effectiveness of dye loading. ATP and 3'-O-(4-benzoyl)benzoyl-ATP significantly increased the signal-to-background ratio compared with the values obtained with the standard dye-loading procedure without ATP, indicating that permeabilization increased SBFI-AM entry into the cells. The permeabilization procedure produced a small decrease in cell viability, as determined with a fluorescent viability assay (ethidium dimer uptake), compared with the standard method of loading SBFI-AM. We used the procedure to measure baseline [Na]i and changes in [Na]i after the administration of ouabain (10(-4) M) and monensin (10(-5) M). Baseline [Na]i with this procedure (19.7 +/- 2.7 mM; n = 15 monolayers) was similar to measurements made in other cell types with the standard method of loading the probe. We conclude that 1) the ATP-induced permeabilization technique is an improved dye-loading method for SBFI-AM in endothelial cell monolayers that facilitates measurement of [Na]i and 2) these data suggest the presence of an ATP4 pore-forming mechanism in this cell type.
荧光探针钠结合苯并呋喃间苯二甲酸酯（SBFI）用于测量细胞内胞质钠浓度（[Na]i）。使用该探针的一个问题是难以将其载入细胞。ATP通过激活四碱基形式的ATP（ATP4-）受体可逆地增加某些细胞的膜通透性。我们研究了ATP诱导的膜通透性对SBFI的乙酰氧基甲酯（AM）形式（SBFI-AM）载入牛肺动脉内皮细胞的影响。将单层细胞置于一系列溶液中，这些溶液可逆地打开孔道、载入荧光探针，最后封闭孔道。使用ATP（1 - 5 mM）或3'-O-(4-苯甲酰基)苯甲酰基-ATP（0.1 - 1 mM，一种对ATP4-受体特异性高30 - 100倍的类似物）使细胞膜通透性增加。细胞内SBFI荧光信号的信噪比用作染料载入有效性的指标。与不使用ATP的标准染料载入程序所获得的值相比，ATP和3'-O-(4-苯甲酰基)苯甲酰基-ATP显著提高了信噪比，表明通透性增加了SBFI-AM进入细胞的量。与载入SBFI-AM的标准方法相比，用荧光活力测定法（碘化丙啶二聚体摄取）测定发现，通透性程序使细胞活力略有下降。我们使用该程序测量基线[Na]i以及给予哇巴因（10(-4) M）和莫能菌素（10(-5) M）后[Na]i的变化。用该程序测得的基线[Na]i（19.7 ± 2.7 mM；n = 15个单层）与用标准探针载入方法在其他细胞类型中测得的结果相似。我们得出结论：1）ATP诱导的通透性技术是一种改进的用于内皮细胞单层中SBFI-AM的染料载入方法，有助于[Na]i的测量；2）这些数据表明在这种细胞类型中存在ATP4孔形成机制。
