Pavenstädt-Grupp I, Grupp C, Kinne R K
Max-Planck-Institut für Systemphysiologie, Dortmund, Federal Republic of Germany.
Pflugers Arch. 1989 Feb;413(4):378-84. doi: 10.1007/BF00584487.
A method was developed to measure the element content of freshly isolated papillary collecting duct (PCD) cells by electron probe microanalysis in a scanning electron microscope. After isolation, the cells were transferred onto a Thermanox support by centrifugation and the extracellular medium was removed by brief exposure to buffered ammonium acetate; cryofixation, freeze-drying, and coating with carbon followed. Under visual control in the scanning electron microscope the Na, Cl, K and P content of cell clusters (about 30 cells/cluster) was then measured by X-ray microanalysis. Cells incubated in control medium showed potassium:sodium ratios identical to those determined previously in cryosections of the same cells. In ouabain-treated cells sodium influx and potassium efflux was demonstrated. Potassium left the cells with a t1/2 of 21.7 min. The t1/2 of Na influx was 12.6 min for the first 15 min of incubation, whereafter further influx was markedly slower. Ouabain-induced sodium influx was inhibited 40% by amiloride. These results indicate that X-ray microanalysis can be applied to analyze the ion content of isolated cell clusters derived from the papillary collecting duct. Using ouabain and amiloride as inhibitors the suitability of the method to identify transport systems is demonstrated.
开发了一种方法，通过扫描电子显微镜中的电子探针微量分析来测量新鲜分离的乳头集合管（PCD）细胞的元素含量。分离后，通过离心将细胞转移到Thermanox载体上，并通过短暂暴露于缓冲醋酸铵中去除细胞外培养基；随后进行冷冻固定、冷冻干燥和碳涂层。在扫描电子显微镜的视觉控制下，然后通过X射线微量分析测量细胞簇（约30个细胞/簇）的Na、Cl、K和P含量。在对照培养基中孵育的细胞显示出的钾：钠比率与先前在相同细胞的冷冻切片中测定的比率相同。在哇巴因处理的细胞中证实了钠内流和钾外流。钾以21.7分钟的半衰期离开细胞。在孵育的前15分钟内，钠内流的半衰期为12.6分钟，此后进一步的内流明显减慢。哇巴因诱导的钠内流被氨氯吡咪抑制40%。这些结果表明，X射线微量分析可用于分析源自乳头集合管的分离细胞簇的离子含量。使用哇巴因和氨氯吡咪作为抑制剂，证明了该方法用于识别转运系统的适用性。
