Weiss E R, Cook J S
Am J Physiol. 1986 Feb;250(2 Pt 1):C199-206. doi: 10.1152/ajpcell.1986.250.2.C199.
Over a period of 2-3 wk after plating, cultured LLC-PK1 (pig kidney) cells develop a high capacity for Na+-dependent accumulation of alpha-methyl-D-glucoside. To further the analysis of this developmental process, we have developed a method for separating transporting from nontransporting cells on the basis of density changes accompanying hexose accumulation and the corresponding uptake of water. Volume regulation was prevented by suspending the cells in a K+-free, Cl(-)-free Na-gluconate medium. Na+-dependent transport was maintained at nearly control levels by addition of low concentrations of (NH4)2SO4, since NH+4 stimulates Na+-K+-ATPase at the K+ site and allows for the extrusion of accumulated Na+; NH+4-stimulated hexose uptake is ouabain sensitive. With volume regulation blocked but with transport near normal, transporting cells exhibited a phlorizin-sensitive density shift in methylglucoside-containing medium and could be separated from nontransporting cells on Percoll gradients.
接种后2至3周内，培养的LLC-PK1（猪肾）细胞对α-甲基-D-葡萄糖苷的钠依赖性积累能力增强。为了进一步分析这一发育过程，我们开发了一种方法，根据伴随己糖积累和相应水分摄取的密度变化，将转运细胞与非转运细胞分离。通过将细胞悬浮在无钾、无氯的葡萄糖酸钠培养基中来防止体积调节。通过添加低浓度的(NH4)2SO4，钠依赖性转运维持在接近对照水平，因为NH4+在钾位点刺激钠钾ATP酶，并允许挤出积累的Na+；NH4+刺激的己糖摄取对哇巴因敏感。在体积调节受阻但转运接近正常的情况下，转运细胞在含甲基葡萄糖苷的培养基中表现出根皮苷敏感的密度变化，并可在Percoll梯度上与非转运细胞分离。
