Mills J W, Macknight A D, Dayer J M, Ausiello D A
Am J Physiol. 1979 Mar;236(3):C157-62. doi: 10.1152/ajpcell.1979.236.3.C157.
Pig kidney cells, LLC-PK1, grown by standard tissue-culture techniques form monolayers and maintain morphological features characteristic of epithelia. Cultures exposed to 2 X 10(-6) M [3H]ouabain for 30 min at 37 degrees C bound 7.77 +/- 0.37 pmol/mg protein. This could be reduced by 58% by incubation in the presence of 45 mM K+. Freeze-dry radioautographic localization of [3H]ouabain-binding sites revealed grains distributed only along that fraction of the plasmalemma directly facing the culture-dish surface. Binding and localization of [3H]ouabain were correlated with an inhibition of the Na+ pump in these cells because analysis of cellular electrolytes in control cultures versus those exposed to 10(-3) M ouabain revealed a fall in K+ from 419 +/- 9 to 173 +/- 4 mmol/kg dry wt with a reciprocal increase in Na+. There was no change in cell H2O. Similarly, oxygen consumption was reduced by 32% after exposure to ouabain. These results provide direct evidence that in epithelial cells in culture the membrane facing the culture dish corresponds to the basolateral membrane of epithelial cells in vivo.
通过标准组织培养技术培养的猪肾细胞LLC-PK1形成单层，并保持上皮细胞特有的形态特征。在37℃下，将培养物暴露于2×10⁻⁶ M [³H]哇巴因中30分钟，每毫克蛋白质结合7.77±0.37皮摩尔。在45 mM K⁺存在下孵育可使其降低58%。[³H]哇巴因结合位点的冷冻干燥放射自显影定位显示，银粒仅沿着质膜直接面向培养皿表面的部分分布。[³H]哇巴因的结合和定位与这些细胞中Na⁺泵的抑制相关，因为对对照培养物与暴露于10⁻³ M哇巴因的培养物中的细胞电解质分析显示，K⁺从419±9降至173±4 mmol/kg干重，而Na⁺则相应增加。细胞含水量没有变化。同样，暴露于哇巴因后，氧气消耗减少了32%。这些结果提供了直接证据，表明在培养的上皮细胞中，面向培养皿的膜对应于体内上皮细胞的基底外侧膜。
