Department of Biomathematics, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic.
Department of Biomathematics, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic.
Biochim Biophys Acta Gen Subj. 2017 May;1861(5 Pt A):1099-1112. doi: 10.1016/j.bbagen.2017.02.011. Epub 2017 Feb 13.
The effect of long-term exposure of live cells to lithium cations (Li) was studied in HEK293 cells cultivated in the presence of 1mM LiCl for 7 or 21days. The alteration of Na/K-ATPase level, protein composition and biophysical state of plasma membrane was determined with the aim to characterize the physiological state of Li-treated cells.
Na/K-ATPase level was determined by [H]ouabain binding and immunoblot assays. Overall protein composition was determined by 2D electrophoresis followed by proteomic analysis by MALDI-TOF MS/MS and LFQ. Li interaction with plasma membrane was characterized by fluorescent probes DPH, TMA-DPH and Laurdan.
Na/K-ATPase was increased in plasma membranes isolated from cells exposed to Li. Identification of Li-altered proteins by 2D electrophoresis, MALDI-TOF MS/MS and LFQ suggests a change of energy metabolism in mitochondria and cytosol and alteration of cell homeostasis of calcium. Measurement of Laurdan generalized polarization indicated a significant alteration of surface layer of isolated plasma membranes prepared from both types of Li-treated cells.
Prolonged exposure of HEK293 cells to 1mM LiCl results in up-regulation of Na/K-ATPase expression, reorganization of overall cellular metabolism and alteration of the surface layer/polar head-group region of isolated plasma membranes.
Our findings demonstrate adaptation of live HEK293 cell metabolism to prolonged exposure to therapeutic concentration of Li manifested as up-regulation of Na/K-ATPase expression, alteration of protein composition and change of the surface layer of plasma membrane.
本研究旨在探讨在存在 1mM LiCl 的情况下培养 HEK293 细胞 7 天或 21 天后，细胞长期暴露于锂离子（Li）的影响。通过测定 Na/K-ATP 酶的水平、质膜的蛋白组成和生物物理状态来改变，以表征 Li 处理细胞的生理状态。
通过[H]哇巴因结合和免疫印迹分析测定 Na/K-ATP 酶的水平。通过二维电泳（2-DE）后进行 MALDI-TOF MS/MS 和 LFQ 分析来确定总蛋白组成。通过荧光探针 DPH、TMA-DPH 和 Laurdan 来表征 Li 与质膜的相互作用。
从暴露于 Li 的细胞中分离的质膜中 Na/K-ATP 酶增加。通过 2-DE、MALDI-TOF MS/MS 和 LFQ 鉴定 Li 改变的蛋白质，表明线粒体和细胞质中的能量代谢发生变化，以及细胞内钙平衡发生改变。Laurdan 广义极化的测量表明，从两种类型的 Li 处理细胞中分离的质膜的表面层发生了显著改变。
HEK293 细胞长期暴露于 1mM LiCl 会导致 Na/K-ATP 酶表达上调、整体细胞代谢重新组织以及分离质膜表面层/极性头部区域的改变。
我们的研究结果表明，活 HEK293 细胞代谢适应长期暴露于治疗浓度 Li 的表现为 Na/K-ATP 酶表达上调、蛋白组成改变和质膜表面层改变。
