分离的大鼠肝细胞的活力控制和特殊性质

Viability control and special properties of isolated rat hepatocytes.

作者信息

Pfaff E, Schuler B, Krell H, Höke H

出版信息

Arch Toxicol. 1980 Mar;44(1-3):3-21. doi: 10.1007/BF00303179.

Abstract

The need for quick viability tests is stressed. Aas these should achieve more than statically categorizing dead or non-dead cells, several procedures are suggested that picture the energetic state of the cells. The almost classical criterion of this category, namely stimulation of respiration by succinate, must be questioned on the basis of the present results. It is shown, that restricted respiration by succinate is not due to limited permeability of the plasma membrane, but to competition by endogenous substrates for uptake into mitochondria. Distribution equilibria for succinate appear to be according to (delta pH)2 with regard to cytoplasm. They are attained within 5-20 s or faster. Uptake is in part regulated by the surface charge density. Permeability changes caused by effectors of surface charge, such as amphiphilic ions, are examplified for succinate, chloride, phosphate, Na+, K+, and Ca2+. Such changes repeatedly also occur after pulses of BSP. They are counterregulated by the cell within a minute in a manner dependent on BSP concentration and the state of the cells. During the preincubation phase, that is the time of readaptation after transfer of cells from 0 degree C to higher temperature, a special labile state transiently occurs, where cyclic permeability changes for Ca2+, Na+, K+ can be caused by substrate addition, especially succinate, and/or ATP. The extent of these changes and their sequence again depend on the energetic state of the cells. In a probably narrow energetic window a sequence of cation movements reminding of that after depolarization of an excitable cell, is observed. Manipulation of the Na+/K+-ratio by variation of preincubation time and by ouabain shows that this is not simply the denominator for reversible calcium uptake. As the surface charge appears to reflect the energetic state, ANS fluorescence is applied to monitor the state of the plasma membrane, though difficulties arising from a slow ANS permeation are not yet solved.

摘要

强调了快速活力测试的必要性。由于这些测试应不仅仅是对死细胞或非死细胞进行静态分类，因此建议采用几种能够描绘细胞能量状态的方法。基于目前的结果，这一类别的几乎经典的标准，即琥珀酸对呼吸的刺激作用，必须受到质疑。结果表明，琥珀酸导致的呼吸受限并非由于质膜通透性有限，而是由于内源性底物竞争进入线粒体。琥珀酸的分布平衡似乎与细胞质的（δpH）² 有关。它们在5 - 20秒内或更快达到平衡。摄取部分受表面电荷密度调节。表面电荷效应剂（如两亲离子）引起的通透性变化以琥珀酸、氯离子、磷酸根、Na⁺、K⁺ 和Ca²⁺ 为例进行了说明。在BSP脉冲后也会反复出现这种变化。细胞会在一分钟内以依赖于BSP浓度和细胞状态的方式对其进行反向调节。在预孵育阶段，即细胞从0摄氏度转移到更高温度后的重新适应时间，会短暂出现一种特殊的不稳定状态，在此期间，添加底物（特别是琥珀酸）和/或ATP可导致Ca²⁺、Na⁺、K⁺ 的循环通透性变化。这些变化的程度及其顺序再次取决于细胞的能量状态。在一个可能很窄的能量窗口内，观察到一系列阳离子运动，这让人想起可兴奋细胞去极化后的情况。通过改变预孵育时间和使用哇巴因对Na⁺/K⁺ 比值进行操作表明，这不仅仅是可逆钙摄取的分母。由于表面电荷似乎反映了能量状态，因此应用ANS荧光来监测质膜状态，尽管ANS渗透缓慢所带来的困难尚未解决。