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Viability control and special properties of isolated rat hepatocytes
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.