Glutathione movements during cold preservation of rat hepatocytes

In this study we have examined the movements of glutathione (GSH) during cold preservation of rat hepatocytes in University of Wisconsin solution. During the preservation process at a low temperature (4 degrees C), with a high extracellular potassium concentration, an extracellular nondiffusible anion (lactobionate), and a Cl(-)-free medium, there is a depletion of metabolites and the development of a time-dependent injury. Also, there is a loss of GSH that is not compensated by transport or synthesis and is basically due to increased catabolic processes. This sensitizes the cells to different forms of oxidative injury, which can play a negative role during transplantation. The addition of GSH improves liver cell preservation but the mechanism is unclear. To elucidate this process we have isolated hepatocytes and preserved them under different conditions: with or without GSH: in the presence of DL-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione synthetase, and acivicine to inhibit the ectoactivity of cellular gammaglutamyl transpeptidase; or by obtaining hepatocytes from rats depleted of GSH by an injection of diethyl maleate. Under all these conditions we evaluated the GSH content of the cells during cold storage. We also report the time course of accumulation of [glycine-2-3H]GSH. Our results show that during hypothermic storage in University of Wisconsin solution, hepatocytes are permeable to GSH, and the mechanism involved is a rapid nonsaturable process, with linear dependence of the extracellular GSH concentration. This finding may have valuable applications in the improvement of the delivery of compounds to cells.