We have previously shown that in cultured liver endothelial cells the mere sequence of hypothermia and rewarming induces a pronounced, iron-dependent apoptosis that is likely to contribute to hepatic preservation injury. Here we study the involvement of proteases in this cold-induced apoptosis.Methods.
Cultured liver endothelial cells were incubated in preservation solutions at 4°C in the absence or presence of protease inhibitors. Cell injury and different protease activities were assessed.Results.
Cold incubation of liver endothelial cells in University of Wisconsin or histidine-tryptophan-ketoglutarate (HTK) solution led to marked cell injury, which was strongly inhibited by the protease inhibitor 3,4-dichloroisocoumarin (DCI) (lactate dehydrogenase release: 86.0±2.6% in HTK and 4.0±0.4% in HTK + DCI after 24 hr of cold incubation). Determination of protease activity showed a doubling in the activity of a Suc-Leu-Leu-Val-Tyr-AMC–cleaving protease and some increase in a relatively low Suc-Ala-Ala-Ala-AMC–cleaving activity after 8 hr of cold incubation in HTK solution or 16 hr in University of Wisconsin solution. Further characterization of the protease activities showed that they belonged to two different proteases, with the major activity being calcium independent, inhibited by DCI, MG-132, and lactacystin, and strongly decreased by immunoprecipitation with an antiproteasome antibody. Preincubation of the cells with the iron chelator deferoxamine prevented the cold-induced increase of both activities.Conclusion.
These results show that (i) the proteasome and possibly, in addition, a serine protease are involved in cold-induced apoptosis of liver endothelial cells and (ii) the protease activation is downstream from the increase in intracellular chelatable iron.