Doeppner Thorsten R, Grune Tilman, de Groot Herbert, Rauen Ursula
Institut für Physiologische Chemie, Universitätsklinikum, Essen, Germany.
Transplantation. 2003 Jun 27;75(12):1946-53. doi: 10.1097/01.TP.0000065291.02855.6A.
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.
Cultured liver endothelial cells were incubated in preservation solutions at 4 degrees C in the absence or presence of protease inhibitors. Cell injury and different protease activities were assessed.
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.
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.
我们之前已经表明，在培养的肝内皮细胞中，单纯的低温及复温过程会诱导一种明显的、铁依赖性的细胞凋亡，这可能导致肝脏保存损伤。在此，我们研究蛋白酶在这种冷诱导细胞凋亡中的作用。
将培养的肝内皮细胞在4℃下于保存液中孵育，保存液中存在或不存在蛋白酶抑制剂。评估细胞损伤和不同的蛋白酶活性。
在威斯康星大学溶液或组氨酸-色氨酸-酮戊二酸（HTK）溶液中对肝内皮细胞进行冷孵育会导致明显的细胞损伤，蛋白酶抑制剂3,4-二氯异香豆素（DCI）可强烈抑制这种损伤（冷孵育24小时后，HTK溶液中乳酸脱氢酶释放率为86.0±2.6%，HTK + DCI溶液中为4.0±0.4%）。蛋白酶活性测定显示，在HTK溶液中冷孵育8小时或在威斯康星大学溶液中冷孵育16小时后，切割Suc-Leu-Leu-Val-Tyr-AMC的蛋白酶活性增加一倍，而切割相对较低的Suc-Ala-Ala-Ala-AMC的活性有一定增加。对蛋白酶活性的进一步表征表明，它们属于两种不同的蛋白酶，主要活性不依赖钙，受DCI、MG-132和乳胞素抑制，并且用抗蛋白酶体抗体进行免疫沉淀后活性大幅降低。用铁螯合剂去铁胺对细胞进行预孵育可防止冷诱导的两种活性增加。
这些结果表明，（i）蛋白酶体以及可能还有一种丝氨酸蛋白酶参与肝内皮细胞的冷诱导凋亡，（ii）蛋白酶激活发生在细胞内可螯合铁增加之后。
