title: Cold-induced apoptosis of rat liver endothelial cells: involvement of the proteasome.
abstract: BACKGROUND
  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 degrees 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institut für Physiologische Chemie, Universitätsklinikum, Essen, Germany.
authors.forename: Thorsten R
authors.initials: TR
authors.lastname: Doeppner
authors.forename: Tilman
authors.initials: T
authors.lastname: Grune
authors.forename: Herbert
authors.initials: H
authors.lastname: de Groot
authors.forename: Ursula
authors.initials: U
authors.lastname: Rauen
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D003080:Cold Temperature; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004727:Endothelium / Q000166:cytology / Q000201:enzymology; D022781:Hepatocytes / Q000166:cytology* / Q000201:enzymology; D008099:Liver / Q000166:cytology* / Q000201:enzymology; D008297:Male; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D017208:Rats, Wistar
chemical_list: D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases