Hypothermic preservation up-regulates calpain expression and increases ubiquitination in cultured vascular endothelial cells: influence of dopamine pretreatment.
BACKGROUND
Prolonged hypothermia, as occurs during solid organ transplantation, negatively influences transplantation outcome. Proteolysis is one of the deleterious events implicated in preservation injury of organ allografts. This strongly affects graft quality and hence immediate organ function. Since donor catecholamine treatment improves transplantation outcome after renal transplantation, the present study was conducted to examine the influence of dopamine (DA) pretreatment on hypothermia induced proteolysis in endothelial cells subjected to prolonged cold storage.


METHODS
Lactate dehydrogenase (LDH) assay, two-dimensional electrophoresis, ubiquitination analysis, intracellular calcium measurement, and Western blot analysis were performed on human umbilical vein endothelial cells (HUVEC) subjected to hypothermic preservation or not.


RESULTS
HUVEC were highly susceptible to cold storage, which was reflected by morphological changes, loss of viability, and by significant changes in cellular proteome. DA pretreatment prevented cell death during cold storage. Western blot analysis demonstrated a time dependent up-regulation of calpain 1 and 2 during cold storage, which could be prevented by addition of EDTA. DA pretreatment abolished autoproteolysis of calpain 1. Analysis of ubiquitination revealed a significant increase in ubiquitinated conjugates after cold storage. This was not prevented by DA pretreatment. Neither proteasome nor calpain inhibitors prevented cell death during cold storage.


CONCLUSIONS
In endothelial cells subjected to cold preservation, activation of the calpain pathway and the ubiquitin proteasome system occur. Although DA pretreatment inhibits the former, calpain inhibition did not protect endothelial cells during cold storage. DA pretreatment might influence proteolysis, but proteolysis is not the major cause of endothelial cell death.
5th Department of Medicine Nephrology, University of Heidelberg, Mannheim, Germany.
Boris
B
Rudic
Hui
H
Song
Annette
A
Breedijk
Paul
P
Brinkkoetter
Grietje
G
Beck
Benito
B
Yard
Norbert
N
Ponelies
D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D015925:Cryopreservation; D004298:Dopamine / Q000494:pharmacology*; D015259:Dopamine Agents / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D019172:Transplantation Conditioning / Q000379:methods*; D019737:Transplants; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination; D014471:Umbilical Veins / Q000166:cytology; D015854:Up-Regulation / Q000502:physiology
D015259:Dopamine Agents; D004791:Enzyme Inhibitors; D020543:Proteome; D025801:Ubiquitin; D002154:Calpain; D002118:Calcium; D004298:Dopamine