Calpain mediates ischemic injury of the liver through modulation of apoptosis and necrosis.
OBJECTIVE
Calpain proteases have been implicated in cell death by necrosis and more recently by apoptosis. Experiments were designed to determine the role of calpain proteases in ischemic rat liver injury by measurement of cytosolic calpain activity after different periods of ischemia-reperfusion and by evaluation of the effects of calpain inhibition on tissue injury and animal survival.


METHODS
Calpain activity was measured in the cytosol using Suc-Leu-Leu-Val-Try-7 amino-4 methyl coumarin, a specific fluorogenic substrate, and Cbz-Leu-Leu-Tyr-CHN2, a specific inhibitor.


RESULTS
Calpain activity increased significantly with the duration of ischemia-reperfusion and was inhibited more than 80% by the inhibitor. Calpain inhibition resulted in a significant decrease in transaminase release and tissue necrosis and converted nonsurvival ischemic conditions to survival conditions. When the in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling assay for apoptosis was used, 35% +/- 6% of nonparenchymal cells and 16% +/- 3% of hepatocytes stained positively after 60 minutes of ischemia and 6 hours of reperfusion. In contrast, animals pretreated with the calpain inhibitor showed minimal evidence of apoptosis. This was further substantiated by gel electrophoresis assay for DNA fragmentation and by electron-microscopic evaluation.


CONCLUSIONS
These data suggest that calpain proteases play a pivotal role in warm ischemia-reperfusion injury of the rat liver through modulation of apoptosis and necrosis.
Kohli
V
V
Hepatobiliary and Liver Transplant Laboratory, Duke University Medical Center, Durham, North Carolina 27710, USA.
Madden
J F
JF
Bentley
R C
RC
Clavien
P A
PA
D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001219:Aspartate Aminotransferases / Q000097:blood; D002154:Calpain / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D053938:DNA Fragmentation; D003978:Diazomethane / Q000031:analogs & derivatives / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D020287:In Situ Nick-End Labeling; D007511:Ischemia / Q000150:complications / Q000473:pathology* / Q000503:physiopathology; D008099:Liver / Q000098:blood supply* / Q000473:pathology; D008107:Liver Diseases / Q000209:etiology / Q000473:pathology* / Q000503:physiopathology; D009336:Necrosis; D009842:Oligopeptides / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000209:etiology / Q000473:pathology* / Q000503:physiopathology
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D009842:Oligopeptides; C090218:benzyloxycarbonylleucyl-leucyl-tyrosine diazomethyl ketone; C095174:N-benzyloxycarbonyl-valyl-phenylalanine methyl ester; D003978:Diazomethane; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase; D002154:Calpain