n-benzyloxycarbonyl-valyl-phenylalanine-methyl-ester and Reperfusion-Injury

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.. 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.. 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.. 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.

Topics: Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; Calpain; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Cytosol; Diazomethane; Dipeptides; DNA Fragmentation; Electrophoresis, Agar Gel; In Situ Nick-End Labeling; Ischemia; Liver; Liver Diseases; Necrosis; Oligopeptides; Rats; Rats, Wistar; Reperfusion Injury

Cold preservation of the liver followed by reperfusion results in sinusoidal endothelial cell (SEC) apoptosis. Calpain-like activity is dramatically increased during reperfusion and inhibition of calpains results in lower graft injury and longer survival. Recently, calpains have been implicated in inducing apoptosis. Our aim was to determine the effect of calpain inhibition on SEC apoptosis.. Livers were stored in the University of Wisconsin solution for 24 hr (survival conditions) and 40 hr (nonsurvival conditions) and ex vivo reperfused for 1 hr at 37 degrees C. Calpain-like activity was inhibited in some experiments using an i.p. injection of a selective inhibitor 2 hr before explantation. Apoptosis was quantified using the terminal deoxynucleotidyl trans. ferase-mediated dUTP nick end-labeling assay. Cross-inhibition by the inhibitor was determined for caspases 1 and 3.. Apoptosis of exclusively the SEC was a key feature of reperfusion injury after both storage periods in University of Wisconsin solution after 1 hr normothermic reperfusion. Inhibition of calpain activity with Cbz-Val-Phe methyl ester resulted in a 50% reduction of apoptotic SEC in the 40-hr preserved liver, and an almost complete abrogation of SEC apoptosis after 24 hr preservation. Only minimal cross-inhibition of caspases was determined at high concentrations in vitro by the calpain inhibitor.. Apoptosis of exclusively SEC is a key feature of reperfusion injury partially mediated through calpain-dependent processes. Calpain inhibition reduces the number of apoptotic SEC. Based on these data and our previous work, calpain inhibition may prove to be useful in clinical transplantation.

Topics: Animals; Apoptosis; Calpain; Cryopreservation; Cysteine Proteinase Inhibitors; Dipeptides; Endothelium; Liver; Organ Preservation; Rats; Rats, Wistar; Reperfusion Injury