V Kohli1, J F Madden, R C Bentley, P A Clavien. 1. Hepatobiliary and Liver Transplant Laboratory, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract
BACKGROUND & AIMS: 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.
BACKGROUND & AIMS: 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 ischemicratliver 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.
Authors: S Cuzzocrea; M C McDonald; E Mazzon; D Siriwardena; I Serraino; L Dugo; D Britti; G Mazzullo; A P Caputi; C Thiemermann Journal: Am J Pathol Date: 2000-12 Impact factor: 4.307
Authors: Pierre-Alain Clavien; Jean Emond; Jean Nicolas Vauthey; Jacques Belghiti; Ravi S Chari; Steven M Strasberg Journal: J Gastrointest Surg Date: 2004 Mar-Apr Impact factor: 3.452
Authors: Jae-Sung Kim; Jin-Hee Wang; Thomas G Biel; Do-Sung Kim; Joseph A Flores-Toro; Richa Vijayvargiya; Ivan Zendejas; Kevin E Behrns Journal: Toxicol Appl Pharmacol Date: 2013-10-12 Impact factor: 4.219
Authors: S Cuzzocrea; M C McDonald; E Mazzon; H Mota-Filipe; T Centorrino; M L Terranova; A Ciccolo; D Britti; A P Caputi; C Thiemermann Journal: Gut Date: 2001-04 Impact factor: 23.059