title: Mitochondrial permeability transition in the switch from necrotic to apoptotic cell death in ischemic rat hepatocytes.
abstract: OBJECTIVE
  Ischemia/reperfusion can initiate both necrotic and apoptotic death of hepatocytes. Previous work showed that onset of the mitochondrial permeability transition (MPT) can initiate necrotic cell death after reperfusion, but the MPT is also implicated in apoptosis. Here, we investigated factors regulating how cell death switches from necrosis to apoptosis after ischemia/reperfusion injury.
  
  
  METHODS
  Overnight cultured rat hepatocytes were incubated in anoxia at pH 6.2 for 4 hours and reoxygenated at pH 7.4 to simulate ischemia/reperfusion. Some cells were incubated with fructose plus glycine just before and then continuously after reperfusion. Development of apoptosis was evaluated by examining chromatin condensation, nuclear DNA fragmentation, and caspase 3 activity.
  
  
  RESULTS
  Reperfusion with the glycolytic substrate fructose plus the cytoprotective amino acid glycine prevented necrotic cell killing. Instead, apoptosis developed within 12 hours as shown by nuclear chromatin changes, TUNEL staining, and caspase 3 activation. This apoptotic cell killing was prevented by cyclosporin A, an MPT blocker, and by pancaspase and caspase 3 inhibition, but not by caspase 8 inhibition. Cyclosporin A also blocked caspase-3 activation. Reperfusion with glycine alone prevented necrotic cell death but did not induce apoptosis and only poorly promoted recovery of ATP, whereas fructose alone during reperfusion promoted both ATP recovery and apoptosis.
  
  
  CONCLUSIONS
  Glycolytic ATP generation after reperfusion prevents necrotic killing of hepatocytes after simulated ischemia/reperfusion despite onset of the MPT. Instead, the MPT promotes caspase-and ATP-dependent apoptosis. Thus, the MPT is a common mechanism responsible for both necrosis and apoptosis after ischemia/reperfusion.
authors.affiliation: Department of Cell and Developmental Biology, School of Medicine, University of North Carolina, Chapel Hill, 27599, USA.
authors.forename: Jae-Sung
authors.initials: JS
authors.lastname: Kim
authors.forename: Ting
authors.initials: T
authors.lastname: Qian
authors.forename: John J
authors.initials: JJ
authors.lastname: Lemasters
mesh_terms: D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D004734:Energy Metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005632:Fructose / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D006019:Glycolysis; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology / Q000502:physiology*; D020287:In Situ Nick-End Labeling; D007511:Ischemia / Q000473:pathology / Q000503:physiopathology*; D008102:Liver Circulation; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism*; D009336:Necrosis; D010539:Permeability; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000473:pathology / Q000503:physiopathology; D013194:Staining and Labeling
chemical_list: D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D005632:Fructose; D016572:Cyclosporine; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005998:Glycine