Calpain activation after mitochondrial permeability transition in microcystin-induced cell death in rat hepatocytes.
Previous studies have shown that microcystin-LR (MLR), a specific hepatotoxin, induces onset of mitochondrial permeability transition (MPT) and apoptosis in cultured rat hepatocytes. Here we attempted to investigate the downstream events after the onset of MPT in MLR-treated hepatocytes. Various mitochondrial electron transport chain (ETC) inhibitors effectively prevented the onset of MPT, suggesting that the mitochondrial ETC plays an important role in MLR-induced MPT. MLR also induced mitochondrial cytochrome c release, which can be prevented by a specific MPT inhibitor (cyclosporin A, CsA), and by various ETC inhibitors. Interestingly, the release of cytochrome c did not activate caspase-9 and -3, the main caspases involved in apoptosis. Instead, MLR activated calpain in rat hepatocytes, probably through the increase of intracellular Ca(2+) released from mitochondria. Both ALLN and ALLM, two calpain inhibitors, significantly blocked MLR-induced calpain activation and subsequent cell death. CsA also prevented MLR-induced calpain activation and cell death, suggesting that the activation of calpain may be a post-mitochondrial event. These data demonstrate for the first time that calpain rather than caspases plays an important role in MLR-induced apoptosis.
Ding
Wen-Xing
WX
Center for Environmental and Occupational Health, Department of Community, Occupational, and Family Medicine, Faculty of Medicine, National University of Singapore, Singapore 117597, Singapore.
Shen
Han-Ming
HM
Ong
Choon-Nam
CN
D000818:Animals; D017209:Apoptosis; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport / Q000187:drug effects; D005434:Flow Cytometry; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008387:Marine Toxins; D008564:Membrane Potentials; D052998:Microcystins; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D010456:Peptides, Cyclic / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010539:Permeability; D051381:Rats; D017207:Rats, Sprague-Dawley
D003574:Cytochrome c Group; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D002154:Calpain; D020169:Caspases; C057862:cyanoginosin LR; D002118:Calcium