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Calpain activation after mitochondrial permeability transition in microcystin-induced cell death in rat hepatocytes
Wen-Xing Ding1, Han-Ming Shen, Choon-Nam Ong
1Center for Environmental and Occupational Health, Department of Community, Occupational, and Family Medicine, Faculty of Medicine, National University of Singapore, Singapore 117597, Singapore.
Abstract:
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.
Insights
Microcystin-LR (MLR) triggers mitochondrial permeability transition (MPT) and apoptosis in liver cells. This study reveals calpain activation, not caspases, mediates MLR-induced cell death, offering new therapeutic targets.
Area of Science:
- Hepatology
- Toxicology
- Cell Biology
Background:
- Microcystin-LR (MLR) is a hepatotoxin known to induce mitochondrial permeability transition (MPT) and apoptosis.
- The precise downstream signaling pathways following MPT in MLR-treated hepatocytes require further elucidation.
Purpose of the Study:
- To investigate the molecular events downstream of MPT in MLR-induced hepatocyte apoptosis.
- To determine the role of caspases and calpains in MLR-induced cell death.
Main Methods:
- Primary rat hepatocyte culture.
- Treatment with MLR, mitochondrial electron transport chain (ETC) inhibitors, cyclosporin A (CsA), and calpain inhibitors (ALLN, ALLM).
- Assessment of MPT onset, cytochrome c release, caspase activation (caspase-9, -3), and calpain activation.
Main Results:
- MLR-induced MPT was prevented by ETC inhibitors, implicating the mitochondrial ETC.
- MLR caused cytochrome c release, which was inhibited by CsA and ETC inhibitors.
- Cytochrome c release did not activate caspases -9 and -3.
- MLR activated calpain, likely via mitochondrially released Ca(2+).
- Calpain inhibitors (ALLN, ALLM) and CsA blocked MLR-induced calpain activation and cell death.
Conclusions:
- Calpain activation, rather than caspase activation, is a critical mediator of MLR-induced apoptosis in rat hepatocytes.
- MLR-induced cell death involves a post-mitochondrial event pathway centered on calpain.
- Targeting calpain may offer a novel therapeutic strategy against MLR toxicity.