Mitochondrial permeability transition in the switch from necrotic to apoptotic cell death in ischemic rat hepatocytes

Opening of a high-conductance pore in the mitochondrial inner membrane induces onset of the mitochondrial permeability transition (mPT). Cyclosporin A and trifluoperazine inhibit this pore and block necrotic cell death in oxidative stress, Ca2+ ionophore toxicity, Reye-related drug toxicity, pH-dependent ischaemia/reperfusion injury and other models of cell injury. Confocal fluorescence microscopy directly visualizes the increased mitochondrial membrane permeability of the mPT from the movement of calcein from the cytosol into the matrix space. Pyridine nucleotide oxidation, increased mitochondrial Ca2+ and mitochondrial generation of reactive oxygen species (ROS) all contribute to the onset of the mPT in situ. Confocal microscopy also shows directly that the mPT is a critical link in apoptotic signalling by tumour necrosis factor-alpha at a point downstream of caspase 8 and upstream of caspase 3. Cyclosporin A blocks this mPT, preventing release of pro-apoptotic cytochrome c from mitochondria and subsequent apoptotic cell killing. Progression to necrosis or apoptosis after the mPT depends on the availability of ATP, which blocks necrosis but promotes the apoptotic programme. Given the pathophysiological importance of the mPT, development of agents to modulate the mPT represents an important new goal for pharmaceutical drug discovery.

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Calpain Activation after Mitochondrial Permeability Transition in Microcystin-Induced Cell Death in Rat Hepatocytes

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.2002.6453 ◽

2002 ◽

Vol 291 (2) ◽

pp. 321-331 ◽

Cited By ~ 80

Author(s):

Wen-Xing Ding ◽

Han-Ming Shen ◽

Choon-Nam Ong

Keyword(s):

Cell Death ◽

Mitochondrial Permeability Transition ◽

Permeability Transition ◽

Rat Hepatocytes ◽

Mitochondrial Permeability ◽

Calpain Activation

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Apoptotic cell death of cultured salamander photoreceptors induced by cccp: CsA-insensitive mitochondrial permeability transition

Journal of Cell Science ◽

10.1242/jcs.114.9.1655 ◽

2001 ◽

Vol 114 (9) ◽

pp. 1655-1664

Author(s):

J.H. Yang ◽

R.L. Gross ◽

S.F. Basinger ◽

S.M. Wu

Keyword(s):

Cell Death ◽

Mitochondrial Permeability Transition ◽

Cell Model ◽

Apoptotic Cell ◽

Permeability Transition ◽

Apoptotic Cell Death ◽

Mitochondrial Inner Membrane ◽

Underlying Mechanisms ◽

Induced Apoptosis

Photoreceptor degeneration is mediated by apoptosis in several animal models, although the underlying mechanisms are yet to be elucidated. We present here an apoptotic model based on a primary cell culture of tiger salamander photoreceptors, in which treatment with carbonyl cyanide m-chlorophenylhydrazone (cccp), a protonophore, induced apoptosis. Cells exposed to cccp showed condensed nuclei and displayed positive TdT-dUTP terminal nick-end labeling (TUNEL). In addition, 10–100 microM cccp rapidly induced a reduction of Delta psi(m) and > or = 30 microM cccp induced a significant leakage of calcein from mitochondria to cytosol and nucleus, indicating a change in mitochondrial inner membrane permeability. Cyclosporin A (CsA), a transition pore blocker, did not prevent the cccp-induced MPT or the cccp-evoked apoptotic cell death, suggesting that cccp-induced apoptotic process was mediated by a CsA-insensitive pathway. This cell model provides an in vitro tool for studying mechanisms of photoreceptor apoptosis in isolated photoreceptors and may provide clues to the etiology of retinal degeneration.

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Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation: role of Ca2+-dependent mitochondrial formation of reactive oxygen species

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00082.2011 ◽

2012 ◽

Vol 302 (7) ◽

pp. G723-G731 ◽

Cited By ~ 51

Author(s):

Jae-Sung Kim ◽

Jin-Hee Wang ◽

John J. Lemasters

Keyword(s):

Reactive Oxygen Species ◽

Cell Death ◽

Mitochondrial Permeability Transition ◽

Ischemia Reperfusion ◽

Permeability Transition ◽

Rat Hepatocytes ◽

Oxygen Species ◽

Mitochondrial Permeability ◽

Ros Formation

Onset of the mitochondrial permeability transition (MPT) is the penultimate event leading to lethal cellular ischemia-reperfusion injury, but the mechanisms precipitating the MPT after reperfusion remain unclear. Here, we investigated the role of mitochondrial free Ca2+ and reactive oxygen species (ROS) in pH- and MPT-dependent reperfusion injury to hepatocytes. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 h and then reoxygenated at pH 7.4 to simulate ischemia-reperfusion. Some cells were loaded with the Ca2+ chelators, BAPTA/AM and 2-[(2-bis-[carboxymethyl]aono-5-methoxyphenyl)-methyl-6-methoxy-8-bis[carboxymethyl]aminoquinoline, either by a cold loading protocol for intramitochondrial loading or by warm incubation for cytosolic loading. Cell death was assessed by propidium iodide fluorometry and immunoblotting. Mitochondrial Ca2+, inner membrane permeability, membrane potential, and ROS formation were monitored with Rhod-2, calcein, tetramethylrhodamine methylester, and dihydrodichlorofluorescein, respectively. Necrotic cell death increased after reoxygenation. Necrosis was blocked by 1 μM cyclosporin A, an MPT inhibitor, and by reoxygenation at pH 6.2. Confocal imaging of Rhod-2, calcein, and dichlorofluorescein revealed that an increase of mitochondrial Ca2+ and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca2+ chelation, but not cytosolic Ca2+ chelation, prevented ROS formation and subsequent necrotic and apoptotic cell death. Reoxygenation with the antioxidants, desferal or diphenylphenylenediamine, also suppressed MPT-mediated cell death. However, inhibition of cytosolic ROS by apocynin or diphenyleneiodonium chloride failed to prevent reoxygenation-induced cell death. In conclusion, Ca2+-dependent mitochondrial ROS formation is the molecular signal culminating in onset of the MPT after reoxygenation of anoxic hepatocytes, leading to cell death.

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