Ca2+ depletion prevents anoxic death of hepatocytes by inhibiting mitochondrial permeability transition

1995 ◽  
Vol 268 (3) ◽  
pp. C676-C685 ◽  
Author(s):  
J. G. Pastorino ◽  
J. W. Snyder ◽  
J. B. Hoek ◽  
J. L. Farber
Keyword(s):  
Culture Medium ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition ◽  
Rat Hepatocytes ◽  
Acetoxymethyl Ester ◽  
Intact Cells ◽  
Mitochondrial Permeability ◽  
Isolated Mitochondria ◽  
The Matrix ◽  
Matrix Concentration

Removal of Ca2+ from the culture medium or treatment with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) prevented the killing of rat hepatocytes by anoxia and rotenone, but not by cyanide. Neither manipulation prevented the loss of the mitochondrial membrane potential or the depletion of ATP. A mitochondrial permeability transition (MPT) was demonstrated in digitonin-permeabilized hepatocytes as an increased [3H]sucrose-accessible space sensitive to cyclosporin A (CyA). Ca2+ depletion by either means prevented the MPT measured in intact cells made anoxic or treated with rotenone. In isolated mitochondria deenergized by rotenone, BAPTA-AM prevented the MPT induced by palmitoyl CoA. By contrast, in isolated mitochondria deenergized by cyanide, BAPTA-AM alone did not prevent the MPT. Rather, BAPTA-AM plus CyA were required. Similarly, the killing of cultured hepatocytes by cyanide was prevented by BAPTA-AM plus CyA, but not by either agent alone. The MPT in intact cells treated with cyanide was also prevented by BAPTA-AM plus CyA. These data define a specific requirement for Ca2+ in the killing of hepatocytes that follows the inhibition of electron transport. A model is presented in which the MPT depends on factors that modulate the sensitivity of the permeability transition to the matrix concentration of Ca2+.

Accelerated Mitochondrial Reactive Oxygen Species Formation Induces OnsẸt of the Mitochondrial Permeability Transition and Mitochondrial Swelling in Cultured Hepatocytes After TNFα Exposure

2001 ◽  
Vol 7 (S2) ◽  
pp. 604-605
Author(s):  
Ting Qian ◽  
John J. Lemasters
Keyword(s):  
Mitochondrial Permeability Transition ◽  
Permeability Transition ◽  
Rat Hepatocytes ◽  
Cultured Hepatocytes ◽  
Intact Cells ◽  
Mitochondrial Permeability ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Ros ◽  
Ros Formation ◽  
Induced Apoptosis

INTRODUCTION: The mitochondrial permeability transition (MPT) is implicated in mediating TNFα-induced apoptosis in cultured hepatocytes. Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the MPT. After the MPT, mitochondria swell, the outer membrane bursts, and pro-apoptotic cytochrome c is released into the cytosol. in isolated mitochondria, ROS formation promotes onset of the MPT. However, how mitochondrial ROS formation regulates the MPT in intact cells in TNFα-induced apoptosis is unknown. AIM: The present study was designed to determine the role of mitochondrial ROS formation in TNFα-induced MPT and apoptosis in cultured rat hepatocytes.METHODS: Hepatocytes expressing an IkB superrepressor were pretreated with 2 μM t-BuOOH 4 hours before TNFα exposure with and without CsA (2 μM, an inhibitor of the PT pore) and the antioxidants, desferal (0.5 mM) or diphenylphenylendiamine (DPPD, 10 μM). Cell viability was monitored by propidium iodide fluorometry.

scholarly journals Metformin inhibits mitochondrial permeability transition and cell death: a pharmacological in vitro study

2004 ◽  
Vol 382 (3) ◽  
pp. 877-884 ◽  
Author(s):  
Bruno GUIGAS ◽  
Dominique DETAILLE ◽  
Christiane CHAUVIN ◽  
Cécile BATANDIER ◽  
Frédéric De OLIVEIRA ◽  
...  
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition ◽  
Cytochrome C Release ◽  
Intact Cells ◽  
Human Carcinoma ◽  
Butyl Hydroperoxide ◽  
Mitochondrial Permeability ◽  
New Findings

Metformin, a drug widely used in the treatment of Type II diabetes, has recently received attention owing to new findings regarding its mitochondrial and cellular effects. In the present study, the effects of metformin on respiration, complex 1 activity, mitochondrial permeability transition, cytochrome c release and cell death were investigated in cultured cells from a human carcinoma-derived cell line (KB cells). Metformin significantly decreased respiration both in intact cells and after permeabilization. This was due to a mild and specific inhibition of the respiratory chain complex 1. In addition, metformin prevented to a significant extent mitochondrial permeability transition both in permeabilized cells, as induced by calcium, and in intact cells, as induced by the glutathione-oxidizing agent t-butyl hydroperoxide. This effect was equivalent to that of cyclosporin A, the reference inhibitor. Finally, metformin impaired the t-butyl hydroperoxide-induced cell death, as judged by Trypan Blue exclusion, propidium iodide staining and cytochrome c release. We propose that metformin prevents the permeability transition-related commitment to cell death in relation to its mild inhibitory effect on complex 1, which is responsible for a decreased probability of mitochondrial permeability transition.

scholarly journals Cyclophilin-D promotes the mitochondrial permeability transition but has opposite effects on apoptosis and necrosis

2004 ◽  
Vol 383 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Yanmin LI ◽  
Nicholas JOHNSON ◽  
Michela CAPANO ◽  
Mina EDWARDS ◽  
Martin CROMPTON
Keyword(s):  
Oxidative Stress ◽  
Cell Injury ◽  
Mitochondrial Permeability Transition ◽  
Adenine Nucleotide ◽  
Permeability Transition ◽  
Cyclophilin D ◽  
Intact Cells ◽  
Inner Membrane ◽  
Mitochondrial Permeability ◽  
Apoptosis And Necrosis

Cyclophilin-D is a peptidylprolyl cis–trans isomerase of the mitochondrial matrix. It is involved in mitochondrial permeability transition, in which the adenine nucleotide translocase of the inner membrane is transformed from an antiporter to a non-selective pore. The permeability transition has been widely considered as a mechanism in both apoptosis and necrosis. The present study examines the effects of cyclophilin-D on the permeability transition and lethal cell injury, using a neuronal (B50) cell line stably overexpressing cyclophilin-D in mitochondria. Cyclophilin-D overexpression rendered isolated mitochondria far more susceptible to the permeability transition induced by Ca2+ and oxidative stress. Similarly, cyclophilin-D overexpression brought forward the onset of the permeability transition in intact cells subjected to oxidative stress. In addition, in the absence of stress, the mitochondria of cells overexpressing cyclophilin-D maintained a lower inner-membrane potential than those of normal cells. All these effects of cyclophilin-D overexpression were abolished by cyclosporin A. It is concluded that cyclophilin-D promotes the permeability transition in B50 cells. However, cyclophilin-D overexpression had opposite effects on apoptosis and necrosis; whereas NO-induced necrosis was promoted, NO- and staurosporine-induced apoptosis were inhibited. These findings indicate that the permeability transition leads to cell necrosis, but argue against its involvement in apoptosis.

scholarly journals Bax-induced Cytochrome C Release from Mitochondria Is Independent of the Permeability Transition Pore but Highly Dependent on Mg2+ Ions

1998 ◽  
Vol 143 (1) ◽  
pp. 217-224 ◽  
Author(s):  
Robert Eskes ◽  
Bruno Antonsson ◽  
Astrid Osen-Sand ◽  
Sylvie Montessuit ◽  
Christoph Richter ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cyclosporin A ◽  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cytochrome C Release ◽  
Mitochondrial Permeability ◽  
Isolated Mitochondria ◽  
Mammalian Homologue

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homologue of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). We now report that Bax is sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A–sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg2+ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A, the other Bax dependent, Mg2+ sensitive but cyclosporin insensitive.

Protectors of the mitochondrial permeability transition pore activated by iron and doxorubicin

2021 ◽  
Vol 21 ◽  
Author(s):  
Tatiana A. Fedotcheva ◽  
Nadezhda I. Fedotcheva
Keyword(s):  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Membrane Damage ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Selective Electrode ◽  
Mitochondrial Permeability ◽  
Isolated Mitochondria ◽  
Mptp Opening ◽  
Dependent Cell

Aim: The study of action of iron, DOX, and their complex on the mitochondrial permeability transition pore (MPTP) opening and the detection of possible protectors of MPTP in the conditions close to mitochondria-dependent ferroptosis. Background: The toxicity of doxorubicin (DOX) is mainly associated with the free iron accumulation and mitochondrial dysfunction. DOX can provoke ferroptosis, iron-dependent cell death driven by the membrane damage. The mitochondrial permeability transition pore (MPTP) is considered as a common pathway leading to the development of apoptosis, necrosis, and, possibly, ferroptosis. The influence of DOX on the Ca2+ -induced opening of MPTP in the presence of iron has not yet been studied. Objective: The study was conducted on isolated liver and heart mitochondria. MPTP and succinate-ubiquinone oxidoreductase were studied as targets of DOX in mitochondria-dependent ferroptosis. Methods: The study was conducted on isolated mitochondria of the liver and heart. Changes of threshold calcium concentrations required for MPTP opening were measured by a Ca2+ selective electrode, mitochondrial membrane potential was registered by tetraphenylphosphonium (TPP+)-selective electrode, and mitochondrial swelling was recorded as a decrease in absorbance at 540 nm. The activity of succinate dehydrogenase (SDH) was determined by the reduction of the electron acceptor DCPIP. Conclusion: MPTP and the respiratory complex II are identified as the main targets of the iron-dependent action of DOX on the isolated mitochondria. All MPTP protectors tested abolished or weakened the effect of iron and a complex of iron with DOX on Ca2+ -induced MPTP opening, acting in different stages of MPTP activation. These data open new approaches to the modulation of the toxic influence of DOX on mitochondria with the aim to reduce their dysfunction

Mitochondrial control of apoptosis: an overview

1999 ◽  
Vol 66 ◽  
pp. 1-15 ◽  
Author(s):  
Guido Kroemer
Keyword(s):  
Transmembrane Potential ◽  
Mitochondrial Permeability Transition ◽  
Ion Selectivity ◽  
Permeability Transition ◽  
Critical Event ◽  
Intact Cells ◽  
Isolated Mitochondria ◽  
Pore Opening ◽  
Mitochondrial Megachannel

The mitochondrial permeability transition (PT) pore, also called the mitochondrial megachannel, is a multiprotein complex formed at the contact site between the mitochondrial inner and outer membranes, exactly the same location at which Bax, Bcl-2 and Bcl-XL are particularly abundant. The PT pore participates in the regulation of matrix Ca2+, pH, transmembrane potential and volume, and functions as a Ca2+-, voltage-, pH- and redox-gated channel with several levels of conductance and little, if any, ion selectivity. We have obtained three independent lines of evidence implicating the mitochondrial PT pore in apoptosis. First, in intact cells, apoptosis is accompanied by an early dissipation of the mitochondrial transmembrane potential, ΔΨm. In several models of apoptosis, specific agents inhibiting the mitochondrial PT pore abolish this dissipation of the ΔΨm and simultaneously prevent activation of downstream caspases and endonucleases, indicating that PT pore opening can be a critical event of the apoptotic process. Secondly, mitochondria are rate-limiting for caspase and nuclease activation in several cell-free systems of apoptosis. Isolated mitochondria release apoptogenic factors capable of activating pro-caspases or endonucleases upon opening of the mitochondrial megachannel in vitro. Thirdly, opening of the purified PT pore complex reconstituted into liposomes is inhibited by recombinant Bcl-2 or Bcl-XL, two apoptosis-inhibitory proteins that also prevent PT pore opening in cells and isolated mitochondria. Altogether, our results suggest that PT pore opening is sufficient and (mostly) necessary for triggering apoptosis. The implications of these findings are examined in the light of pharmacological interventions in apoptosis.

scholarly journals Hepatitis B Virus X Protein Modulates Apoptosis in Primary Rat Hepatocytes by Regulating both NF-κB and the Mitochondrial Permeability Transition Pore

2009 ◽  
Vol 83 (10) ◽  
pp. 4718-4731 ◽  
Author(s):  
Amy J. Clippinger ◽  
Tricia L. Gearhart ◽  
Michael J. Bouchard
Keyword(s):  
Hepatitis B Virus ◽  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Rat Hepatocytes ◽  
Mitochondrial Permeability ◽  
Primary Rat Hepatocytes ◽  
B Virus ◽  
Apoptotic Pathways

ABSTRACT The hepatitis B virus (HBV) X protein (HBx) is a multifunctional protein that regulates numerous cellular signal transduction pathways, including those that modulate apoptosis. However, different HBx-dependent effects on apoptosis have been reported; these differences are likely the consequence of the exact conditions and cell types used in a study. Many of the previously reported studies that analyzed HBx regulation of apoptosis were conducted in immortalized or transformed cells, and the alterations that have transformed or immortalized these cells likely impact apoptotic pathways. We examined the effects of HBx on apoptotic pathways in cultured primary rat hepatocytes, a biologically relevant system that mimics normal hepatocytes in the liver. We analyzed the effects of HBx on apoptosis both when HBx was expressed in the absence of other HBV proteins and in the context of HBV replication. HBx stimulation of NF-κB inhibited the activation of apoptotic pathways in cultured primary rat hepatocytes. However, when HBx-induced activation of NF-κB was blocked, HBx stimulated apoptosis; blocking the activity of the mitochondrial permeability transition pore inhibited HBx activation of apoptosis. These results suggest that HBx can be either proapoptotic or antiapoptotic in hepatocytes, depending on the status of NF-κB, and confirm previous studies that link some HBx activities to modulation of the mitochondrial permeability transition pore. Overall, our studies define apoptotic pathways that are regulated by HBx in cultured primary hepatocytes and provide potential mechanisms for the development of HBV-associated liver cancer.

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