Ceramide induces cytochrome c release from isolated mitochondria

1999 ◽  
Vol 66 ◽  
pp. 27-31 ◽  
Author(s):  
Christoph Richter ◽  
Pedram Ghafourifar
Keyword(s):  
Cytochrome C ◽  
Membrane Integrity ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Cytochrome C Release ◽  
Mitochondrial Oxygen Consumption ◽  
Mitochondrial Functions ◽  
Cyt C ◽  
C6 Ceramide

This chapter addresses the role of mitochondria in apoptosis. Emphasis is put on the recently observed influence of ceramides on mitochondrial functions. We report here that N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide) and, to a much lesser extent, C2-dihydroceramide, induce cytochrome c (cyt c) release from isolated rat liver mitochondria. Ceramide-induced cyt c release is prevented by a low concentration of Bcl-2. The release takes place when cyt c is oxidized, but not when it is reduced. Upon cyt c release mitochondrial oxygen consumption, mitochondrial transmembrane potential (ΔΨm) and Ca2+ retention are diminished. Bcl-2 prevents, and addition of cyt c reverses, the alteration of these mitochondrial functions. In ATP-energized mitochondria ceramides do not alter ΔΨm, neither when cyt c is oxidized nor when it is reduced. This rules out a non-specific disturbance by ceramides of mitochondrial-membrane integrity. It is concluded that some of the apoptogenic properties of ceramides are mediated via their interaction with mitochondrial cyt c followed by its release.

Cytochrome c release from rat liver mitochondria is compromised by increased saturated cardiolipin species induced by sucrose feeding

2015 ◽  
Vol 309 (9) ◽  
pp. E777-E786 ◽  
Author(s):  
Angélica Ruiz-Ramírez ◽  
Miguel-Angel Barrios-Maya ◽  
Ocarol López-Acosta ◽  
Dora Molina-Ortiz ◽  
Mohammed El-Hafidi
Keyword(s):  
Cytochrome C ◽  
Superoxide Radical ◽  
Lipid Membrane ◽  
Liver Mitochondria ◽  
Membrane Lipid ◽  
Rat Liver Mitochondria ◽  
Ros Generation ◽  
Cytochrome C Release ◽  
Mitochondrial Inner Membrane ◽  
Sucrose Feeding

Cytochrome c release from mitochondria has been described to be related to reactive oxygen species (ROS) generation. With ROS generation being increased in fatty liver from sucrose-fed (SF) rats, we hypothesized that cytochrome c release might be positively associated with H2O2 generation from SF mitochondria. Surprisingly, cytochrome c release from mitochondria of SF liver was found to be significantly lower compared with control (C) mitochondria oxidizing pyruvate/malate or succinate. Exposure of mitochondria to exogenous superoxide radical generated by the xanthine/xanthine oxidase system elicits a dose-response cytochrome c release in both control and SF mitochondria, but cytochrome c release remains lower in SF mitochondria compared with C mitochondria. Furthermore, the addition of ebselen, PEG-catalase, or catalase, a H2O2 scavenger, significantly reduces cytochrome c release from C and SF mitochondria. Our results suggest that both intra- and extramitochondrial H2O2 are involved in cytochrome c release, but the persisting difference between C and SF levels can be attributed to the differences in cardiolipin compositions. Indeed, the ratio of palmitic acid-rich cardiolipin species was found to be increased in lipid membrane from SF mitochondria compared with C mitochondria, whereas that of linoleic acid-rich cardiolipin species was found decreased. In addition, the content of tafazzin, a protein responsible for cardiolipin remodeling, was decreased in SF mitochondria. Therefore, we conclude that the changes observed in the composition of cardiolipin molecular species in SF mitochondria may be involved in cytochrome c interaction with mitochondrial inner membrane lipid and in its reduced release from SF mitochondria.

Role of calcium and superoxide dismutase in sensitizing mitochondria to peroxynitrite-induced permeability transition

2004 ◽  
Vol 286 (1) ◽  
pp. H39-H46 ◽  
Author(s):  
Paul S. Brookes ◽  
Victor M. Darley-Usmar
Keyword(s):  
Superoxide Dismutase ◽  
Cytochrome C ◽  
Rat Liver ◽  
Permeability Transition ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Isolated Rat Liver ◽  
Ptp Opening ◽  
Isolated Rat

The mitochondrial permeability transition pore (PTP) is a membrane protein complex assembled and opened in response to Ca2+ and oxidants such as peroxynitrite (ONOO–). Opening the PTP is mechanistically linked to the release of cytochrome c, which participates in downstream apoptotic signaling. However, the molecular basis of the synergistic interactions between oxidants and Ca2+ in promoting the PTP are poorly understood and are addressed in the present study. In isolated rat liver mitochondria, it was found that the timing of the exposure of the isolated rat liver mitochondria to Ca2+ was a critical factor in determining the impact of ONOO– on PTP. Specifically, addition of Ca2+ alone, or ONOO– and then Ca2+, elicited similar low levels of PTP opening, whereas ONOO– alone was ineffective. In contrast, addition of Ca2+ and then ONOO– induced extensive PTP opening and cytochrome c release. Interestingly, Cu/Zn-superoxide dismutase enhanced pore opening through a mechanism independent of its catalytic activity. These data are consistent with a model in which Ca2+ reveals a molecular target that is now reactive with ONOO–. As a test of this hypothesis, tyrosine nitration was determined in mitochondria exposed to ONOO– alone or to Ca2+ and then ONOO–, and mitochondrial membrane proteins were analyzed using proteomics. These studies suggest protein targets revealed by Ca2+ include dehydrogenases and CoA-containing enzymes. These data are discussed in the context of the role of mitochondria, Ca2+, and ONOO– in apoptotic signaling.

Palladacycles catalyse the oxidation of critical thiols of the mitochondrial membrane proteins and lead to mitochondrial permeabilization and cytochrome c release associated with apoptosis

2008 ◽  
Vol 417 (1) ◽  
pp. 247-256 ◽  
Author(s):  
Débora P. Santana ◽  
Priscila A. Faria ◽  
Edgar J. Paredes-Gamero ◽  
Antonio C. F. Caires ◽  
Iseli L. Nantes ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Cytochrome C ◽  
Disulfide Bonds ◽  
Mitochondrial Membrane ◽  
Apoptotic Cell ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Cytochrome C Release ◽  
Mitochondrial Permeabilization

Permeabilization of the mitochondrial membrane has been extensively associated with necrotic and apoptotic cell death. Similarly to what had been previously observed for B16F10-Nex2 murine melanoma cells, PdC (palladacycle compounds) obtained from the reaction of dmpa (N,N-dimethyl-1-phenethylamine) with the dppe [1,2-ethanebis(diphenylphosphine)] were able to induce apoptosis in HTC (hepatoma, tissue culture) cells, presenting anticancer activity in vitro. To elucidate cell site-specific actions of dmpa:dppe that could respond to the induction of apoptosis in cancer cells in the present study, we investigated the effects of PdC on isolated RLM (rat liver mitochondria). Our results showed that these palladacycles are able to induce a Ca2+-independent mitochondrial swelling that was not inhibited by ADP, Mg2+ and antioxidants. However, the PdC-induced mitochondrial permeabilization was partially prevented by pre-incubation with CsA (cyclosporin A), NEM (N-ethylmaleimide) and bongkreic acid and totally prevented by DTT (dithiothreitol). A decrease in the content of reduced thiol groups of the mitochondrial membrane proteins was also observed, as well as the presence of membrane protein aggregates in SDS/PAGE without lipid and GSH oxidation. FTIR (Fourier-transform IR) analysis of PdC-treated RLM demonstrated the formation of disulfide bonds between critical thiols in mitochondrial membrane proteins. Associated with the mitochondrial permeabilization, PdC also induced the release of cytochrome c, which is sensitive to inhibition by DTT. Besides the contribution to clarify the pro-apoptotic mechanism of PdC, this study shows that the catalysis of specific protein thiol cross-linkage is enough to induce mitochondrial permeabilization and cytochrome c release.

Distribution of protein disulphide isomerase in rat liver mitochondria

2001 ◽  
Vol 356 (2) ◽  
pp. 567-570 ◽  
Author(s):  
Maria Pia RIGOBELLO ◽  
Arianna DONELLA-DEANA ◽  
Luca CESARO ◽  
Alberto BINDOLI
Keyword(s):  
Thioredoxin Reductase ◽  
Immunoblot Analysis ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Specific Marker ◽  
Protein Disulphide Isomerase ◽  
Mitochondrial Functions ◽  
The Matrix ◽  
Molecular Masses

Here we report the localization of protein disulphide isomerase (PDI) in the mitochondrial compartments, comparing it with that of thioredoxin reductase. The latter enzyme is present mostly in the matrix, whereas PDI is located at the level of the outer membrane. We characterize the different submitochondrial fractions with specific marker enzymes. PDI, whether isolated from whole mitochondria or from purified outer membranes, exhibits the same electrophoretic mobility, indicating identical molecular masses. Moreover, immunoblot analysis with monoclonal anti-PDI antibody shows immunoreactivity only with the microsomal PDI, indicating the specificity of the mitochondrial isoform. The significance of these findings is discussed with reference to the potential role of PDI and thioredoxin reductase in regulating the mitochondrial functions dependent on the thiol–disulphide transition.

scholarly journals Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of contact sites

2000 ◽  
Vol 348 (2) ◽  
pp. 343-350 ◽  
Author(s):  
Elena DORAN ◽  
Andrew P. HALESTRAP
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Adenine Nucleotide ◽  
Permeability Transition ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Voltage Dependent Anion Channel ◽  
Cytochrome C Release ◽  
Membrane Rupture ◽  
Contact Sites

Percoll-purified rat liver mitochondria were shown to contain BAX dimer and rapidly (< 2 min) release 5-10% of their cytochrome c when incubated in a standard KCl incubation medium under energized conditions. This release was not accompanied by release of adenylate kinase (AK), another intermembrane protein, and was not inhibited by Mg2+, dATP, inhibitors of the permeability transition or ligands of the peripheral benzodiazepine receptor. However, release was greatly reduced by the presence of 5% (w/v) dextran (40 kDa), which caused a decrease in the light scattering (A520) of mitochondrial suspensions. Dextran also inhibited both mitochondrial oxidation of exogenous ferrocytochrome c in the presence of rotenone and antimycin, and respiratory-chain-driven reduction of exogenous ferricytochrome c. Hypo-osmotic medium or digitonin treatment of mitochondria caused a large additional release of both cytochrome c and AK that was not blocked by dextran. Polyaspartate, which stabilizes the low conductance state of the voltage-dependent anion channel (VDAC), increased cytochrome c release. VDAC and BAX are both found at the contact sites between the inner and outer membranes and dextran is known to stabilize these contact sites in isolated mitochondria. Thus our data suggest that regulation of a specific permeability pathway for cytochrome c may be mediated by changes in protein-protein interactions within contact sites. The adenine nucleotide translocase is known to bind to VDAC and thus provides an additional link between the specific cytochrome c release pathway and the permeability transition.

Quantitation of cytochrome c release from rat liver mitochondria

2003 ◽  
Vol 317 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Elliott D. Crouser ◽  
Martha E. Gadd ◽  
Mark W. Julian ◽  
Jennifer E. Huff ◽  
Kimberly M. Broekemeier ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Cytochrome C Release

scholarly journals Synthetic and natural polyanions induce cytochrome c release from mitochondria in vitro and in situ

2011 ◽  
Vol 300 (5) ◽  
pp. C1193-C1203 ◽  
Author(s):  
Boris F. Krasnikov ◽  
Nickolay S. Melik-Nubarov ◽  
Lubava D. Zorova ◽  
Alevtina E. Kuzminova ◽  
Nickolay K. Isaev ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Mitochondrial Permeability Transition ◽  
Hydrophobic Interactions ◽  
Liver Mitochondria ◽  
Mitochondrial Swelling ◽  
Molar Ratio ◽  
Rat Liver Mitochondria ◽  
Cytochrome C Release

A synthetic polyanion composed of styrene, maleic anhydride, and methacrylic acid (molar ratio 56:37:7) significantly inhibited the respiration of isolated rat liver mitochondria in a time-dependent fashion that correlated with 1) collapse of the mitochondrial membrane potential and 2) high amplitude mitochondrial swelling. The process is apparently Ca2+ dependent. Since it is blocked by cyclosporin A, the process is ascribed to induction of the mitochondrial permeability transition. In mitoplasts, i.e., mitochondria lacking their outer membranes, the polyanion rapidly blocked respiration. After incubation of rat liver mitochondria with the polyanion, cytochrome c was released into the incubation medium. In solution, the polyanion modified by conjugation with fluorescein formed a complex with cytochrome c. Addition of the polyanion to cytochrome c-loaded phosphatidylcholine/cardiolipin liposomes induced the release of the protein from liposomal membrane evidently due to coordinated interplay of Coulomb and hydrophobic interactions of the polymer with cytochrome c. We conclude that binding of the polyanion to cytochrome c renders it inactive in the respiratory chain due to exclusion from its native binding sites. Apparently, the polyanion interacts with cytochrome c in mitochondria and releases it to the medium through breakage of the outer membrane as a result of severe swelling. Similar properties were demonstrated for the natural polyanion, tobacco mosaic virus RNA. An electron microscopy study confirmed that both polyanions caused mitochondrial swelling. Exposure of cerebellar astroglial cells in culture to the synthetic polyanion resulted in cell death, which was associated with nuclear fragmentation.

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