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Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process.

Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD, Green DR - J. Cell Biol. (2001)

Bottom Line: After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production.Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled.These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.

ABSTRACT
During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.

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Apoptosis and cytochrome c release proceed in the absence of hyperpolarization. (A) Jurkat cells were treated with etoposide (40 μM) or actinomycin D (500 nM), and Cc-GFP-HeLa cells were treated with UV (180 mJ/cm2) or actinomycin D (1 μM) in the presence or absence of FCCP (5 μM) for the times indicated. The cells were analyzed for phosphatidylserine exposure as a measure of apoptosis. (B) Similar cells to those assayed in A were analyzed for cytochrome c release (Bi) by western blotting (Jurkat) or cytochrome c–GFP release (Bii) by flow cytometry (Cc-GFP-HeLa). (C) Untreated Cc-GFP-HeLa cells or Cc-GFP-HeLa cells treated with UV (180 mJ/cm2) in the presence or absence of the concentrations of FCCP indicated were harvested at 6 h and assayed for phosphatidylserine exposure (annexin V-FITC binding) or cytochrome c–GFP release (CLAMI assay) by flow cytometry.
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Figure 6: Apoptosis and cytochrome c release proceed in the absence of hyperpolarization. (A) Jurkat cells were treated with etoposide (40 μM) or actinomycin D (500 nM), and Cc-GFP-HeLa cells were treated with UV (180 mJ/cm2) or actinomycin D (1 μM) in the presence or absence of FCCP (5 μM) for the times indicated. The cells were analyzed for phosphatidylserine exposure as a measure of apoptosis. (B) Similar cells to those assayed in A were analyzed for cytochrome c release (Bi) by western blotting (Jurkat) or cytochrome c–GFP release (Bii) by flow cytometry (Cc-GFP-HeLa). (C) Untreated Cc-GFP-HeLa cells or Cc-GFP-HeLa cells treated with UV (180 mJ/cm2) in the presence or absence of the concentrations of FCCP indicated were harvested at 6 h and assayed for phosphatidylserine exposure (annexin V-FITC binding) or cytochrome c–GFP release (CLAMI assay) by flow cytometry.

Mentions: Uncouplers such as FCCP, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and 2,4, dinitrophenol (DNP) dissipate the transmembrane potential by preventing the generation of a proton gradient. We examined the effects of FCCP on cytochrome c release and apoptosis in Jurkat cells and Cc-GFP-HeLa cells treated with various inducers of apoptosis. In no case did dissipation of the transmembrane potential block, diminish, or delay cytochrome c release or apoptosis and often slightly increased sensitivity to proapoptotic agents (Fig. 6). Similar results were observed when CCCP or DNP were used in place of FCCP (data not shown).


Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process.

Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD, Green DR - J. Cell Biol. (2001)

Apoptosis and cytochrome c release proceed in the absence of hyperpolarization. (A) Jurkat cells were treated with etoposide (40 μM) or actinomycin D (500 nM), and Cc-GFP-HeLa cells were treated with UV (180 mJ/cm2) or actinomycin D (1 μM) in the presence or absence of FCCP (5 μM) for the times indicated. The cells were analyzed for phosphatidylserine exposure as a measure of apoptosis. (B) Similar cells to those assayed in A were analyzed for cytochrome c release (Bi) by western blotting (Jurkat) or cytochrome c–GFP release (Bii) by flow cytometry (Cc-GFP-HeLa). (C) Untreated Cc-GFP-HeLa cells or Cc-GFP-HeLa cells treated with UV (180 mJ/cm2) in the presence or absence of the concentrations of FCCP indicated were harvested at 6 h and assayed for phosphatidylserine exposure (annexin V-FITC binding) or cytochrome c–GFP release (CLAMI assay) by flow cytometry.
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Related In: Results  -  Collection

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Figure 6: Apoptosis and cytochrome c release proceed in the absence of hyperpolarization. (A) Jurkat cells were treated with etoposide (40 μM) or actinomycin D (500 nM), and Cc-GFP-HeLa cells were treated with UV (180 mJ/cm2) or actinomycin D (1 μM) in the presence or absence of FCCP (5 μM) for the times indicated. The cells were analyzed for phosphatidylserine exposure as a measure of apoptosis. (B) Similar cells to those assayed in A were analyzed for cytochrome c release (Bi) by western blotting (Jurkat) or cytochrome c–GFP release (Bii) by flow cytometry (Cc-GFP-HeLa). (C) Untreated Cc-GFP-HeLa cells or Cc-GFP-HeLa cells treated with UV (180 mJ/cm2) in the presence or absence of the concentrations of FCCP indicated were harvested at 6 h and assayed for phosphatidylserine exposure (annexin V-FITC binding) or cytochrome c–GFP release (CLAMI assay) by flow cytometry.
Mentions: Uncouplers such as FCCP, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and 2,4, dinitrophenol (DNP) dissipate the transmembrane potential by preventing the generation of a proton gradient. We examined the effects of FCCP on cytochrome c release and apoptosis in Jurkat cells and Cc-GFP-HeLa cells treated with various inducers of apoptosis. In no case did dissipation of the transmembrane potential block, diminish, or delay cytochrome c release or apoptosis and often slightly increased sensitivity to proapoptotic agents (Fig. 6). Similar results were observed when CCCP or DNP were used in place of FCCP (data not shown).

Bottom Line: After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production.Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled.These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.

ABSTRACT
During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.

Show MeSH
Related in: MedlinePlus