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Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis.

Ricci JE, Gottlieb RA, Green DR - J. Cell Biol. (2003)

Bottom Line: Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II.Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment.Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

View Article: PubMed Central - PubMed

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

ABSTRACT
During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (Delta Psi m) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, Delta Psi m generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

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ROS production in the presence of malate/palmitate or succinate is enhanced by caspase-3. Jurkat cells (106) were permeabilized and incubated in the presence of dihydroethidium (2 μM) and caspase-3 (0.5 μg/ml), tBid (20 μg/ml), or Bcl-xL-ΔC (20 μg/ml) for 30 min at 37°C. (A) Control; (B) in the presence of malate/palmitate, and (C) in the presence of succinate. Then the ROS production was monitored by flow cytometry. Note that the scales differ for each condition.
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fig6: ROS production in the presence of malate/palmitate or succinate is enhanced by caspase-3. Jurkat cells (106) were permeabilized and incubated in the presence of dihydroethidium (2 μM) and caspase-3 (0.5 μg/ml), tBid (20 μg/ml), or Bcl-xL-ΔC (20 μg/ml) for 30 min at 37°C. (A) Control; (B) in the presence of malate/palmitate, and (C) in the presence of succinate. Then the ROS production was monitored by flow cytometry. Note that the scales differ for each condition.

Mentions: One consequence of a caspase-mediated disruption in electron transport may be the zVAD-fmk–inhibitable generation of ROS discussed above (Fig. 1). Therefore, we examined if substrates for complex I (Fig. 6 B) or complex II (Fig. 6 C) drive caspase-dependent ROS generation in digitonin-permeabilized Jurkat cells. Addition of substrates for complexes I or II fueled the production of ROS in untreated mitochondria, and this was not increased by treatment with tBid. In contrast, treatment with caspase-3 (with or without addition of recombinant tBid) resulted in significant ROS production with either substrate (Fig. 6, B and C) (but not without substrates; Fig. 6 A). The increase was inhibited by Bcl-xL-ΔC, probably via inhibition of the caspase-activated, Bid-mediated permeabilization of the mitochondrial outer membrane as discussed above. Therefore, it is likely that the caspase-mediated disruption of complex I and complex II function contributes to high ROS production during apoptosis. This would account for the effect of caspase inhibition on apoptosis-associated ROS generation we observed in the experiment in Fig. 1.


Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis.

Ricci JE, Gottlieb RA, Green DR - J. Cell Biol. (2003)

ROS production in the presence of malate/palmitate or succinate is enhanced by caspase-3. Jurkat cells (106) were permeabilized and incubated in the presence of dihydroethidium (2 μM) and caspase-3 (0.5 μg/ml), tBid (20 μg/ml), or Bcl-xL-ΔC (20 μg/ml) for 30 min at 37°C. (A) Control; (B) in the presence of malate/palmitate, and (C) in the presence of succinate. Then the ROS production was monitored by flow cytometry. Note that the scales differ for each condition.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172744&req=5

fig6: ROS production in the presence of malate/palmitate or succinate is enhanced by caspase-3. Jurkat cells (106) were permeabilized and incubated in the presence of dihydroethidium (2 μM) and caspase-3 (0.5 μg/ml), tBid (20 μg/ml), or Bcl-xL-ΔC (20 μg/ml) for 30 min at 37°C. (A) Control; (B) in the presence of malate/palmitate, and (C) in the presence of succinate. Then the ROS production was monitored by flow cytometry. Note that the scales differ for each condition.
Mentions: One consequence of a caspase-mediated disruption in electron transport may be the zVAD-fmk–inhibitable generation of ROS discussed above (Fig. 1). Therefore, we examined if substrates for complex I (Fig. 6 B) or complex II (Fig. 6 C) drive caspase-dependent ROS generation in digitonin-permeabilized Jurkat cells. Addition of substrates for complexes I or II fueled the production of ROS in untreated mitochondria, and this was not increased by treatment with tBid. In contrast, treatment with caspase-3 (with or without addition of recombinant tBid) resulted in significant ROS production with either substrate (Fig. 6, B and C) (but not without substrates; Fig. 6 A). The increase was inhibited by Bcl-xL-ΔC, probably via inhibition of the caspase-activated, Bid-mediated permeabilization of the mitochondrial outer membrane as discussed above. Therefore, it is likely that the caspase-mediated disruption of complex I and complex II function contributes to high ROS production during apoptosis. This would account for the effect of caspase inhibition on apoptosis-associated ROS generation we observed in the experiment in Fig. 1.

Bottom Line: Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II.Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment.Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

View Article: PubMed Central - PubMed

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

ABSTRACT
During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (Delta Psi m) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of Delta Psi m and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, Delta Psi m generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of Delta Psi m) and generate ROS through effects of caspases on complex I and II in the electron transport chain.

Show MeSH
Related in: MedlinePlus