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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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Caspase-3 treatment of permeabilized cells causes loss of ΔΨm. (A) MEFs from wild-type or Bid−/− cells were permeabilized with digitonin and incubated in the presence of caspase-3 (0.5 μg/ml), tBid (20 μg/ml), and/or cytochrome c (100 μM) as indicated plus TMRE at 37°C for 30 min in the presence of succinate as substrate. (B–E) Permeabilized HeLa cells (106) were incubated with or without caspase-3 (0.5 μg/ml) in the presence of cytochrome c (100 μM) and TMRE with or without zVAD-fmk (100 μM) or BclXL-Δc (20 μg/ml) as indicated. (B) ΔΨm generated by the incubation of permeabilized cells with the substrates. (C) Effect of caspase-3 on ΔΨm in the presence of malate/palmitate. (D) Effect of caspase-3 on ΔΨm in the presence of rotenone and succinate. (E) Effect of caspase-3 on ΔΨm in the presence of antimycin A and TMPD/ascorbate. Substrates and inhibitors were added at the concentrations described in the legend to Fig. 3 B. Cells were analyzed for ΔΨm by flow cytometry. In each case, the MFI for cells treated with FCCP to dissipate ΔΨm was set as 0.
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fig5: Caspase-3 treatment of permeabilized cells causes loss of ΔΨm. (A) MEFs from wild-type or Bid−/− cells were permeabilized with digitonin and incubated in the presence of caspase-3 (0.5 μg/ml), tBid (20 μg/ml), and/or cytochrome c (100 μM) as indicated plus TMRE at 37°C for 30 min in the presence of succinate as substrate. (B–E) Permeabilized HeLa cells (106) were incubated with or without caspase-3 (0.5 μg/ml) in the presence of cytochrome c (100 μM) and TMRE with or without zVAD-fmk (100 μM) or BclXL-Δc (20 μg/ml) as indicated. (B) ΔΨm generated by the incubation of permeabilized cells with the substrates. (C) Effect of caspase-3 on ΔΨm in the presence of malate/palmitate. (D) Effect of caspase-3 on ΔΨm in the presence of rotenone and succinate. (E) Effect of caspase-3 on ΔΨm in the presence of antimycin A and TMPD/ascorbate. Substrates and inhibitors were added at the concentrations described in the legend to Fig. 3 B. Cells were analyzed for ΔΨm by flow cytometry. In each case, the MFI for cells treated with FCCP to dissipate ΔΨm was set as 0.

Mentions: Caspase-3 can cleave and activate Bid and in this manner trigger mitochondrial outer membrane permeabilization (Li et al., 1998), and it was therefore likely that treatment of digitonin-treated cells with caspase-3 would induce cytochrome c release. Therefore, we compared Bid+/+ and Bid−/− cells to further assess the possible role of tBid in the dissipation of ΔΨm induced by caspase-3. We observed previously that activated Bid (tBid) can induce loss of ΔΨm in digitonin-permeabilized cells, and this was restored by addition of exogenous cytochrome c (Waterhouse et al., 2001b) consistent with the lack of effect of tBid on electron transport function we have observed here (Fig. 3 B). In the experiment shown in Fig. 5 A, caspase-3 induced a loss of ΔΨm but only in cells containing Bid; Bid−/− cells did not show a drop in ΔΨm in response to caspase-3 treatment. This is consistent with our observations that caspase-3 did not act directly on isolated mitochondria to disrupt ΔΨm (Fig. 2) or respiration (Fig. 3). Addition of tBid plus caspase-3 induced a loss of ΔΨm in both wild-type and Bid−/− cells, which was not restored by addition of cytochrome c. Thus, caspase-3 appeared to have two effects: activation of Bid to permeabilize the outer membrane without disrupting ΔΨm, as described (Waterhouse et al., 2001b), and an action on the permeabilized mitochondria to disrupt ΔΨm.


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

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

Caspase-3 treatment of permeabilized cells causes loss of ΔΨm. (A) MEFs from wild-type or Bid−/− cells were permeabilized with digitonin and incubated in the presence of caspase-3 (0.5 μg/ml), tBid (20 μg/ml), and/or cytochrome c (100 μM) as indicated plus TMRE at 37°C for 30 min in the presence of succinate as substrate. (B–E) Permeabilized HeLa cells (106) were incubated with or without caspase-3 (0.5 μg/ml) in the presence of cytochrome c (100 μM) and TMRE with or without zVAD-fmk (100 μM) or BclXL-Δc (20 μg/ml) as indicated. (B) ΔΨm generated by the incubation of permeabilized cells with the substrates. (C) Effect of caspase-3 on ΔΨm in the presence of malate/palmitate. (D) Effect of caspase-3 on ΔΨm in the presence of rotenone and succinate. (E) Effect of caspase-3 on ΔΨm in the presence of antimycin A and TMPD/ascorbate. Substrates and inhibitors were added at the concentrations described in the legend to Fig. 3 B. Cells were analyzed for ΔΨm by flow cytometry. In each case, the MFI for cells treated with FCCP to dissipate ΔΨm was set as 0.
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Related In: Results  -  Collection

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fig5: Caspase-3 treatment of permeabilized cells causes loss of ΔΨm. (A) MEFs from wild-type or Bid−/− cells were permeabilized with digitonin and incubated in the presence of caspase-3 (0.5 μg/ml), tBid (20 μg/ml), and/or cytochrome c (100 μM) as indicated plus TMRE at 37°C for 30 min in the presence of succinate as substrate. (B–E) Permeabilized HeLa cells (106) were incubated with or without caspase-3 (0.5 μg/ml) in the presence of cytochrome c (100 μM) and TMRE with or without zVAD-fmk (100 μM) or BclXL-Δc (20 μg/ml) as indicated. (B) ΔΨm generated by the incubation of permeabilized cells with the substrates. (C) Effect of caspase-3 on ΔΨm in the presence of malate/palmitate. (D) Effect of caspase-3 on ΔΨm in the presence of rotenone and succinate. (E) Effect of caspase-3 on ΔΨm in the presence of antimycin A and TMPD/ascorbate. Substrates and inhibitors were added at the concentrations described in the legend to Fig. 3 B. Cells were analyzed for ΔΨm by flow cytometry. In each case, the MFI for cells treated with FCCP to dissipate ΔΨm was set as 0.
Mentions: Caspase-3 can cleave and activate Bid and in this manner trigger mitochondrial outer membrane permeabilization (Li et al., 1998), and it was therefore likely that treatment of digitonin-treated cells with caspase-3 would induce cytochrome c release. Therefore, we compared Bid+/+ and Bid−/− cells to further assess the possible role of tBid in the dissipation of ΔΨm induced by caspase-3. We observed previously that activated Bid (tBid) can induce loss of ΔΨm in digitonin-permeabilized cells, and this was restored by addition of exogenous cytochrome c (Waterhouse et al., 2001b) consistent with the lack of effect of tBid on electron transport function we have observed here (Fig. 3 B). In the experiment shown in Fig. 5 A, caspase-3 induced a loss of ΔΨm but only in cells containing Bid; Bid−/− cells did not show a drop in ΔΨm in response to caspase-3 treatment. This is consistent with our observations that caspase-3 did not act directly on isolated mitochondria to disrupt ΔΨm (Fig. 2) or respiration (Fig. 3). Addition of tBid plus caspase-3 induced a loss of ΔΨm in both wild-type and Bid−/− cells, which was not restored by addition of cytochrome c. Thus, caspase-3 appeared to have two effects: activation of Bid to permeabilize the outer membrane without disrupting ΔΨm, as described (Waterhouse et al., 2001b), and an action on the permeabilized mitochondria to disrupt ΔΨm.

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