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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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Oxygen consumption in apoptotic cells. (A) Jurkat cells were treated for 18 h with etoposide (10 μM) or staurosporine (0.05 μM) in the presence or absence of 100 μM of zVAD-fmk as indicated. The cells were then permeabilized with digitonin. The equivalent of 400 μg of protein was loaded into respiratory chambers and oxygen consumption in the presence of different substrates and inhibitors (malate/palmitate for complex I, rotenone with succinate for complex II, antimycin A with TMPD/ascorbate for cytochrome c/complex IV) was measured as in the legend to Fig. 3 B. (B) HeLa cells were treated with staurosporine (0.05 μM) for 18 h, then treated with digitonin, and analyzed for oxygen consumption as in A.
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fig8: Oxygen consumption in apoptotic cells. (A) Jurkat cells were treated for 18 h with etoposide (10 μM) or staurosporine (0.05 μM) in the presence or absence of 100 μM of zVAD-fmk as indicated. The cells were then permeabilized with digitonin. The equivalent of 400 μg of protein was loaded into respiratory chambers and oxygen consumption in the presence of different substrates and inhibitors (malate/palmitate for complex I, rotenone with succinate for complex II, antimycin A with TMPD/ascorbate for cytochrome c/complex IV) was measured as in the legend to Fig. 3 B. (B) HeLa cells were treated with staurosporine (0.05 μM) for 18 h, then treated with digitonin, and analyzed for oxygen consumption as in A.

Mentions: Oxygen consumption in apoptotic cells followed a similar pattern. Jurkat cells were treated with etoposide or staurosporine to induce apoptosis. The cells were then digitonin-permeabilized to provide access of substrates to the mitochondria. We found that oxygen consumption in the presence of complex I or complex II substrates was destroyed by the apoptotic process (Fig. 8 A). This effect was caspase dependent, as it was blocked by the caspase inhibitor zVAD-fmk. In contrast, oxygen consumption by complex IV remained largely intact after caspase activation. However, there was a small but reproducible drop in complex IV activity that was seen in this assay, and this was also blocked by zVAD-fmk. This small decrease in complex IV activity (versus large decreases in those of complex I and II) was similarly seen in apoptotic HeLa cells (Fig. 8 B). This small caspase-dependent effect is likely to be indirect, based on our results in isolated mitochondria (Fig. 3 D) or may involve caspases other than caspase-3. Further, this may represent a small decrease in oxygen consumption without a decrease in ΔΨm, since ΔΨm did not decrease under the same conditions (Fig. 7). Similar results were obtained in HeLa cells treated with staurosporine to induce apoptosis (Fig. 8 B). Again, oxygen consumption in the presence of complex I or complex II substrates was destroyed by the apoptotic process, although the function of complex IV remained largely intact.


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

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

Oxygen consumption in apoptotic cells. (A) Jurkat cells were treated for 18 h with etoposide (10 μM) or staurosporine (0.05 μM) in the presence or absence of 100 μM of zVAD-fmk as indicated. The cells were then permeabilized with digitonin. The equivalent of 400 μg of protein was loaded into respiratory chambers and oxygen consumption in the presence of different substrates and inhibitors (malate/palmitate for complex I, rotenone with succinate for complex II, antimycin A with TMPD/ascorbate for cytochrome c/complex IV) was measured as in the legend to Fig. 3 B. (B) HeLa cells were treated with staurosporine (0.05 μM) for 18 h, then treated with digitonin, and analyzed for oxygen consumption as in A.
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fig8: Oxygen consumption in apoptotic cells. (A) Jurkat cells were treated for 18 h with etoposide (10 μM) or staurosporine (0.05 μM) in the presence or absence of 100 μM of zVAD-fmk as indicated. The cells were then permeabilized with digitonin. The equivalent of 400 μg of protein was loaded into respiratory chambers and oxygen consumption in the presence of different substrates and inhibitors (malate/palmitate for complex I, rotenone with succinate for complex II, antimycin A with TMPD/ascorbate for cytochrome c/complex IV) was measured as in the legend to Fig. 3 B. (B) HeLa cells were treated with staurosporine (0.05 μM) for 18 h, then treated with digitonin, and analyzed for oxygen consumption as in A.
Mentions: Oxygen consumption in apoptotic cells followed a similar pattern. Jurkat cells were treated with etoposide or staurosporine to induce apoptosis. The cells were then digitonin-permeabilized to provide access of substrates to the mitochondria. We found that oxygen consumption in the presence of complex I or complex II substrates was destroyed by the apoptotic process (Fig. 8 A). This effect was caspase dependent, as it was blocked by the caspase inhibitor zVAD-fmk. In contrast, oxygen consumption by complex IV remained largely intact after caspase activation. However, there was a small but reproducible drop in complex IV activity that was seen in this assay, and this was also blocked by zVAD-fmk. This small decrease in complex IV activity (versus large decreases in those of complex I and II) was similarly seen in apoptotic HeLa cells (Fig. 8 B). This small caspase-dependent effect is likely to be indirect, based on our results in isolated mitochondria (Fig. 3 D) or may involve caspases other than caspase-3. Further, this may represent a small decrease in oxygen consumption without a decrease in ΔΨm, since ΔΨm did not decrease under the same conditions (Fig. 7). Similar results were obtained in HeLa cells treated with staurosporine to induce apoptosis (Fig. 8 B). Again, oxygen consumption in the presence of complex I or complex II substrates was destroyed by the apoptotic process, although the function of complex IV remained largely intact.

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