Permeabilization of the mitochondrial outer mitochondrial membrane, regulated by Bcl-2 family members, is an integral event during apoptosis. Upon permeabilization, cytochrome c is released to facilitate formation of the apoptosome and caspase activation. The mechanism of outer membrane permeabilization remains controversial, and the consequences of this event on mitochondrial and cellular metabolism—other than activation of caspases—are not well understood. In this paper, we have shown that after the permeabilization of the mitochondrial outer membrane, ΔΨm transiently decreases. When caspase activation is blocked, ΔΨm then recovers and is maintained by the low levels of cytochrome c diffuse throughout the cell. In cells that have released cytochrome c in the absence of caspase activation, the maintenance of ΔΨm is sufficient for the mitochondria to generate ATP.
Several hypotheses for the mechanism of mitochondrial outer membrane permeabilization predict an increase (hyperpolarization) or decrease (permeability transition) in ΔΨm before the permeabilization event. However, in single-cell analysis, using cytochrome c–GFP as an indicator of mitochondrial outer membrane permeabilization and TMRE to follow ΔΨm, we never observed a loss of ΔΨm before cytochrome c release (). Similarly, hyperpolarization did not occur uniformly before cytochrome c release and was only observed in a small subset of cells. In support of these observations, neither cytochrome c release nor the apoptotic response was affected by the addition of uncouplers that dissipate ΔΨm and thereby prevent any hyperpolarization (). Indeed, the presence of these uncouplers in no way affected the function of proapoptotic or antiapoptotic Bcl-2 family members (). These data preclude a requirement for large-scale changes (either an increase or decrease) in ΔΨm as a general mechanism for mitochondrial outer membrane permeabilization during apoptosis.
Although there were no significant changes in ΔΨm before cytochrome c release, in our single-cell experiments we observed a caspase-independent loss of ΔΨm shortly after cytochrome c release (). The loss of ΔΨm after cytochrome c release that we observed in single-cell analysis appeared to contrast with our results obtained by bulk cell analysis, which showed that ΔΨm was maintained after cytochrome c release (; Bossy-Wetzel et al. 1998
). This paradox was resolved by the observation that in the presence of a caspase inhibitor or in the absence of Apaf-1 (which prevents caspase activation through the apoptosome), ΔΨm regenerated during the 60 min after cytochrome c release and sustained subsequently (). Since permeabilization of the mitochondrial outer membrane occurs at different times in different cells, this loss and recovery of ΔΨm appears as an overall maintenance of ΔΨm at the bulk cell level.
We found that after cytochrome c release, the ΔΨm was maintained by the electron transport chain rather than by reversal of the ATP synthase (). In digitonin-treated cells, 10 μM cytochrome c was sufficient to maintain electron transport after permeabilization of the mitochondrial outer membrane during apoptosis (). We calculated that, if cytochrome c was evenly dispersed throughout the Cc-GFP-HeLa cells, it would result in a concentration of ~10 μM. This is consistent with previous estimations that the concentration of cytochrome c in the intermembrane space is between 0.5 and 5 mM (Forman and Azzi 1997
). Since the intermembrane space represents ~10% of the mitochondria and mitochondria can occupy 10–30% of the total cell volume, we can estimate that the final concentration of cytochrome c throughout a cell would range between 5 and 150 μM.
Although there is sufficient cytochrome c to maintain ΔΨm after permeabilization of the mitochondrial outer membrane, we observe a transient depolarization that then recovers in the absence of caspase activity. The mechanism of this depolarization is unknown. One possibility is based on changes in respiration in response to ADP. At high ADP concentrations, mitochondria increase respiration (state 3), resulting in a decrease in ΔΨm. As the ADP is converted to ATP, the ratio of ADP/ATP decreases, and mitochondria enter state 4, with decreased respiration and increased ΔΨm.
Therefore, the drop in ΔΨm we observe after cytochrome c release may represent a shift from state 4 to 3 respiration, induced by a sudden increase in ADP/ATP. This would be followed by an increase in ΔΨm as the ADP is converted to ATP. This is consistent with a model suggesting that the voltage-dependent anion channel closes during apoptosis, and therefore ATP accumulates in the mitochondria, whereas ADP accumulates in the cytosol (Vander Heiden et al. 1999
, Vander Heiden et al. 2000
). Upon outer membrane permeabilization, ADP levels at the mitochondria would therefore suddenly increase. However, for these transitions to state 3 and 4 to occur, both electron transport and the function of the F0/F1 ATPase are required. Since oligomycin, which inhibits the F0/F1 ATPase, had no effect on either the drop or recovery of ΔΨm after cytochrome c release (), this explanation is unlikely. Another possibility is a transient opening of the adenosine nucleotide translocator, which temporarily dissipates ΔΨm. However, cyclosporin A (≤400 μM), which delays adenosine nucleotide translocator opening (Crompton et al. 1998
), had no effect on the kinetics or extent of the depolarization or the recovery of ΔΨm after cytochrome c release (data not shown). A third possibility is that the decrease in cytochrome c concentration results in a transient production of reactive oxygen species that temporarily disrupt electron transport. Cytochrome c, at the high levels found in mitochondria, is a potent antioxidant (Forman and Azzi 1997
), and this effect may be compromised as the levels drop upon outer membrane permeabilization.
Although we do not know the underlying mechanism of this ΔΨm depolarization, the effects of temperature on this phenomenon are potentially informative. We have previously reported that the release of cytochrome c from the mitochondria is rapid and temperature independent. That is, reduction of the temperature to 24°C did not affect the interval between initial and complete release of cytochrome c (Goldstein et al. 2000
). However, we found that dissipation of ΔΨm to 10% of initial levels in cells incubated at 24°C took more than twice as long as cells incubated at 37°C. This prolonged kinetics suggests that temperature-dependent enzymatic effects are involved in the process by which ΔΨm is lost but do not play a role in cytochrome c release.
In addition to maintenance of ΔΨm, we found that in the absence of caspase activation ATP synthesis by the mitochondria (i.e., oligomycin-sensitive maintenance of ATP levels) continues in cells despite mitochondrial outer membrane permeabilization (). These data show that even though the outer mitochondrial membrane has been permeabilized, the mitochondrial inner membrane remains intact, the electron transport chain and ATP synthase remain functional, and the cytochrome c within the cytosol is sufficient to drive oxidative phosphorylation. These conclusions are supported by previous studies that show that isolated mitochondria treated with tBid maintain their ability to import protein after cytochrome c release (von Ahsen et al. 2000
Various aspects of mitochondrial metabolism have been reported to impact on the apoptotic process. Apoptosis is an active (ATP-requiring) process and, if sufficient ATP is not present, the cell death deviates from an apoptotic to a necrotic phenotype (Eguchi et al. 1997
; Nicotera et al. 1998
). The proton gradient across the mitochondrial inner membrane is essential for the production of ATP via oxidative phosphorylation, and a reduction in this gradient will impact on the potential of the cell to produce ATP and survive. Recent studies have reported that sympathetic neurons can be rescued from caspase-independent cell death, even after mitochondrial outer membrane permeabilization and cytochrome c release, however, recovery was not possible after ΔΨm had dissipated (Deshmukh et al. 2000
). Similarly, we observe an eventual loss of ΔΨm even in the presence of caspase inhibitors, and this precedes a drop in ATP and death of the cell (; data not shown). Before this drop, however, it remains possible that during the period over which ΔΨm is maintained, nonneuronal cells may have the potential to recover after cytochrome c release. This is consistent with the recent observations that defects in Apaf-1 or caspase-9, which should act downstream of mitochondrial outer membrane permeabilization, can contribute to oncogenic transformation (Soengas et al. 1999
In conclusion, our results suggest that before or in the absence of caspase activation, mitochondria can maintain several functions, including the generation of ATP, and may contribute to survival of the cells for prolonged periods after cytochrome c release.