Figure 6.

Exogenous cytochrome c failed to reverse inhibition of respiratory function caused by MOMP. 293T BimS-ER/GFP cells were collected at various time points after treatment with vehicle (control), zVAD-fmk, or OHT plus zVAD-fmk. Cells were then treated with a low concentration of digitonin to permeabilize the plasma membrane, while the mitochondrial membranes remained intact (see Materials and Methods). (A) Representative oxygen consumption curves. Maximal respiration (in the presence of glutamate/malate and FCCP) was followed before and after the addition of exogenous cytochrome c in control, zVAD, or zVAD/OHT-treated digitonin-permeabilized cells. As a positive control, N/C-Bid (14 nM) was added to a separate sample of zVAD-treated cells 30 min after digitonin incubation. Numbers under the curves indicate respiratory rates (nmol O₂/min/10⁶ cells). As expected, respiration in the control cells was not stimulated by exogenous cytochrome c, as MOMs were intact. In contrast, N/C-Bid caused permeabilization of the MOM, and exogenous cytochrome c was therefore able to gain access to respiratory complexes to stimulate respiration. (B) Summary of effects of cytochrome c on respiration, expressed as a percentage of the maximal increase expected, based on the measured percentage of cells having undergone MOMP. Exogenous cytochrome c partially restored respiration 4 h after OHT addition, but not at later times. Data show the average change in the rate of maximal respiration after addition of exogenous cytochrome c, measured 4, 8, 24, and 72 h after zVAD/OHT treatment. Error bars, SD from three to five independent experiments.