Mitochondrial respiratory control is lost during growth factor deprivation

Abstract

The ability of cells to maintain a bioenergetically favorable ATP/ADP ratio confers a tight balance between cellular events that consume ATP and the rate of ATP production. However, after growth factor withdrawal, the cellular ATP/ADP ratio declines. To investigate these changes, mitochondria from growth factor-deprived cells isolated before the onset of apoptosis were characterized in vitro. Mitochondria from growth factor-deprived cells have lost their ability to undergo matrix condensation in response to ADP, which is accompanied by a failure to perform ADP-coupled respiration. At the time of analysis, mitochondria from growth factor-deprived cells were not depleted of cytochrome c and cytochrome c-dependent respiration was unaffected, demonstrating that the inhibition of the respiratory rate is not due to loss of cytochrome c. Agents that disrupt the mitochondrial outer membrane, such as digitonin, or maintain outer membrane exchange of adenine nucleotide, such as Bcl-x(L), restored ADP-dependent control of mitochondrial respiration. Together, these data suggest that the regulation of mitochondrial outer membrane permeability contributes to respiratory control.

Figure 1

Mitochondria isolated from IL-3-deprived cells respond to reducing substrates but not to ADP. Cells were maintained in the presence or absence of IL-3 for 12 h. Mitochondria were isolated and placed in a reaction buffer either alone (no substrates = state 1) or together with succinate (+succinate = state 4), or succinate and ADP (+succinate +ADP = state 3) for 5 min followed by fixation and EM analysis. The dark regions (higher electron density) correspond to the matrix.

Figure 2

Mitochondria from IL-3-deprived cells display a reduction in ADP-coupled respiration. (A) Cells were maintained in the presence or absence of IL-3 for 12 h followed by mitochondria isolation. Mitochondria (500 μg) were diluted into reaction buffer containing rotenone and succinate, and oxygen levels were recorded. State 4 respiration was recorded for 200 s followed by the addition of ADP to induce state 3 respiration. (B) The rate of oxygen consumption, in states 4 and 3, of mitochondria isolated from control (+IL-3) or IL-3-deprived (-IL-3) cells. The results are the average and standard deviation of 3 independent experiments.

Figure 3

Cytochrome c loss does not account for the decrease in RCR after IL-3 withdrawal. (A) Cytochrome c-dependent respiration was measured using mitochondria from cells growing in the presence or absence of IL-3 for 12 h. After isolation, mitochondria (500 μg) were diluted into reaction buffer in the presence of antimycin A. Where indicated, TMPD and ascorbate, as electron donors for cytochrome c, were added to the chamber. (B) The RCR of succinate-mediated respiration of mitochondria isolated from control (+IL-3) or IL-3-deprived (−IL-3) cells in the presence or absence of exogenous cytochrome c. Mitochondria were prepared and analyzed as in Fig. 2. (C Upper) Mitochondria from cells growing in the presence of IL-3 were isolated and incubated with or without tBid for 5 min. (Lower) tBid-treated mitochondria were analyzed in the presence or absence of exogenous cytochrome c. After adding rotenone and succinate, samples were analyzed in the respirometer as in Fig. 2. (D) The RCR of mitochondria treated or untreated with tBid, with or without exogenous cytochrome c as shown in C.

Figure 4

Digitonin causes disruption of the outer mitochondrial membrane but does not cause a significant release of cytochrome c. (A) Mitochondria were isolated from control (+IL-3) or IL-3-deprived (−IL-3) cells and maintained in the isolation buffer (MIB) without substrates. Where indicated, digitonin was added and mitochondria were treated as described in Materials and Methods and subjected to EM analysis. Arrows indicate different types of lesions to the outer membrane including 1) membrane pinching, 2) small gaps, 3) large gaps. (B) Cytochrome c (cyt c) levels and cytochrome c oxidase (COX IV) levels, as a loading control, were assessed by Western blot analysis of mitochondria isolated from cells growing in the presence or absence of IL-3 for 12 h with or without digitonin treatment.

Figure 5

Digitonin treatment restores ADP response to mitochondria isolated from IL-3-deprived cells. Mitochondria were isolated from cells growing in the presence or absence of IL-3 for 12 h. Mitochondria were either treated or untreated with digitonin and analyzed in the respirometer as described in Fig. 2. Respiration was performed in the absence of exogenously added cytochrome c. (A) Mitochondria from control (+IL-3) cells. (B) Mitochondria from cells deprived of IL-3 for 12 h. (C) RCR of mitochondria isolated from cells growing in the presence or absence of IL-3 and incubated with or without digitonin. The results are the average and standard deviation of 3 independent experiments.

Figure 6

Recovery of uncoupled respiration of mitochondria from IL-3-deprived cells by a membrane permeant electron donor and prevention of the decrease in coupled respiration by Bcl-x_L expression. (A) Respiration analysis of uncoupled mitochondria by using duroquinol as an electron donor. Mitochondria (500 μg) from control or IL-3-deprived cells were incubated in the reaction buffer in the presence of rotenone and carbonylcyanide m-chlorophenlyhydrazone. Where indicated, 100 μM duroquinol (DQH2) was added to the chamber. (B) FL5.12 cells transfected with control vector (Neo) or with vector encoding Bcl-x_L (XL) were incubated in the presence or absence of IL-3 for 12 h. Mitochondria (500 μg) were diluted into reaction buffer in the presence of rotenone and oxygen consumption was measured. State 1 respiration (no substrate) was recorded for 100 seconds followed by the addition of succinate. ADP was added 200 s later to induce state 3 respiration.