Bistability of Mitochondrial Respiration Underlies Paradoxical Reactive Oxygen Species Generation Induced by Anoxia
Figure 4
Experimental observations of bistability in mitochondrial electron transport and their simulation in the model.
(A) shows the registration of ROS accumulation (traces “ros”) and membrane potential (traces “mp”) and in the suspension of isolated rat brain mitochondria incubated with 5 mM of succinate in the absence (blue traces) and in the presence of 1mM ADP (red traces). In the absence of ADP mitochondria are in high ROS producing state; the presence of ADP switches them to low ROS producing mode. Within the first 3 min initially present ADP is completely converted into ATP; the completion of ATP synthesis could be seen as an increase of membrane potential measured as the quenching of safranin O fluorescence (red trace “mp,ADP”). After the transformation of ADP into ATP and return to the state 4 of respiration, ROS production remained low (red trace “ros,ADP”). Under the conditions where ADP was absent (blue traces), 1 mM of ATP together with 1 uM of oligomycin was added and it did not decrease the rate of ROS production. The reason for addition of oligomycin together with ATP was to avoid effect of small amounts of ADP, which are usually present in preparations of ATP (blue traces). Thus, as the model predicts (Figures 3B and 3C), under the same conditions there exist two different states of ROS production. The complete set of experiments is presented in Text S1. (B) The experiments similar to that shown in (A) performed for various succinate concentrations revealed that two branches of steady state ROS production existed for the same experimental conditions in accordance with the model's prediction (Figures 3B and 3C). Upper branch (asterisks) corresponds to the measured initial rate of ROS production in state 4 (blue curve “ros” in Figure 4A), then 1 mM of ADP was added and, after 3 min of incubation with ADP, oligomycin was added to assure the transition back to state 4 of respiration. The temporal ATP synthesis switched the respiratory chain into low ROS production mode persisted after return to state 4 of respiration. The rate measured in this state (corresponded to the red trace “ros,ADP” in Figure 4A) constituted the lower branch (squares). Lines are the model simulations where the relative succinate concentration is transformed into mM using a scaling factor (the same for all points), chosen so that the threshold for stepwise shift to high ROS production in the upper branch in simulation corresponds to the one obtained experimentally. The succinate dehydrogenase is considered here as a Michaelis' function of not only Q, but also succinate (see eq (17) in “Methods”). Upper steady states were reached in the model when initially the system was in a high ROS producing state, lower steady states were reached when initially the system was in a low ROS producing state.