Exclusive activation of glutamate-dependent state 3 respiration of brain mitochondria by extramitochondrial Ca²⁺ in the nanomolar range.

<p>(A,E) Respirograms of rat brain mitochondria were obtained by high-resolution respirometry. (A) Isolated rat brain mitochondria were incubated in EGTA medium (Ca²⁺_free = 0.15 µM) in the presence of 10 mM glutamate and 2 mM malate as substrates. Additions: M, 0.06 mg/ml brain mitochondria, A, 2.5 mM ADP to activate the phosphorylation-related respiration (state 3); Ca²⁺_4,9, 4.9 µM Ca²⁺_free; S, 10 mM succinate as substrate of respiratory chain complex II; C, 5 µM carboxyatractyloside to block the adenine nucleotide translocase. Blue lines indicate the oxygen concentration and red lines represent respiration rates (nmol O₂/mg mitochondrial protein/min). (B) Means of state 3 respiration±S.E. as measured in experiments shown in A without (black columns, n = 6) or with 250 nM RR, an inhibitor of mitochondrial Ca²⁺ uptake (red columns, n = 6). First group of columns, state 3 at Ca²⁺_free = 0.15 µM. Second group, state 3 with Ca²⁺_free = 4.9 µM. Third group, state 3 with Ca²⁺_free = 4.9 µM in the additional presence of 10 µM succinate. *, p<0.05. (C) As B, but derived from experiments with 10 mM pyruvate + 2 mM malate as substrates. *, p<0.05. (D) As B, but derived from experiments with 10 mM succinate + 2 µM rotenone as substrate. (E) Ca²⁺ titration of state 3_glu/mal by stepwise increase of Ca²⁺ as indicated either without (E,F) or with (F) 250 nM RR. (F) Incremental accretions of Ca²⁺-induced state 3_glu/mal were plotted against the fluorimetrically measured Ca²⁺ activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008181#pone-0008181-g001" target="_blank">Fig. 1F</a>), allowing the calculation of the half-activation constant (S_0.5) and the maximum velocity (V_max) using the SigmaPlot kinetic module as given in the text. (G) Rates of state 3_glu/mal respiration obtained by Ca²⁺ titrations under various conditions. (○) Control mitochondria were investigated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008181#pone-0008181-g001" target="_blank">Fig. 1E</a>. (□) As (○), but in the additional presence of 10% dextran 20. (▿) As (○), but in the additional presence of 1 mM CsA. (▵) as (○), but mitochondria isolated without digitonin were used. (◊) as (○), but mitoplasts were used. () as (○), but mitochondria were uncoupled by 50 nM FCCP from the beginning of experiments, and then Ca²⁺ titration was performed. (▴) as (○), but Ca²⁺ was adjusted at the beginning of experiments as indicated. Thereafter, 100 µM ADP was added, causing short transitions between the active and resting states of respiration. After reaching state 4 respiration, FCCP titrations were performed to uncouple respiration and ATP generation. Maximum respiration rates were obtained at 60 or 80 nM FCCP and were plotted against the Ca²⁺_free value for the respective incubation. Data are means±S.E. of 4 independent experiments.</p