The glutamate-dependent respiration of isolated BM (brain mitochondria) is regulated by Ca2+cyt (cytosolic Ca2+) (S0.5=225±22 nM) through its effects on aralar. We now also demonstrate that the α-glycerophosphate-dependent respiration is controlled by Ca2+cyt (S0.5=60±10 nM). At higher Ca2+cyt (>600 nM), BM accumulate Ca2+ which enhances the rate of intramitochondrial dehydrogenases. The Ca2+-induced increments of state 3 respiration decrease with substrate in the order glutamate>α-oxoglutarate>isocitrate>α-glycerophosphate>pyruvate. Whereas the oxidation of pyruvate is only slightly influenced by Ca2+cyt, we show that the formation of pyruvate is tightly controlled by Ca2+cyt. Through its common substrate couple NADH/NAD+, the formation of pyruvate by LDH (lactate dehydrogenase) is linked to the MAS (malate–aspartate shuttle) with aralar as a central component. A rise in Ca2+cyt in a reconstituted system consisting of BM, cytosolic enzymes of MAS and LDH causes an up to 5-fold enhancement of OXPHOS (oxidative phosphorylation) rates that is due to an increased substrate supply, acting in a manner similar to a ‘gas pedal’. In contrast, Ca2+mit (intramitochondrial Ca2+) regulates the oxidation rates of substrates which are present within the mitochondrial matrix. We postulate that Ca2+cyt is a key factor in adjusting the mitochondrial energization to the requirements of intact neurons.
- intramitochondrial dehydrogenase
- lactate dehydrogenase
- mitochondrial gas pedal
- mitochondrial substrate supply
- pyruvate precursor
The Ca2+-dependent regulation of OXPHOS (oxidative phosphorylation) is one of the major issues of cell biology. According to a generally accepted paradigm, Ca2+cyt (cytosolic Ca2+) enters the matrix space of mitochondria via the Ca2+ uniporter and there activates α-KGDH [α-KG (α-oxoglutarate) dehydrogenase], ICDH [IC (isocitrate) dehydrogenase] and PDH (pyruvate dehydrogenase) [1,2]. However, a model considering activation of these dehydrogenases by Ca2+mit (intramitochondrial Ca2+) does not fully comply with findings made in vivo  and is inconsistent with results of computer simulations . Moreover, several studies have revealed that mitochondria respond to elevated workloads in vivo even if the Ca2+ uniporter is inhibited by RR (Ruthenium Red) [5,6]. On the other hand, it has been shown that Ca2+cyt activates the glutamate/aspartate carriers aralar and citrin [7,8], as well as the mitochondrial α-GPDH [α-GP (α-glycerophosphate) dehydrogenase], all of which are located in the mitochondrial inner membrane [7,8]. Since their regulatory Ca2+-binding sites are exposed to the mitochondrial intermembrane space, these enzymes can sense Ca2+cyt. Aralar is the key component of the MAS (malate–aspartate shuttle) , responsible for the Ca2+cyt-dependent transport of reducing hydrogen generated by NADH from the cytosol into mitochondria [7,8]. Similarly, α-GPDH is the central enzyme of the α-glycerophosphate shuttle transporting electrons to ubiquinone . The consequences of these Ca2+-dependent mechanisms for regulation of OXPHOS in brain mitochondria have not previously been addressed.
Recently, we discovered that in BM (brain mitochondria) the rate of OXPHOS can be increased reversibly by elevation of Ca2+cyt within the nanomolar range in the presence of glutamate and malate as substrates [9–11]. In contrast, Ca2+cyt exerts a low stimulatory effect on OXPHOS in the presence of pyruvate/malate, and no effect at all with succinate [9–11]. For this reason, the Ca2+cyt-specific regulation of OXPHOS was attributed to the activation of aralar [9–11].
The main metabolic fuels of BM are the pyruvate precursors lactate and glucose [12,13] and to a small extent glycerol . Each precursor can be metabolized into pyruvate by the involvement of the oxidizing enzymes GAPDH (glyceraldehyde-3-phosphate dehydrogenase), LDH (lactate dehydrogenase) or α-GPDH . These oxidations, however, can occur only if NAD+ is regenerated by the MAS.
In the present study we hypothesized that metabolic coupling of the MAS-mediated transport of reducing hydrogen into mitochondria to the formation of pyruvate enables the cell to control the substrate supply to BM by Ca2+cyt. Therefore the aims of the present study were (i) to assess in detail the contributions of intra- and extra-mitochondrial Ca2+ to the regulation of OXPHOS in BM, and (ii) to investigate the influence of Ca2+cyt on the substrate supply of isolated BM in the presence of the complete MAS, as reconstructed by the involvement of lactate and the pyruvate-generating enzyme LDH in the medium. We investigated kinetically the mitochondrial oxidation of glutamate/malate, α-GP, pyruvate/malate, α-KG/malate and IC/malate. Measurements were performed at low (11 nM) Ca2+cyt and compared with those at 700 nM Ca2+cyt which was high enough to allow Ca2+ to accumulate in the BM.
We found that in isolated BM the Ca2+cyt stimulation of state 3Glu/Mal exceeds the effect of Ca2+mit on state 3 with other substrate pairs α-KG/malate, IC/malate and pyruvate/malate. Whereas oxidation of pyruvate is only slightly influenced by Ca2+cyt, we show that the formation of pyruvate via LDH is tightly controlled by Ca2+cyt. A rise in Ca2+cyt in a reconstituted system consisting of BM, cytosolic enzymes of the MAS [GOT (glutamate oxaloacetate transaminase) and MDH (malate dehydrogenase)] and LDH causes an up to 5-fold enhancement of OXPHOS rates because an increased substrate supply acts in a manner similar to a ‘gas pedal’. In contrast, at low Ca2+cyt the pyruvate substrate supply to BM was greatly reduced. Lower mitochondrial membrane potential (ΔΨ) and lower rates of glutamate-dependent respiration were observed at diminished Ca2+cyt, indicating the occurence of substrate-limited states in vitro.
Thus it is envisaged that Ca2+cyt acts as a key factor regulating the pyruvate supply to BM through regulation of the MAS, and that this mechanism, together with the regulation of intramitochondrial substrate oxidation rates, controls the energization of BM.
BM, containing synaptosomal and non-synaptosomal fractions, were isolated from 3–4-month-old mice . The isolation and incubation medium did not contain BSA. Before final suspension, the mitochondrial Ca2+ content was routinely reduced by two extractions with nitriloacetic acid . All research and animal care procedures were performed according to European guidelines.
Mitochondrial respiration was measured with a Clark-type oxygen electrode and high-resolution respirometry [17,18] using an OROBOROS Oxygraph-2k instrument at 30°C. Respiration of mitochondria (0.06 mg of protein/ml) was measured in an EGTA medium containing 120 mM mannitol, 40 mM Mops, 5 mM KH2PO4, 60 mM KCl, 5 mM MgCl2 and 1 mM EGTA (pH 7.4). The Ca2+ concentration in the medium (Ca2+cyt) was adjusted either by up to six sequential Ca2+ additions (each of 200 μM) or, alternatively, by one single Ca2+ addition (640 μM) into the EGTA medium. In both cases, free Ca2+cyt was verified by Fura-2 measurements. The following mixtures were used as substrates: 10 mM glutamate+2 mM malate, 10 mM α-GP, 10 mM pyruvate+2 mM malate, 10 mM α-KG+2 mM malate and 10 mM IC+2 mM malate. In some experiments, substrate concentrations were varied as indicated.
Measurement of Ca2+free in EGTA medium
Ca2+free in the medium was measured fluorimetrically after appropriate Ca2+ additions to EGTA medium containing 2 mM ADP and 0.06 mg of BM/ml, using Fura-2 (10 μM) as described previously . The dissociation constant (Kd) of the Ca2+–Fura-2 complex was assumed to be 0.19 μM .
Mitochondrial membrane potential (ΔΨ) measurements
ΔΨ was monitored by extramitochondrial safranine (10 μM) fluorescence , at 495 nm excitation and 586 nm emission, with a Cary Eclipse fluorimeter (Varian) in stirred and thermostatically controlled (30°C) cells. Measurements were performed in EGTA medium with isolated mitochondria (0.25 mg of protein/ml) and additions as indicated.
Mitochondrial protein concentrations were determined by the bicinchoninic acid assay  with BSA as a standard.
Substrate-dependent OXPHOS of mouse BM
According to Figures 1(A)–1(E), respirometric measurements of isolated mouse BM were performed with various substrates (glutamate/malate, α-GP, pyruvate/malate, α-KG/malate, IC/malate) and stepwise increases of Ca2+cyt. All experiments were performed at saturating substrate concentrations (10 mM) except malate (2 mM). Incubations were made in the presence of EGTA (1 mM) in order to adjust basal Ca2+cyt concentrations in the low nanomolar range. The addition of 2 mM ADP to BM, with glutamate and malate as substrates (Figure 1A), resulted in a very low rate of state 3Glu/Mal respiration, since glutamate uptake of BM via aralar was not activated at such a low Ca2+cyt as 11 nM. A stepwise increase in Ca2+cyt within the nanomolar range caused a 5-fold rise in state 3Glu/Ma with S0.5=225±22 nM of Ca2+cyt. A further increase in Ca2+cyt caused a noteworthy inhibition of state 3Glu/Mal due to mitochondrial Ca2+ overload.
Next, we investigated the effect of Ca2+cyt on the oxidation of α-GP, which donates electrons through flavoprotein-linked α-GPDH of the inner mitochondrial membrane to CoQ in BM. As illustrated in Figure 1(B), the level of state 3α-GP respiration was very low at 11 nM Ca2+cyt, but cumulative addition of Ca2+ led to an 8-fold stimulation as compared with the basal state before Ca2+ addition. No inhibition of state 3α-GP respiration at elevated Ca2+cyt was detectable. Thus, in contrast with state 3Glu/Mal, the regulation of state 3α-GP was characterized by higher sensitivity to Ca2+cyt (S0.5=60±10 nM), by a lower Vmax (Figure 1B) and by a higher stability of state 3α-GP at elevated Ca2+cyt.
Figure 1(C) shows that pyruvate oxidation was almost fully activated already at the basal Ca2+cyt of 11 nM, as any further increase in Ca2+cyt caused a slight stimulation of state 3Pyr/Mal respiration only, whereas at larger Ca2+cyt concentrations increasing inhibition occurred.
The state 3α-KG/Mal respiration was lower than state 3Pyr/Mal respiration (Figure 1D) but, remarkably, was also stimulated strongly by Ca2+cyt. Finally, state 3IC/Mal was lower than state 3α-KG/mal, although the extent of Ca2+cyt activation was larger than in the case of state 3α-KG/Mal. Clearly, the activation of OXPHOS by Ca2+cyt exhibits a substrate-specific nature.
Next, we measured the free Ca2+cyt concentration in incubations as used in Figure 1(A) (see the legend) in dependence on the added total Ca2+cyt (Figure 1F). Measurements were performed either with or without mitochondria in order to obtain the information at which Ca2+cyt concentration the mitochondria start to accumulate Ca2+. Up to a Ca2+total concentration of 600 μM (reached in the third Ca2+ addition), the free Ca2+cyt was similar in the two kinds of incubations, although there was already a tendency towards slightly decreased Ca2+cyt levels in those containing mitochondria. With higher Ca2+total (at 700 nM), the Ca2+cyt differences became larger and significant, indicating that isolated BM are able to take up Ca2+cyt under these conditions. However, we did not observe an inhibition of state 3, therefore we used this Ca2+cyt (700 nM) for comparison of the substrate-specific state 3 activation by Ca2+cyt and Ca2+mit.
Next, we measured the total capacity of complex I-dependent respiration of isolated BM using three substrates: 10 mM pyruvate, 10 mM glutamate and 2 mM malate (state 3Glu/Pyr/Mal=253±10 nmol of O2/min per mg) at 700 nM Ca2+cyt (Table 1). It was found that the state 3Pyr/Mal (89%) makes up the largest part of total complex I-dependent respiration, followed by state 3α-KG/Mal (63%), state 3Glut/Mal (60%) and state 3IC/Mal (52%) (Table 1).
Kinetic analysis of Ca2+cyt-dependent activation of substrate-specific OXPHOS
In order to study Ca2+cyt effects on the kinetics of mitochondrial substrate oxidation, the respective substrate concentrations were varied in the presence of low (10 nM) and elevated (700 nM) Ca2+cyt concentrations (Figures 1G–1K). We did not use higher Ca2+cyt levels, as we wished to avoid mitochondrial Ca2+ overload. At both Ca2+ levels used, the rate of glutamate oxidation increased continuously with rising substrate concentrations (Figure 1G). However, Vmax of state 3Glu/Mal was approximately 3-fold higher at 700 nM Ca2+cyt (152±4 nmol of O2/mg per min) than at 10 nM Ca2+cyt (56±2 nmol of O2/mg per min) (Table 1). In contrast with the marked increase in Vmax, the Km for glutamate remained unaffected by Ca2+cyt (Km,11 nM Ca2+=620±62 μM, Km,700 nM Ca2+=640±34 μM; Table 1). Inhibition of the mitochondrial Ca2+ uptake via the uniporter by RR did not affect the kinetics of OXPHOS at 700 nM Ca2+cyt (Figure 1G), confirming our earlier observation that Ca2+ activation of state 3Glu/Mal is exclusively an extramitochondrial phenomenon [9–11].
Effects of Ca2+cyt on the shape of α-GP titration curves (Figure 1H) were completely different from those seen with glutamate (Figure 1G). At 11 nM Ca2+cyt, state 3α-GP started to increase only after the application of high α-GP concentrations (>4 mM) owing to a large Km (Km,11 nM Ca2+=12.7±0.4 mM). Increasing Ca2+cyt to 700 nM resulted in a substantial decrease in the Km for α-GP (Km,700 nM Ca2+=2.1±0.2 mM) without any effect on Vmax (Figure 1H and Table 1). Since the Ca2+ activation of state 3α-GP was not altered by RR (Figure 1H), the α-GP-dependent OXPHOS must also be regulated exclusively by Ca2+cyt in BM.
As illustrated in Figure 1(I), Ca2+cyt had only a minor, but nevertheless statistically significant, effect on the kinetics of pyruvate utilization in brain mitochondria: Vmax was increased by 16%, i.e. from 194±3 to 225±11 nmol of O2/mg per min, whereas the affinity for the substrate was not affected by Ca2+ (Km,11 nM Ca2+=30±3 μM, Km,700 nM Ca2+=29±8 μM; Table 1). Confirming previous findings , the Km value for pyruvate was at least one order of magnitude lower than for all the other mitochondrial substrates tested in the present study (Table 1) and corresponds to an estimated cytosolic pyruvate concentration range in the low micromolar range , thus underlining the role of pyruvate as a preferred substrate for BM.
The kinetic analysis of mitochondrial α-KG oxidation (Figure 1J) revealed that Ca2+cyt did significantly stimulate state 3α-KG/Mal under saturating substrate concentrations, i.e. from 95±4 to 159±5 nmol of O2/mg per min (Table 1). This change was parallelled by a decrease in Km for α-KG from 1373±98 μM to 403±25 μM at 700 nM Ca2+cyt. In the presence of RR, a clear shift of Ca2+-dependent state 3α-KG respiration towards higher substrate concentrations was observed (Figure 1J). This finding underlines the idea that Ca2+cyt has to be accumulated by BM before it can activate the α-KGDH, an observation that is in agreement with results of earlier studies by Denton and McCormack [1,2].
Using the mitochondrial substrate pair IC/malate, a clear Ca2+cyt-dependent stimulation of respiration, characterized by significantly increased Vmax and decreased Km values, was observed (Table 1 and Figure 1K). Analogous to state 3α-KG/Mal, both parameters were markedly affected by RR. On the other hand, RR was not able to reverse completely the activation by Ca2+cyt of α-KG- and IC-dependent respiration.
Ca2+cyt-induced activation of state 3Glu/Mal: greater than for all other substrates
In order to compare the absolute extent of Ca2+cyt activation for various mitochondrial substrates, the increments between stimulated and non-stimulated respiration rates (Figures 1G–1K) were calculated and plotted against the respective substrate concentrations (Figure 1L). The largest increase in Ca2+cyt-dependent state 3 respiration was found with glutamate/malate (+120 nmol of O2/mg per min, 100%) followed by α-KG/malate (+76 nmol of O2/mg per min, 63%) and isocitrate/malate (+56 nmol of O2/mg per min, 47%), whereas the stimulation in the presence of all other substrates was clearly lower (state 3α-GP, +39 nmol of O2/mg per min (32%); state 3Pyr/Mal, +24 nmol of O2/mg per min (20%). Ca2+ activation of state 3Glu/Mal respiration increased continuously with substrate dose and reached its maximum at the highest glutamate concentration tested in the present study (Figure 1L). In contrast, stimulation of state 3α-KG/Mal and state 3α-GP were highest at intermediate substrate concentrations and then decreased, giving the response curve a bell-shaped profile. The stimulation by Ca2+ is a consequence of a Ca2+-induced decrease in the respective Michaelis constants (Table 1).
MAS reconstitution studies
The next experiments were designed to reconstitute the complete MAS and to check to what extent its function and its ability to provide pyruvate for BM are controlled by Ca2+cyt. As illustrated in Figure 2(A), the complete MAS can be reconstituted by incubation of isolated BM with the purified enzymes GOT, MDH and LDH. From the scheme it is evident that operation of the MAS can be launched by addition of either glutamate/malate or α-KG/aspartate. In the presence of LDH, lactate and NADH, the MAS is coupled to pyruvate formation through LDH and therefore ensures a pyruvate supply to fuel mitochondrial respiration. In this system, pyruvate supply should be amplified secondarily by Ca2+cyt through its primary activating effect on aralar.
A typical experiment aimed at checking such a function of Ca2+cyt is shown in Figure 2(B). After pre-incubation of isolated BM in an EGTA-containing medium in the presence of LDH and its substrates lactate (5 mM) and NADH (100 μM), as well as GOT and MDH, 2 mM of ADP was added to induce state 3 respiration. The rate of the latter process was negligible in the absence of glutamate and remained very low even after the addition of glutamate because aralar was not activated by basal Ca2+cyt (11 nM) (Figure 2B, upper trace). After Ca2+cyt addition (230 nM) did we observe a substantial increase in the state 3Glu/Mal respiration rate. In contrast, Ca2+cyt failed to activate state 3 respiration in the second incubation when no glutamate was added (Figure 2B, lower trace). After stationary rates of state 3 respiration had been attained, 10 mM pyruvate was added so as to reach the same maximum rates of complex I-dependent state 3 respiration in both incubations. Plotting the rates of state 3 respiration measured in similar experiments against Ca2+cyt concentrations revealed that the largest stimulation of state 3 occurred in the low nanomolar Ca2+cyt concentration range (<500 nM). This is most probably caused by activation of aralar, causing a secondary activation of pyruvate supply by the complete MAS (Figure 2C). At higher Ca2+cyt levels (>500 nM), mitochondrial Ca2+ accumulation allowed the additional activation of the matrix dehydrogenases PDH, α-KGDH and ICDH by Ca2+mit. At sufficiently high Ca2+cyt, the complete MAS reached maximum efficacy and the final pyruvate additions did not further increase the rate of respiration (Figure 2C). The respiratory rate under these conditions was, with 252±22 nmol of O2/mg per min, clearly higher than with glutamate/malate alone (Table 1) indicating that pyruvate oxidation is included. This point of view was further supported by incubations without LDH where the rates of respiration were significantly decreased (results not shown). In further incubations without glutamate no pyruvate could be formed by LDH (no cytosolic NAD+ regeneration) and, owing to the missing hydrogen transport into mitochondria, no Ca2+ stimulation could be observed.
Figure 2(D) shows the experiments aimed at testing the assumption that if the complete MAS supplies the BM with reducing hydrogen and pyruvate, an inhibition of the mitochondrial pyruvate uptake should substantially reduce the rate of respiration. Indeed, when BM were incubated under conditions of complete MAS activation (achieved by the addition of 5 mM α-KG plus 5 mM aspartate), the addition of cinnamate (which is an inhibitor of the mitochondrial pyruvate uptake), caused a 53% decrease in the state 3 respiration rate (Figure 2D, black trace) which was statistically significant (Figure 2E). The observed effects of cinnamate were not related to altered control by adenylates, as CAT (carboxyatractyloside), a blocker of ANT (adenine nucleotide translocase), effectively reduced the rate of ADP-dependent respiration. Clearly, the cinnamate-inhibition-induced shift from the MAS in its complete state, up-regulated by Ca2+, to an incomplete MAS resulted in a strongly reduced ability of the MAS to energize the mitochondria specifically due to suppression of pyruvate supply. Thus these results demonstrate that (i) through activation of MAS Ca2+cyt strongly up-regulates the rate of pyruvate supply, but not the mitochondrial ability to oxidize substrate, and that (ii) the capacity of the complete MAS is sufficient to support maximum rates of complex I-dependent oxidative phosphorylation.
Control of state 3Glu/Mal and state 4Glu/Mal via ΔΨ of isolated BM by Ca2+cyt
After having investigated the role of Ca2+cyt and the MAS in the regulation of BM energization, we asked whether the low state 3Glu/Mal respiration observed at basal Ca2+cyt (11 nM) was indeed caused by a limited supply of substrate to the BM. In this case, it can be predicted that the ΔΨ of BM oxidizing glutamate/malate will be significantly decreased at low Ca2+cyt. To test this hypothesis, mitochondrial respiration rates (Figures 3A and 3B) and the fluorescence of the ΔΨ indicator safranine (Figures 3C and 3D) were measured in parallel at various levels of OXPHOS activation, adjusted by alterations in the concentrations of ADP and Ca2+. BM were exposed to glutamate (10 mM) and malate (2 mM) at low Ca2+cyt (11 nM; Figure 3, black lines and black bars) or high Ca2+cyt (700 nM; Figure 3, grey lanes and grey bars). After the addition of ADP (200 μM), a transient activation of respiration (intermediate state, i) was observed, which, however, became higher and shorter-lasting after the Ca2+cyt was increased to 700 nM (Figures 3A and 3B). The subsequent addition of ADP at a saturating concentration (2 mM), allowed the adjustment of the active state 3Glu/Mal (a), the level of which was twice as high in the presence of 700 nM Ca2+cyt than with 11 nM Ca2+cyt (Figures 3A and 3B). The subsequent increase in Ca2+cyt from 11 nM to 700 nM enhanced the state 3Glu/Mal to a level similar to that at permanently high (700 nM) Ca2+cyt (Figure 3A).
Parallel measurements of safranin fluorescence revealed a consistently higher fluorescence signal and, accordingly, a lower ΔΨ at 11 nM Ca2+cyt compared with that at 700 nM Ca2+cyt (Figures 3C and 3D). After the increase in Ca2+cyt to 700 nM, the safranine fluorescence of BM fell immediately to the same value as obtained in the parallel measurement at 700 nM Ca2+cyt (Figure 3C). This means that the rise in Ca2+cyt promoted an increase in ΔΨ. As expected, the subsequent addition of the mitochondrial uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone; 1 μM) resulted in a ΔΨ collapse. The corresponding fluorescence signal was used for calculation of ΔΨ values. This analysis revealed significantly diminished ΔΨ values for each metabolic state of glutamate/malate-dependent respiration, i.e. at resting, intermediate and active states at 11 nM and 700 nM Ca2+cyt (Figure 3D).
Collectively, these data suggest that Ca2+cyt activates mitochondrial substrate supply in a concentration-dependent manner, a process that in turn builds up a higher ΔΨ and thereby regulates OXPHOS.
Regulation of OXPHOS by Ca2+cyt and by Ca2+mit
We have previously shown that increased Ca2+cyt causes a 2-fold activation of the glutamate-dependent respiration of rat BM [9–11]. The present study shows that under optimized experimental conditions, including the routine extraction of Ca2+ with nitriloacetic acid during the preparation of mitochondria  and increasing the Ca2+cyt from 11 nM to 1300 nM, a 5-fold reinforcement of state 3Glu/Mal (Figure 1A) can be observed. At the same time, the Km of glutamate oxidation of BM remained constant, similar to that observed for heart mitochondria . The half-activation constant for this Ca2+ activation (S0.5=225±25 nM Ca2+cyt) corresponds fairly well to the normal range of Ca2+cyt in neurons (50–300 nM) [25,26]. The influence of Ca2+cyt on OXPHOS is fully reversible, as chelation of Ca2+cyt with EGTA suppresses the glutamate-dependent respiration . These observations point to the physiological relevance of regulation of mitochondrial function by fluctuations of Ca2+cyt in vivo.
The present study shows that state 3α-GP is also regulated by Ca2+cyt (Figures 1B and 1G and Table 1). Like aralar, the mitochondrial α-GPDH can be activated by Ca2+cyt [27,28] through its regulatory Ca2+-binding site in the intermembrane space . However, the low Vmax of state 3α-GP and the very high Km for α-GP (2.1–12.7 mM, Table 1) suggest that the importance of mitochondrial α-GP oxidation in the brain is not high. It appears more likely that, analogous to the oxidation of lactate to pyruvate by LDH, α-GP is preferentially oxidized by cytosolic α-GPDH (Km=400 μM)  to form the pyruvate precursor DHAP (dihydroxyacetone phosphate).
Oxidation of pyruvate/malate, the most important substrate of BM, was slightly, but significantly, activated by Ca2+cyt (+16%, Figures 1C, 1I and 1L, and Table 1); as the Km for pyruvate did not change, this effect was due to increased Vmax (Table 1).
In contrast, the oxidation of α-KG/malate was found to be largely dependent on Ca2+cyt, as the Vmax clearly increased (by 67%), together with enhancement of the affinity of α-KGDH towards α-KG in response to increased Ca2+cyt (Figures 1D and 1J, and Table 1). Also the oxidation rates of IC/malate were remarkably activated by Ca2+cyt (by +55%) combined with an increased affinity of ICDH to IC (Figures 1K and 1L, and Table 1). As shown in Figure 1(L), the stimulation by Ca2+ of state 3α-KG/Mal and state 3IC/Mal respiration revealed a bell-shaped characteristic, as it was considerably elevated at decreasing substrate concentrations down from 10 mM reaching a maximum activation at 1 mM (Figure 1J), which is the same as previous findings in heart mitochondria by McCormack and Denton .
Similar to observations made by Denton and McCormack , we also found that in the presence of RR the Ca2+cyt effects were clearly diminished, indicating that the oxidation of α-KG/malate and IC/malate is related to intramitochondrial effects of Ca2+, which accelerates the α-KGDH and ICDH reactions in the tricarboxylic acid cycle. On the other hand, RR did not completely suppress the Ca2+cyt-induced activations, although under such conditions Ca2+ should not be taken up by BM . The reason for RR-independent activation in oxidation of α-KG is not clear. RR inhibits all presently known Ca2+-uptake pathways, including uniporter , Ram [31,32], RyR (ryanodine receptor)-sensitive Ca2+ transporter  and Letm 1  (see  for a review). Therefore the RR-insensitive state 3 activation recorded suggests the existence of hitherto unknown RR-independent Ca2+cyt-mediated signalling or of a non-specific Ca2+ entry into the matrix space; in either case it requires further investigation.
For a long time, the activation of intramitochondrial dehydrogenases by Ca2+mit was the only known mechanism underlying the stimulation by Ca2+ of mitochondrial substrate consumption (Figure 4) [1,2]. However, since it has become evident that Ca2+cyt regulates the rates of aralar [7,8] and GPDH [7,27] independently of the accumulation of Ca2+ ions into the matrix, a new additional mechanism must be considered for the regulation of mitochondrial energization through the substrate supply to mitochondria [7–11,36,37]. Indeed, before matrix dehydrogenases can be activated, Ca2+cyt has to be accumulated by mitochondria. The Ca2+mit, however, only speeds up the oxidation of those substrates (α-KG, IC and pyruvate) which are already present in the matrix space. In contrast, the activation of aralar by Ca2+cyt enhances the rate of substrate supply and transport into mitochondria, thus acting similarly to a ‘gas’ pedal in the car.
To compare the role and quantitative importance of the two mechanisms, we performed Ca2+ titrations of respiration in BM, starting at 11 nM Ca2+cyt (Figures 1A–1E). From the measurements of free Ca2+cyt concentrations in these incubations a significant mitochondrial Ca2+ uptake was detected at ≥700 nM free Ca2+cyt (Figure 1F). This finding agrees with results reported by Chalmers and Nicholls , who established a threshold of 500 nM Ca2+cyt for Ca2+ uptake by BM. Therefore we can assume that the Ca2+ titrations from 500 to 700 nM in Figures 1(A)–1(E) meet the conditions where matrix Ca2+ increases sufficiently to activate fully the intramitochondrial dehydrogenases. Consequently, we can rule out any possibility that the relatively low levels of activation are caused by artificial conditions where the Ca2+mit is not sufficiently increased.
We found that the Ca2+ activation of substrate oxidation was at its highest when we used glutamate/malate under saturating conditions (5–20 mM; Figure 1L); this corresponds to physiological glutamate levels in the cytosol of neurons . The highest Ca2+-activations of α-KG- and IC-dependent respiration rates were only slightly lower (63% and 52% respectively) than that of glutamate/malate, but occurred at non-saturating substrate concentration (1 mM). In contrast, the extent of maximal Ca2+ stimulation of the other substrate oxidations tested (α-GP, pyruvate/malate) was lower (Figure 1L) Therefore we conclude that the Ca2+-controlled energization of isolated BM is realized mainly by aralar, operating through Ca2+cyt, as well as by α-KGDH and ICDH, both operating through Ca2+mit (Figure 4).
In all of our Ca2+-titration experiments with complex I-dependent substrates (Figures 1A and 1C–1E) an increasing level of inhibition was observed at the highest Ca2+cyt concentrations. These inhibitions are probably caused by opening of the permeability transition pore since CsA (cyclosporin A) can nearly completely abolish the Ca2+-induced inhibition of state 3 respiration performed under similar conditions . The decreasing respiratory rates at permeability transition pore opening have been shown to be connected with a CsA-sensitive release of NAD+/NADH  explaining why, after the addition of NADH, the respiratory rates increased again and why the succinate-dependent respiration is not so much affected by permeability transition . In agreement with that conclusion, the α-GP-dependent respiration is also not inhibited by excess Ca2+ (Figure 1B).
The complete MAS acts as a ‘gas pedal’
The present study was undertaken to test our hypothesis that Ca2+cyt exerts two effects on the energy metabolism of BM: primary stimulation of the activity of the MAS and secondary enhancement of the rate of cytosolic pyruvate generation. Two lines of evidence support this hypothesis. (i) Reconstitution experiments with BM and the complete MAS (including the pyruvate formation catalysed by LDH) in the presence of excess ADP revealed a clear dependence of the state 3 respiration on Ca2+cyt (Figure 2C). The most pronounced stimulation of state 3 respiration occurred in the Ca2+cyt concentration range of <500 nM. If pyruvate was added at the end of such incubations, a further increase in state 3 respiration was observed, indicating that Ca2+cyt mainly up-regulates the rates of pyruvate formation without exerting an influence on the total capacity of BM to oxidize pyruvate. At higher Ca2+cyt (>500 nM), the BM began to substantially accumulate Ca2+; it follows that the stimulation of the state 3 respiration in this concentration range was mainly caused by Ca2+mit operating as an activator of substrate oxidation through α-KGDH, ICDH and PDH [1,2]. (ii) Under conditions where the complete MAS is functioning, the decrease in respiration by 56% under the influence of cinnamate, a potent inhibitor of the pyruvate transporter , directly demonstrated that the enhanced substrate supply capacity of the complete MAS stems from the parallel formation of pyruvate.
The complete MAS is characterized by certain energetic advantages. (i) By coupling of aralar activity to extramitochondrial pyruvate production it ensures pyruvate regeneration which is dependent on Ca2+cyt concentration. This mechanism is required in vivo to ensure a continuous respiratory substrate supply, mostly by glycolysis. (ii) Whereas only one NADH molecule is generated intramitochondrially per molecule of glutamate and malate transported into mitochondria by the simple MAS (without pyruvate formation), the complete MAS supplies, in addition, one pyruvate molecule, which is then able to produce five NADH/FADH2 molecules. (iii) The complete MAS is able to supply the BM with substrates sufficiently to attain the maximum complex I-dependent state 3 respiration (Table 1). (iv) Both the mitochondrial uptake of reducing hydrogen through the MAS and the pyruvate uptake are driven by the electrochemical proton gradient which ‘pumps’ the substrates into the mitochondria. Therefore the uptake does not need the respective substrate gradients. All of these properties are prerequisites for the use of the complete MAS as a Ca2+cyt-controlled ‘gas pedal’.
Although the functional coupling of the MAS to pyruvate-generating reactions has been known for a long time , the MAS function has been assessed in several studies under conditions which do not allow parallel pyruvate formation to take place [8,44,45]. Studies of the hydrogen transport capacity of the MAS without parallel pyruvate formation (the incomplete MAS)  have shown that elevated Ca2+cyt triggers Ca2+mit accumulation via the Ca2+ uniporter, which in turn activates α-KGDH. The activated α-KGDH competes increasingly with the MAS for α-KG. As a consequence, MAS activity is inhibited by a reduced export of α-KG from mitochondria . However, this phenomenon is probably only detectable in the absence of cytosolic pyruvate formation, and this is therefore a non-physiological condition.
Mitochondrial energization should be adjusted to metabolic needs in order to avoid possible negative consequences of permanent activation or even over-energization of mitochondria. Such a negative consequence as an increased ROS (reactive oxygen species) formation [45–48] could be possibly avoided if a decreasing Ca2+cyt diminishes the pyruvate supply to BM.
The present study raises an important question about the cell-type-specificity of the presence and role of the MAS. The experiments described in the present paper were performed with mitochondria that had been isolated from total mouse brains and therefore consisted of nearly equal amounts of neuronal and non-neuronal mitochondria . It is known that glial cells contain much less, or even none, aralar than do neurons [50–52]; consequently, the MAS activity is also low in, or absent from, glial cells [50–52]. Therefore an inhibition of the MAS cannot influence the oxidative metabolism in intact astrocytes. This can be taken as indirect evidence of the existence of the α-glycerophosphate shuttle in these cells [52–54]. Moreover, Pellerin et al.  assumed that glia cells preferentially produce lactate from glucose (aerobic glycolysis). This lactate leaves the glia cells via a monocarboxylate carrier and can be accumulated by neurons (astrocyte–neuron lactate shuttle) . Neurons convert the lactate into pyruvate, since they have an active MAS coupled to pyruvate formation. This point of view is further supported by experiments performed in our group showing that the state 3Glu/Mal of astrocytes is low and cannot be stimulated by Ca2+cyt (Z. Gizatullina and F. N. Gellerich, unpublished work). Therefore, despite the fact that we performed our measurements in a mixture of neuronal and non-neuronal mitochondria, it is reasonable to conclude that the MAS is most probably a property of neurons, but not of glial cells.
Although we have shown that the ‘gas pedal’ described above also regulates the energization of mitochondria in other tissues such as skeletal muscle (E. Seppet and F.N. Gellerich, unpublished work), it appears to play an especially important role in BM, which, being unable to oxidize fatty acids , rely exclusively on pyruvate oxidation.
Zemfira Gizatullina was involved in all experiments and discussions. Sonata Trumbekaite performed experiments exploring the kinetic of substrate oxidation. Timur Gaynutdinov performed the Ca2+ measurements. Bernard Korzeniewski contributed conceptionally to the MAS experiments and wrote the paper. Frank-Norbert Gellerich, Enn Seppet, Frank Striggow, Stefan Vielhaber and Hans-Jochen Heinze were involved in the conceptual work and wrote the paper.
This work was supported by the Federal Ministry of Trade and Commerce [project Mitoscreen number IWO 072052] and the Foundation of Medical Science (F.N.G.); the “Excellence programme” of the state of Sachsen–Anhalt and the Foundation of Medical Science (Z.G.); the DZNE joint project and the Foundation of Medical Science (S.V.); the Estonian Ministry of Education and Research [grant number SF0180114As08] and the Estonian Science Foundation [grant numbers 7117 and 7823] (E.S.); the DAAD (German Academic Exchange Service) (S.T. and T.G.); the Federal Ministry of Education and Research [BMBF grant numbers 0315638C and 03IS2211I] (F.S.).
We thank Veronica Wöllner and Ellen Fröhlich for skilful technical assistance, Katja Zschibsch for respirometric measurements, and Doreen Jerzembeck and Aurelius Zimkus for measuring the mitochondrial membrane potential.
Abbreviations: BM, brain mitochondria; Ca2+cyt, cytosolic Ca2+; Ca2+mit, intramitochondrial Ca2+; CAT, carboxyatractyloside; CsA, cyclosporin A; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GOT, glutamate oxaloacetate transaminase; α-GP, α-glycerophosphate; α-GPDH, α-GP dehydrogenase; IC, isocitrate; ICDH, IC dehydrogenase; α-KG, α-oxoglutarate; α-KGDH, α-KG dehydrogenase; LDH, lactate dehydrogenase; MAS, malate–aspartate shuttle; MDH, malate dehydrogenase; OXPHOS, oxidative phosphorylation; PDH, pyruvate dehydrogenase; RR, Ruthenium Red
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