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.

INTRODUCTION

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 [3] and is inconsistent with results of computer simulations [4]. 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) [7], 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 [7]. 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 [911]. In contrast, Ca2+cyt exerts a low stimulatory effect on OXPHOS in the presence of pyruvate/malate, and no effect at all with succinate [911]. For this reason, the Ca2+cyt-specific regulation of OXPHOS was attributed to the activation of aralar [911].

The main metabolic fuels of BM are the pyruvate precursors lactate and glucose [12,13] and to a small extent glycerol [14]. 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 [13]. 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.

EXPERIMENTAL

Mitochondria

BM, containing synaptosomal and non-synaptosomal fractions, were isolated from 3–4-month-old mice [15]. 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 [16]. All research and animal care procedures were performed according to European guidelines.

Respirometry

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 [10]. The dissociation constant (Kd) of the Ca2+–Fura-2 complex was assumed to be 0.19 μM [19].

Mitochondrial membrane potential (ΔΨ) measurements

ΔΨ was monitored by extramitochondrial safranine (10 μM) fluorescence [20], 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.

Protein determination

Mitochondrial protein concentrations were determined by the bicinchoninic acid assay [21] with BSA as a standard.

RESULTS

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.

Substrate-specific effects of Ca2+cyt on the active respiration of isolated BM

Figure 1
Substrate-specific effects of Ca2+cyt on the active respiration of isolated BM

(AE) Ca2+ titration of substrate-specific rates of state 3 respiration. BM (0.06 mg/ml) isolated under Ca2+-depletion conditions [16,33] were incubated in EGTA medium (Ca2+cyt=11 nM) in the presence of (A) 10 mM glutamate+2 mM malate, (B) 10 mM α-GP, (C) 10 mM pyruvate+2 mM malate, (D) 10 mM α-KG+2 mM malate, and (E) 10 mM IC+2 mM malate. Additions indicated: ADP, 2 mM ADP; Ca2+, 200 μM Ca2+total was added at each arrow (in this way the free Ca2+cyt was increased stepwise from 11 nM to 110 nM, 291 nM, 601 nM, 1364 nM, 1500 nM and, finally, 1600 nM). Each addition of Ca2+ was made when a constant rate of respiration had been reached. Respiration was measured with OROBOROS high-resolution oxygraphs [11,17,18]. Thin lines, oxygen concentration; thick lines, rate of mitochondrial oxygen consumption in nmol of O2/min per mg of mitochondrial protein. Representative measurements from greater than six experiments are shown. (F) Fluorimetrically measured Ca2+free in incubations as used in (A) with (○) and without (▽) mitochondria. Data are means±S.E.M. for n>16 individual measurements. The inset shows the data points between 600 and 800 nM Ca2+free at higher magnification. (GL) Kinetics of substrate oxidation by BM at low and elevated Ca2+cyt. BM (0.06 mg/ml) were incubated in EGTA medium without Ca2+ addition (○, Ca2+cyt=11 nM) or in the presence of 700 nM Ca2+cyt (Δ,□), without (□) or with (Δ) RR (250 nM). Experiments were started by the addition of 2 mM ADP, followed by substrate titrations, (G) 0.1–32 mM glutamate at constant malate (2 mM), (H) 0.4–72 mM α-GP, (I) 0.01–32 mM pyruvate and 2 mM malate, (J) 0.1–32 mM α-KG and 2 mM malate, and (K) 0.1–32 mM IC and 2 mM malate. Rates of state 3 respiration were plotted against substrate concentrations. Data are means±S.E.M. for n>6 experiments. Arrows indicate a Ca2+-induced change of kinetic properties of substrate oxidation caused by increased Vmax (↑). Note that, in contrast with the other substrates, pyruvate oxidation started at 10 μM pyruvate. (L) Substrate-specific differences between Ca2+cyt-induced increments of state 3 respiration. Ca2+ stimulation of state 3 respiration was calculated by subtraction of respiratory rates measured in the presence of 11 nM Ca2+cyt from state 3 rates obtained with 700 nM Ca2+cyt for the substrates glutamate/malate (▽), α-KG/malate (○), α-GP (Δ), pyruvate/malate (□) and isocitrate/malate ().

Figure 1
Substrate-specific effects of Ca2+cyt on the active respiration of isolated BM

(AE) Ca2+ titration of substrate-specific rates of state 3 respiration. BM (0.06 mg/ml) isolated under Ca2+-depletion conditions [16,33] were incubated in EGTA medium (Ca2+cyt=11 nM) in the presence of (A) 10 mM glutamate+2 mM malate, (B) 10 mM α-GP, (C) 10 mM pyruvate+2 mM malate, (D) 10 mM α-KG+2 mM malate, and (E) 10 mM IC+2 mM malate. Additions indicated: ADP, 2 mM ADP; Ca2+, 200 μM Ca2+total was added at each arrow (in this way the free Ca2+cyt was increased stepwise from 11 nM to 110 nM, 291 nM, 601 nM, 1364 nM, 1500 nM and, finally, 1600 nM). Each addition of Ca2+ was made when a constant rate of respiration had been reached. Respiration was measured with OROBOROS high-resolution oxygraphs [11,17,18]. Thin lines, oxygen concentration; thick lines, rate of mitochondrial oxygen consumption in nmol of O2/min per mg of mitochondrial protein. Representative measurements from greater than six experiments are shown. (F) Fluorimetrically measured Ca2+free in incubations as used in (A) with (○) and without (▽) mitochondria. Data are means±S.E.M. for n>16 individual measurements. The inset shows the data points between 600 and 800 nM Ca2+free at higher magnification. (GL) Kinetics of substrate oxidation by BM at low and elevated Ca2+cyt. BM (0.06 mg/ml) were incubated in EGTA medium without Ca2+ addition (○, Ca2+cyt=11 nM) or in the presence of 700 nM Ca2+cyt (Δ,□), without (□) or with (Δ) RR (250 nM). Experiments were started by the addition of 2 mM ADP, followed by substrate titrations, (G) 0.1–32 mM glutamate at constant malate (2 mM), (H) 0.4–72 mM α-GP, (I) 0.01–32 mM pyruvate and 2 mM malate, (J) 0.1–32 mM α-KG and 2 mM malate, and (K) 0.1–32 mM IC and 2 mM malate. Rates of state 3 respiration were plotted against substrate concentrations. Data are means±S.E.M. for n>6 experiments. Arrows indicate a Ca2+-induced change of kinetic properties of substrate oxidation caused by increased Vmax (↑). Note that, in contrast with the other substrates, pyruvate oxidation started at 10 μM pyruvate. (L) Substrate-specific differences between Ca2+cyt-induced increments of state 3 respiration. Ca2+ stimulation of state 3 respiration was calculated by subtraction of respiratory rates measured in the presence of 11 nM Ca2+cyt from state 3 rates obtained with 700 nM Ca2+cyt for the substrates glutamate/malate (▽), α-KG/malate (○), α-GP (Δ), pyruvate/malate (□) and isocitrate/malate ().

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).

Table 1
Kinetic constants of mitochondrial substrate oxidation

Isolated BM were investigated by high-resolution respirometry as described in Figure 1. Kinetic constants (Vmax and Km) were determined at low (11 nM) and high (700 nM) Ca2+cyt in the presence or absence of 250 nM RR. State 3glu/pyr/mal=253±11 nmol of O2/min per mg (n=6) was measured at Ca2+cyt=700 nM with the complex I-dependent substrates glutamate (10 mM), pyruvate (10 mM) and malate (2 mM). Data are means±S.E.M. from at least six independent experiments. Units of Vmax, nmol of O2/mg per min; units of Km, μM except for α-GP (mM). */†/‡, significant differences between marked data pairs of Vmax or Km respectively within the same column (P<0.01).

Condition Glutamate/malate α-GP Pyruvate/malate α-KG/malate IC/malate 
Vmax, state 3, 10 nM Ca2+ 56±2*† 56±4 194±3*† 95±4*† 85±4*† 
Vmax state 3, 700 nM Ca2+ 152±2* 58±4 225±11* 159±5*‡ 132±4*‡ 
Vmax state 3, 700 nM Ca2+,RR 160±4† 55±8 217±6† 131±5†‡ 102±3†‡ 
Km,11 nM Ca2+ 620±62 12.7±0.4*† 30±3 1373±98*† 707±23*† 
Km,700 nM Ca2+ 640±34 2.1±0.2* 29±8 403±25*‡ 495±18*‡ 
Km,700 nM Ca2+,RR 910±93 2.5±0.2† 33±4 502±48†‡ 690±15†‡ 
Condition Glutamate/malate α-GP Pyruvate/malate α-KG/malate IC/malate 
Vmax, state 3, 10 nM Ca2+ 56±2*† 56±4 194±3*† 95±4*† 85±4*† 
Vmax state 3, 700 nM Ca2+ 152±2* 58±4 225±11* 159±5*‡ 132±4*‡ 
Vmax state 3, 700 nM Ca2+,RR 160±4† 55±8 217±6† 131±5†‡ 102±3†‡ 
Km,11 nM Ca2+ 620±62 12.7±0.4*† 30±3 1373±98*† 707±23*† 
Km,700 nM Ca2+ 640±34 2.1±0.2* 29±8 403±25*‡ 495±18*‡ 
Km,700 nM Ca2+,RR 910±93 2.5±0.2† 33±4 502±48†‡ 690±15†‡ 

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 [911].

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,700nM 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,11nM Ca2+=30±3 μM, Km,700nM Ca2+=29±8 μM; Table 1). Confirming previous findings [22], 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 [23], 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.

Control by Ca2+cyt acting through the complete MAS of the substrate supply to BM

Figure 2
Control by Ca2+cyt acting through the complete MAS of the substrate supply to BM

(A) Metabolic scheme of complete MAS including pyruvate formation by LDH. 1, aralar; 2, pyruvate transporter; 3, α-KG–malate carrier; Asp, aspartate; Lac, lactate; Pyr, pyruvate. The MAS can be triggered by the addition of glutamate+malate or of α-KG+asparate. For reconstitution experiments BM were incubated with lactate, LDH and NADH, and GOT and MDH. Pyruvate formation requires NAD+ formation by the MAS. Cinnamate inhibits the mitochondrial pyruvate carrier, switching the complete MAS to an incomplete one while still ensuring NADH regeneration by LDH. (B) Ca2+cyt-dependent stimulation of state 3 respiration due to activation of the complete MAS. A complete MAS was reconstituted by supplementation of EGTA medium with 40 units/ml LDH, 40 units/ml GOT, 40 units/ml MDH, 5 mM malate, 5 mM lactate and 100 μM NADH in the presence of a low concentration (11 nM) of Ca2+. BM (0.06 mg protein/ml) were incubated in medium with complete MAS in the presence (black line) or the absence (grey line) of glutamate. Additions indicated: ADP, 2 mM ADP; Glu, 10 mM glutamate (only to the complete MAS experiment, black line); Ca2+, 230 nM Ca2+; Pyr, 10 mM pyruvate. (C) Ca2+cyt activates the state 3 respiration of BM via the complete MAS. State 3 respiration rates as measured in (B) [complete MAS, black line (○); absence of glutamate, grey line (Δ) and after a final addition of 10 mM pyruvate ()] but at various Ca2+cyt concentrations plotted against Ca2+free. (D) The incomplete MAS is less effective in energizing isolated BM than the complete MAS. Incubation of BM (0.06 mg/ml) with components of the complete MAS either in the absence (black line) or in the additional presence of 100 μM cinnamate an inhibitor of the mitochondrial pyruvate transporter. Additions indicated: ADP, 2 mM ADP, α-KG+Asp (Ca2+free=230 nM). 5 mM α-KG+5 mM aspartate; Cinnamate, 100 μM cinnamate; CAT, 10 μM CAT. Cinnamate shifts the system from being a complete MAS to being a incomplete one. (E) Inhibition of mitochondrial pyruvate uptake suppresses the state 3 respiration of BM by 50%. Stationary rates of respiration were measured as shown in (D) with and without 100 μM cinnamate. Data are means±S.E.M. of five measurements, ★ Significantly different to measurements without cinnamate (P < 0.01).

Figure 2
Control by Ca2+cyt acting through the complete MAS of the substrate supply to BM

(A) Metabolic scheme of complete MAS including pyruvate formation by LDH. 1, aralar; 2, pyruvate transporter; 3, α-KG–malate carrier; Asp, aspartate; Lac, lactate; Pyr, pyruvate. The MAS can be triggered by the addition of glutamate+malate or of α-KG+asparate. For reconstitution experiments BM were incubated with lactate, LDH and NADH, and GOT and MDH. Pyruvate formation requires NAD+ formation by the MAS. Cinnamate inhibits the mitochondrial pyruvate carrier, switching the complete MAS to an incomplete one while still ensuring NADH regeneration by LDH. (B) Ca2+cyt-dependent stimulation of state 3 respiration due to activation of the complete MAS. A complete MAS was reconstituted by supplementation of EGTA medium with 40 units/ml LDH, 40 units/ml GOT, 40 units/ml MDH, 5 mM malate, 5 mM lactate and 100 μM NADH in the presence of a low concentration (11 nM) of Ca2+. BM (0.06 mg protein/ml) were incubated in medium with complete MAS in the presence (black line) or the absence (grey line) of glutamate. Additions indicated: ADP, 2 mM ADP; Glu, 10 mM glutamate (only to the complete MAS experiment, black line); Ca2+, 230 nM Ca2+; Pyr, 10 mM pyruvate. (C) Ca2+cyt activates the state 3 respiration of BM via the complete MAS. State 3 respiration rates as measured in (B) [complete MAS, black line (○); absence of glutamate, grey line (Δ) and after a final addition of 10 mM pyruvate ()] but at various Ca2+cyt concentrations plotted against Ca2+free. (D) The incomplete MAS is less effective in energizing isolated BM than the complete MAS. Incubation of BM (0.06 mg/ml) with components of the complete MAS either in the absence (black line) or in the additional presence of 100 μM cinnamate an inhibitor of the mitochondrial pyruvate transporter. Additions indicated: ADP, 2 mM ADP, α-KG+Asp (Ca2+free=230 nM). 5 mM α-KG+5 mM aspartate; Cinnamate, 100 μM cinnamate; CAT, 10 μM CAT. Cinnamate shifts the system from being a complete MAS to being a incomplete one. (E) Inhibition of mitochondrial pyruvate uptake suppresses the state 3 respiration of BM by 50%. Stationary rates of respiration were measured as shown in (D) with and without 100 μM cinnamate. Data are means±S.E.M. of five measurements, ★ Significantly different to measurements without cinnamate (P < 0.01).

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).

Adjustment of substrate-limitation states of BM and lowering of ΔΨ by low Ca2+cyt

Figure 3
Adjustment of substrate-limitation states of BM and lowering of ΔΨ by low Ca2+cyt

(A) Ca2+cyt-dependent respiration rates of BM using 10 mM glutamate and 2 mM malate as substrates. BM were incubated with low (11 nM, black line) or high (700 nM, grey line) Ca2+cyt and with 10 mM glutamate and 2 mM malate. Additions indicated: BM, 0.1 mg of protein/ml; ADP200, 200 μM ADP; ADP2000, 2000 μM ADP; Ca2+, 700 nM Ca2+ [only in low-Ca2+cyt experiments (black line)]. Curves represent mitochondrial respiration rates in nmol of O2/mg per min from one typical experiment out of eight independent experiments. (B) Elevated Ca2+cyt caused a significant increase in glutamate respiration. Stationary respiration rates from measurements as shown in (A) at three metabolic states: r, resting state (state 4) after phosphorylation of the added 200 μM ADP, i, intermediate state at a maximum phosphorylation rate of 200 μM ADP; a, active respiration (state 3Glu/Mal) in the presence of 2000 μM ADP. Data are means±S.E.M., n=8. (C) Ca2+cyt-dependent safranin fluorescence in a BM incubation with glutamate and malate as substrates measured as in (A). In addition, safranin (2 μM) was used. Additions as in (A), and FCCP, 1 μM FCCP. Curves reflect the safranin fluorescence given in arbitrary units from one typical experiments out of eight independent experiments. (D) Ca2+cyt-induced increase in ΔΨ of glutamate/malate utilizing BM despite elevated respiration rates. Stationary ΔΨ values calculated from measurements shown in (C), in the three metabolic states r, i and a, as explained in (B). Data are means±S.E.M. of ΔΨ in mV as calculated from fluorescence data shown in (C), n=8.

Figure 3
Adjustment of substrate-limitation states of BM and lowering of ΔΨ by low Ca2+cyt

(A) Ca2+cyt-dependent respiration rates of BM using 10 mM glutamate and 2 mM malate as substrates. BM were incubated with low (11 nM, black line) or high (700 nM, grey line) Ca2+cyt and with 10 mM glutamate and 2 mM malate. Additions indicated: BM, 0.1 mg of protein/ml; ADP200, 200 μM ADP; ADP2000, 2000 μM ADP; Ca2+, 700 nM Ca2+ [only in low-Ca2+cyt experiments (black line)]. Curves represent mitochondrial respiration rates in nmol of O2/mg per min from one typical experiment out of eight independent experiments. (B) Elevated Ca2+cyt caused a significant increase in glutamate respiration. Stationary respiration rates from measurements as shown in (A) at three metabolic states: r, resting state (state 4) after phosphorylation of the added 200 μM ADP, i, intermediate state at a maximum phosphorylation rate of 200 μM ADP; a, active respiration (state 3Glu/Mal) in the presence of 2000 μM ADP. Data are means±S.E.M., n=8. (C) Ca2+cyt-dependent safranin fluorescence in a BM incubation with glutamate and malate as substrates measured as in (A). In addition, safranin (2 μM) was used. Additions as in (A), and FCCP, 1 μM FCCP. Curves reflect the safranin fluorescence given in arbitrary units from one typical experiments out of eight independent experiments. (D) Ca2+cyt-induced increase in ΔΨ of glutamate/malate utilizing BM despite elevated respiration rates. Stationary ΔΨ values calculated from measurements shown in (C), in the three metabolic states r, i and a, as explained in (B). Data are means±S.E.M. of ΔΨ in mV as calculated from fluorescence data shown in (C), n=8.

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.

DISCUSSION

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 [911]. The present study shows that under optimized experimental conditions, including the routine extraction of Ca2+ with nitriloacetic acid during the preparation of mitochondria [16] 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 [24]. 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 [10]. These observations point to the physiological relevance of regulation of mitochondrial function by fluctuations of Ca2+cytin 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 [7]. 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) [29] 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 [2].

Similar to observations made by Denton and McCormack [1], 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 [10]. The reason for RR-independent activation in oxidation of α-KG is not clear. RR inhibits all presently known Ca2+-uptake pathways, including uniporter [30], Ram [31,32], RyR (ryanodine receptor)-sensitive Ca2+ transporter [33] and Letm 1 [34] (see [35] 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 [711,36,37]. 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.

Ca2+-regulation of mitochondrial energization consists of Ca2+cyt-dependent control of mitochondrial pyruvate supply and the regulation of oxidation rates of intramitochondrial substrates by Ca2+mit

Figure 4
Ca2+-regulation of mitochondrial energization consists of Ca2+cyt-dependent control of mitochondrial pyruvate supply and the regulation of oxidation rates of intramitochondrial substrates by Ca2+mit

The formation of pyruvate from its precursors is coupled to NAD+ regeneration via MAS. The activity of aralar, the glutamate/aspartate carrier as the central enzyme of the MAS, is controlled by Ca2+cyt [7,8]. Aralar transports glutamate into the mitochondria, but under steady-state conditions this glutamate is not metabolized since it will be regenerated within the complete MAS (Figure 2A). Whereas mitochondrial pyruvate uptake and pyruvate metabolism are only slightly dependent on Ca2+cyt, the formation of pyruvate is strongly controlled by Ca2+cyt. Owing to its low Km and high Vmax, pyruvate is the preferred substrate of BM. Because of the electrogenic nature of the glutamate transport via the MAS and the coupling of the PC (pyruvate carrier) to the electrochemical proton gradient, this pathway can actively increase intramitochondrial NADH and pyruvate like a ‘gas pedal’. The biological importance of this mechanism particularly targets BM, since BM cannot use fatty acids as alternative oxidation substrates [55]. After the uptake of Ca2+cyt by the mitochondrial Ca2+ uniporter (UP), Ca2+mit activates PDH, α-KGH and ICDH [1,2], thus increasing the oxidation rates of their substrates, which are already within the matrix space. Thus Ca2+cyt regulates the substrate supply to mitochondria, whereas Ca2+mit regulates the oxidation rates of the intramitochondrial dehydrogenases.

Figure 4
Ca2+-regulation of mitochondrial energization consists of Ca2+cyt-dependent control of mitochondrial pyruvate supply and the regulation of oxidation rates of intramitochondrial substrates by Ca2+mit

The formation of pyruvate from its precursors is coupled to NAD+ regeneration via MAS. The activity of aralar, the glutamate/aspartate carrier as the central enzyme of the MAS, is controlled by Ca2+cyt [7,8]. Aralar transports glutamate into the mitochondria, but under steady-state conditions this glutamate is not metabolized since it will be regenerated within the complete MAS (Figure 2A). Whereas mitochondrial pyruvate uptake and pyruvate metabolism are only slightly dependent on Ca2+cyt, the formation of pyruvate is strongly controlled by Ca2+cyt. Owing to its low Km and high Vmax, pyruvate is the preferred substrate of BM. Because of the electrogenic nature of the glutamate transport via the MAS and the coupling of the PC (pyruvate carrier) to the electrochemical proton gradient, this pathway can actively increase intramitochondrial NADH and pyruvate like a ‘gas pedal’. The biological importance of this mechanism particularly targets BM, since BM cannot use fatty acids as alternative oxidation substrates [55]. After the uptake of Ca2+cyt by the mitochondrial Ca2+ uniporter (UP), Ca2+mit activates PDH, α-KGH and ICDH [1,2], thus increasing the oxidation rates of their substrates, which are already within the matrix space. Thus Ca2+cyt regulates the substrate supply to mitochondria, whereas Ca2+mit regulates the oxidation rates of the intramitochondrial dehydrogenases.

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 [38], 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 [39]. 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 [40]. The decreasing respiratory rates at permeability transition pore opening have been shown to be connected with a CsA-sensitive release of NAD+/NADH [41] 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 [40]. 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 [42], 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 [43], 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) [8] 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 [8]. 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 [4548] 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 [49]. It is known that glial cells contain much less, or even none, aralar than do neurons [5052]; consequently, the MAS activity is also low in, or absent from, glial cells [5052]. 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 [5254]. Moreover, Pellerin et al. [13] 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) [13]. 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 [55], rely exclusively on pyruvate oxidation.

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

AUTHOR CONTRIBUTION

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.

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.

FUNDING

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.).

References

References
1
Denton
R. M.
McCormack
J. G.
Ca2+ as a second messenger within mitochondria of the heart and other tissues
Annu. Rev. Physiol.
1990
, vol. 
52
 (pg. 
451
-
466
)
2
McCormack
J. G.
Denton
R. M.
The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex
Biochem. J.
1979
, vol. 
180
 (pg. 
533
-
544
)
3
Korzeniewski
B.
Regulation of ATP supply during muscle contraction: theoretical studies
Biochem. J.
1998
, vol. 
330
 (pg. 
1189
-
1195
)
4
Korzeniewski
B.
Regulation of oxidative phosphorylation through parallel activation
Biophys. Chem.
2007
, vol. 
129
 (pg. 
93
-
110
)
5
Katz
L. A.
Koretzky
A. P.
Balaban
R. S.
Activation of dehydrogenase activity and cardiac respiration: a 31P-NMR study
Am. J. Physiol.
1988
, vol. 
255
 (pg. 
H185
-
H188
)
6
Garcia-Rivas
Gde J.
Carvial
K.
Correa
F.
Zazutea
C.
Ru360, a specific mitochondrial calcium uptake inhibitor, improves cardiac post-ischaemic functional recovery in rats in vivo
Br. J. Pharmacol.
2006
, vol. 
149
 (pg. 
829
-
837
)
7
Satrústegui
J.
Pardo
B.
Del Arco
A.
Mitochondrial transporters as novel targets for intracellular calcium signaling
Physiol. Rev.
2007
, vol. 
87
 (pg. 
29
-
67
)
8
Contreras
L.
Satrústegui
J.
Calcium signaling in brain mitochondria: interplay of malate aspartate NADH shuttle and calcium uniporter/mitochondrial dehydrogenase pathways
J. Biol. Chem.
2009
, vol. 
28
 (pg. 
7091
-
7099
)
9
Gellerich
F. N.
Gizatullina
Z.
Nguyen
H. P.
Trumbeckaite
S.
Vielhaber
S.
Seppet
E.
Zierz
S.
Landwehrmeyer
B.
Ries
O.
von Hoersten
S.
Striggow
F.
Impaired regulation of brain mitochondria by extramitochondrial Ca2+ in transgenic Huntington disease rats
J. Biol. Chem.
2008
, vol. 
283
 (pg. 
30715
-
30724
)
10
Gellerich
F. N.
Gizatullina
Z.
Arandarcikaite
O.
Jerzembek
D.
Vielhaber
S.
Seppet
E.
Striggow
F.
Extramitochondrial Ca2+ in the nanomolar range regulates glutamate-dependent oxidative phosphorylation on demand
PLoS ONE
2009
, vol. 
4
 pg. 
e8181
 
11
Gellerich
F. N.
Gizatullina
Z.
Trumbeckaite
S.
Nguyen
H. P.
Pallas
T.
Arandarcikaite
O.
Vielhaber
S.
Seppet
E.
Striggow
F.
The regulation of OXPHOS by extramitochondrial calcium
Biochim. Biophys. Acta
2010
, vol. 
1797
 (pg. 
1018
-
1027
)
12
Mangia
S.
Giove
F.
Bianciardi
M.
Di Salle
F.
Garreffa
G.
Maraviglia
B.
Issues concerning the construction of a metabolic model for neuronal activation
J. Neurosci. Res.
2003
, vol. 
71
 (pg. 
463
-
467
)
13
Pellerin
L.
Pellegri
G.
Bittar
P. G.
Charnay
Y.
Bouras
C.
Martin
J. L.
Stella
N.
Magistretti
P. J.
Evidence supporting the existence of an activity-dependent astrocyte-neuron lactate shuttle
Dev. Neurosci.
1998
, vol. 
20
 (pg. 
291
-
299
)
14
Tildon
J. T.
Stevenson
J.H.
Jr
Ozand
P. T.
Mitochondrial glycerol kinase activity in rat brain
Biochem. J.
1976
, vol. 
157
 (pg. 
513
-
516
)
15
Kudin
A. P.
Bimpong-Buta
N. Y.
Vielhaber
S.
Elger
C. E.
Kunz
W. S.
Characterization of superoxide-producing sites in isolated brain mitochondria
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
4127
-
4135
)
16
Johnston
J. G.
Brand
M. D.
Biochem
Soc. Trans.
1986
, vol. 
14
 (pg. 
1182
-
1185
)
17
Kuznetsov
A. V.
Veksler
V.
Gellerich
F. N.
Saks
V.
Margreiter
R.
Kunz
W. S.
Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells
Nat. Protoc.
2008
, vol. 
3
 (pg. 
965
-
976
)
18
Gnaiger
E.
Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply
Resp. Phys.
2001
, vol. 
128
 (pg. 
277
-
297
)
19
Konishi
M.
Olson
A.
Hollingworth
S.
Baylor
S. M.
Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements
Biophys. J.
1988
, vol. 
54
 (pg. 
1089
-
104
)
20
Zanotti
A.
Azzone
G. F.
Safranine as membrane potential probe in rat liver mitochondria
Arch. Biochem. Biophys.
1980
, vol. 
201
 (pg. 
255
-
265
)
21
Wiechelman
K. J.
Braun
R. D.
Fitzpatrick
J. D.
Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation
Anal. Biochem.
1988
, vol. 
175
 (pg. 
231
-
237
)
22
Nicklas
W. J.
Clark
J. B.
Williamson
J. R.
Metabolism of rat brain mitochondria. Studies on the potassium ion-stimulated oxidation of pyruvate
Biochem. J.
1971
, vol. 
123
 (pg. 
83
-
95
)
23
Sharma
N.
Okere
I. C.
Brunengraber
D. Z.
McElfresh
T. A.
King
K. L.
Sterk
J. P.
Huang
H.
Chandler
M. P.
Stanley
W. C.
Regulation of pyruvate dehydrogenase activity and citric acid cycle intermediates during high cardiac power generation
J. Physiol.
2005
, vol. 
562
 (pg. 
593
-
603
)
24
Contreras
L.
Gomez-Puertas
P.
Iijima
M.
Kobayashi
K.
Saheki
T.
Satrústegui
J.
Ca2+ activation kinetics of the two aspartate-glutamate mitochondrial carriers, aralar and citrin: role in the heart malate-aspartate NADH shuttle
J. Biol. Chem.
2007
, vol. 
282
 (pg. 
7098
-
70106
)
25
Supnet
C.
Bezprozvanny
I.
The dysregulation of intracellular calcium in Alzheimer disease
Cell Calcium
2010
, vol. 
47
 (pg. 
183
-
189
)
26
Kuchibhotla
K. V.
Goldman
S. T.
Lattarulo
C. R.
Wu
H. Y.
Hyman
B. T.
Bacskai
B. J.
Aβ plaques lead to aberrant regulation of calcium homeostasis in vivo resulting in structural and functional disruption of neuronal networks
Neuron
2008
, vol. 
59
 (pg. 
214
-
225
)
27
Klingenberg
M.
Localization of the glycerol-phosphate dehydrogenase in the outer phase of the mitochondrial inner membrane
Eur. J. Biochem.
1970
, vol. 
13
 (pg. 
247
-
252
)
28
MacDonald
M. J.
Brown
L. J.
Calcium activation of mitochondrial glycerol phosphate dehydrogenase restudied
Arch. Biochem. Biophys.
1996
, vol. 
326
 (pg. 
79
-
84
)
29
Lin
E. C.
Glycerol utilization and its regulation in mammals
Annu. Rev. Biochem.
1977
, vol. 
46
 (pg. 
765
-
795
)
30
Montero
M.
Lobaton
C. D.
Moreno
A.
Alvarez
J.
A novel regulatory mechanism of the mitochondrial Ca2+ uniporter revealed by the p38 mitogen-activated protein kinase inhibitor SB202190
FASEB J.
2002
, vol. 
16
 (pg. 
1955
-
1957
)
31
Pinton
P.
Leo
S.
Wieckowski
M. R.
Di Benedetto
G.
Rizzuto
R.
Long-term modulation of mitochondrial Ca2+ signals by protein kinase C isozymes
J. Cell Biol.
2004
, vol. 
165
 (pg. 
223
-
232
)
32
Koncz
P.
Szanda
G.
Fulop
L.
Rajki
A.
Spat
A.
Mitochondrial Ca2+ uptake is inhibited by a concerted action of p38MAPK and protein kinase D
Cell Calcium
2009
, vol. 
46
 (pg. 
122
-
129
)
33
Szanda
G.
Koncz
P.
Rajki
A.
Spat
A.
Participation of p38 MAPK and a novel-type protein kinase C in the control of mitochondrial Ca2+ uptake
Cell Calcium
2008
, vol. 
43
 (pg. 
250
-
259
)
34
Rizzuto
R.
Pinton
P.
Carrington
W.
Fay
F. S.
Fogarty
K. E.
Lifshitz
L. M.
Tuft
R. A.
Pozzan
T.
Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses
Science
1998
, vol. 
280
 (pg. 
1763
-
1766
)
35
Santo-Domingo
J.
Demaurex
N
Calcium uptake mechanisms of mitochondria
Biochim. Biophys. Acta
2010
, vol. 
1797
 (pg. 
907
-
1012
)
36
Ramos
M.
del Arco
A.
Pardo
B.
Martínez-Serrano
A.
Martínez-Morales
J. R.
Kobayashi
K.
Yasuda
T.
Bogónez
E.
Bovolenta
P.
Saheki
T.
Satrústegui
J.
Developmental changes in the Ca2+-regulated mitochondrial aspartate-glutamate carrier aralar1 in brain and prominent expression in the spinal cord
Dev. Brain Res.
2003
, vol. 
143
 (pg. 
33
-
46
)
37
Lasorsa
F. M.
Pinton
P.
Palmieri
L.
Fiermonte
G.
Rizzuto
R.
Palmieri
F.
Recombinant expression of the Ca2+-sensitive aspartate/glutamate carrier increases mitochondrial ATP production in agonist-stimulated Chinese hamster ovary cells
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
38686
-
38692
)
38
Chalmers
S.
Nicholls
D. G.
The relationship between free and total calcium concentrations in the matrix of liver and brain mitochondria
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
19062
-
19070
)
39
Daikhin
Y.
Yudkoff
M.
Compartmentation of brain glutamate metabolism in neurons and glia
J. Nutr.
2000
, vol. 
130
 (pg. 
1026S
-
10231S
)
40
Gizatullina
Z. Z.
Gaynutdinov
T. M.
Svoboda
H.
Jerzembek
D.
Knabe
A.
Vielhaber
S.
Fischer
G.
Striggow
F.
Gellerich
F. N.
Effects of cyclosporin A and its immunesupressive/non-immunesuppressive analogues D-Ser8-CsA and Cs9 on oxidative phosphorylation and Ca2+ accumulation of mitochondria from different brain regions
Mitochondrion
2010
, vol. 
11
 (pg. 
421
-
429
)
41
Di Lisa
F.
Menabò
R.
Canton
M.
Barile
M.
Bernardi
P.
Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
2571
-
2575
)
42
Halestrap
A. P.
Cinnamate inhibitor: the mechanism of the inhibition of the mitochondrial pyruvate transportater by α-cyanocinnamate derivatives
Biochem. J.
1976
, vol. 
156
 (pg. 
181
-
183
)
43
Safer
B.
Smith
C. M.
Williamson
J. R.
Control of the transport of reducing equivalents across the mitochondrial membrane in perfused rat heart
J. Mol. Cell. Cardiol.
1971
, vol. 
2
 (pg. 
111
-
124
)
44
Occhipinti
R.
Somersalo
E.
Calvetti
D.
Energetics of inhibition: insights with a computational model of the human GABAergic neuron-astrocyte cellular complex
J. Cereb. Blood Flow Metab.
2010
, vol. 
30
 (pg. 
1834
-
1846
)
45
Lu
M.
Zhou
L.
Stanley
W. C.
Role of the malate-asparate shuttle on the metabolic response to myocardial ischemia
J. Theor. Biol.
2008
, vol. 
254
 (pg. 
466
-
475
)
46
Toime
L. J.
Brand
M. D.
Uncoupling protein-3 lowers reactive oxygen species production in isolated mitochondria
Free Radical Biol. Med.
2010
, vol. 
49
 (pg. 
606
-
611
)
47
Papa
S.
Skulachev
V. P.
Reactive oxygen species, mitochondria, apoptosis and aging
Mol. Cell. Biochem.
1997
, vol. 
174
 (pg. 
305
-
319
)
48
Brand
MD.
Uncoupling to survive? The role of mitochondrial inefficiency in ageing
Exp. Gerontol.
2000
, vol. 
35
 (pg. 
811
-
820
)
49
Azevedo
F. A.
Carvalho
L. R.
Grinberg
L. T.
Farfel
J. M.
Ferretti
R. E.
Leite
R. E.
Jacob Filho
W.
Lent
R.
Herculano-Houzel
S.
Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain
J. Comp. Neurol.
2009
, vol. 
10
 (pg. 
532
-
541
)
50
Berkich
D. A.
Ola
M. S.
Cole
J.
Sweatt
A. J.
Hutson
S. M.
LaNoue
K. F.
Mitochondrial transport proteins of the brain
J. Neurosci. Res.
2007
, vol. 
85
 (pg. 
3367
-
3377
)
51
Xu
Y.
Ola
M. S.
Berkich
D. A.
Gardner
T. W.
Barber
A. J.
Palmieri
F.
Hutson
S. M.
LaNoue
K. F.
Energy sources for glutamate neurotransmission in the retina: absence of the aspartate/glutamate carrier produces reliance on glycolysis in glia
J. Neurochem.
2007
, vol. 
10
 (pg. 
120
-
131
)
52
Cahoy
J. D.
Emery
B.
Kaushal
A.
Foo
L. C.
Zamanian
J. L.
Christopherson
K. S.
Xing
Y.
Lubischer
J. L.
Krieg
P. A.
Krupenko
S. A.
, et al. 
A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function
J. Neurosci.
2008
, vol. 
2
 (pg. 
264
-
278
)
53
O'Brien
J.
Kla
K. M.
Hopkins
I. B.
Malecki
E. A.
McKenna
M. C.
Kinetic parameters and lactate dehydrogenase isozyme activities support possible lactate utilization by neurons
Neurochem. Res.
2007
, vol. 
32
 (pg. 
597
-
607
)
54
McKenna
M. C.
Waagepetersen
H. S.
Schousboe
A.
Sonnewald
U.
Neuronal and astrocytic shuttle mechanisms for cytosolic-mitochondrial transfer of reducing equivalents: current evidence and pharmacological tools
Biochem. Pharmacol.
2006
, vol. 
71
 (pg. 
399
-
407
)
55
Yang
S. Y.
He
X. Y.
Schulz
H.
Fatty acid oxidation in rat brain is limited by the low activity of 3-ketoacyl-coenzyme A thiolase
J. Biol. Chem.
1987
, vol. 
262
 (pg. 
13027
-
13032
)