The Relationship between Free and Total Calcium Concentrations in the Matrix of Liver and Brain Mitochondria*

Susan Chalmers and David G. Nicholls {ddagger}

From the Buck Institute for Age Research, Novato, California 94945

Received for publication, December 12, 2002 , and in revised form, February 19, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Three sequential phases of mitochondrial calcium accumulation can be distinguished: matrix dehydrogenase regulation, buffering of extramitochondrial free calcium, and finally activation of the permeability transition. Relationships between these phases, free and total matrix calcium concentration, and phosphate concentration are investigated in rat liver and brain mitochondria. Slow, continuous calcium infusion is employed to avoid transient bioenergetic consequences of bolus additions. Liver and brain mitochondria undergo permeability transitions at precise matrix calcium loads that are independent of infusion rate. Cytochrome c release precedes the permeability transition. Cyclosporin A enhances the loading capacity in the presence or absence of acetoacetate. A remarkably constant free matrix calcium concentration, in the range 1–5 µM as monitored by matrix-loaded fura2-FF, was observed when total matrix calcium was increased from 10 to at least 500 nmol of calcium/mg of protein. Increasing phosphate decreased both the free matrix calcium and the matrix calcium-loading capacity. Thus the permeability transition is not triggered by a critical matrix free calcium concentration. The rate of hydrogen peroxide detection by Amplex Red decreased during calcium infusion arguing against a role for oxidative stress in permeability pore activation in this model. A transition between a variable and buffered matrix free calcium concentration occurred at 10 nmol of total matrix calcium/mg protein. The solubility product of amorphous Ca3(PO4)2 is consistent with the observed matrix free calcium concentration, and the matrix pH is proposed to play the major role in maintaining the low matrix free calcium concentration.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolated mitochondria from a variety of sources possess a large but finite capacity to accumulate and retain Ca2+ (for reviews see Refs. 1 and 2). When this limit is exceeded, mitochondria assemble a pore in their inner membrane that is non-selectively permeable to ions and solutes up to 1.4 kDa (for review see Ref. 3). It is unclear what exactly defines Ca2+ overload and how this triggers the permeability transition. Phosphate (Pi) is required for massive Ca2+ loading of the matrix and a variety of evidence suggests that a calcium phosphate complex is formed within the matrix once about 10 nmol of Ca2+/mg has been accumulated. First, phosphate is required for extensive matrix Ca2+ loading that can exceed 1 M total Ca2+ concentration before swelling can be observed (1, 2). Second, over a wide range of total matrix Ca2+ ({Sigma}Ca2+m)1 the activity of the Ca2+ efflux pathways in both liver and brain mitochondria are inversely related to the free Pi concentration but independent of {Sigma}Ca2+m, consistent with a matrix free Ca2+, [Ca2+]m, that is governed by the solubility product of a calcium phosphate complex and is suboptimal for maximal activity of the efflux pathways of liver or brain mitochondria (4, 5). Third, mitochondria can maintain a remarkably constant setpoint when {Sigma}Ca2+m is varied widely (6), indicating that the activity of the efflux pathway, and hence [Ca2+]m, is not varying. Fourth, mitochondria accumulating Ca2+ in the presence of excess phosphate extrude 1 H+ per Ca2+ accumulated, consistent with the formation of Ca3(PO4)2 in the matrix (7). Finally, Pi-depleted mitochondria in the presence of acetate as permeant anion show an increasing efflux activity with increasing Ca2+ load and fail to maintain a setpoint that is independent of {Sigma}Ca2+m (4).

All of these observations are consistent with a matrix Ca2+ phosphate complex that obeys mass-action relationships to maintain a low [Ca2+]m. However, any complex must be freely reversible, since the physiological complex is capable of dissociating into Ca2+ and Pi immediately following mitochondrial depolarization, allowing the two ions to exit the mitochondrion on their individual carriers (5).

Mitochondrial Ca2+ loading has almost invariably been investigated by the addition of one or more bolus additions of Ca2+, each of which induces a non-steady-state condition in which mitochondrial respiration, membrane potential and redox status change with time. Even multiple, relatively small, Ca2+ additions result in repetitive partial mitochondrial depolarizations and bioenergetic demands. In addition, it is highly likely that the calcium phosphate complex formed in response to a rapid increase in [Ca2+]m following a bolus addition may differ from that formed when Ca2+ is slowly accumulated, since many forms of calcium phosphate are thermodynamically unstable and spontaneously interconvert (8).

In this study we first investigate the capacity of liver and brain mitochondria to accumulate Ca2+ when the cation is slowly infused into the incubation, allowing the mitochondria to accumulate the cation continuously, with minimal bioenergetic demands. Second we monitor the matrix free Ca2+ concentrations during such loading in order to establish whether the supposed matrix buffering actually occurs, and finally we attempt to determine whether any relationship exists between matrix free Ca2+ and the initiation of the permeability transition.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Reagents—Calcium Green 5N (CaG5N), fura2-FFAM, tetramethylrhodamine methylester (TMRM), and Amplex Red were obtained from Molecular Probes (Eugene, OR). Anti-cytochrome c (7HB) was obtained from BD PharMingen (San Diego, CA). All other reagents were obtained from Sigma Chemical Co.

Preparation of Rat Liver Mitochondria (RLM)—Mitochondria were isolated from 6-week-old Wistar rats. The liver was homogenized in 125 mM sucrose, 125 mM mannitol, 5 mM Hepes, 1 mM EGTA, pH 7.2 and centrifuged at 1000 x g for 10 min at 4 °C. The supernatant was then centrifuged for 10 min at 8 000 x g at 4 °C. The resulting pellet was resuspended and centrifuged for 10 min at 8500 x g at 4 °C. The upper layer of this pellet was removed by gentle pipetting and the lower mitochondrial pellet resuspended in 0.5 ml of 250 mM sucrose, 16 µM BSA, 5 mM Hepes, pH 7.2.

Preparation of Rat Brain Mitochondria (RBM)—The cerebral cortices of two 6-week-old rats were rapidly removed into 20 ml of ice-cold isolation buffer (320 mM sucrose, 5 mM Tes, 1 mM EGTA, pH 7.2) and homogenized. The homogenate was centrifuged at 1000 x g for 5 min at 4 °C. The supernatant was centrifuged at 8500 x g for 10 min, and the resulting pellet resuspended in 1 ml of isolation buffer. This was layered onto a discontinuous gradient consisting of 1 ml of 6% Ficoll, 0.5 ml of 9% Ficoll, and 4.5 ml of 12% Ficoll (all prepared in isolation buffer) and centrifuged at 75,000 x g for 30 min. The myelin, synaptosomal, and free mitochondrial fractions formed respectively above the 6% layer, as a doublet within the 9% layer and as a pellet. The pellet was resuspended in 250 mM sucrose, 16 µM bovine serum albumin, 10 mM Tes, pH 7.2 and centrifuged at 8000 x g before being resuspended in this last buffer to 10–20 mg of protein/ml by the Bradford protein assay.

Mitochondrial Ca2+ Accumulation—0.1 mg of mitochondrial protein was suspended in 2 ml of incubation medium (100 mM NaCl, 25 mM Hepes, pH 7.2, 1 µg/ml oligomycin, 2 mM sodium phosphate, and 0.5 mM ADP unless otherwise stated. A NaCl-based medium was used as this was shown in an earlier study (4) to give optimal respiratory control. Experiments with RLM utilized 2 mM succinate as substrate in the presence of 1 µM rotenone; those with RBM utilized 5 mM glutamate plus 5 mM malate or 5 mM pyruvate plus 5 mM malate. Experiments were performed at 37 °C in stirred cuvettes in a PerkinElmer LS-50B fluorimeter. CaCl2 additions were made either as bolus additions or as gradual infusions with a Braun Perfusor (FT Scientific Instruments, Glos., UK) modified to take a Hamilton microsyringe (5).

Depletion of Endogenous Phosphate—Isolated mitochondria were depleted of endogenous phosphate by incubation with 1 mM glucose, 0.75 units/ml hexokinase, 1 mM MgCl2, 5 mM glutamate, 5 mM malate,and 0.5 mM ADP in incubation medium at 37 °C for 5 min in the presence of substrate as previously described (5).

Mitochondrial Free Ca2+RBM (0.1 mg of protein) were incubated in 25 µl of 250 mM sucrose, 10 mM Tes, 80 µM fura2-FF AM, 16 µM BSA, pH 7.2 on ice for 30 min and then at room temperature for 30 min. Any contaminating synaptosomes were permeabilized by addition of 1 ml of 250 mM sucrose, 16 µM albumin, 5 mM Hepes, pH 7.2 containing 0.01% digitonin for the last 5 min of the incubation. The mitochondria were then centrifuged for 1 min at 10,000 x g, the pellet resuspended in 1 ml of 250 mM sucrose, 16 µM BSA, 5 mM Hepes, pH 7.2 and recentrifuged. The pellet was finally resuspended in 20 µl of the same medium and used immediately.

Estimation of [Ca]m in Equilibrium with Amorphous Tricalcium Phosphate as a Function of pHmThe following dissociation constants were taken for phosphate, pK1 = 2.13, pK2 = 7.2, pK3 = 12.39 (10). A representative value for the Ksp for amorphous Ca3(PO4)2 of 3 x 1030 was assumed, as published values from vary from 1026 to 1033. Equilibrium was assumed across the membrane via the phosphate carrier for an antiport (11). As the calculation was intended to show how [Ca2+]m in equilibrium with Ca3(PO4)2 could vary with pH and [Pi]e rather than report definitive values, no attempt was made to correct for activity coefficients of the charged species in the highly non-ideal conditions within the matrix.

Mitochondrial Light Scattering, Membrane Potential, H2O2 Production, and NAD(P) Pool Redox Status—All parameters were monitored in a stirred cuvette at 37 °C in the LS50B fluorimeter. Mitochondrial light scattering was monitored at 625 nm. Changes in membrane potential were followed qualitatively by the fluorescence quenching of tetramethylrhodamine methyl ester (549 nm excitation, 575 nm emission). H2O2 production was monitored fluorimetrically with 1 µM Amplex Red and 0.75 units/ml horseradish peroxidase (563 nm excitation, 587 nm emission). Changes in NAD(P)H fluorescence were monitored at 350 nm excitation, 450 nm emission.

Cytochrome c Release—Release of cytochrome c was monitored by removing 100-µl samples from the cuvette during an experiment and centrifuging to separate mitochondrial pellet and supernatant. Samples were separated and visualized by Western blotting. The blot was blocked with 5% albumin in TBS-Tween (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween 20) and then incubated with 1:2500 mouse anti-cytochrome c antibody in TBS-Tween plus 1% BSA followed by 1:2500 sheep anti-mouse antibody-horseradish peroxidase conjugate in TBS-Tween containing 1% BSA prior to enhanced chemiluminescence (ECL) detection (Amersham Biosciences).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Rat Liver Mitochondrial Ca2+-loading Capacity during Steady Ca2+ Infusion—The capacity of RLM to accumulate Ca2+ in the presence of phosphate, and ADP is dependent upon the mode of addition (Fig. 1). A single bolus addition of 450 nmol of Ca2+/mg initiates a permeability transition within 15 min, whereas 12 smaller additions of 50 nmol of Ca2+/mg can be made to a parallel preparation, while the steady infusion of Ca2+ at a rate of 85 nmol/mg/min allowed 800 nmol/mg to be accumulated before the permeability transition was observed.



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FIG. 1.
Effect of the mode of Ca2+ addition upon the capacity of liver mitochondria to accumulate Ca2+ before permeability transition initiation. 0.1 mg of RLM were suspended in 2 ml of incubation buffer (100 mM NaCl, 25 mM Hepes pH 7.2, 16 µM BSA, 2 mM NaPO4,2mM succinate, 0.2 mM ADP, 1 µM rotenone, 1 µg/ml oligomycin and 0.2 µM CaG5N). CaCl2 was added as a single bolus addition of 450 nmol/mg (22.5 µM) (a), as multiple additions of 50 nmol/mg each (2.5 µM) (b), or as a continuous infusion of 85 nmol/mg/min (42 µM/min) (c).

 

As previously demonstrated (5) the steady infusion of Ca2+ into a mitochondrial incubation results in an initial increase in external Ca2+ concentration ([Ca2+]e) until the uniporter is sufficiently active to accumulate Ca2+ at a rate equal to that at which Ca2+ is being added to the external medium (i.e. the infusion rate plus the activity of the mitochondrial Ca2+ efflux pathway). Subsequently, and for as long as the activities of the uniporter and efflux pathway remain constant, Ca2+ is accumulated into the matrix at the same rate that it is delivered to the cuvette. The onset of the permeability transition is signaled by the increase in [Ca2+]e.

A would be predicted from the catastrophic consequences of removing the proton-impermeability of the inner membrane, permeability pore opening in individual mitochondria is associated with a virtually instantaneous collapse of membrane potential (12, 13). The resultant discharge of Ca2+ into the medium may trigger Ca2+ overload in adjacent mitochondria, leading to a chain reaction in which the entire mitochondrial population is seen to undergo the permeability transition in a short period of time. In order to minimize this effect in the present study, and also to ensure that anoxia did not occur during prolonged Ca2+ infusions, a very low mitochondrial protein concentration (0.05 mg/ml incubation) was used in most of these experiments.

Changes in mitochondrial membrane potential, NAD(P)+ reduction, light scattering and cytochrome c retention were monitored in parallel with the Ca2+ infusion (Fig. 2). At the single mitochondrion level, the inner membrane permeabilization associated with the permeability transition results in an immediate collapse in membrane potential (13) and consequent loss of matrix Ca2+. A complicating factor therefore in conventional studies with mitochondrial populations is that this released Ca2+ can be taken up by adjacent mitochondria, either masking the initiation of the permeability transition, or triggering a chain reaction by inducing Ca2+ overload. Since the release of cytochrome c is an irreversible consquence of outer membrane rupture following the permeability transition, the experiment depicted in Fig. 2d, was performed to monitor the time course of release of the cytochrome during continuous infusion. Samples were taken from the cuvette during the Ca2+ infusion and rapidly centrifuged to pellet the mitochondria through silicone oil. Extensive cytochrome c release is first seen just before the massive release of matrix Ca2+ due to the permeability transition (sample iv). This suggests that a significant proportion of the mitochondria in the cuvette have undergone the transition by this stage. The modest increase in external free Ca2+ indicates that surrounding mitochondria accumulate Ca2+ from their depolarized neighbors, which would be predicted to facilitate in turn their depolarization and propagation of the transition throughout the incubation. A permeability transition-independent release of cytochrome c from rat brain mitochondria has been reported previously (14), although in that case it was induced by a single massive bolus addition of Ca2+ (3200 nmol/mg) and could therefore be due to transient osmotic swelling of the matrix prior to formation of the osmotically inactive calcium phosphate complex.



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FIG. 2.
{Delta}{Psi}m loss, mitochondrial swelling, NAD(P)-pool reduction, and cytochrome c release during Ca2+ accumulation. Rat liver mitochondria suspensions were infused with Ca2+ as in Fig. 1c and simultaneous measurements were made of: membrane potential with 1 µM TMRM and 549/575 nm excitation/emission (a), mitochondrial swelling with light scattering at 625 nm (b), NAD(P) pool autofluorescence at 350/450 nm (c), or cytochrome c release by removing 100-µl samples (i–v) at the arrows (d), centrifuging to pellet the mitochondria and Western blotting the supernatants for cytochrome c. Note that the TMRM+ trace is inverted so that an upward deflection corresponds to an increase in membrane potential.

 

No change in the extent of TMRM+ quenching could be detected until shortly before the onset of the permeability transition (Fig. 2a). In contrast to Ca2+, whose distribution is the consequence of a dynamic balance between independent uptake and efflux pathways, TMRM+ responds in a Nernstian manner to the membrane potential. A permeability transition in a fraction of the mitochondria is therefore not masked by reuptake of the membrane potential indicator by the residual mitochondria. For example, collapse in the membrane potential in 50% of the mitochondria would halve the aggregate matrix volume in which the TMRM+ is accumulated, releasing the probe and decreasing the quenching in the cuvette.

Light scattering increased steadily during the infusion (Fig. 2b). Since this parameter is a function of the difference in refractive index between the matrix and medium, it is likely that what is being observed is an increase in matrix refractive index due to the formation of a calcium phosphate complex rather than a physical contraction of the matrix (14).

NAD(P)H fluorescence also increases as Ca2+ is infused (Fig. 2c). As will be seen below (Fig. 8) [Ca2+]m is almost invariant over the range where the increased fluorescence signal is observed and so it is unlikely that the signal increases as a result of Ca2+-dependent substrate dehydrogenase activation (15). Since fluorescence is sensitive to the environment, it is equally possible that the fluorescence change reflects a change in matrix environment rather than increased reduction.



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FIG. 8.
Effect of matrix Ca2+ accumulation in the presence of Pi upon [Ca2+]m. RBM were loaded with fura2-FF as described under "Experimental Procedures." a, fura2-FF-loaded mitochondria were added to 2 ml of 100 mM NaCl, 25 mM Hepes pH 7.2, 5 mM NaPO4, 0.5 mM ADP, 5 mM glutamate, 5 mM malate, 1 µg/ml oligomycin, and 0.2 µM CaG5N. CaCl2 was infused into this suspension and [Ca2+]m monitored. b, the above experiment was repeated monitoring [Ca2+]e. c, the ratio of bound/free Ca2+ in the matrix at any point was then calculated assuming a matrix volume of ~1 µl/mg: (Ca infusedt – [Ca2+]et – [Ca2+]e0 – [Ca2+]mt)/[Ca2+]mt). Where Ca infusedt = the total amount of Ca2+ infused at time point t; [Ca2+]et = the extramitochondrial [Ca2+] at point t; [Ca2+]e0 = the initial extramitochondrial [Ca2+]; and [Ca2+]mt = the matrix free [Ca2+] at point t.

 

By altering the speed of the infusion pump it is possible to determine whether the onset of the permeability transition is influenced by the rate or the extent of matrix Ca2+ loading. Varying the infusion rate from 42.5 to 170 nmol of Ca2+/mg/min had no effect upon the capacity of the liver mitochondria to accumulate Ca2+ (Fig. 3a). In the presence of the permeability transition inhibitor cyclosporin A (Fig. 3b) the capacity increased almost 3-fold and again seemed independent of the rate of infusion, although a slight decrease in capacity was observed for the slowest infusion.



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FIG. 3.
Effect of the rate of mitochondrial Ca2+ accumulation and presence of CsA on Ca2+ accumulation capacity. Rat liver mitochondrial suspensions were infused with Ca2+ as in Fig. 1c at a rate of 170, 85, or 42.5 nmol/min/mg in the absence (a and c) or presence (b and d) of 1 µM cyclosporin A. Data was plotted as a function of time (a and b) or Ca2+ infusion (c and d).

 

Oxidation of RLM matrix NADH by acetoacetate in the absence of exogenous adenine nucleotides facilitates the permeability transition in response to bolus additions of Ca2+ (16). It has recently been reported (17) that acetoacetate activates a low conductance form of the permeability transition pore in RLM, that is sensitive to cyclosporin A and manifests itself as an increase in the rate of Ca2+ efflux observed following ruthenium red addition, but without a detectable loss of membrane potential. Under the present conditions, NADH oxidation induced by acetoacetate does not increase the steady-state rate of Ca2+ efflux from the mitochondria, since that would be reflected in an increased steady-state [Ca2+]e and none can be detected (Fig. 4), but instead the capacity of the mitochondria to accumulate Ca2+ is considerably decreased. Importantly this effect of acetoacetate is still observed in the presence of 1 µM cyclosporin and ADP (Fig. 4).



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FIG. 4.
Effect of acetoacetate upon mitochondrial Ca2+ accumulation capacity. Rat liver mitochondria were suspended and infused with Ca2+ as in Fig. 1c with the inclusion of 1 µM CsA where shown. 2 mM lithium acetoacetate was added 2 min after the start of Ca2+ infusion (arrow) where shown (AA).

 

Relationship between Free and Total Matrix Ca2+ Concentrations during Ca2+ Infusion—Preincubation of isolated mitochondria with fluorescent Ca2+ indicators can allow sufficient loading to occur to monitor matrix free Ca2+ concentrations (15, 18, 19, 20, 21, 22, 23, 24). In our hands it was difficult to achieve a reliable extent of loading with RLM and so subsequent experiments were performed with RBM.

The low affinity fura analog fura2-FF (Kd = 5.5 µM) was found to load into the mitochondrial matrix as the acetoxymethyl ester and to be hydrolyzed sufficiently to enable changes in [Ca2+]m to be followed. Since mitochondrial autofluorescence, due primarily to NAD(P)H at the wavelengths employed, changes at the onset of the permeability transition (Fig. 2) in most experiments this was corrected for by parallel determinations in the absence of loaded dye.

The hypothesis on which these studies were based was that changes in [Ca2+]m in the presence of excess phosphate would be largely buffered by the formation of calcium phosphate complexes. In order initially to assess the ability of the loaded fura2-FF to detect changes in [Ca2+]m therefore, mitochondria were extensively depleted of endogenous Pi by preincubation with ADP, glucose and hexokinase, as previously described (5), and acetate was added as permeant anion to prevent build up of a pH gradient. Since calcium acetate is highly soluble it would be predicted that [Ca2+]m would rise with Ca2+ load. Fig. 5 shows the increase in [Ca2+]m of RBM loaded with fura2-FF following four bolus additions of 10 µM Ca2+ to the cuvette. It is notable that the matrix free Ca2+ rose stepwise with each addition. In contrast, [Ca2+]e, determined in parallel with external CaG5N, partially recovered after each bolus addition as Ca2+ was accumulated into the matrix. Subsequent addition of 1 µM FCCP to depolarize the mitochondria resulted in an extrusion of Ca2+ from the matrix to the suspension medium, a decrease in [Ca2+]m and increase in [Ca2+]e, corresponding to Ca2+ efflux via the uniporter.



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FIG. 5.
Free [Ca2+]m changes upon accumulation of bolus Ca2+ additions and upon {Delta}{Psi}m depolarization in the absence of Pi and presence of acetate. Fura2-FF was loaded into the matrix of RBM as described under "Experimental Procedures." 0.1 mg of mitochondria were then depleted of endogenous Pi by incubation at 37 °C in 2 ml of 100 mM NaCl, 25 mM Hepes pH 7.2, 0.5 mM ADP, 5 mM glutamate, 5 mM malate, 1 mM glucose, and 0.75 units/ml hexokinase for 5 min. 1 µg/ml oligomycin and 2 mM sodium acetate was then added to stop ADP depletion and to act as a permeant anion respectively. Fura2-FF fluorescence was monitored as four bolus Ca2+ additions were made of 200 nmol/mg (10 µM). 1 µM FCCP was added followed by 1 mM Ca2+ and 5 mM EGTA to obtain maximum and minimum fluorescence respectively. a, the raw fluorescence traces are shown along with the autofluorescence traces (AF) of mitochondria similarly treated but sham loaded with vehicle (Me2SO) instead of fura2-FF. b, the autofluorescence values were subtracted from the fura2-FF values and these then converted to matrix [Ca2+]free using the Grynkowitz equation. c, 0.1 mg of fura2-FF-loaded and Pi-depleted mitochondria were treated with exactly the same additions but in the presence of 0.2 µM CaG5N to monitor [Ca2+]e.

 

There is compelling evidence that the independent Ca2+ efflux pathway in heart mitochondria (15, 22) is not saturated by [Ca2+]m under conditions of limited Ca2+ loading when Ca2+-dependent changes in dehydrogenase activation can be observed. Thus the steady-state [Ca2+]m determined both from matrix-loaded fura2 and indirectly by the activation state of matrix dehydrogenases increases with [Ca2+]e (15) while under extensive loading conditions the activity of the efflux pathways is invariant with matrix Ca2+ load but varies inversely with the matrix free phosphate (5).

Fig. 6 shows the transition between these two phases of matrix Ca2+ loading. When isolated mitochondria are suspended in incubation media similar to that used here, there is sufficient contaminating Ca2+ in the medium and residual Ca2+ in the matrix such that the initial loading condition when [Ca2+]m varies with Ca2+ load is not seen. On addition of 10–30 µM EGTA, a sharp decline in the steady-state [Ca2+]e was observed as {Sigma}Cam decreased from 10 to 3 nmol/mg (Fig. 6), consistent with a decrease in free matrix Ca2+ (5). In contrast, additions of Ca2+ caused little increase in steady-state [Ca2+]e, indicating that the setpoint had been attained.



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FIG. 6.
Effect of {Sigma}Cam upon the steady-state [Ca2+]e maintained by rat liver mitochondria. For each data point four simultaneous, identical experiments were performed, with one used to monitor [Ca2+]e and mitochondrial samples removed from the other three. 0.5 mg of rat liver mitochondria were added to 2 ml of incubation buffer identical to that in Fig. 1 with the omission of CaG5N and the inclusion of 1 µM fluo-4 (Kd for Ca2+ of 0.345 µM). [Ca2+]e was allowed to come to a steady-state level and then an addition of either 10, 20, or 30 µM EGTA; 2.5, 6.25, or 12.5 µM CaCl2 or vehicle (H2O) was made to alter total [Ca2+]m. The mitochondria were then allowed to come to their new steady-state [Ca2+]e. 1 mM CaCl2 followed by 5 mM EGTA was added to one experiment (to obtain maximum and minimum fluo-4 fluorescence) while 1.5 ml was removed from each of the other three and centrifuged to obtain a mitochondrial pellet. The pellets were resuspended in 1 ml of 25 mM Hepes pH 7.2 pre-treated with Chelex-100 resin to remove contaminating Ca2+. Mitochondria were subjected to two cycles of freeze-thawing and were sonicated for 4 x 3s each before {Sigma}Ca2+m was measured fluorimetrically. The [Ca2+]e values measured were confirmed by repetition of the same EGTA or Ca2+ additions with a second mitochondrial preparation.

 

Since inefficiency of the [Ca2+]e buffering in the presence of acetate contrasts so much with that seen in the presence of excess Pi (Fig. 1), the inference is that the latter allows [Ca2+]m to be essentially independent of {Sigma}Cam over the range where a setpoint is observed, in the present preparation from 20 nmol/mg (Fig. 6) to 500 nmol/mg {Sigma}Cam (Fig. 1). Studies to investigate this were performed on RBM. Since RBM have been reported to respond to Ca2+ addition differently from liver mitochondria (38) the basic infusion was repeated (Fig. 7). RBM maintain a high membrane potential and NAD(P)H reduction until the onset of the permeability transition in the same way as liver mitochondria.



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FIG. 7.
Rat brain mitochondria maintain membrane potential and NAD(P)H reduction until the permeability transition during continuous Ca2+ loading. RBM (0.1 mg of protein/2 ml) were incubated in the presence of 5 mM glutamate, 5 mM malate, 8 mM Pi, 0.5 mM ADP, and 1 µg/ml oligomycin with the addition of 0.2 µM CaGreen-5N or 100 nM TMRM+. Ca2+ was infused at 87 nmol/min/mg. Note that the TMRM+ trace is inverted so that an upward deflection corresponds to an increase in membrane potential.

 

This was tested in the experiments depicted in Fig. 8a. Ca2+ was steadily infused at a rate of 85 nmol/min/mg, while [Ca2+]e was monitored with CaGreen5N and [Ca2+]m by fura2-FF. Three phases of [Ca2+]m maintenance can be distinguished in the presence of 5 mM Pi. No increase in [Ca2+]m was detected during the infusion of the first 120 nmol/mg of Ca2+. For the next 360 nmol/mg Ca2+ infusion a slow increase in [Ca2+]m can be observed, while above a {Sigma}Ca2+m of 480 nmol/mg [Ca2+]m starts to increase at a rate comparable to that seen initially in Pi-depleted mitochondria (not shown), where [Ca2+]m increases immediately and uniformly.

The discontinuity in the [Ca2+]m trace after infusion of 480 nmol/mg corresponds to the point at which [Ca2+]e monitored by CaG5N starts to increase (Fig. 8b), and this may represent the initiation of the permeability transition, which would allow matrix fura2-FF to be released and equilibrate with [Ca2+]e. The relationship between [Ca2+]m and {Sigma}Ca2+m is shown in more detail in Fig. 8c. Based on a matrix volume of 1 µl/mg the ratio of bound/free Ca2+ in the matrix can reach 150,000 in Ca2+-loaded rat brain mitochondria.

It is frequently assumed that the permeability transition is triggered by an increase in matrix free Ca2+, however increasing external phosphate decreases the capacity of mitochondria to accumulate Ca2+ before the permeability transition is activated (25). If the assumption is correct that [Ca2+]m is defined by the solubility product of a calcium phosphate complex, then [Ca2+]m would be buffered at lower values by the increased matrix Pi. This is confirmed in Fig. 9. Increasing external Pi from 2 to 5 mM decreases the capacity of brain mitochondria to buffer [Ca2+]e by almost 50%; however, the [Ca2+]m at which the permeability transition was activated in the presence of 5 mM Pi was substantially lower than that in the presence of 2 mM Pi. It can be concluded that the permeability transition is not triggered by an increased [Ca2+]m. Consistent with this, cyclosporin A has no effect on the relationship between [Ca2+]m and {Sigma}Ca2+m during Ca2+ infusion (Fig. 10). Cyclosporin A more than doubled the capacity of the mitochondria to accumulate and retain Ca2+ without affecting the relationship between {Sigma}Ca2+m and [Ca2+]m until the permeability transition is activated.



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FIG. 9.
Effect of elevated buffer [Pi] on [Ca2+]m and mitochondrial Ca2+ accumulation capacity. Fura2-FF-loaded RBM were suspended as in Fig. 8b with 0 (thin line/small filled squares),2mM Na-PO4 (medium line/open squares), or 5 mM NaPO4 (thick line/large filled squares). CaCl2 was then infused at 85 nmol/min/mg and [Ca2+]m and [Ca2+]e monitored simultaneously. The point of permeability transition is shown (a) for 5 mM Pi and (b) for 2 mM Pi.

 


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FIG. 10.
Effect of CsA upon [Ca2+]m. Fura2-FF-loaded RBM were suspended as in Fig. 8b in the absence (thin line/open squares) or presence (thick line/filled squares) of 1 µM CsA. CaCl2 was infused at 85 nmol/min/mg as shown and [Ca2+]m and [Ca2+]e monitored simultaneously.

 

The release of hydrogen peroxide from brain mitochondria oxidizing NAD-linked substrates is membrane potential-dependent, and bolus additions of Ca2+ decrease H2O2 release monitored by Amplex Red to an extent that can be correlated with a decrease in membrane potential (26). Since slow Ca2+ infusion occurs independently of any significant depolarization (Fig. 2), H2O2-dependent Amplex Red oxidation in the presence of cyclosporin A was monitored during infusion of Ca2+ (Fig. 11). Compared with a parallel blank infusion there was a slow decline in the rate. It is thus not evident that the permeability transition can be ascribed to a Ca2+-dependent oxidative stress.



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FIG. 11.
Effect of gradual Ca2+ accumulation on the rate of H2O2 production. Rat liver mitochondria were suspended as in Fig. 1 with the addition of 1 µM Amplex Red plus 0.75 units/ml horseradish peroxidase. 85 nmol/mg Ca2+ (+Ca2+) or an equal volume of H2O (no Ca2+) was then infused and H2O2 monitored as an increase in 563/587 nm fluorescence – lower traces. Each line is an average of three experiments. An additional infusion was carried out with 0.2 µM CaG5N in place of Amplex Red plus horseradish peroxidase to indicate the point of PT – upper trace, offset upwards by 10 units for clarity.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix Free Ca2+A series of studies performed 10–15 years ago established that free Ca2+ concentrations within the matrices of isolated liver and heart mitochondria varied over a range from 0.1 to 2 µM under conditions of limited matrix Ca2+ loading, i.e. <10 nmol/mg of protein (15, 23, 24, 25, 26, 27). Three approaches were taken, a null-point technique in which extramitochondrial free Ca2+ was adjusted until the addition of the Ca2+ ionophore A23187 [GenBank] caused no net flux of Ca2+ (18, 28), determination from the activation state of Ca2+-dependent matrix dehydrogenases (15, 23) and loading or entrapment of optical Ca2+ indicators (15, 19, 20, 23, 27). Under these conditions matrix free Ca2+ varied with external free Ca2+ in a way consistent with physiological control of matrix dehydrogenase activities (for review see Ref. 29).

In recent years there have been numerous studies with both intact and digitonin-permeabilized cells that have been loaded with the cationic Ca2+ indicator Rhod-2. Under appropriate conditions the positive charge localizes the ester to the mitochondrial matrix where it is hydrolyzed to generate Rhod-2, a Ca2+ indicator with a Kd of 0.57 µM. The ability of this high affinity probe to detect changes in [Ca2+]m within intact cells indicates that this parameter can vary in situ over the range where Rhod-2 is responsive, say 0.06–6 µM. Substantially higher estimates of transient [Ca2+]m elevations have been reported using matrix-targeted aequorins (30), although the in vitro calibration technique employed has recently been criticized (31), since with an in situ calibration, values close to those obtained with Rhod-2 have been obtained (31). However, since aequorin is consumed by Ca2+, it is not suitable for the measurement of long term steady-state concentrations as studied here.

Matrix Total Ca2+A limitation with the above studies is that total Ca2+ is generally not determined in parallel, and so it is not possible to draw conclusions about the chelation of matrix Ca2+ or how this changes with matrix load. In 1978 the first studies were performed in which steady-state extramitochondrial free Ca2+ concentrations ([Ca2+]e) were monitored during extensive matrix Ca2+ loading (4, 5, 31). In the presence of excess Pi and physiological concentrations of adenine nucleotides, liver (6), and brain (4) mitochondria were able to restore a [Ca2+]e that was independent of matrix Ca2+ load from 10 to almost 1000 nmol of Ca2+/mg.

Analysis of the individual kinetics of the Ca2+ uniporter and efflux pathways indicated that this setpoint was the consequence of a kinetic balance between the activities of a uniporter highly dependent upon [Ca2+]e and an efflux pathway activity apparently totally insensitive to changes in total matrix Ca2+ over this range (5). This latter in turn could be due either to saturation of the efflux pathway by [Ca2+]m or to a [Ca2+]m that was essentially independent of matrix Ca2+ load over this range. Two experiments supported the latter alternative and indicated that complexation of Ca2+ with matrix Pi helped to maintain a constant [Ca2+]m. First the activity of the RLM efflux pathway was inversely related to the Pi concentration (5) and second when acetate replaced Pi as permeant anion the Ca2+ efflux activity, and hence the setpoint increased continuously with matrix load (5).

While such studies were initially criticized as being nonphysiological, the advent of digital Ca2+ imaging demonstrated that cytoplasmic free Ca2+ ([Ca2+]c) in excitable cells undergoes large excursions far exceeding the setpoint values obtained with isolated mitochondria (33), and it is now established that in situ mitochondria can accumulate large amounts of Ca2+ when the local [Ca2+]c is above 0.5 µM and release Ca2+ to the cytoplasm when [Ca2+]c is restored to basal values (e.g. Ref. 34). It is however apparent that monitoring [Ca2+]m of in situ mitochondria provides little information on the extent of total Ca2+ accumulation within the matrix. Thus in repetitively stimulated lizard motor nerve terminals the sustained clearance of cytoplasmic Ca2+ by mitochondria fails to increase [Ca2+]m above 1 µM regardless of stimulation frequency or the amount of accumulated Ca2+ (35). The present study shows (Fig. 8) that [Ca2+]m of isolated rat brain mitochondria changes by less than 20% when total matrix Ca2+ is increased from about 10 to 400 nmol/mg.

These two aspects of mitochondrial Ca2+ accumulation are not mutually incompatible, and as shown in Fig. 6, there is a smooth transition from the region where [Ca2+]m varies with Ca2+ load to the invariant region associated with a constant setpoint and buffered [Ca2+]m.

The Nature of the Matrix Calcium Phosphate Complex— Although much evidence points to the presence of a calcium phosphate complex within the matrix of loaded mitochondria, its nature remains obscure, due largely to the considerable complexity and variability of biologically relevant calcium phosphate forms (for review see Ref. 10). Additionally, investigations are hindered by the ability of the presumed matrix complex instantly to dissociate into Ca2+ and Pi when the Ca2+-loaded mitochondria are depolarized, so that Ca2+ exits via the reversal of the uniporter, and Pi exits via the Pi transporter (5).

The formation of a calcium phosphate complex within the matrix that is in equilibrium with 5 mM free phosphate but with only 2 µM [Ca2+]m (Fig. 8) has to be reconciled with the ability to prepare physiological extracellular buffers containing similar phosphate concentrations but one-thousand times higher free Ca2+ concentrations. Similarly, external Ca2+ electrodes or low affinity extramitochondrial Ca2+ indicators show that Ca2+ released from Ca2+-loaded mitochondria into Pi-containing media remains in solution even though the [Ca2+]e is much higher than the [Ca2+]m indicated by the matrix-loaded fura2-FF (Figs. 9 and 10). The contrast between the ability of polarized mitochondria to retain enormous amounts of Ca2+ within their matrices and the rapid and almost complete release of the ion when the mitochondria are depolarized is additionally puzzling since the concentration gradients of free Ca2+ and Pi across the polarized membrane appear to be so small (Fig. 8), [Ca2+]m being limited by the solubility product of the complex and [Pi]m being defined from the external Pi and the transmembrane pH gradient.

Hydroxyapatite can be detected in fixed and dessicated samples, but it is generally accepted that this is an artifact (36). In an artificial cytoplasm in the presence of ATP, amorphous Ca3(PO4)2 is initially formed when millimolar Ca2+ is titrated, particularly at pH 7.5–8.0 (corresponding to the matrix pH) and is stable for prolonged periods (8). At external pH 7.0, the mitochondrial uptake of one Ca2+ in the presence of excess Pi is accompanied by the net extrusion of one proton (7), consistent with the rapid formation of Ca3(PO4)2 (Fig. 12).



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FIG. 12.
Possible stoichiometries of Ca-Pi complex formation in the mitochondrial matrix. Ion transport stoichiometries are shown for respiration-linked mitochondrial Ca2+ accumulation in the presence of excess phosphate. The external pH is assumed to be 7.2 such that there are equal concentrations of and . The phosphate carrier is assumed to function as a H3PO4 uniporter, this is equivalent to a antiport. a, amorphous tricalcium phosphate, Ca3(PO4)2, forms in the matrix, a H+/Ca2+ ratio of 1 is seen in the external medium. b, CaHPO4 formation gives a H+/Ca2+ ratio of 0.5. c, Ca(HPO4)2 formation gives a H+/Ca2+ ratio of –1. d, hydroxyapatite formation would be associated with a H+/Ca2+ ratio of 1.1. e, matrix free Ca2+ concentration ([Ca]m) in equilibrium with either amorphous tricalcium phosphate (TCA, Ksp = 3 x 1030)or hydroxyapatite (HAP, Ksp = 1 x 1059) for two concentrations of total external phosphate (2 and 5 mM) and as a function of matrix pH. The three dissociation constants for phosphate were taken as: pK1 = 2.13, pK2 = 7.2, pK3 = 12.39 (10). Note that the gradient of the anion varies as the third power of the pH gradient across the inner membrane, thus matrix acidification from pH 8.0 to pH 7.0 would decrease the matrix concentration by a factor of 1000. At a constant solubility product for Ca3(PO4)2, this would raise the [Ca2+]m in equilibrium with the complex by 100-fold. In the presence of 5 mM total Pi, an indicated [Ca2+]m of 2.1 µM would correspond to a matrix pH close to 7.7, i.e. 0.5 pH units alkaline with respect to the medium.

 

Reported values for the ion activity product (solubility product) of amorphous Ca3(PO4)2 differ widely in the literature, from 2 x 1033 to 1.6 x 1025. It is not easy to find the original references in which these constants were determined, and in any case the mitochondrial matrix is perhaps as far from an ideal solution as it is possible to imagine, but nevertheless the solubility properties of this calcium salt provides a way to understand a number of apparently conflicting aspects of calcium storage in the mitochondrial matrix. Thus it is not immediately apparent how micromolar free Ca2+ concentrations could form such a complex with Pi within the mitochondrial matrix, while much higher free Ca2+ concentrations can coexist with millimolar Pi in physiological incubation media without precipitation. Secondly, if the gradient of free Ca2+ concentration across the inner membrane is really so small (1–4-fold) as is indicated by this and other studies, why is a high membrane potential required to retain Ca2+ within the matrix and why does the addition of protonophore lead to such a massive and rapid efflux of Ca2+ and Pi? The answer may lie in the high pH dependence of the Ca2+ concentration that is in equilibrium with amorphous Ca3(PO4)2 (Fig. 12). The highly active phosphate carrier equilibrates the transported species with OH (11) and thus accumulates the monoanion as a function of {Delta}pH. The further dissociations of to , and of to , the species that complexes with Ca2+ to form Ca3(PO4)2, are each dependent on pHm, with the result that the concentration of the species increases as the third power of pHm. Maintenance of the solubility product for Ca3(PO4)2 thus means that the concentration of free matrix Ca2+ in equilibrium with the complex decreases as the second power of the matrix pH and as the two-thirds power of the total external phosphate concentration (Fig. 12). The collapse or even temporary reversal of {Delta}pH following addition of a protonophore would thus be predicted to facilitate dissociation of the complex, liberating free Ca2+ and facilitating its rapid efflux via reversal of the uniporter in response to the concomitant collapse of {Delta}{Psi}m.

The Relationship between Bolus Ca2+ Additions and Continuous Infusion—This is the second study in which we have utilized slow, continuous Ca2+ infusion in order to investigate mitochondrial Ca2+ transport. In the first (5) we determined the kinetics of the rat liver mitochondrial Ca2+ uniporter by infusing the cation at different rates and determining the value at which [Ca2+]e stabilized, i.e. when the uniporter activity exactly balanced that of the infusion rate plus the activity of the efflux pathway. Uniporter activity was found to increase as an exponential function of [Ca2+]e until respiration became rate-limiting (5).

It is extraordinarily complex to analyze mitochondrial Ca2+ transport and sequestration in response to a bolus addition of the cation when [Ca2+]e, {Delta}{Psi}m, light scattering, phosphate transport, and calcium phosphate complex formation are all changing rapidly with time (see e.g. Ref. 38). The present study removes much of this complexity by monitoring Ca2+ sequestration in the matrix when the cation is infused sufficiently slowly to minimize the bioenergetic load, and hence effects on {Delta}{Psi}m. This is of particular relevance since it has been proposed that the permeability transition pore is activated by a lowering of the membrane potential (39). Infusion also allows ample time for calcium phosphate complexes to form in the matrix; it is possible that the permeability-transition-independent release of cytochrome c seen when large Ca2+ boluses (400–2100 nmol/mg of protein) are given to brain mitochondria (38) is a consequence of a high free matrix Ca2+ before complex formation occurs, particularly since phosphate transport follows Ca2+ entry and is initiated by the enhanced transmembrane pH gradient.

Relevance to in Vivo Mitochondrial Ca2+ Transport—It is becoming increasingly apparent that brain mitochondria in situ can accumulate Ca2+ under a variety of physiological and pathological conditions such as epilepsy (40), ischemia (41, 42), and concussive brain injury (43). At the same time there is active debate as to whether the permeability transition participates in neuronal ischemic death and whether permeability transition inhibitors are neuroprotective (9, 32, 37, 44, 45, 46). It is clearly important to establish the factors that define the Ca2+-loading capacity of brain mitochondria. Two scenarios may be envisaged, one in which in situ brain mitochondria load with Ca2+ rapidly in response to a sudden dramatic increase in free cytoplasmic Ca2+, for example due to repetitive firing of voltage-activated Ca2+ channels, and one in which they slowly accumulate the cation in response to a slow uncompensated inward leak of the cation across the plasma membrane. The present study may be of relevance to the latter. It is apparent that phosphate plays a major role in defining the capacity of the mitochondria; it would of importance to determine whether cytoplasmic phosphate is in excess or is limiting under the above conditions of mitochondrial Ca2+ loading.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 1-415-209-2095; Fax: 1-415-209-2232; E-mail: dnicholls{at}buckinstitute.org.

1 The abbreviations used are: {Sigma}Ca2+m, total matrix Ca2+; [Ca2+]m, matrix free Ca2+ concentration; [Ca2+]e, extramitochondrial free Ca2+ concentration; Ksp, solubility product; CaG5N, Calcium Green 5N, TMRM+, tetramethylrhodamine methyl ester; RLM, rat liver mitochondria; RBM, rat brain mitochondria; BSA, bovine serum albumin; Tes, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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