Regulation of Oxidative Phosphorylation in Mitochondria by External Free Ca2+ Concentrations*

The rate of oxidative phosphorylation was studied in rat liver mitochondria incubated with free Ca2+ con- centrations that range from lo-’ to 5 % lo-‘ M. The highest rate was observed between 0.5-1.0 PM Ca2+. ATP synthesis was measured by polarographic and spectrophotometric techniques and by uptake of radio-active inorganic phosphate. The concentration of Ca2+ at which maximal rates of ATP synthesis take place is modified by Mg2+ and phosphate. The dependence of oxidative phosphorylation on Ca2+ was observed with a-ketoglutarate, glutamate + malate, and succinate, but not with B-hydroxybutyrate. At lo-’ M Ca2+ there is a continuous exit of endogenous Ca2+, while with lo-‘ M Ca2+, intramitochondrial Ca2+ levels remained constant throughout time. Apparently the control of the level of internal Ca2+ by external Ca2+ modulates the rate of oxidative phosphorylation. Uncoupler-stim-ulated respiration also depends on Ca2+ concentration, even though at lo-’ to lo-‘ M Ca2+ the rate of oxidative phosphorylation is lower than the rate of uncoupled respiration. The contribution of the ADP/ATP carrier and the ATP synthase to the kinetic regulation of ATP synthesis at lo-’ and lo-‘ M Ca2+ was evaluated by titrations with carboxyatractyloside and oligomycin, respectively. The contribution of the carrier and the synthase to the regulation of the final rate of ATP synthesis was different at the two concentrations of Ca2+; therefore, the concentration of extramitochondrial Ca2+ influences the overall kinetics of oxidative phosphorylation.

cleotides (13) which apparently diminishes the activity of the translocator, thus inducing low rates of oxidative phosphorylation in Ca2+-loaded mitochondria (13). It has also been suggested that Ca2+ prevents the release of inhibiting action of the ATPase inhibitor protein from mitochondrial ATPase (14,15).
Interestingly, Robertson et al. (16) reported that in heart mitochondria external Ca2+ in the range of 10-9-10-5 M induces a dual effect on oxidative phosphorylation, either an activation of approximately 25% (between 0.1-1.0 PM) and a strong inhibiting effect with >1.0 PM regardless of the presence of M$+. In this work the effect of external Ca2+ concentrations lower than 1.0 PM on oxidative phosphorylation was studied. The results showed that by varying external Ca2+ concentrations, it is possible to observe different rates of steady-state ATP synthesis.
At these different rates, the contribution of the adenine nucleotide translocase and the ATP synthase to the control of rate of oxidative phosphorylation (17, 18) was evaluated. It was found that the level of external Ca2+ induces important modifications of the overall kinetics of oxidative phosphorylation.

MATERIALS AND METHODS
Preparation of Mitochondria-Female Wistar rats weighing 180-230 g and fasted for 38-44 h were killed by decapitation. The liver was extracted and washed twice and homogenized with 250 mM sucrose, 5 mM HEPES,' 0.5 mM EGTA (SHE), pH 7.0, in the cold. The homogenate was centrifuged to 7000 X g for 10 min. The mitochondrial pellet was washed with SHE, resuspended, and incubated with 0.5% bovine serum albumin for 5 min in ice with occasional stirring; subsequently the mixture was diluted 20 times and centrifuged to 7000 X g for 10 min. The sediment was washed and resuspended in SHE to a concentration of  mg/ml.
Oxygen Consumption-Oxygen uptake of mitochondria incubated in 3 ml of incubation basis mixture that contained 130 mM KCl, 20 mM HEPES, 2 mM MgC12, 2 mM K phosphate, 5 mM succinate, and Ca2+-EGTA buffer, pH 7.20, was recorded by means of an oxygen electrode (Yellow Springs Instrument Co.) Ca2+-EGTA Buffers-To calculate the free Ca2+ concentration, the stability constants and the program described by Fabiato and Fabiato (19) were used. This program considers the contribution of all the ligands and metals ions that were included in the experiments. The final concentration of EGTA was 2.0 mM, and the pH of the fresh medium was carefully adjusted to 7.20.
Sartorius filters of a pore diameter of 0.22 pm. The filter was washed with 1 ml of cold 130 mM KC1, 5 mM Tris-HC1, pH 7.4, and counted for radioactivity after solubilization with 5 ml of the scintillation liquid Tritosol (20).
lntramitochondrial Ca2+ Content-This measurement was made by atomic absorption spectrophotometry, using a Perkin-Elmer (model 560) spectrophotometer. Mitochondria were separated from the medium by rapid centrifugation in a Beckman microfuge B. The pellet was extracted for Ca2+ determination with 1.5% (v/v) HCI, 1% (w/v) LaCl3, 25 mM KCl, and denatured protein eliminated by centrifugation. The supernatant was used for the assays.
Spectrophotometric ATP Determination-This was carried out according to Lamprecht and Traustschold (21). Mitochondria were incubated under the conditions described under "Results." At fixed times aliquots of 2 ml were transferred to cuvettes that contained the ATP (21).
necessary compounds for the spectrophotometric determination of Uptake of 32Pi into ATP-Mitochondria were incubated under the indicated conditions with 32Pi (approximately 0.5 pCi/pmol). At predetermined times the reaction was stopped with 6% trichloroacetic acid (final concentration). After centrifugation of denatured protein an aliquot of the supernatant was withdrawn and 32Pi extracted as described originally by Lindberg and Ernster (22) with n-butyl acetate. The aqueous phase was used for assay of 32Pi incorporated into ATP by assay of Cerenkov radiation. Table I   concentrations Mitochondria (3 mg) were incubated in 3 ml of basic medium (see "Materials and Methods") with the CaZ+ concentrations shown. At the times indicated, an aliquot was centrifuged in a Microfuge, the supernatant discarded, and Ca2+ determined in the sediment by atomic spectrophotometric absorption. This was corrected by the trapped volume which was measured with ["C]sucrose. At 5 min 250 p~ ADP was added. The numbers in parenthesis indicate the number of determinations made. Synthesis of ATP at Various Ca2+ Concentrations-Mitochondria incubated in state 4 conditions for 5 min with different oxidizable substrates and then given ADP show a distinct rate of state 3 respiration that depends on the concentration of Ca2+ in the medium (Table 11). With succinate, glutamate + malate, and a-ketoglutarate as oxidizable substrates, higher rates of oxygen uptake are observed with M than at lo-' M Ca2+. With all these substrates, 5 X 10"j M Ca2+ induces a strong diminution of the state 3 respiration rate; with P-hydroxybutyrate the diminution starts to be observed at M ca2+. Fig. 1 shows a curve of Ca2+ concentration versus rate of respiration with succinate as substrate. Maximal stimulation of state 3 respiration was observed with M Ca2+ (Fig. L4). From preparation to preparation, the maximum was found to vary between 0.5-1.0 p~ Ca2+, but in all cases, at higher Ca2+ concentrations the rate of state 3 respiration falls sharply. Even though state 4 respiratory rates increase progressively showed that ATP formation parallels the rate of oxygen consumption (Fig. 1B). For example, with the polarographic method it was observed that state 3 respiration is approximately 33% higher at M than at M Ca2+, while the spectrophotometric assay of ATP, in the presence of 10 mM AMP, indicated that ATP synthesis was 25% higher at 5 x  (Table 111). Thus the results of Fig. 1 and Tables I to 111 indicate that there is a critical concentration of Ca2+ at which oxidative phosphorylation takes place at maximum rates. The strong inhibition of oxidative phosphorylation by high Ca2+ concentrations (5 x M) observed in Fig. 1 and Tables I1 and 111 is probably due to a massive accumulation of Ca2+, a phenomenon which has been extensively documented (Refs. 3-16 and see also Table I).

Ca2+ Uptake at Various External Free Ca2+ Concentrations-The experiments detailed in
Characteristics of the Effect of External Ca2+ on Oxidative Phosphorylation-The rate of oxidative phosphorylation de-

I1
Effect of Caz+ on rate of state 3 respiration with several Oxidizable substrates Mitochondria (3 mg) were incubated in 3 ml of basic medium (see "Materials and Methods") with the Ca2+ concentrations shown. At 5 min 300 phi ADP was added, and the rates of respiration were recorded. The concentration of all oxidizable substrates was 5 mM.  Spectrophotometric ATP determination was made according to Ref. 21. Mitochondria (1 mg/ml) were incubated for 5 min, and aliquots were transferred to cuvettes with the necessary compounds plus 0.5 mM ADP for ATP determination (21). Respiration was carried out at 30 "C and ATP determination at 23 "C. The unit of x axis of the inset is 1 M Ca2+; ATP synthesis is nmol min" mg".

TABLE I11
Effect of Cu2+ on uptake of "Pi into ATP Mitochondria (1 mg/ml) were incubated for the time and Ca2+ concentrations shown. Aliquots were transferred to vessels which contained 250 PM ADP. After 1 min, the reaction was stopped and the uptake of 32Pi was determined in the supernatant as described under "Materials and Methods." pends on the time of exposure of mitochondria to different concentrations of external Ca2+ (Fig. 2, see also Table 111). In mitochondria incubated with lo-' M Ca", the rate of state 3 respiration decreases with the length of state 4 conditions; with M Ca2+, the rate of state 3 is affected, but to a lower extent. In both cases, the rate of state 3 respiration ceases t o be modified after 10 min of incubation. ATP synthesis as assayed by the spectrophotometric technique and by 32Pi uptake yielded the same results (Fig. 2, Table 111).
Similar results are obtained when mitochondria are exposed to consecutive ADP additions at different Ca2+ concentrations (Table IV), with two substrates that were assayed, succinate and glutamate-malate. Again, with lo-' M Ca2+, there is a time-dependent diminution of state 3 respiration, while with M Ca2+, the diminution is less pronounced. The same pattern of results was obtained with succinate or glutamatemalate as substrates. Interestingly, the rate of state 4 respiration also decreases in a time-dependent manner with lo-' M Ca2+ and stays constant with M Ca2+ (Table IV,  In rat liver mitochondria it is established that Mg2+ inhibits    It was also observed that the effect of Ca2+ on oxidative phosphorylation is modified by the concentration of inorganic phosphate. With the optimal Ca2+ concentration (lo-' M), the rate of state 3 respiration was further increased when inorganic phosphate was varied from 0.5 to 10 mM. The rate of resting respiration decreased approximately 30% in the same range of inorganic phosphate (data not shown). Thus from the experiments described, it appears that there is a level of endogenous Ca2+ which would be essential for maximal rates of oxidative phosphorylation.
Uncoupled Respiration of Mitochondria Incubated with lo-' and lov6 M Ca2+"In an attempt to understand why the rate of oxidative phosphorylation is higher at than lo-' M external Ca2+, the rate of uncoupler-stimulated respiration was studied (Table V). It was observed that in mitochondria exposed to lo-' M Ca2+ the rate of respiration decreased as the time of preincubation was increased. In mitochondria incubated with M Ca2+, the rate of respiration remained more or less constant regardless of the length of the preincubation. These observations indicate that external Ca2+ has an effect on the rate of electron flow and/or the transport of oxidizable substrates into the mitochondria. However. this does not seem to account completely for the different rates of ATP synthesis observed at the two Ca2+ concentrations, since the rate of uncoupled-stimulated respiration is higher than that of state 3 (Table V), notwithstanding the concentration of Ca2+. This suggests that the presently described effect of different Ca2+ concentrations on the rate of ATP synthesis is most likely due to an action on the phosphorylation of ADP or on the adenine nucleotide translocation.
Control of the Rate of ATP Synthesis by the Adenine Nucleotide Translocator and by the ATP Synthase-It has been previously proposed that the rate of oxidative phosphorylation is limited by the activity of the adenine nucleotide translocator (29-32). However, this postulation has not been entirely accepted (Refs. 33, and 34; for a review see Ref. 35). Since different steady-state rates of ATP synthesis can be induced by varying external Ca2+ concentrations, it was possible to explore the contribution of the adenine nucleotide translocator to the process, in an attempt to define the role of Ca2+ in the control of the rate of ATP synthesis. To this purpose a titration of the rate of state 3 respiration with a specific irreversible inhibitor of the translocator (36) was carried out.   control of a step in a metabolic pathway may be estimated by measuring the initial slope of the inhibition curve and relating it to the uninhibited and fully inhibited activity. The results of the calculations are shown in Table VI. Clearly there is significant difference in the control of the rate of oxidative phosphorylation by the translocator at the two Ca2+ concentrations studied, independently of the substrate used. Its contribution to the overall control of the rate of oxidative phosphorylation is about 4.6 and 2.4 times higher with succinate and with glutamate-malate, respectively, at M than at 10-~ M Ca2+.
The contribution of the ATP synthase to the control of oxidative phosphorylation was judged by assaying the sensitivity of state 3 respiration to oligomycin of mitochondria incubated with lo-' and M Ca2+. The results of Fig. 4 show that regardless of the rate of state 3 respiration, the inhibition curve by increasing concentrations of oligomycin is highly sigmoidal; nevertheless, it is of interest to point out that lo-' M Ca2+, the degree of control of oxidative phosphorylation exerted by the ATP synthase is higher than at M Ca2+. Moreover, according to the criterion employed to estimate the control exerted by the ATP synthase and the translocase, it would appear that with M external Ca2+, the two enzymes possess a similar quantitative effect on the regulation of the rate of ATP synthesis.

DISCUSSION
The effect of Ca2+ on oxidative phosphorylation has been extensively studied, and there is general agreement that at relatively high amounts of Ca2+ accumulated, oxidative phosphorylation is inhibited (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). In this work these results have been confirmed, but in addition it was found that there is a concentration of Ca2+ at which maximal rates of ATP synthesis (as determined by the polarographic method or by direct assay) take place. In our standard experimental conditions this is around M Ca2+, but it may vary with the presence or absence of M e and with the concentration of phosphate in the medium.
It may be considered that the lower rates of ATP synthesis observed at lo-' M Ca2+ in comparison to those observed with M Ca2+ are due to damage of mitochondria. In this respect, it is to be noted that between IO-' and M external Ca2+ concentration, similar ADP/O ratios are attained, which indicates that the permeability properties of the mitochondria are not affected.
The modulation of the rates of oxidative phosphorylation by concentrations of external Ca2+ below 1.0 p~ is of interest, since this is apparently the range in which Ca2+ concentration varies within the cell under physiological conditions (26). As to how these concentrations of external Ca2+ regulate the rate of ATP synthesis, the results of this work also show that upon exposure of mitochondria to concentrations of external Ca2+ of lo-' M (a concentration at which oxidative phosphorylation is about 30% lower than at M Ca"), the amount of internal Ca2+ falls to a level of about 10 nmol/mg of protein (after 5 min of incubation). While during incubation with M external Ca2+, the amount of intramitochondrial Ca2+ is poised at a level of approximately 20 nmol mg", a concentration approximately equal to that of the starting preparation. Following Nicholls (23), at concentrations of IO-' M external Ca2+, a membrane potential of -192 mV would be required to retain internal Ca2+ (as derived from the Nernst equation assuming an activity coefficient of internal Ca2+ of 0.1). Since in conditions similar to those of the present work, the mitochondrial membrane potential is of the order of 120-170 mV negative inside (37,38), Ca2+ release would be a thermodynamically favored process. Using the same considerations, in mitochondria incubated with M external Ca2+ and in which maximal rates of ATP synthesis were detected, the inward and the outward movements of Ca2+ would be in near equilibrium.
The present data show that the rate of ATP synthesis in mitochondria decreases in a time-dependent process when incubated at M Ca2+ in comparison to that attained at M Ca2+. Apparently as Ca2+ leaks out of the mitochondria, the rate of oxidative phosphorylation gradually decreases. Moreover, in conditions in which internal Ca2+ is maintained at a constant level of approximately 20 nmol/mg of protein, the rate of respiration is not ostensibly affected. Thus it would appear that the level of internal Ca2+ as modulated by the extramitochondrial Ca2+ concentrations influences the rate of oxidative phosphorylation.
An attempt has been made to explore how the internal concentration of Ca2+ affects the overall kinetics of ATP synthesis. It has been observed that, similar to the state 3 respiratory rates, electron transport of uncoupled mitochondria is faster at than at lo-' M Ca2+. This Ca2+-dependent process can be related to the higher rate of ATP synthesis observed with M Ca2+, but it is noteworthy that the rate of state 3 respiration is lower than uncoupled respiration, regardless of the Ca2+ concentration in the medium. This indicates a priori that state 3 respiration is more importantly limited by either the phosphorylation of ADP and/or by the activity of the adenine nucleotide translocator.
An analysis of the inhibition curve of state 3 respiration as induced by oligomycin revealed that the control exerted by the ATP synthase was higher at lo-' than at M Ca2+. However, it was also observed that to attain a steep change in the slope of the inhibition curve more oligomycin was required at M Ca2+. The latter would suggest that a different number of active enzymes would be operating at the two concentrations of Ca2+ studied; indeed it has been previ-

Ca2+ and Oxidative
Phosphorylation 4033 ously reported that Ca2+ levels may affect the interaction of the inhibitor protein with the ATP synthase (14, 15), thus affecting the number of functional enzymes. Therefore, the overall data obtained with oligomycin suggest that the analysis of the control exerted by an enzyme on a metabolic pathway as described by Kacser and Burns (39,40) and by Heinrich and Rapoport (41,42) would seem to require an experimental evaluation of the extent to which an enzyme that undergoes reversible transitions affects its degree of control on the pathway. The ATP synthase would seem to be a good system to test the reported theoretical considerations.
The titrations of state 3 respiration with carboxyatractyloside showed that the degree of control exerted by the translocase is significantly different in the presence of lo-' than with M Ca", being several times higher at than at lo-' M Ca2+. Thus, the modification of the rate of oxidative phosphorylation parallels a change of the kinetic control exerted by the translocase, which suggests that other kinetic transitions are occurring in other steps of coupled ATP synthesis when the amount of mitochondrial Ca2+ is varied. In other words, if the degree of control exerted by the ADP/ATP carrier is different at the two levels of Ca2+ studied, it follows that the quantitative contribution of the other steps of oxidative phosphorylation would also necessarily undergo modification. In fact, it is relevant that the Ca2+-dependent variations in the control, as evaluated from the initial slope of inhibition curves (17,18, [39][40][41][42], exerted by the ATP synthase and the translocase occur in opposite directions, i.e. when the control exerted by the ATP synthase is higher (lo-' M external Caz+), the control by the translocator is relatively low. The kind of kinetic relation where a change in the degree to which one enzyme is rate limiting results in an opposite directed change in the degree to which the other enzyme becomes limiting has been studied by Stoner and Sirak (43). They concluded that such a sequential coupling relation is established between ADP transport and the phosphorylation reaction during oxidative phosphorylation.
Moreover, the observed difference between the rates of uncoupled electron transport and state 4 respiration at and lo-' M external Ca2+ suggests the existence of a Ca2+induced modification of substrate transport and/or electron transfer. Therefore, it is suggested that variations in the extramitochondrial Ca2+ concentration, within the limits that exist in living cells (26), modify quantitatively the contribution of the multiple control points (17, 18) that are involved in the overall kinetics of ATP synthesis poising the system a t various rates of oxidative phosphorylation.
Along this line, measurements of the activity of several intramitochondrial dehydrogenases (44-46) and electron probe analysis of heart and other muscle cells (47) indicate that the possible physiological range of intramitochondrial Ca2+ is of the order of 1-5 nmol/mg of mitochondrial protein.
In our experimental conditions, the Ca2+ levels are near the aforementioned values which suggests that Ca2+ exerts a physiological role on the regulation of oxidative phosphorylation. Moreover, a dependence of the activity of carbamoyl phosphate synthase and pyruvate carboxylase on internal Caz+ levels in a range similar to that attained here has been reported (11, 48).
At the moment it is not possible to ascertain the mechanism through which the internal concentrations of Ca2+ induce the aforementioned changes of the kinetics of oxidative phosphorylation, but it is interesting that there are reports that indicate that the amount of intramitochondrial adenine nucleotides is affected by the internal concentration of Ca2+ (13, 49, 50), and recently it was reported that prolonged incuba-tions of mitochondria increase the control strength exerted by the adenine nucleotide translocator (51) which apparently is associated with a diminution in the intramitochondrial pool of adenine nucleotides. However, it is necessary to consider that variations in the level of other endogenous components may account for the presently described observations. With respect to M$+, it is important to point out that in liver mitochondria with a content of approximately 30 nmol of MgZ+/mg of protein incubated for 5 min in mixtures similar to these employed here, Masini et al. (52) observed a loss of approximately 3 nmol of M%+/mg. Thus variations in Mg2+ levels would not seem to be an important factor in the presently described effects of Ca2+ on oxidative phosphorylation.
In relation to the contribution of the ATP synthase to the overall process of ATP synthesis, it must be recalled that the action of the natural ATPase inhibitor protein on the ATP synthase appears to be modulated by the concentrations of Ca2+ in the mitochondria (14, 15). Therefore, it would seem that Ca2+ concentration in the exterior and/or interior of the mitochondria induces important modifications of the kinetic characteristics of many of the steps involved in the total process of ATP synthesis.