The Ba2+ sensitivity of the Na+-induced Ca2+ efflux in heart mitochondria: the site of inhibitory action

The Na+-induced Ca2+ release from rat heart mitochondria was measured in the presence of Ruthenium red. Ba2+ effectively inhibited the Na+-induced Ca2+ release. At 10 mM Na+ 50% inhibition was reached by 1.51 +/- 0.48 (S.D., n = 8) microM Ba2+ in the presence of 0.1 mg/ml albumin and by 0.87 +/- 0.25 (S.D., n = 3) microM Ba2+ without albumin. In order to inhibit, it was not required that Ba2+ ions enter the matrix. 140Ba2+ was not accumulated in the mitochondrial matrix space; further, in contrast to liver mitochondria, Ba2+ inhibition was immediate. The Na+-induced Ca2+ release was inhibited by Ba2+ non-competitively, with respect of the extramitochondrial Na+. The double inhibitor titration of the Na+-Ca2+ exchanger with Ba2+ in the presence and absence of extramitochondrial Ca2+ revealed that the exchanger possesses a common binding site for extramitochondrial Ca2+ and Ba2+, presumably the regulatory binding site of the Na+-Ca2+ exchanger, which was described by Hayat and Crompton (Biochem. J. 202 (1982) 509-518). All these observations indicate that Ba2+ acts at the cytoplasmic surface of the inner mitochondrial membrane. The inhibitory properties of Ba2+ on the Na+-dependent Ca2+ release in heart mitochondria are basically different from those found on Na+-independent Ca2+ release in liver mitochondria (Lukács, G.L. and Fonyó, A. (1985) Biochim. Biophys. Acta 809, 160-166).     

Interaction of alkaline earth and transition metals with Na+/Ca2+-plasma membrane exchanger in secretory cells of the gastric gland

Investigation of Na(+)-dependent Ca2+ uptake into the secretory cells of isolated gastric glands from guinea pig with the use of calcium isotope (45Ca2+) has been performed. The presence of Na+/Ca(2+)-exchanger in the cells membrane was established. Ca2+ uptake into the cells through Na+/Ca(2+)-exchanger was competitively inhibited by the number of alkaline earthy and transient metals" cations. Potency of inhibition increases in such an order (Ki, mM): Ba2+ (117.7) < Sr2+ (53.4) < < Mn2+ (15.2) < < Co2+ (12.8) < Cd2+ (8.6). By one-factor dispersion analysis it was shown that potency of inhibition depends on ionic radii and hydration enthalpy of metals" cations (hx2 = 93.93-94.15%) and also on stability constants of their complexes with oxygen-containing bioligands (acetic, aspartic and glutamic acid) (hx2 = 82.32-82.47%). Dependence of inhibitory constants from ionic radii is most adequately described by the parabolic equation, such dependence from hydration enthalpy and stability constants with oxygen-containing bioligands--by exponential or multiplicative equations. The conclusion has been made that velocity of Ca2+ transport through Na+/Ca(2+)-exchanger and potency of its inhibition by metals" cations is determined by the interaction between energy of their interaction with cation-binding sites of transport system and energy of hydration. Energetics of such interactions mainly depends on the steric conformity between the metal cation and cation-binding sites of the exchanger.     

Regulation of Ca2+ efflux from kidney and liver mitochondria by unsaturated fatty acids and Na+ ions.

The effects of fatty acids and monovalent cations on the Ca2+ efflux from isolated liver and kidney mitochondria were investigated by means of electrode techniques. It was shown that unsaturated fatty acids and saturated fatty acids of medium chain length (C12 and C14) induced a Ca2+ efflux from mitochondria which was not inhibited by ruthenium red, but was specifically inhibited by Na+ and Li+. The Ca2+-releasing activity of unsaturated fatty acids did not correlate with their uncoupling activity. In kidney mitochondria a spontaneous, temperature-dependent Ca2+ efflux was observed which was inhibited either by albumin or by Na+. It is suggested that the net Ca2+ accumulation by mitochondria depends on the operation of independent pump and leak pathways. The pump is driven by the membrane potential and can be inhibited by ruthenium red, the leak depends on the presence of unsaturated fatty acids and is inhibited by Na+ and Li+. It is suggested that the unsaturated fatty acids produced by mitochondrial phospholipase A2 can be essential in the regulation of the Ca2+ retention in and the Ca2+ release from the mitochondria.     

In squid nerve fibers monovalent activating cations are not cotransported during Na+/Ca2+ exchange

Squid axons display a high activity of Na+/Ca2+ exchange which is largely increased by the presence of external K+, Li+, Rb+ and NH+4. In this work we have investigated whether this effect is associated with the cotransport of the monovalent cation along with Ca2+ ions. 86Rb+ influx and efflux have been measured in dialyzed squid axons during the activation (presence of Ca2+i) of Ca2+o/Na+i and Ca2+i/Ca2+o exchanges, while 86Rb+ uptake was determined in squid optic nerve membrane vesicles under equilibrium Ca2+/Ca2+ exchange conditions. Our results show that although K+o significantly increases Na+i-dependent Ca2+ influx (reverse Na+/Ca2+ exchange) and Rb+i stimulates Ca2+o-dependent Ca2+ efflux (Ca2+/Ca2+ exchange), no sizable transport of rubidium ions is coupled to calcium movement through the exchanger. Moreover, in the isolated membrane preparation no 86Rb+ uptake was associated with Ca2+/Ca2+ exchange. We conclude that in squid axons although monovalent cations activate the Na+/Ca2+ exchange they are not cotransported.     

Dependence of the inhibitory effect of metal cations on rat liver mitochondrial Ca2+ uptake on their physico-chemical properties

It has been found that Sr2+, La3+ Mn2+ (10-50 microM) inhibit Ca2+ transport into mitochondria in a competitive manner. Cd2+ ions show the mixed type inhibition of this transport. The inhibitory constants (Ki, microM) of the metals cations effect on Ca2+ transport increases in such a sequence: La3+ (2,11), Cd2+ (10,36), Mn2+ (49,29), Sr2+ (66,43). The metals cations inhibitory effect has an insignificant dependence on their ionic radii. But it is good correlated with the series of metals cations, based on the stability constants of their complexes with acetate (r = -0.96), aspartic (r = -0.91) and glutaminic acids and their hydratation enthalpy (r = -0.78). These data reveal that hydratation of metals cations and their interaction with carboxyles of Ca(2+)-uniporter plays an important role in the process of Ca2+ transport into mitochondrial matrix space and its inhibition by the metals cations. The mixed type inhibition of mitochondrial Ca2+ uptake by Cd2+ seems to be caused by the partial de-energization of mitochondria owing to Cd2+ interaction with SH-containing respiratory chain components and pore-forming ligands of mitochondrial membrane.     

Effects of variation in the structure of spermine on the association with DNA and the induction of DNA conformational changes.

Manganese shares the uniport mechanism of mitochondrial calcium influx, accumulates in mitochondria and is cleared only very slowly from brain. Using dual-label isotope techniques, we have investigated both Mn2+ and Ca2+ mitochondrial efflux kinetics. We report that (1) there is no significant Na(+)-dependent Mn2+ efflux from brain mitochondria; (2) Mn2+ inhibits both Na(+)-dependent and Na(+)-independent Ca2+ efflux in brain, in a mode that appears to be primarily competitive and with apparent Ki values of 5.1 and 7.9 nmol/mg respectively; and (3) Ca2+ does not appear to inhibit Mn2+ efflux from brain mitochondria. Findings (1) and (2) suggest the possibility of mitochondrial accumulation of both Mn2+ and Ca2+ in Mn2(+)-intoxicated brain.     

Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A

Ca(2+) plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca(2+) is controlled primarily by the mitochondrial Ca(2+) uniporter and the mitochondrial Na(+)/Ca(2+) exchanger, influencing NADH production through Ca(2+)-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca(2+)-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca(2+) release. Here we selectively measure Ca(2+) influx rate through the mitochondrial Ca(2+) uniporter and Ca(2+) efflux rates through Na(+)-dependent and Na(+)-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na(+)/Ca(2+) exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ(m) loss, Ca(2+) release, NADH oxidation, swelling) of high extramitochondrial Ca(2+) additions, allowing mitochondria to tolerate total mitochondrial Ca(2+) loads of >400nmol/mg protein. For Ca(2+) pulses up to 15μM, Na(+)-independent Ca(2+) efflux through the permeability transition pore accounted for ~5% of the total Ca(2+) efflux rate compared to that mediated by the mitochondrial Na(+)/Ca(2+) exchanger (in 5mM Na(+)). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na(+)/Ca(2+) exchanger-mediated Ca(2+) efflux at higher concentrations (IC(50)=2μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~40% at 10μM cyclosporine A, while having no effect on the mitochondrial Ca(2+) uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca(2+) load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Interaction of metal cations with Ca2+-transport sites of the plasma membrane Ca2+ pump of secretory cells of gastric glands

Investigation of Ca2+ transport by calcium pump of the cell plasma membrane of the gastric glands isolated from guinea pigs and its inhibition by metal cations has been performed. The mainly competitive type of Ca2+ translocation inhibition by the calcium pump by metals cations (0.025-1.00 mM) was determined. Potency of inhibition increases in such an order (I50, mM): Ba2+ (0.336) < Sr2+ (0.251) < Mn2+ (0.099) < Co2+ (0.029) < Cd2+ (0.016). It was shown by one-factor dispersion analysis that potency of inhibition depends on ionic radii and hydration enthalpy of metal cations and also on stability constants of their complexes with oxygen-containing bioligands (acetic, aspartic and glutamic acid) (hx2 = 83.73-85.95). Dependence of the inhibition constants (I50) on ionic radii is most adequately described by the parabolic equation, such a dependence on hydration enthalpy and stability constants with oxygen-containing bioligands--by exponential or multiplicative equations. The conclusion has been made that selective Ca2+ translocation by the calcium pump and its inhibition by metal cations is determined by the interaction between energy of their interaction with cation-binding sites of the transport system and energy of hydration. Energetics of such interactions depends on the steric factors. The physicochemical model of the Ca2+ selective translocation by calcium pump and its inhibition by metal cations has been proposed.     

Functional differences of Na+/Ca2+ exchanger expression in Ca2+ transport system of smooth muscle of guinea pig stomach

The plasma membrane ATP-dependent Ca2+ pump and the Na+/Ca2+ exchanger (NCX) are the major means of Ca2+ extrusion in smooth muscle. However, little is known regarding distribution and function of the NCX in guinea pig gastric smooth muscle. The expression pattern and distribution of NCX isoforms suggest a role as a regulator of Ca2+ transport in cells. Na+ pump inhibition and the consequent to removal of K+ caused gradual contraction in fundus. In contrast, the response was significantly less in antrum. Western blotting analysis revealed that NCX1 and NCX2 are the predominant NCX isoforms expressed in stomach, the former was expressed strongly in antrum, whereas the latter displayed greater expression in fundus. Isolated plasma membrane fractions derived from gastric fundus smooth muscle were also investigated to clarify the relationship between NCX protein expression and function. Na+-dependent Ca2+ uptake increased directly with Ca2+ concentration. Ca2+ uptake in Na+-loaded vesicles was markedly elevated in comparison with K+-loaded vesicles. Additionally, Ca2+ uptake by the Na+- or K+-loaded vesicles was substantially higher in the presence of A23187 than in its absence. The result can be explained based on the assumption that Na+ gradients facilitate downhill movement of Ca2+. Na+-dependent Ca2+ uptake was abolished by the monovalent cationic ionophore, monensin. NaCl enhanced Ca2+ efflux from vesicles, and this efflux was significantly inhibited by gramicidin. Results documented evidence that NCX2 isoform functionally contributes to Ca2+ extrusion and maintenance of contraction-relaxation cycle in gastric fundus smooth muscle.     

In dialyzed squid axons Ca2+i activates Ca2+o-Na+i and Na+o-Na+i exchanges in the absence of Ca chelating agents

We used internally dialyzed squid axons to explore whether the reported activatory effect of Ca2+i on the partial reactions of the Na+-Ca2+ exchange (essential activator) is secondary to the presence of Ca2+ chelating agents in the internal medium. The effect of Ca2+i pulses on both the reverse (Ca2+o-dependent Na+ efflux) and Na+-Na+ exchange (Na+o-dependent Na+ efflux) modes of the Na+-Ca2+ exchange was studied in axons dialyzed without EGTA. For these experiments a substantial inhibition of the Ca2+ buffer capacity of the axoplasm was achieved by the use of Ruthenium red (10-20 microM), cyanide (1 mM) and vanadate (1 mM) in the dialysis solution. Our results indicate that the Ca2+i requirement of the reverse and Na+-Na+ exchange can not be explained by a direct inhibition of the Na+-Ca2+ exchanger by EGTA. In fact, both modes of operation of the exchanger can be activated by internal Ca2+ ions in the complete absence of Ca2+ chelating agents thus indicating that the 'catalytic' effect of Ca2+i on the Na+-Ca2+ exchanger is a real phenomenon.     

Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.     

Asymmetrical properties of the Na-Ca exchanger in voltage-clamped, internally dialyzed squid axons under symmetrical ionic conditions

In this work we have investigated whether the asymmetrical properties of the Na/Ca exchange process found in intact preparations are intrinsic to the exchange protein(s) or the result of the asymmetric ionic environment normally prevailing in living cells. The activation of the Na/Ca exchanger by Ca2+ ions, monovalent cations, ATP gamma S and the effect of membrane potential on the different operational modes of the exchanger (Nao/Cai, Cao/Nai, Cao/Cai, and Nao/Nai) was studied in voltage-clamped squid giant axons externally perfused and internally dialyzed with symmetrical ionic solutions. Under these conditions: (a) Ca ions activate with higher affinity from the inside (K1/2 = 22 microM) than from the outside (K1/2 = 300 microM); (b) experiments measuring the Cao-dependent Ca efflux in the conditions Lio-Trisi, Lio-Lii, Triso-Trisi, and Triso-Lii, show that the activating monovalent cation site on the exchanger faces the external surface; (c) ATP gamma S activates the Cao-dependent Ca efflux (Cao/Cai exchange) only at nonsaturating [Ca2+]i. Its effect appears to be on the Ca transport site since no alteration in the apparent affinity of the activating monovalent cation site was observed. The above results show that the Na/Ca exchange process is indeed a highly asymmetric transport mechanism. Finally, the voltage dependence of the components of the different exchange modes was measured over the range of +20 to -40 mV. The voltage dependence (approximately 26% change/25 mV) was found to be similar for all modes of operation of the exchanger except Nao/Nai exchange, which was found to be voltage insensitive. The sensitivity of the Cao/Cai exchange to voltage was found to be the same in the presence and in the complete absence of monovalent cations. This finding does not support the proposition that the voltage sensitivity of the Cao/Cao exchange is induced by the binding and transport of an external monovalent cation.     

Roles of Na(+)-Ca2+ exchange and of mitochondria in the regulation of presynaptic Ca2+ and spontaneous glutamate release

The release of neurotransmitter from presynaptic terminals depends on an increase in the intracellular Ca2+ concentration ([Ca2+]i). In addition to the opening of presynaptic Ca2+ channels during excitation, other Ca2+ transport systems may be involved in changes in [Ca2+]i. We have studied the regulation of [Ca2+]i in nerve terminals of hippocampal cells in culture by the Na(+)-Ca2+ exchanger and by mitochondria. In addition, we have measured changes in the frequency of spontaneous excitatory postsynaptic currents (sEPSC) before and after the inhibition of the exchanger and of mitochondrial metabolism. We found rather heterogeneous [Ca2+]i responses of individual presynaptic terminals after inhibition of Na(+)-Ca2+ exchange. The increase in [Ca2+]i became more uniform and much larger after additional treatment of the cells with mitochondrial inhibitors. Correspondingly, sEPSC frequencies changed very little when only Na(+)-Ca2+ exchange was inhibited, but increased dramatically after additional inhibition of mitochondria. Our results provide evidence for prominent roles of Na(+)-Ca2+ exchange and mitochondria in presynaptic Ca2+ regulation and spontaneous glutamate release.     

Pathways for Ca2+ efflux in heart and liver mitochondria.

1. Two processes of Ruthenium Red-insensitive Ca2+ efflux exist in liver and in heart mitochondria: one Na+-independent, and another Na+-dependent. The processes attain maximal rates of 1.4 and 3.0 nmol of Ca2+.min-1.mg-1 for the Na+-dependent and 1.2 and 2.0 nmol of Ca2+.min-1.mg-1 for the Na+-independent, in liver and heart mitochondria, respectively. 2. The Na+-dependent pathway is inhibited, both in heart and in liver mitochondria, by the Ca2+ antagonist diltiazem with a Ki of 4 microM. The Na+-independent pathway is inhibited by diltiazem with a Ki of 250 microM in liver mitochondria, while it behaves as almost insensitive to diltiazem in heart mitochondria. 3. Stretching of the mitochondrial inner membrane in hypo-osmotic media results in activation of the Na+-independent pathway both in liver and in heart mitochondria. 4. Both in heart and liver mitochondria the Na+-independent pathway is insensitive to variations of medium pH around physiological values, while the Na+-dependent pathway is markedly stimulated parallel with acidification of the medium. The pH-activated, Na+-dependent pathway maintains the diltiazem sensitivity. 5. In heart mitochondria, the Na+-dependent pathway is non-competitively inhibited by Mg2+ with a Ki of 0.27 mM, while the Na+-independent pathway is less affected; similarly, in liver mitochondria Mg2+ inhibits the Na+-dependent pathway more than it does the Na+-independent pathway. In the presence of physiological concentrations of Na+, Ca2+ and Mg2+, the Na+-independent and the Na+-dependent pathways operate at rates, respectively, of 0.5 and 1.0 nmol of Ca2+.min-1.mg-1 in heart mitochondria and 0.9 and 0.2 nmol of Ca2+.min-1.mg-1 in liver mitochondria. It is concluded that both heart and liver mitochondria possess two independent pathways for Ca2+ efflux operating at comparable rates.     

Transmembrane Ca2+ exchange in depolarized rat myometrium mitochondria

Polarization of the inner membrane is the key factor in maintenance of the physiologically significant cations accumulation, in particular Ca2+, in the mitochondria. It has been well established that mitochondria accumulate calcium through the uniporter, driven by the mitochondrial membrane potential. Nevertheless, it has been shown that depolarized mitochondria also accumulate Ca2+. The aim of this paper is to investigate free Ca level in depolarized myometrium mitochondria. As we have shown previously Ca2+ addition to the incubation medium, that did not contain K-phosphate, ATP and Mg2+, led to inner mitochondrial membrane depolarization. Nevertheless Ca2+ addition to such medium led to the concentration-dependent accumulation of this cation in the matrix. RuR or Mg addition to the incubation medium led to the higher elevation of mitochondrial Ca2+ level in depolarized mitochondria. Mitochondrial Ca2+ level was not affected by 5 microM cyclosporine A. It was suggested that H+/Ca2+ exchanger could provide calcium accumulation in depolarized mitochondria. The elevation of mitochondrial Ca2+ level after addition of Mg2+ and RuR may be due to inhibition of Ca2+- efflux through Ca2+ uniporter.     

The interrelations between the transport of sodium and calcium in mitochondria of various mammalian tissues.

Addition of ruthenium red to mitochondria isolated from brain, adrenal cortex, parotid gland and skeletal muscle inhibits further uptake of Ca2+ by these mitochondria but induces little or no net Ca2+ efflux; the further addition of Na+, however, induces rapid efflux of Ca2+. The velocity of the Na+-induced efflux of Ca2+ from these mitochondria exhibits a sigmoidal dependence on the [Na+]. Addition of Na+ to mitochondria exhibiting the most active Na+-dependent efflux of Ca2+ (brain and adrenal cortex) also releases Ca2+ in the absence of ruthenium red and, under these conditions, the mitochondria become uncoupled. It is concluded that the efflux of Ca2+ from these mitochondria occurs via a Na+-dependent pathway, possibly a Na+-Ca2+ antiporter, that is distinct from the ruthenium-red-sensitive carrier that catalyses energy-linked Ca2+-influx. The possible role of the Na+-dependent efflux process in the distribution of Ca2+ between the mitochondria and the cytosol is discussed. In contrast, mitochondria from liver, kidney, lung, uterus muscle and ileum muscle exhibit no Na+-dependent efflux of Ca2+.     

Alcohol stimulates Na+/Ca2+ exchange in brain mitochondria

Ethanol, at low concentrations, specifically stimulates the Na(+)-dependent Ca2(+)-efflux in brain mitochondria. In addition, at higher concentrations, ethanol inhibits the Na(+)-independent Ca2(+)-efflux. The electrogenic Ca(+)-uptake system is not affected by ethanol. The specific stimulation of Na+/Ca2+ exchange reaches a maximum of 60% stimulation, with half-maximal stimulation at 130 mM ethanol. The inhibition of the Na(+)-independent efflux is proportional to the ethanol concentration, becoming significant only above 200 mM, with 50% inhibition at 0.5 M. The inhibition of the Na(+)-independent efflux is, in large part, due to an inhibition of the activation of the Cyclosporin-sensitive pore. Long-term ethanol-feeding had no effect on the Ca2+ transport systems and their sensitivity to acute ethanol treatment. It is suggested that the stimulation of the Na(+)-dependent Ca2(+)-efflux, which is the dominant Ca2+ efflux pathway in brain mitochondria, contributes to the intoxicating effects of ethanol.     

Inhibition and stimulation of respiration-linked Mg2+ efflux in rat heart mitochondria

Respiration-driven Mg²⁺ efflux from rat heart mitochondria has been studied in different conditions. Almost total release of Mg²⁺ from the mitochondria occurs upon addition of a proton/bivalent cation exchanger, A23187. The content of Mg²⁺ remaining in mitochondria after A23187 treatment is the same if part of the mitochondrial Mg²⁺ has already been extruded through the energy-linked mechanism. Some inhibition of Mg²⁺ efflux is observed in the presence of high concentrations of La³⁺ (100 µM). A proton/monovalent cation exchanger, nigericin, completely prevents Mg²⁺ efflux, whereas a cation conductor, valinomycin, considerably stimulates it. The results indicate that the main part of mitochondrial Mg²⁺ is present in a membrane-bounded compartment, probably in the matrix space. The driving force of the Mg²⁺ efflux appears to be the proton gradient (pH) created by mitochondrial respiration.     

The influence of pH on Ca2+ exchange in ferret heart mitochondria

The effect of pH changes on Ca2+ transport by isolated heart mitochondria was measured. Two components of Ca2+ transport were identified, an accumulation dependent on mitochondrial respiration and a Na+-dependent efflux. A decrease of pH over the range 7.7-6.7 reduced the initial rate and the total amount of respiration dependent Ca2+ accumulation. At pH 7.2 the [Na+] required to activate half-maximal efflux, k1/2, was 7.5 +/- 1.1 mM. Decreasing the pH over the range 7.7 to 6.9 increased the k1/2 from 3.6 to 11.6. The effect of acidosis was more profound on the respiration dependent Ca2+ uptake than the Na+-dependent efflux.     

Mechanism of sodium independent calcium efflux from rat liver mitochondria

On the basis of primarily two types of observations, it has been suggested that the Na+-independent Ca2+ efflux mechanism of rat liver mitochondria is a passive Ca2+-2H+ exchanger. First, when a pulse of acid is added to a suspension of mitochondria loaded with Ca2+, a pulse of intramitochondrial Ca2+ is often released, even in the presence of the inhibitor of mitochondrial Ca2+ influx, ruthenium red. Second, at a pH near 7, the stoichiometry of Ca2+ released to H+ taken up by Ca2+-loaded mitochondria, following treatment with ruthenium red, has been observed to be 1:2. This evidence for a Ca2+-2H+ exchanger is reexamined here by studying the release of Ca2+ upon acidification of the medium by addition of buffer, the dependence of liver mitochondrial Ca2+ efflux on external medium pH and intramitochondrial pH, and the Ca2+-Ca2+ exchange properties of the Ca2+ efflux mechanism. These studies show no pulse of mitochondrial Ca2+ efflux when pH is abruptly lowered by addition of buffer. The stoichiometry between Ca2+ and H+ fluxes is found to be highly pH dependent. The reported 1:2 stoichiometry between Ca2+ efflux and H+ influx is only observed at one pH. Furthermore, the rate of Ca2+ efflux from mitochondria is found to increase only very slightly at most as suspension pH is decreased. The rate of Ca2+ efflux is not found to increase with increasing intramitochondrial pH. Finally, no Ca2+-Ca2+ isotope exchange can be demonstrated over the Na+-independent efflux mechanism (i.e., in the presence of ruthenium red). It is concluded that these data do not support the hypothesis that the Na+-independent Ca2+ efflux mechanism is a passive Ca2+-2H+ exchanger.