Role of Ca2+ in pyruvate dehydrogenase interconversion in brain mitochondria and synaptosomes.

The steady-state content of active (dephospho) pyruvate dehydrogenase (PDHA) of suspensions of coupled rat brain mitochondria oxidizing succinate was found to be markedly increased with increasing free Ca2+ ion concentration of the medium, with a half-maximal effect at 10(-6.43) M Ca2+. Other ions were present in these studies at concentrations appropriate for the cytosol. Depolarization of the plasma membrane of synaptosomes caused an increase in the steady-state content of PDHA, with veratridine giving a larger increase than depolarization by 33 mM-KCl. Values were 68 +/- 1% (n = 13) and 81 +/- 1% (n = 19) of maximal activity, for control incubations and incubations in the presence of 30 microM-veratridine, respectively. Measurements of cytosolic free Ca2+ concentrations ([Ca2+]cyt.) in these suspensions of synaptosomes, with the use of the fluorescent Ca2+-indicator Quin-2, indicated an increase on depolarization, with the change due to 30 microM-veratridine being larger in extent than that due to 33 mM-KCl. Values were 217 +/- 21 nM (n = 15), 544 +/- 48 nM (n = 15) and 783 +/- 75 nM (n = 14) for control, KCl-depolarized and veratridine-depolarized synaptosomes respectively. Experiments in which synaptosomes were treated with Ruthenium Red, an inhibitor of mitochondrial Ca2+ uptake, gave much lower resting contents of PDHA (42 +/- 2% of maximal), but failed to prevent totally an increase on depolarization. Addition of an excess of EGTA to the synaptosomal suspension just before the addition of veratridine resulted in a partial diminution in the response of PDHA content. Parallel studies with Quin-2 indicated no increase in [Ca2+]cyt. on addition of veratridine, under these conditions. Thus an increase in [Ca2+]cyt. forms only a part of the mechanism whereby pyruvate dehydrogenase interconversion responds to depolarization. A decrease in the ATP/ADP ratio may also be important, as inferred from the results of experiments with ouabain, which inhibits the Na+ + K+-dependent ATPase.     

Effect of micromolar concentrations of manganese ions on calcium-ion cycling in rat liver mitochondria.

The effects of micromolar concentrations of Mn2+ on the rat liver mitochondrial Ca2+ cycle were investigated. It was found that the addition of Mn2+ to mitochondria which were cycling 45Ca2+ led to a rapid dose dependent decrease in the concentration of extramitochondrial 45Ca2+ of about 1 nmol/mg of protein. The effect was complete within 30 s, was half maximal with 10 microM Mn2+ and was observed in the presence of 3 mM Mg2+ and 1 mM ATP. It occurred over a broad range of incubation temperatures, pH and mitochondrial Ca2+ loads. It was not observed when either Mg2+ or phosphate was absent from the incubation medium, or in the presence of Ruthenium Red. These findings indicate that micromolar concentrations of Mn2+ stimulate the uptake of Ca2+ by rat liver mitochondria, and provide evidence for an interaction between Mg2+ and Mn2+ in the control of mitochondrial Ca2+ cycling.     

Regulation of the pyruvate dehydrogenase complex by Ca2+ within toluene-permeabilized heart mitochondria

(1) Rat heart mitochondria, permeabilized to all low Mr solutes by toluene treatment, have been used to study the regulation in situ of the phosphatase and kinase components of the pyruvate dehydrogenase complex (PDH) by Ca2+. (2) Inactivation of the complex, resulting from phosphorylation by the kinase, and reactivation induced by the phosphatase, were both apparent first-order processes. This behaviour of the phosphatase differs from that observed with toluene-permeabilized adipose tissue mitochondria (Midgley, P.J.W., Rutter, G.A. and Denton, R.M. (1987) Biochem. J. 241, 271-377) where a 'lag phase' preceded reactivation of inactive complex. Further, reactivation due to phosphatase activity was stimulated by Ca2+ only at subsaturating Mg2+ concentrations, in contrast with the extracted enzyme which is stimulated by Ca2+ at all Mg2+ concentrations. (3) Maximum values of half-times observed for inactivation and reactivation were about 10 and 15 s, respectively, at 30 degrees C. (4) At Mg2+ concentrations where effects of Ca2+ on the activity of the phosphatase were apparent, no effect of Ca2+ on the activity of the kinase could be detected. (5) The sensitivity of the phosphatase to [Ca2+] was essentially unchanged in the presence of either ADP or ATP, with half-maximal effects at 0.7 microM in each case.     

Role of calcium ions in the regulation of intramitochondrial metabolism. Effects of Na+, Mg2+ and ruthenium red on the Ca2+-stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria.

1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2--3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed.     

Relative importance of pyruvate dehydrogenase interconversion and feed-back inhibition in the effect of fatty acids on pyruvate oxidation by rat heart mitochondria.

Rat heart mitochondria have been incubated with concentrations of pyruvate from 50 to 500 μm as substrate in the presence or absence of an optimal concentration of palmitoylcarnitine and with respiration limited by phosphate acceptor. The rate of pyruvate utilization has been determined and compared with the amount of active (dephosphorylated) pyruvate dehydrogenase measured in samples of mitochondria taken throughout the experiments and assayed under V_max conditions. At a given pyruvate concentration, differences in pyruvate utilization as a proportion of the content of active pyruvate dehydrogenase are attributed to differences in feed-back inhibition and interpreted in terms of the ratios of mitochondrial $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ and CoA/acetyl-CoA. Under most conditions, differences in inhibition can be attributed to differences in the CoA/acetyl-CoA ratio. Feed-back inhibition is very pronounced in the presence of palmitoylcarnitine. These parameters are also examined in the presence of dichloroacetate, used to raise the steady-state levels of active pyruvate dehydrogenase in the absence of changing the pyruvate concentration, and in the presence of various ratios of carnitine/acetylcarnitine, which predominantly change the mitochondrial CoA/acetyl-CoA ratio. The latter experiment provides evidence that a decrease in mitochondrial $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio from 3.5 to 2.2 essentially balances an increase in CoA/acetyl-CoA ratio from 0.67 to 12 in modulating enzyme interconversion, whereas the change in CoA/acetyl-CoA ratio is preponderant in effecting feed-back inhibition. Increasing the free Ca²⁺ concentration of incubation media from 10^?7 to 10^?6m using CaCl₂-ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffers is shown to increase the steady-state level of active pyruvate dehydrogenase in intact mitochondria, in the absence of added ionophores. Moreover, this activation is reversible. Effects of Ca²⁺ ions are dependent upon the poise of the enzyme interconversion system and were only seen in these experiments in the presence of palmitoylcarnitine.     

Dependence of cardiac mitochondrial pyruvate dehydrogenase activity on intramitochondrial free Ca2+ concentration.

(1) The free Ca2+ concentration of the matrix of rat heart mitochondria ([Ca2+]m) was determined from the fluorescence of internalized indo-1. The value of the Kd of indo-1-Ca2+ in the mitochondrial matrix was determined to be 95 nM, on the basis of equilibration of [Ca2+]m with the extramitochondrial free Ca2+ ([Ca2+]o) in the presence of rotenone, nigericin, valinomycin and Br-A23187. (2) [Ca2+]m responded to energization/de-energization protocols, the inhibition of Ca2+-uptake by Ruthenium Red and the potentiation of Ca2+-efflux by Na+ in a manner which was consistent with the known kinetic properties of the mitochondrial Ca2+-transport processes. (3) The concentration gradient [Ca2+]m/[Ca2+]o was found to be near unity (0.82 +/- 0.18) when mitochondria were incubated in media containing 10 mM-Na+; the additional presence of 1 mM-Mg2+ reduced the gradient to values below unity (0.26 +/- 0.03). The polyamine spermine increased the Ca2+ concentration gradient in the presence of 1 mM-Mg2+. (4) The fraction of pyruvate dehydrogenase in the active form (PDHA) was found to increase with [Ca2+]m, with a K0.5 for activation of approximately 300 nM-Ca2+. This value of the activation constant was not affected by conditions, e.g. addition of Mg2+, which changed the [Ca2+]m/[Ca2+]o concentration gradient, and the presence of different oxidizable substrates, which changed the [NADH/NAD+]m concentration ratio. Thus pyruvate dehydrogenase interconversion responds directly to changes in [Ca2+]m, as inferred in earlier work.     

Parallel measurement of oxoglutarate dehydrogenase activity and matrix free Ca2+ in fura-2-loaded heart mitochondria

The entrapment of the Ca2+-sensitive fluorescence indicators fura-2 or quin2 in the matrix space of isolated heart mitochondria renders possible the direct monitoring of the matrix free Ca2+ [( Ca2+]m) [(1987) Biochem J. 248, 609-613]. In this paper the correlation between the [Ca2+]m and the in situ activity of oxoglutarate dehydrogenase (OGDH) in fura-2-loaded mitochondria is shown. At the initial value of [Ca2+]m, 64 nM, which corresponded to 0.36 nmol/mg mitochondrial Ca content, the OGDH activity was 12% of the maximal. Half-maximal and maximal activation were attained at 0.8 and 1.6 microM [Ca2+]m, respectively. The results indicate that an increase of the mitochondrial Ca content in the physiological range enhances the OGDH activity by means of elevation of [Ca2+]m.     

The relationship between the free concentrations of Ca2+ and Ca2+-calmodulin in intact cells

Using stably expressed fluorescent indicator proteins, we have determined for the first time the relationship between the free Ca2+ and Ca2+-calmodulin concentrations in intact cells. A similar relationship is obtained when the free Ca2+ concentration is externally buffered or when it is transiently increased in response to a Ca2+-mobilizing agonist. Below a free Ca2+ concentration of 0.2 microM, no Ca2+-calmodulin is detectable. A global maximum free Ca2+-calmodulin concentration of approximately 45 nM is produced when the free Ca2+ concentration exceeds 3 microM, and a half-maximal concentration is produced at a free Ca2+ concentration of 1 microM. Data for fractional saturation of the indicators suggest that the total concentration of calmodulin-binding proteins is approximately 2-fold higher than the total calmodulin concentration. We conclude that high-affinity calmodulin targets (Kd /= 100 nM) occurs only where free Ca2+-calmodulin concentrations can be locally enhanced.     

Ba2+ ions inhibit the release of Ca2+ ions from rat liver mitochondria

The release of Ca2+ from respiring rat liver mitochondria following the addition of either ruthenium red or an uncoupler was measured by a Ca2+-selective electrode or by 45Ca2+ technique. Ba2+ ions are asymmetric inhibitors of both Ca2+ release processes. Ba2+ ions in a concentration of 75 microM inhibited the ruthenium red and the uncoupler induced Ca2+ release by 80% and 50%, respectively. For the inhibition, it was necessary that Ba2+ ions entered the matrix space: Ba2+ ions did not cause any inhibition of Ca2+ release if addition of either ruthenium red or the uncoupler preceded that of Ba2+. The time required for the development of the inhibition of the Ca2+ release and the time course of 140Ba2+ uptake ran in parallel. Ba2+ accumulation is mediated through the Ca2+ uniporter as 140Ba2+ uptake was competitively inhibited by extramitochondrial Ca2+ and prevented by ruthenium red. Due to the inhibition of the ruthenium red insensitive Ca2+ release, Ba2+ shifted the steady-state extramitochondrial Ca2+ concentration to a lower value. Ba2+ is potentially a useful tool to study mitochondrial Ca2+ transport.     

Effects of Ca2+ and Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within toluene-permeabilized mitochondria.

Mitochondria from rat epididymal white adipose tissue were made permeable to small molecules by toluene treatment and were used to investigate the effects of Mg2+ and Ca2+ on the re-activation of pyruvate dehydrogenase phosphate by endogenous phosphatase. Re-activation of fully phosphorylated enzyme after addition of 0.18 mM-Mg2+ showed a marked lag of 5-10 min before a maximum rate of reactivation was achieved. Increasing the Mg2+ concentration to 1.8 mM (near saturating) or the addition of 100 microM-Ca2+ resulted in loss of the lag phase, which was also greatly diminished if pyruvate dehydrogenase was not fully phosphorylated. It is concluded that, within intact mitochondria, phosphatase activity is highly sensitive to the degree of phosphorylation of pyruvate dehydrogenase and that the major effect of Ca2+ may be to overcome the inhibitory effects of sites 2 and 3 on the dephosphorylation of site 1. Apparent K0.5 values for Mg2+ and Ca2+ were determined from the increases in pyruvate dehydrogenase activity observed after 5 min. The K0.5 for Mg2+ was diminished from 0.60 mM at less than 1 nM-Ca2+ to 0.32 mM at 100 microM-Ca2+; at 0.18 mM-Mg2+, the K0.5 for Ca2+ was 0.40 microM. Ca2+ had little or no effect at saturating Mg2+ concentrations. Since effects of Ca2+ are readily observed in intact coupled mitochondria, it follows that Mg2+ concentrations within mitochondria are sub-saturating for pyruvate dehydrogenase phosphate phosphatase and hence less than 0.5 mM.     

Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat heart. Evidence from studies with isolated mitochondria that adrenaline activates the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes by increasing the intramitochondrial concentration of Ca2+.

Increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase which result from the perfusion of rat hearts with adrenaline were still evident during the preparation of mitochondria in sucrose-based media containing EGTA (at 0 degrees C) and their subsequent incubation at 30 degrees C in Na+-free KCl-based media containing respiratory substrates and EGTA. The differences from control values gradually diminished with time of incubation, but were still present after 8 min. Similar increases resulting from an increase in the concentration of Ca2+ in the perfusing medium also persisted. However, similar increases caused by 5 mM-pyruvate were only maintained during the preparation of mitochondria, not their incubation. Parallel increases, within incubated mitochondria, were found in the activity of the 2-oxoglutarate dehydrogenase complex assayed at a non-saturating concentration of 2-oxoglutarate. The enhancement of the activities of both of these Ca2+-sensitive enzymes within incubated mitochondria as a result of perfusion with adrenaline or a raised concentration of Ca2+ in the medium could be abolished within 1 min by the presence of 10 mM-NaCl. This effect of Na+ was blocked by 300 microM-diltiazem, which has been shown to inhibit Na+-induced egress of Ca2+ from rabbit heart mitochondria [Vághy, Johnson, Matlib, Wang & Schwartz (1982) J. Biol. Chem. 257, 6000-6002]. The enhancements could also be abolished by increasing the extramitochondrial concentration of Ca2+ to a value where it caused maximal activation of the enzymes within control mitochondria. The results are consistent with the hypothesis that adrenaline activates rat heart pyruvate dehydrogenase by increasing the intramitochondrial concentration of Ca2+ and that this increase persists through to incubated mitochondria. Support for this conclusion was obtained by the yielding of a similar set of results from parallel experiments performed on control mitochondria that had firstly been preincubated (under conditions of steady-state Ca2+ cycling across the inner membrane) with sufficient proportions of Ca-EGTA buffers to achieve a similar degree of Ca2+-activation of pyruvate dehydrogenase (as caused by adrenaline) and had then undergone the isolation procedure again.     

Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of (14)CO(2) from [1-(14)C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K(i) of 100mum. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

Studies on the interactions of Ca2+ and pyruvate in the regulation of rat heart pyruvate dehydrogenase activity. Effects of starvation and diabetes.

1. Previous studies showed that the activation of pyruvate dehydrogenase within intact rat heart mitochondria of pyruvate is much diminished in mitochondria from starved or diabetic animals [see Kerbey, Randle, Cooper, Whitehouse, Pask & Denton (1976) Biochem. J. 154, 327-348]. In the present study, diminished responses to added Ca2+ and ADP were also found in these mitochondria. 2. Starvation or diabetes did not affect the mitochondrial respiratory control ratio of the ATP content. Moreover, starvation and diabetes did not alter the response of the intramitochondrial Ca2+-sensitive enzyme, 2-oxoglutarate dehydrogenase, to changes in the extramitochondrial concentration of Ca2+ and 2-oxoglutarate, thus indicating that there were no appreciable changes in the distribution of Ca2+ and H+ across the mitochondrial inner membrane. 3. Pyruvate, Ca2+ and ADP were found to have synergistic effects on pyruvate dehydrogenase activity, particularly in mitochondria from starved and diabetic rats. 4. The results suggest that the effects of diabetes and starvation on pyruvate dehydrogenase are not brought about by changes in the distribution of these effectors across the mitochondrial inner membrane or by changes in the intrinsic sensitivity of the kinase or phosphatase of the pyruvate dehydrogenase system to pyruvate, Ca2+ or ADP; rather it is probably that there is an increase in the maximum activity of kinase relative to that of the phosphatase. 6. The results also lend further support to the hypothesis that adrenaline may bring about the activation of pyruvate dehydrogenase in the rat heart by an increase in the intramitochondrial concentration of Ca2+.     

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+ concentrations that range from 10(-9) to 5 X 10(-6) M. The highest rate was observed between 0.5-1.0 microM Ca2+. ATP synthesis was measured by polarographic and spectrophotometric techniques and by uptake of radioactive 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 alpha-ketoglutarate, glutamate + malate, and succinate, but not with beta-hydroxybutyrate. At 10(-9) M Ca2+ there is a continuous exit of endogenous Ca2+, while with 10(-6) 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-stimulated respiration also depends on Ca2+ concentration, even though at 10(-9) to 10(-6) 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 10(-9) and 10(-6) 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.     

Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system.     

Kinetic behaviour and properties of aldehyde dehydrogenase from rat testis mitochondria. Effect of Mg2+ ions.

1. Mitochondrial aldehyde dehydrogenase is purified to near homogeneity by hydroxylapatite-, affinity- and hydrophobic interaction-chromatography. 2. The enzyme is an oligomeric protein and its molecular weight, as determined by gel-filtration, is 117,000 +/- 5000. 3. Active only in the presence of exogenous sulfhydryl compounds and NAD(+)-dependent, aldehyde dehydrogenase works optimally with linear-chain aliphatic aldehydes and is practically inactive with benzaldehyde. The pH-optimum is at about pH 8.5. 4. Km-Values for aliphatic aldehydes (C2-C6) range between 0.17 and 0.32 microM. The Km for NAD+ increases from 16 microM with acetaldehyde to 71 microM with capronaldehyde. 5. Millimolar concentrations of Mg2+ promote high increases of both V and Km for NAD+. At the same time, saturation curves with C4-C6 aldehydes can be simulated with a substrate inhibition model. 6. Inhibition by NADH is competitive: with capronaldehyde, the inhibition constant for NADH is 52 microM in the absence of Mg2+ and 14 microM in the presence of 4 mM Mg2+; with acetaldehyde, the inhibition constant is about three times higher (36 and 159 microM, respectively).     

Effect of pH and Ca2+ on the retention of Ca2+ by rat liver mitochondria.

A transient Ca²⁺ release from preloaded mitochondria can be induced by a sudden decrease in the pH of the outer medium from 8.0 or 7.4 to 6.8. In the presence of inorganic phosphate the released Ca²⁺ is not taken up again. Upon Ca²⁺ addition to respiring mitochondria the mitochondrial membrane potential (Δ♀) decreases to a new resting level. A further decrease in Δ♀ occurs after the decrease in pH from 7.4 to 6.8, concomitant with the reuptake phase of the Ca²⁺ release. Phosphate, EGTA, and ruthenium red restore Δ♀ to its initial level. If phosphate is present initially, only transient changes in Δ♀ occur upon addition of Ca²⁺ or H⁺ ions. Only a small transient change in Δ♀ upon H⁺ ion addition is seen in the absence of accumulated Ca²⁺. La³⁺, a competitive inhibitor of Ca²⁺ transport, prevents the H⁺ ion-induced Ca²⁺ efflux, whereas this is not the case in the presence of the noncompetitive inhibitor ruthenium red. Ruthenium red, however, prevents the reuptake phase. Mg²⁺, an inhibitor of the surface binding of Ca²⁺, has no or only a slight effect on the H⁺ ion-induced Ca²⁺ release. Mitochondria preloaded with Ca²⁺ release a small fraction of Ca²⁺ during the subsequent uptake of another pulse of Ca²⁺. The results indicate that at least one pool of mitochondrial Ca²⁺ exists in a mobile state. The possible existence of a $\text{H}{}^{\mathrm{+}}\text{Ca}{}^{\mathrm{2+}}$ exchanger in the mitochondrial membrane is discussed.     

Synergistic effects of micromolar concentrations of Zn2+ and Ca2+ on membrane fusion

Resonance Energy Transfer between N-(7-nitro-2,1,3 benzoxadiazol -4 yl) phosphatidyl ethanolamine and N-Lissamine-Rhodamine B sulfonyl) phosphatidyl ethanolamine embedded in two different populations of small unilamellar vesicles made of phosphatidyl serine has been used to study the fusion process induced by Zn2+ and Ca2+. Lipid intermixing demonstrating fusion of liposome membranes can already be observed at 125 and 250 mumol/l of Zn2+. After short time pre-incubations with micromolar concentrations of Zn2+ as low as 150 mumol/l, Ca2+ induces an instantaneous increase of vesicle fusion. The lipid intermixing induced by micromolar concentrations of Ca2+ (250-500 mumol/l) could be increased up to 4 times when pre-incubated with 150 or 200 mumol/l of Zn2+. The effect of 1 mM of Ca2+ alone on lipid intermixing can be mimicked by 150 mumol/l of Zn2+ followed by 500 mumol/l of Ca2+. Our data demonstrate that Zn2+ and Ca2+ act synergistically to affect cation-induced membrane fusion. We suggest that Zn2+ specifically alters the physical state of phospholipid membranes making them more prone to calcium-triggered fusion.