Effect of Ca2+ on induction of the mitochondrial pore opening in the rat myocardium

The mitochondrial role opening (MPT) induced by Ca2+ has been studied in isolated rat heart mitochondria. MPT was characterized as cyclosporine A-inhibited swelling accompanied by the loss of membrane potential (deltapsim) and Ca2+ efflux after the Ca2+ -loading which was followed spectrophotometrically after the Ca2+ -arsenaso-III complex formation. It has been shown that in suspension of isolated mitochondria MPT was activated by low (with maximum at about 20 microM Ca2+) and high concentrations of Ca2+ (the concentration curve shows a saturation at about 1.0-1.5 mM). In all the cases an access of Ca2+ ions to the matrix space of the mitochondria was necessary for MPT induction. MPT activated by low concentrations of Ca2+ was accompanied by slow decrease of deltapsim and slow release of Ca2+, enhanced by ruthenium red (RR), and was independent of the substrate used (glutamate or succinate). It had not been observed if the respiratory chain was inhibited, even if the Ca2+ access to the inner mitochondrial membrane was provided by Ca2+ -ionophore A23187. At high Ca2+ concentrations rapid Ca2+ -uptake and release via Ca2+ -uniporter (inhibited by ruthenium red) followed by extensive swelling (pore formation) have been observed. It had been supposed that rapid MPT at high concentrations of Ca2+ was the result of Ca2+ entrance to the mitochondrial matrix and depolarisation of the mitochondrial membrane. The data obtained show two different mechanisms of Ca2+ -induced MPT. The one is sensitive to the redox-state of the electron transport chain and is abolished if the respiration is inhibited. The other is independent of mitochondrial respiration and needs only Ca2+ access to the inner mitochondrial membrane and Ca2+ binding to some specific sites leading to MPT opening.     

Effects of glucagon and N6O2''-dibutyryladenosine 3'':5''-cyclic monophosphate on calcium transport in isolated rat liver mitochondria.

1. The administration of glucagon to fed rats by intraperitoneal injection, or the perfusion of livers from fed rats with glucagon by the method of Mortimore [Mortimore (1963) Am.J. Physiol. 204, 699--704] was associated with increases of 15- and 5-fold respectively, in the time for which a given load of exogenous Ca2+ is retained by mitochondria subsequently isolated from the liver. This effect of glucagon was (a) also induced by N6O2'-dibutyryl cyclic AMP, (b) completely blocked by cycloheximide, (c) relatively slow in onset (15--60 min) and (d) associated with a stimulation of about 20% in the rates of ADP-stimulated oxygen utilization and Ca2+ transport measured in the presence of succinate. 2. Perfusion of livers with glucagon resulted in the isolation of mitochandria which showed a 50% increase, no significant change and a 40% increase in the concentrations of endogenous Ca, Mg and Pi respectively, when compared with mitochondria isolated from control perfused livers. 3. The administration of insulin or adrenaline to fed rats induced increases of 10- and 8-fold respectively, in the time for which Ca2+ is retained by isolated liver mitochondria. Perfusion of livers with insulin had no effect on mitochondrial Ca2+ retention time. 4. The perfusion of livers from starved rats with glucagon, or the administration of either glucagon or insulin to starved rats, increased by about 2.5- and 15-fold respectively, the time for which isolated mitochondria retain Ca2+. 5. Mechanisms which may be responsible for the observed alterations in Ca2+-retention time are discussed.     

Undesirable feature of safranine as a probe for mitochondrial membrane potential

This communication describes experiments showing that safranine, at the concentrations usually employed as a probe of mitochondrial membrane potential, causes significant undesirable side effects on Ca2+ transport by liver mitochondria. The major observations are: (i) safranine potentiates the spontaneous Ca2+ release from liver mitochondria induced by phosphate or acetoacetate. This is paralelled by potentiation of the release of state-4 respiration and of the rate of mitochondrial swelling, indicating a generalized effect of the dye on the mitochondrial membrane; (ii) the efflux of mitochondrial Ca2+ stimulated by hydroperoxide is irreversible in the presence of safranine even if membrane stabilizers such as Mg2+ and ATP are present. It is concluded that the use of safranine to monitor the changes in membrane potential during Ca2+ transport by mitochondria should be avoided or special care be taken.     

Regulation of pyruvate transport in mitochondria by thyroid hormones

During thyroidectomy, the stimulating action of the catalytic amounts of a thermostable fraction of rat liver and diaphragm cytoplasm on Ca2+ transport in mitochondria, which indicates the decrease of the activity of an insulin-dependent cytoplasmic regulator (IDR) in insulin target organs. Thyroidectomized rats also manifested a decrease in blood insulin and glucose concentrations. Administration of the physiological doses of thyroxine produced an increase in both blood glucose concentration and IDR activity in the liver and diaphragm of thyroidectomized rats. Experiments with measuring the kinetics of the swelling of deenergized mitochondria in isoosmotic solution of ammonium pyruvate demonstrated the inhibition of liver mitochondrial swelling in thyroidectomized rats.     

The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes

We have examined the effects of extracellular and intracellular Ca2+ concentrations upon basal and insulin-stimulated 2-deoxyglucose uptake in isolated rat adipocytes. In the absence of extracellular Ca2+, both basal and insulin-stimulated glucose uptake were significantly reduced. Insulin-stimulated glucose transport was optimal at 1 and 2 mM Ca2+. Further increases in extracellular Ca2+ concentration (3 mM) significantly diminished insulin-stimulated glucose uptake. When intracellular Ca2+ concentrations were augmented by ionomycin (1 microM), insulin-stimulated glucose uptake was significantly reduced at extracellular Ca2+ concentrations of 2 and 3 mM. The levels of intracellular free Ca2+ concentrations were then measured with Ca2+ indicator fura-2. The correlation between the levels of intracellular free Ca2+ and the magnitude of insulin-stimulated glucose uptake revealed that the optimal effect of insulin is observed at Ca2+ levels between 140 and 370 nM. At both extremes outside of this window, both low and high levels of intracellular Ca2+ result in diminished cellular responsiveness to insulin. These data suggest that intracellular calcium concentrations may exert a dual role in the regulation of cellular sensitivity to insulin. First, there must exist a minimal concentration of intracellular calcium to promote insulin action. Second, increased levels of intracellular calcium may provide a critical signal for diminution of insulin action.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

Effects of hormones and N6O2''-dibutyryl-adenosine 3'' :5''-cyclic monophosphate, administered in vivo, on phosphate transport and metabolism in isolated rat liver mitochondria.

1. The administration of glucagon or N6O2'-dibutyryl cyclic AMP to fed rats by intraperitoneal injection was associated with a 2-fold increase in the amounts of endogenous Pi and ATP, and an increase in the rate and extent of transport of exogenous Pi (measured in either the presence or the absence of Ca2+) in mitochondria subsequently isolated from the liver. No change was observed in either the maximum rate of transport of exogenous Pi or in the rate of 32Pi exchange. 2. The changes induced by glucagon and dibutyryl cyclic AMP were markedly decreased by the co-administration of cycloheximide. 3. The administration of insulin to rats resulted in an increase of about 1.3-fold in the concentration of endogenous mitochondrial Pi 4. The amounts of endogenous Pi in mitochondrial isolated from the livers of starved rats were 3 times those in mitochondria isolated from fed animals. 5. It is concluded that the liver mitochondrial phosphatetransport system may be an important site of hormone action. 6. In the course of these experiments, it was shown that Ca2+ markedly stimulates mitochondrial phosphate transports.     

Spermine. A regulator of mitochondrial calcium cycling

Steady-state free Ca2+ concentrations have been measured with a Ca2+ electrode using suspensions of isolated rat liver mitochondria or saponin-treated hepatocytes. Mitochondria, when incubated in the presence of Mg2+ and MgATP2-, maintain a steady-state pCa2+ (-log [Ca2+]) of approximately 6.1 (0.8 microM). Addition of spermine lowered this value to a pCa2+ of 6.6 (0.25 microM). Spermine was the most effective polyamine, giving half-maximal effects at 170 microM and maximal effects at 400 microM. With saponin-permeabilized hepatocytes, spermine addition similarly showed that the mitochondria buffered the steady-state medium-free Ca2+ at a level approximating the cytosolic free Ca2+ concentration of intact hepatocytes. The initial rate of Ca2+ uptake by the mitochondrial Ca2+ uniporter was investigated using Ca2+-depleted mitochondria incubated in the presence of succinate and 0.3 mM free Mg2+. Under control conditions, Ca2+ uptake was not observed at free Ca2+ concentrations below 0.5 microM. Spermine (350 microM) increased the rate of Ca2+ uptake at all Ca2+ concentrations below 4.5 microM, but at higher Ca2+ concentrations, it was inhibitory. Spermine also affected mitochondrial Ca2+ efflux by decreasing the apparent Km from 16 to 3.8 nmol of Ca2+/mg of mitochondrial protein with no change of Vmax. Experiments with 45Ca2+ confirmed that spermine increased mitochondrial Ca2+ cycling at 0.2 microM free Ca2+. Hepatic spermine contents are reported to be about 1 mumol/g, wet weight, suggesting that this polyamine may have an important physiological role in intracellular calcium homeostasis.     

The effect of extracellular Ca and Mg on mitochondrial ultrastructure in isolated intact neurons

The ultrastructural transformations of mitochondria in isolated crayfish neurons were studied after incubation of the cells in saline media containing different Ca2+ and Mg2+ concentrations. Incubation in a 5-fold higher Ca concentration resulted in the swelling of mitochondria that was prevented by the addition of the calcium channel blocker, verapamil. Exposure of the cells to Mg2+-depleted medium induced swelling of all the mitochondria, followed by substantial shrinkage of most of them. The absence of Ca as well as the presence of verapamil in Mg2+-free medium led to the inhibition of mitochondrial swelling and to a strong contraction of the mitochondria after 1 h incubation. The omission of Ca2+ from the saline medium or the addition of Ca2+-ionophore A23187 in the presence of Ca2+ resulted in strong mitochondrial shrinkage. These structural alterations of mitochondria are interpreted as an osmotic response of the inner mitochondrial membranes to changes in their potassium transport, induced by a disturbance in the cellular and mitochondrial Ca2+-Mg2+ homeostasis.     

Chronic exposure to beta-hydroxybutyrate inhibits glucose-induced insulin release from pancreatic islets by decreasing NADH contents

To investigate the effects of chronic exposure to ketone bodies on glucose-induced insulin secretion, we evaluated insulin release, intracellular Ca2+ and metabolism, and Ca2+ efficacy of the exocytotic system in rat pancreatic islets. Fifteen-hour exposure to 5 mM d-beta-hydroxybutyrate (HB) reduced high glucose-induced insulin secretion and augmented basal insulin secretion. Augmentation of basal release was derived from promoting the Ca2+-independent and ATP-independent component of insulin release, which was suppressed by the GDP analog. Chronic exposure to HB affected mostly the second phase of glucose-induced biphasic secretion. Dynamic experiments showed that insulin release and NAD(P)H fluorescence were lower, although the intracellular Ca2+ concentration ([Ca2+](i)) was not affected 10 min after exposure to high glucose. Additionally, [Ca2+](i) efficacy in exocytotic system at clamped concentrations of ATP was not affected. NADH content, ATP content, and ATP-to-ADP ratio in the HB-cultured islets in the presence of high glucose were lower, whereas glucose utilization and oxidation were not affected. Mitochondrial ATP production shows that the respiratory chain downstream of complex II is not affected by chronic exposure to HB, and that the decrease in ATP production is due to decreased NADH content in the mitochondrial matrix. Chronic exposure to HB suppresses glucose-induced insulin secretion by lowering the ATP level, at least partly by inhibiting ATP production by reducing the supply of NADH to the respiratory chain. Glucose-induced insulin release in the presence of aminooxyacetate was not reduced, which implies that chronic exposure to HB affects the malate/aspartate shuttle and thus reduces NADH supply to mitochondria.     

Calcium ion fluxes induced by the action of alpha-adrenergic agonists in perfused rat liver.

Phenylephrine (2.0 microM) induces an alpha 1-receptor-mediated net efflux of Ca2+ from livers of fed rats perfused with medium containing physiological concentrations (1.3 mM) of Ca2+. The onset of efflux (7.1 +/- 0.5 s; n = 16) immediately precedes a stimulation of mitochondrial respiration and glycogenolysis. Maximal rates of efflux are observed between 35 s and 45 s after alpha-agonist administration; thereafter the rate decreases, to be no longer detectable after 3 min. Within seconds of terminating phenylephrine infusion, a net transient uptake of Ca2+ by the liver is observed. Similar effects were observed with vasopressin (1 m-unit/ml) and angiotensin (6 nM). Reducing the perfusate [Ca2+] from 1.3 mM to 10 microM had little effect on alpha-agonist-induced Ca2+ efflux, but abolished the subsequent Ca2+ re-uptake, and hence led to a net loss of 80-120 nmol of Ca2+/g of liver from the tissue. The administration at 5 min intervals of short pulses (90 s) of phenylephrine under these conditions resulted in diminishing amounts of Ca2+ efflux being detected, and these could be correlated with decreased rates of alpha-agonist-induced mitochondrial respiration and glucose output. An examination of the Ca2+ pool mobilized by alpha-adrenergic agonists revealed that a loss of Ca2+ from mitochondria and from a fraction enriched in microsomes accounts for all the Ca2+ efflux detected. It is proposed that the alpha-adrenergic agonists, vasopressin and angiotensin mobilize Ca2+ from the same readily depleted intracellular pool consisting predominantly of mitochondria and the endoplasmic reticulum, and that the hormone-induced enhanced rate of mitochondrial respiration and glycogenolysis is directly dependent on this mobilization.     

The calcium sensitive dehydrogenases of vertebrate mitochondria

Three important dehydrogenases in vertebrate mitochondria are activated by Ca2+ ions with half-maximal effects at about 1 microM. These are pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase. Activation of these enzymes can also be demonstrated within intact mitochondria when extramitochondrial Ca2+ is increased within the range of concentrations generally considered to occur in the cytoplasm of vertebrate cells. It is argued that the main role of the calcium transport system in the inner membrane of vertebrate mitochondria is to relay changes in the cytoplasmic concentration of Ca2+ into the mitochondrial matrix. In this way, hormones and other extracellular stimuli which stimulate ATP-requiring processes such as contraction and secretion through increases in the cytoplasmic concentration of Ca2+ may also increase intramitochondrial oxidative metabolism and hence the replenishment of ATP.     

The mechanism of palmitoyl-CoA inhibition of Ca2+ uptake in liver and heart mitochondria.

The mechanism by which palmitoyl-CoA inhibits Ca2+ uptake in liver and heart mitochondria was examined. At a given concentration of palmitoyl-CoA, the extent of inhibition is inversely related to the concentration of the respiratory substrate succinate. Palmitoyl-CoA inhibition of uncoupler-stimulated respiration and respiration stimulated by ionophore-A23187-induced Ca2+ cycling is also relieved by high succinate concentrations. These effects of palmitoyl-CoA and succinate concentration are distinct from the increase in inner-membrane permeability, which can be produced by palmitoyl-CoA and Ca2+ [Beatrice, Palmer & Pfeiffer (1980) J. Biol. Chem. 255, 8663-8671]. The apparent K0.5 of the mitochondrial Ca2+ pump is not altered by palmitoyl-CoA. No or negligible effects of palmitoyl-CoA on the Ca2+-uptake rate are observed when ascorbate replaces succinate as an energy source. These findings, together with the known activity of palmitoyl-CoA as a competitive inhibitor of the dicarboxylate carrier [Morel, Lauquin, Lunardi, Duszynski & Vignais (1974) FEBS Lett. 39, 133-138], indicate that palmitoyl-CoA inhibits energy-linked Ca2+ transport by limiting the rate of electron transport through limitation of succinate entry into the mitochondria rather than by directly inhibiting the Ca2+ carrier.     

Effects of Pentobarbitone on 45Ca2+ Transport by Rat Brain Mitochondria

The effects of pentobarbitone on the transport of 45Ca2+ by rat brain mitochondria were studied, using the Ruthenium Red-EGTA quench technique. In the presence of succinate and inorganic phosphate, mitochondria rapidly accumulate 45Ca2+. Pentobarbitone (0.1-1.0 mM) stimulates the initial rate of Ca2+ transport. In contrast, pentobarbitone (1 mM) did not affect the NaCl (50 mM)-induced efflux of 45Ca2+ from mitochondria. Dibucaine (60 micro M), a clinically used local anaesthetic, inhibits both 45Ca2+ uptake an efflux. The results suggest that barbiturate stimulation of mitochondrial Ca2+ uptake may, in combination with effects on other Ca2+ sequestering processes, contribute to the inhibitor of transmitter release observed at a number of synapses.     

Requirement for aralar and its Ca2+-binding sites in Ca2+ signal transduction in mitochondria from INS-1 clonal beta-cells

Aralar, the mitochondrial aspartate-glutamate carrier present in beta-cells, is a component of the malate-aspartate NADH shuttle (MAS). MAS is activated by Ca2+ in mitochondria from tissues with aralar as the only AGC isoform with an S0.5 of approximately 300 nm. We have studied the role of aralar and its Ca2+-binding EF-hand motifs in glucose-induced mitochondrial NAD(P)H generation by two-photon microscopy imaging in INS-1 beta-cells lacking aralar or expressing aralar mutants blocked for Ca2+ binding. Aralar knock-down in INS-1 beta-cell lines resulted in undetectable levels of aralar protein and complete loss of MAS activity in isolated mitochondria and in a 25% decrease in glucose-stimulated insulin secretion. MAS activity in mitochondria from INS-1 cells was activated 2-3-fold by extramitochondrial Ca2+, whereas aralar mutants were Ca2+ insensitive. In Ca2+-free medium, glucose-induced increases in mitochondrial NAD(P)H were small (1.3-fold) and unchanged regardless of the lack of aralar. In the presence of 1.5 mm Ca2+, glucose induced robust increases in mitochondrial NAD(P)H (approximately 2-fold) in cell lines with wild-type or mutant aralar. There was a approximately 20% reduction in NAD(P)H response in cells lacking aralar, illustrating the importance of MAS in glucose action. When small Ca2+ signals that resulted in extremely small mitochondrial Ca2+ transients were induced in the presence of glucose, the rise in mitochondrial NAD(P)H was maintained in cells with wild-type aralar but was reduced by approximately 50% in cells lacking or expressing mutant aralar. These results indicate that 1) glucose-induced activation of MAS requires Ca2+ potentiation; 2) Ca2+ activation of MAS represents a larger fraction of glucose-induced mitochondrial NAD(P)H production under conditions where suboptimal Ca2+ signals are associated with a glucose challenge (50 versus 20%, respectively); 3) inactivation of EF-hand motifs in aralar prevents activation of MAS by small Ca2+ signals. The results suggest a possible role for aralar and MAS in priming the beta-cell by Ca2+-mobilizing neurotransmitter or hormones.     

Reversibility of energy-dependent Ca2+ accumulation in mitochondria

Ca2+ accumulation in energized rat liver mitochondria has been studied after the blockage of mitochondrial permeability transition pore (MPTP) by cyclosporin A. It is shown that Ca2+ transport is coupled to the countertransport of protons: from the matrix of mitochondria in the medium in the course of Ca2+ accumulation, and, on the contrary, from the medium to mitochondrial matrix after membrane depolarization. In standard incubation medium containing K+, Cl-, oxidation substrate (glutamate) and inorganic phosphate (H2PO4(-)) the observed stoichiometry of the exchange is 1Ca2+ : 1H+. In accordance with this exchange ratio, proton, as well as cation, transport follows the same first-order kinetics, which is characterized in both cases by very close values of reaction half-times and rate constants. It is shown that reversion of Ca2+ -uniporter, sensitive to ruthenium red, is necessary for Ca2+ - efflux from the matrix ofdeenergized mitochondria when MPTP is blocked by cyclosporin A. It is also shown that Ca2+ -uniporter reversion takes place only after membrane depolarization and permeabilization by protonophore CCCP. Calcium release from mitochondria in the presence of CCCP is accompanied by proton flow into the matrix. Both calcium and proton fluxes are sensitive to Ca2+ uniporter blocker, ruthenium red, which gives the evidence of the identity of Ca2+ -efflux and influx pathways. The data obtained lead to the conclusion that calcium-proton exchange is necessary for Ca2+ -uniporter reversion and the reversibility of energy-dependent Ca2+ -uptake in mitochondria.     

Aspects of energy-linked calcium accumulation by rat heart mitochondria.

When intact rat heart mitochondria were pulsed with 150 nmol of CaCl2/mg of mitochondrial protein, only a marginal stimulation of the rate of oxygen consumption was observed. This result was obtained with mitochondria isolated in either the presence or absence of nagarse. In contrast, rat liver mitochondria under similar conditions demonstrated a rapid, reversible burst of respiration associated with energy-linked calcium accumulation. Direct analysis of calcium retention using 45Ca and Millipore filtration indicated that calcium was accumulated by heart mitochondria under the above conditions via a unique energy-dependent process. The rate of translocation by heart mitochondria was less than that of liver mitochondria; likewise the release of bound calcium back into the medium was also retarded. These results suggest that the slower accumulation and release of calcium is characteristic of heart mitochondria. The amound of calcium bound was independent of penetrant anions at low calcium concentrations. Above 100 nmol/mg of mitochondrial protein, the total calcium bound was increased by the presence of inorganic phosphate. Under nonrespiring conditions, a biphasic Scatchard plot indicative of binding sites with different affinities for Ca2+ was observed. The extrapolated constants are 7.5 nmol/mg bound with an apparent half-saturation value of 75 muM and 42.5 nmol/mg bound with half-saturation at 1.15 mM. The response of the reduced State 4 cytochrome b to pulsed additions of Ca2+ was used to calculate an energy-dependent half-saturation constant of 40 muM. When the concentration of free calcium was stabilized at low levels with Ca2+-EGTA buffers, the spectrophotometrically determined binding constant decreased two orders of magnitude to an apparent affinity of 4.16 X 10(-7) M. Primary of calcium transport over oxidative phosphorylation was not observed with heart mitochondria. The phosphorylation of ADP competed with Ca2+ accumulation, depressed the rates of cation transport, and altered the profile of respiration-linked H+ movements. Consistent with these result was the observation that with liver mitochondrial the magnitude of the cytochrome b oxidation-reduction shift was greater for Ca2+ than for ADP, whereas calcium responses never surpassed the ADP response in heart mitochondria. Furthermore, Mg2+ ingibited calcium accumulation by heart mitochondria while having only a slight effect upon calcium transport in liver mitochondria. The unique energetics of heart mitochondrial calcium transport are discussed relative to the regulated flux of cations during the cardiac excitation-relaxation cycle.     

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.     

The effect of ferric iron complex on isolated rat liver mitochondria. II. Ion movements

It has been found that addition of iron(III)-gluconate complex to rat liver mitochondria disturbed the mitochondrial Ca2+ transport. Indirect evidence when the changes in the membrane potential during the transport of Ca2+ were followed, as well as direct evidence, when the fluxes of Ca2+ were monitored by a Ca2+-selective electrode, indicated that this iron complex induced an efflux of Ca2+ from liver mitochondria. The mechanisms by which iron induced Ca2+ release appeared to be linked to the induction of lipoperoxidation of mitochondrial membrane. The mitochondrial membrane, however, did not become irreversibly damaged under these conditions, as indicated by its complete repolarization. It was also shown that the induction by iron of lipoperoxidation brought about an efflux of K+ from mitochondria.