[Purification of the mitochondrial calcium uniporter from the beef heart and characterization of its properties].

A low-molecular-weight component (LMC) inducing selective transport of calcium across the bilayer lipid membrane has been isolated from mitochondria of the bovine heart by the method developed in our laboratory, which excludes the use of detergents and proteolytic enzymes. It was shown that, in the presence of 10 mM CaCl2, LMC forms conduction channels in the membrane multiples of 5 pS. The specific inhibitor of mitochondrial calcium uniporter, ruthenium red, closes Ca2(+)-induced channels formed in the membrane by LMC. In the absence of calcium or in the presence of potassium ions only, the component is incapable of forming channels of conduction. It was shown using nuclear magnetic resonance that LMC is a complex consisting of lipids, amino acids, and sugars with a molecular weight of 1-2 kDa.

Microbial toxin's effect on mitochondrial survival by increasing K+ uptake.

We studied the effects of toxins, which inhibited the motility of boar spermatozoa, on rat liver mitochondria. The toxins studied were originally from bacteria isolated from moisture-damaged buildings where inhabitants exhibited symptoms, or from food causing poisoning. Some strains of Bacillus cereus and Streptomyces griseus produced potassium ionophoric peptides cereulide and valinomycin (Mikkola, et al., European Journal of Biochemistry 1999; 263: 112-117). Of interest is that channels were formed in black-lipid membranes (BLM) with a selectivity of K(+) > Na(+) at a concentration of 26 nM. Recently, bafilomycin A1--an inhibitor of V-H(+)ATPases--was found also to be a K(+)-specific ionophore active at nanomolar concentrations (Teplova, et al., J Bioenerg Biomembr 2007; 39: 321-329), while B. amyloliquefaciens produced amylosin, a cation channel-forming peptide with a higher selectivity for K(+) over Na(+) at around 200 nM concentrations (Mikkola, et al., Toxicon 2007; 49: 1158-1171). Of interest is that channels were formed in BLM with a selectivity of K(+) > Na(+) at a concentration of 26 nM. The ionophores and the channel-forming amylosin caused swelling of energized mitochondria due to uptake of K(+), loss of membrane potential, inhibition of maximal respiration rates due to loss of pyridine nucleotides, and inhibition of ATP synthesis. Various cell types may have different sensitivities to the effects of the ionophores. Thus, the mitochondrial membrane potential in neuronal cells was more sensitive to cereulide than in differentiated Paju cells (Teplova, et al., Acta Biochimica Polonica 2004; 51: 539-544). Swelling causes release of proapoptotic factors from mitochondria, which explains that undifferentiated neuronal cells were sensitive, while differentiated Paju cells were resistant, which probably is due to them having an increased expression of the antiapoptotic protein Bcl-2 and the neuroprotective stanniocalcin.

A historical review of cellular calcium handling, with emphasis on mitochondria.

Calcium ions are of central importance in cellular physiology, as they carry the signal activating cells to perform their programmed function. Ca(2+) is particularly suitable for this role because of its chemical properties and because its free concentration gradient between the extra-cellular and the cytosolic concentrations is very high, about four orders of magnitude. The cytosolic concentration of Ca(2+) is regulated by binding and chelation by various substances and by transport across plasma and intracellular membranes. Various channels, transport ATPases, uniporters, and antiporters in the plasma membrane, endoplasmic and sarcoplasmic reticulum, and mitochondria are responsible for the transport of Ca(2+). The regulation of these transport systems is the subject of an increasing number of studies. In this short review, we focus on the mitochondrial transporters, i.e. the calcium uniporter used for Ca(2+) uptake, and the antiporters used for the efflux, i.e. the Ca(2+)/Na(+) antiporter in mitochondria and the plasma membrane of excitable cells, and the Ca(2+)/nH(+) antiporter in liver and some other mitochondrial types. Mitochondria are of special interest in that Ca(2+) stimulates respiration and oxidative phosphorylation to meet the energy needs of activated cells. The studies on Ca(2+) and mitochondria began in the fifties, but interest in mitochondrial Ca(2+) handling faded in the late seventies since it had become apparent that mitochondria in resting cells contain very low Ca(2+). Interest increased again in the nineties also because it was discovered that mitochondria and Ca(2+) had a central role in apoptosis and necrosis. This is of special interest in calcium overload and oxidative stress conditions, when the opening of the mitochondrial permeability transition pore is stimulated.

In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus.

The in vitro boar spermatozoon test was compared with the LC ion trap MS analysis for measuring the cereulide content of a pasta dish, implemented in serious emetic food poisoning caused by Bacillus cereus. Both assays showed that the poisonous food contained approximately 1.6 microg of cereulide g(-1) implying the toxic dose in human as < or =8 microg kg(-1) body weight. The threshold concentration of cereulide provoking visible mitochondrial damage in boar sperm exposed in vitro was 2 ng of cereulide ml(-1) of extended boar sperm. The same threshold value was found for cereulide extracted from the food and from the cultured bacteria. This shows that other constituents of the food did not enhance or mask the effects of cereulide. Exposure of four human cell lines (HeLa, Caco-2, Calu-3 and Paju) to cereulide showed that the threshold concentration for the loss of mitochondrial membrane potential in human cells was similar to that observed in boar sperm. Human cells and boar sperm were equally sensitive to cereulide. The results show that boar spermatozoan assay is useful for detecting cereulide concentrations toxic to humans. Spermatozoa in commercially available extended fresh boar and cryopreserved bull semen were compared, boar sperms were 100 times more sensitive to cereulide than bull sperms.

Levosimendan is a mitochondrial K(ATP) channel opener.

Levosimendan, a new inodilator developed for the treatment of heart failure has been shown to have a vasodilatory effect via opening of K(ATP) channels in the plasma membrane of vascular smooth muscle cells. In this study, we investigated the effects of levosimendan on the mitochondrial K(ATP) channel. This compound did not influence mitochondrial transmembrane potential (DeltaPsi), and at up to 2.2 microM had no effect on the respiration rate of rat liver mitochondria, respiring on 5 mM succinate (+5 microM rotenone). A sensitive method was developed for assessing K(ATP) channel opening activity employing rat liver mitochondria, respiring only on endogenous substrates in the presence of 400 microM ATP and 1 microg oligomycin/mg mitochondrial protein. In this model, levosimendan (0.7-2.6 microM) decreased DeltaPsi by 6.5-40.4% (n=3, incubation time 15 min). This effect was dependent on the K+ concentration in the incubation medium and was abolished by the selective blocker of the mitochondrial K(ATP) channel-5-hydroxydecanoate (200 microM). Our results indicate that levosimendan opens mitochondrial K(ATP) channels.

Palmitic and stearic acids bind Ca2+ with high affinity and form nonspecific channels in black-lipid membranes. Possible relation to Ca2+-activated mitochondrial pores.

A mitochondrial hydrophobic component that forms Ca2+-induced nonspecific ion channels in black-lipid membranes (Mironova et al., 1997) has been purified and its nature elucidated. It consists of long-chain saturated fatty acids--mainly palmitic and stearic. These fatty acids, similar to the mitochondrial hydrophobic component, bind Ca2+ with high affinity in comparison with unsaturated fatty acids, saturated fatty acids with shorter aliphatic chains, phospholipids, and other lipids. Ca2+-binding is inhibited by Mg2+ but not by K+. For palmitic acid, the Kd for Ca2+ was 5 microM at pH 8.5 and 15 microM at pH 7.5, with the Bmax of 0.48 +/- 0.08 mmol/g. This corresponds to one Ca2+ ion for eight palmitic acid molecules. The data of IR spectroscopy confirm that Ca2+ does not form ionic bonds with palmitic and stearic acids under hydrophobic conditions. It has been found that in the presence of Ca2+, palmitic and stearic acids, but not unsaturated FFA induce a nonspecific permeability in black-lipid membranes. Addition of Ca2+ in order to induce the permeability transition, increases the extractable amount of palmitic and stearic acids, the effect being prevented by a phospholipase A2 inhibitor. The possible involvement of palmitic and stearic acids in the mitochondrial nonspecific permeability is discussed.

Ca(2+) efflux in mitochondria from the yeast Endomyces magnusii.

Calcium release pathways in Ca(2+)-preloaded mitochondria from the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca(2+) was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca(2+) release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca(2+) and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca(2+) efflux set in after uptake of approximately 150 nmol of Ca(2+)/mg of protein. The rate of this efflux was proportional to the Ca(2+) load and insensitive to aeration, protonophorous uncouplers, and Na(+) ions. Ca(2+) efflux was inhibited by La(3+), Mn(2+), Mg(2+), tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca(2+) efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca(2+) efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na(+)-independent pathway, in many ways similar to the one in mammalian mitochondria.

Mechanism of dihydrolipoate stimulation of the mitochondrial permeability transition: effect of different respiratory substrates.

The stimulation of the mitochondrial permeability transition (MPT) by dihydrolipoate (DHLA) was studied in rat liver mitochondria in the presence of different respiratory substrates. The Ca2+ threshold for the induction of MPT was lowest for pyruvate, followed by 2-hydroxybutyrate, 2-oxoglutarate, glutamate plus malate, and succinate plus rotenone, both in the presence and absence of DHLA. DHLA was not able to induce MPT in the absence of Ca2+, in the presence of cyclosporin A, or rotenone with pyridine nucleotide-dependent substrates. The difference in sensitivity of MPT to DHLA with various substrates was correlated with the redox state of pyridine nucleotides but not the redox state of glutathione. These findings demonstrate that DHLA induced MPT pore opening through the P-site thiol. The similarities between the effect of DHLA and that of production of reactive oxygen species found in model experiments suggest that DHLA stimulates MPT by production of reactive oxygen species that exhaust the antioxidant defence.

Ca(2+)-modulated phosphorylation of a low-molecular-mass polypeptide in rat liver mitochondria: evidence that it is identical with subunit c of F(0)F(1)-ATPase.

A 3.5-kDa polypeptide associated with the inner membrane of rat liver was found to be phosphorylated by [gamma-(32)P]ATP, presumably via a cAMP-dependent kinase. The phosphorylation was modulated by [Ca(2+)] in the physiological range, with a minimum at 1 microM and rising fourfold toward lower (10 nM) and higher (10 microM) concentrations. Further characterization of the 3.5-kDa component showed that the polypeptide has the same electrophoretic mobility as subunit c of F(0)F(1)-ATPase and that it selectively binds to antibodies against subunit c.

Magnesium. An update on physiological, clinical and analytical aspects.

There is an increased interest in the role of magnesium ions in clinical medicine, nutrition and physiology. The characteristics of the binding of magnesium and calcium ions to various components, macromolecules and biological membranes are described. Magnesium affects many cellular functions, including transport of potassium and calcium ions, and modulates signal transduction, energy metabolism and cell proliferation. The mechanism of cellular uptake and efflux of magnesium, its intracellular transport, intestinal absorption, renal excretion and the effect of hormones on these are reviewed. Magnesium deficiency is not uncommon among the general population: its intake has decreased over the years especially in the western world. The magnesium supplementation or intravenous infusion may be beneficial in various diseased states. Of special interest is the magnesium status in alcoholism, eclampsia, hypertension, atherosclerosis, cardiac diseases, diabetes, and asthma. The development of instrumentation for the assay of ionized magnesium is reviewed, as are the analytical procedures for total magnesium in blood and free magnesium in the cytosol. The improved procedures for the assay of different magnesium states are useful in understanding the role of magnesium in health and disease.

Calcium-binding properties of the mitochondrial channel-forming hydrophobic component.

A hydrophobic, low-molecular weight component extracted from mitochondria forms a Ca2+-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.3-8.5, the component has a high-affinity binding site for Ca2+ with a Kd of 8 x 10(-6) M, while at pH 7.5 this Kd was decreased to 9 x 10(-5) M. Bmax for the Ca2+-binding site did not change significantly with pH. In the range studied, 0.2 +/- 0.06 mmol Ca2+/g component were bound or one calcium ion to eight molecules of the component. The Ca2+ binding was strongly decreased by 50-100 mM Na+, but not by K+. Treatment of mitochondria with CaCl2 prior to ethanolic extraction resulted in a high level of Ca2+-binding capacity of the partially purified component. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition, when added to the mitochondrial suspension, decreased the Ca2+-binding activity of the purified extract severalfold. The calcium-binding capability of the partially purified component correlates with its calcium-channel activity. This indicates that the channel-forming component might be involved in the permeability transition that stimulates its formation.

Ionophoretic properties and mitochondrial effects of cereulide: the emetic toxin of B. cereus.

The emetic toxin of Bacillus cereus, found to cause immobilization of spermatozoa and swelling of their mitochondria, was purified and its structure found to be identical to the earlier known toxin cereulide. It increased the conductance in black-lipid membranes in KCl solutions in an ionophore-like manner. It formed adducts with K+, Na+, and NH4+ but the conductance was highly selective for K+ in relation to Na+ and H+ (three orders of magnitude). The increase in the kinetics of conductance indicated a stoichiometric ratio between the cereulide and K+. Its ionophoretic properties are thus similar to those of valinomycin. In addition, its effects on rat liver mitochondria were similar: it stimulated swelling and respiration in respiring mitochondria in the presence but not in the absence of K+, it reduced the transmembrane potential under these conditions. In nonrespiring mitochondria, swelling was seen in KNO3- but not in NaNO3-containing media, less in acetate. In NaNO3 media addition of the cereulide caused a transient diffusion potential which was reduced by adding K+. It is concluded that the toxic effects of cereulide are due to it being a K+ ionophore.

The Ca2+ threshold for the mitochondrial permeability transition and the content of proteins related to Bcl-2 in rat liver and Zajdela hepatoma mitochondria.

Zajdela hepatoma mitochondria were able to accumulate two to five times more Ca2+ than rat liver mitochondria before the permeability transition was induced. Pulses of Ca2+ were given in series to determine the Ca2+ threshold by recording changes in [Ca2+] and membrane potential, the permeability transition causing the release of accumulated Ca2+ and collapse of the membrane potential. Hepatoma mitochondria had lower Ca2+ efflux rates, higher net Ca2+ uptake rates and lower phosphorylation rates than liver mitochondria. Since the differences in regard to induction of the permeability transition might be due to higher expression of the Bcl-2 protein in hepatoma cells than in hepatocytes, the transcription of Bcl-2 and the proteins reacting with a Bcl-2 polyclonal antiserum were estimated by Northern and Western blotting, respectively. Hepatoma cells had two Bcl-2 specific mRNA bands of 7 and 2.4 kb, and substantial amounts of the Bcl-2 protein, whereas in liver cells and mitochondria these were not detected. Both cell lines had a reactive band at 19-20 kDa, and hepatocytes a small band at 31-32 kDa. Bcl-2 antibodies stimulated the permeability transition potently in hepatoma mitochondria.

The high calcium ion uptake capacity of Ehrlich ascites tumour cell mitochondria is due to inhibition of the permeability transition and phospholipase A2 activity by magnesium.

Tumour cells frequently have a high Ca2+ threshold for the mitochondrial permeability transition which occurs when a large pore in the inner membrane is opened. We studied whether this was due to the known high content of Mg2+ in Ehrlich ascites tumour cell mitochondria or to the increased expression of the protooncogene bcl-2. The latter was found not to be the case. Mg2+ potently inhibited the permeability transition and the binding of Ca2+ to the inner membrane. Also, phospholipase A2 activity was reduced by Mg2+. It is concluded that the high Ca2+ threshold is due to the high Mg2+ content in these tumour mitochondria.

Effect of prooxidants on mitochondrial permeability transition and cell death in Ehrlich ascites tumour cells.

Ca2+ retention in mitochondria, opening of the Cysclosporin A- sensitive permeability transition pore and cell death were studied in Ehrlich ascites tumour cells in the presence of different prooxidants. Low concentrations (1-20 microM) of the prooxidants (menadione, cumenehydroperoxide, t-butylhydroperoxide) induced pore-opening in permeabilized cells at threshold Ca2+ load. Incubation of cells with low concentrations of prooxidants was able to induce cell cycle disturbance and cell death. Under the prooxidant effect, mitochondrial membrane potential drop and Ca2+ retention decrease in mitochondria were found to precede death of Ehrlich ascites tumour cells.

Stimulation of the yeast mitochondrial calcium uniporter by hypotonicity and by ruthenium red.

The Ca2+ uptake by mitochondria from the yeast Endomyces magnusii has earlier been found to be driven by the membrane potential and to be stimulated by spermine. It thus functions in a similar fashion as the animal mitochondrial calcium uniporter. Here, it is shown that the uptake is stimulated, i.e., Ca2+ can be accumulated from lower [Ca2+], under hypotonic conditions. Ruthenium Red, an inhibitor of the animal uniporter, under certain conditions, stimulates the yeast uniporter. The mechanism of the stimulation by hypotonicity and Ruthenium Red is discussed.

Characterization of a high capacity calcium transport system in mitochondria of the yeast Endomyces magnusii.

The Ca2+ transport system of Endomyces magnusii mitochondria has been shown previously to be activated by spermine. Here we report it to be regulated also by low, physiological ADP concentrations, by the intramitochondrial NADH/NAD+ ratio, and by Ca2+ ions. The combination of all these physiological modulators induced high initial rates of Ca2+ uptake and high Ca2+-buffering capacity of yeast mitochondria, enabling them to lower the medium [Ca2+] to approximately 0.2 microM. The mechanisms of stimulation by these agents are discussed.

The stimulation of the mitochondrial permeability transition by dihydrolipoate and alpha-lipoate.

The effect of alpha-lipoate and dihydrolipoate on the mitochondrial permeability transition was investigated. Both substances promoted the permeability transition in isolated rat liver mitochondria and in permeabilized hepatocytes, dihydrolipoate most potently in spite of it being a dithiol. The stimulation was prevented by Cyclosporin A or hydroxybutyltoluene but not by ascorbate. It is suggested that the greater potency of dihydrolipoate could be due to formation of a radical.

Effect of butylhydroxytoluene and related compounds on permeability of the inner mitochondrial membrane.

Mitochondrial inner membrane contains a latent pore (PTP) that when opened uncouples mitochondrial energy transduction and allows rapid equilibration of low-molecular-weight solutes between the matrix and exterior. Based on sensitivity of the PTP to well-known free radical scavenger butylhydroxytoluene (BHT), it has been proposed that increased steady-state level of oxygen radicals, and subsequent radical attack of proteins and lipids, is a central event in activation of this pore (Novgorodov et al., J. Bioenerg. Biomembr. 19, 191-202, 1987; Carbonera and Azzone, Biochim. Biophys. Acta 943, 245-255, 1988). Present studies revealed that DBT, a derivative of BHT devoid of radical scavenging activity, exerts an analogous effect on the permeability of the inner membrane. Inhibition of the Ca2+-induced PTP opening is essentially complete at dose range of 50-60 nmol/mg protein with IC50 values of about 32 and 23 nmol/mg protein for DBT and BHT, respectively. Electron microscopy and osmotic experiments utilizing polyethylene glycols with different Stokes radii showed that the apparent lack of inhibition seen at high concentrations of these compounds results from cyclosporin A- and Ca2+-insensitive pore formation in the inner membrane. Experiments employing antioxidants with similar structure but dissimilar hydrophobicity provided evidence for localization of the antioxidant binding sites within the hydrophobic zone of the inner membrane or in the matrix space. The data obtained do not refute the notion that oxygen radicals modulate the PTP, but rather indicate that BHT operates independently of its free radical scavenging activity. Overall, the sensitivity to BHT and other antioxidants is not always a reliable criterion for the involvement of free radical reactions in the processes under study.

On the mechanism of rebounding of calcium in liver mitochondria.

Rat liver mitochondria are able to temporarily lower the steady state concentration of external Ca2+ after having accumulated a pulse of added Ca2+. This could be due to inhibition of efflux or/and stimulation of influx of Ca2+. This question has been addressed in mitochondria respiring on succinate +/- malonate. In the presence of malonate the depression of the membrane potential during Ca2+ uptake is more extensive and the rate of Ca2+ uptake slower. There were no discernible differences in the rates of efflux either after inhibition of the calcium uniporter by Ruthenium Red or by studying efflux of preloaded 45Ca-labeled Ca2+. The efflux was not changed by diltiazem or cyclosporin A to inhibit Ca2+ exchange on the Ca2+/nNA+ antiporter or efflux through the permeability transition pore. It is concluded that the rebounding is due mainly to stimulation of the calcium uniporter.