Mitochondrial calcium release as induced by Hg2+

Addition of Hg2+ to mitochondria of rat kidney induces efflux of intramitochondrial Ca2+. This reaction is accompanied by a diminution of the NAD(P)H/NAD(P) ratio and a decrease of the internal negative membrane potential. These effects were enhanced by dithiothreitol. The binding of mercuric ions to mitochondria saturates with a maximal binding of 9 nmol min-1 mg-1. The stoichiometry between Ca2+ released and Hg2+ bound showed that in the presence of dithiothreitol, the binding of approximately 1 nmol of Hg2+/mg of protein suffices to induce the release of the accumulated Ca2+. In the electrophoretic analysis of Hg-labeled mitochondrial proteins it was found that 203Hg2+ bound mainly to proteins that have molecular masses of 20 and 30 kDa. It is proposed that Hg2+-induced Ca2+ release is due to modification of--SH groups of these latter proteins.     

Characteristics of Ca2+ transport by Trypanosoma cruzi mitochondria in situ

The use of digitonin to permeabilize Trypanosoma cruzi plasma membrane has allowed the study of Ca2+ transport and oxidative phosphorylation in mitochondria in situ (R. Docampo and A. E. Vercesi (1989) J. Biol. Chem. 264, 108-111). The present results show that these mitochondria are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Ca2+ uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Addition of Ca2+ or ethylene glycol bis (beta-aminoethyl ether) N-N'-tetraacetic acid to these preparations under steady-state conditions was followed by Ca2+ uptake or release, respectively, tending to restore the original Ca2+ "set point" at about 0.9 microM. In addition, large amounts of Ca2+ were retained by T. cruzi mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butyl hydroperoxide, diamide, and the 1,2-naphthoquinone beta-lapachone. However, when ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine in the presence of antimycin A was used as subtrate, beta-lapachone caused pyridine nucleotide oxidation, and Ca2+ accumulation by these mitochondria was considerably lower than in control preparations, this effect being dose-dependent.     

Parallel efflux of Ca2+ and Pi in energized rat liver mitochondria.

Addition of Ruthenium Red to energized rat liver mitochondria that have previously accumulated Ca2+ and phosphate from the external medium induces a parallel efflux of both these ions. Mersalyl or dithioerythritol, which decrease Ruthenium Red-insensitive Ca2+ efflux, also decrease phosphate efflux to the same extent. Conversely diazenedicarboxylic acid bis(NN-dimethylamide) (DDBA), which increases the Ruthenium Red-induced Ca2+ efflux concurrently increases phosphate release. Dithioerythritol and DDBA, reducing and oxidizing agents of thiol groups respectively, modify Ca2+ and Pi efflux without penetrating the mitochondrial inner membrane. Under all the adopted conditions the membrane potential is preserved. The release of resting respiration and the parallel efflux of Mg2+ and adenine nucleotides, events closely correlated to Ca2+ cycling, are equally prevented either by mersalyl, which inhibits phosphate transport, or dithioerythritol; DDBA has the opposite effect. These findings and the observation that suggest that Ca2+ and phosphate transport in energized liver mitochondria are closely related and dependent on the redox state of membrane-bound thiol groups.     

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+.     

Bax affects intracellular Ca2+ stores and induces Ca2+ wave propagation

In the present study, we evaluated proapoptotic protein Bax on mitochondria and Ca2+ homeostasis in primary cultured astrocytes. We found that recombinant Bax (rBax, 10 and 100 ng/ml) induces a loss in mitochondrial membrane potential (Delta Psi m). This effect might be related to the inhibition of respiratory rates and a partial release of cytochrome c, which may change mitochondrial morphology. The loss of Delta Psi m and a selective permeabilization of mitochondrial membranes contribute to the release of Ca2+ from the mitochondria. This was inhibited by cyclosporin A (5 microM) and Ruthenium Red (1 microg/ml), indicating the involvement of mitochondrial Ca2+ transport mechanisms. Bax-induced mitochondrial Ca2+ release evokes Ca2+ waves and wave propagation between cells. Our results show that Bax induces mitochondrial alteration that affects Ca2+ homeostasis and signaling. These changes show that Ca2+ signals might be correlated with the proapoptotic activities of Bax.     

The role of inorganic phosphate in the release of Ca2+ from rat-liver mitochondria

The effect of inorganic phosphate on Ca2+ retention has been investigated using phosphate-depleted liver mitchondria. Phosphate induces the release of Ca2+ through an efflux route insensitive to ruthenium red. This effect is not due to functional or structural damage, since mitochondria maintain their membrane potential during phosphate-induced Ca2+ efflux. Direct enzymatic measurement of mitochondria pyridine nucleotides has established that changes in their redox state (i.e. increased oxidation) do not play a role in the phosphate-effect. The phosphate-induced Ca2+ efflux requires transport of phosphate out of mitochondria. However, the fluxes of Ca2+ and phosphate do not coincide: the release of phosphate preceeds that of Ca2+.     

Silymarin-induced mitochondrial Ca2+ release

The effect of silymarin on different functions of mitochondria isolated from rat kidneys was studied. Addition of silymarin to mitochondria oxidizing succinate, induced stimulation of the respiratory State 4; while in mitochondria oxidizing NAD-dependent substrates, the drug produced inhibition of the oxygen consumption. It is also shown that silymarin induces mitochondrial swelling, a drop in the transmembrane potential, as well as Ca2+ release. It is proposed that due to its hydrophobic character, silymarin produces an alteration in the lipidic milieu of the inner membrane which is conductive to an inhibition of the electron transport in the NAD-CoQ span of the respiratory chain, as well as to the loss of the energy dependent accumulated Ca2+.     

Zn~(2+)对PTP开放和细胞色素c释放的浓度依赖性双向调节

研究Zn2+对Ca2+介导线粒体通透过渡孔道(PTP)开放和线粒体细胞色素c释放的影响,及其与线粒体膜电位(ΔΨm)和Ca2+介导的线粒体Ca2+释放(mCICR)之间的关系.提取大鼠肝线粒体,通过紫外分光光度仪检测不同浓度Zn2+作用下Ca2+介导的PTP开放状态;采用荧光分光光度仪测定不同浓度Zn2+作用下线粒体膜电位的变化;采用双波长双光束紫外分光光度仪检测不同浓度Zn2+作用下测试体系内Ca2+浓度的变化,以反映线粒体Ca2+的转运情况(即mCICR);通过免疫印迹法检测不同浓度Zn2+作用下Ca2+介导的线粒体细胞色素c的释放.高浓度Zn2+完全抑制Ca2+介导的PTP开放和细胞色素c释放.一定浓度的Zn2+部分抑制Ca2+介导的PTP开放和细胞色素c释放.适当浓度Zn2+自身介导PTP开放和细胞色素c释放.低浓度Zn2+加速Ca2+介导PTP开放和Ca2+释放;高浓度和一定浓度Zn2+分别完全或部分破坏ΔΨm;高浓度Zn2+完全抑制mCICR.当抑制mCICR时,Ca2+和Zn2+对PTP开放和细胞色素c释放的作用完全抑制.结果表明,Zn2+以浓度依赖方式双向调节PTP开放和细胞色素c释放.Zn2+的作用可能与Zn2+破坏ΔΨm和影响mCICR相关.     

Interaction of genistein with the mitochondrial electron transport chain results in opening of the membrane transition pore

Genistein, a natural isoflavone present in soybeans, is a potent agent in the prophylaxis and treatment of cancer. Addition of genistein to isolated rat liver mitochondria (RLM) induces swelling, loss of membrane potential and release of accumulated Ca2+. These changes are Ca2+-dependent and are prevented by cyclosporin A (CsA) and bongkrekic acid (BKA), two classical inhibitors of the mitochondrial permeability transition (MPT). Induction of the MPT by genistein is accompanied by oxidation of thiol groups and pyridine nucleotides. The reducing agent dithioerythritol and the alkylating agent N-ethylmaleimide (NEM) completely prevent the opening of the transition pore, thereby emphasizing that the effect of the isoflavone correlates with the mitochondrial redox state. Further analyses showed that genistein induces the MPT by the generation of reactive oxygen species (ROS) due to its interaction with the respiratory chain at the level of mitochondrial complex III.     

Further studies on the effect of phosphoenolpyruvate on respiration-dependent calcium transport by rat heart mitochondria.

Phosphoenolpyruvate was found to depress extra oxygen consumption associated with Ca2+ -induced respiratory jump by rat heart mitochondria. Addition of phosphoenolpyruvate to mitochondria which have accumulated Ca2+ in the presence of glutamate and inorganic phosphate causes the release of Ca2+ from mitochondria. The phosphoenolpyruvate-stimulated Ca2+ efflux can be observed with mitochondria loaded with low initial Ca2+ concentration (0.12 mM) in the incubation medium. Measurements of mitochondrial H+ translocation produced by addition of Ca2+ to respiring mitochondria show that phosphoenolpyruvate depresses H+ ejection and enhances H+ uptake by mitochondria. The Ca2+ -releasing effect of phosphoenolpyruvate was found to be significantly stronger than that produced by rotenone when added to mitochondria loaded with Ca2+ in the presence of glutamate and inorganic phosphate. Dithiothreitol cannot overcome the effect of phosphoenolpyruvate on mitochondrial Ca2+ transport.     

The role of hydroperoxides as calcium release agents in rat brain mitochondria

Hydroperoxides have previously been shown to induce Ca2+ release from intact rat liver mitochondria via a specific release pathway. Here it is reported that, in rat brain mitochondria, a hydroperoxide-induced Ca2+ release is also operative but is of minor importance. Hydroperoxide stimulates Ca2+ release in the presence of ruthenium red about twofold at a Ca2+ load of 40 nmol/mg mitochondrial protein. After addition of hydroperoxide, Ca2+ release from brain mitochondria can still be evoked by Na+. In the presence of succinate and rotenone, hydroperoxide induces only a very limited oxidation of pyridine nucleotides, most probably due to the low level of glutathione peroxidase (EC 1.11.1.9) and glutathione reductase (EC 1.6.4.2) found in brain mitochondria. Similar to liver mitochondria, a NADase (EC 3.2.2.5) activity is found in brain mitochondria. Its localization and sensitivity toward ADP and ATP, however, is different from that of the liver mitochondrial enzyme.     

Inositol 1,4,5-trisphosphate- and arachidonic acid-induced calcium mobilization in T and B lymphocytes

Inositol triphosphate (IP3) formation and increase in intracytoplasmic calcium are mediators of signal transduction in lymphocytes. It has been proposed that IP3 induces Ca2+ release from intracellular stores. It is in order to study the relationship between these two events that we have analyzed the effect of IP3 addition on Ca2+ mobilization in permeabilized resting T and B lymphocytes, EBV-B lymphocytes, and HTLV1-T lymphocytes. IP3 induces a rapid and significant release of Ca2+ from the endoplasmic reticulum in a dose-dependent manner. Ca2+ release is more sensitive to IP3 addition in cycling cells (EBV-B lymphocytes and HTLV1-T lymphocytes) than in resting T and B lymphocytes. Arachidonic acid (AA) induces Ca2+ release from the endoplasmic reticulum (ER) in a manner similar to that of IP3. Neither component has an effect on Ca2+ accumulated in mitochondria, and they have no additive effects suggesting that they act on a similar Ca2+ pool. These results directly demonstrate that in T and B human lymphocytes IP3 mobilizes Ca2+ from ER as in other cellular systems and that other potential second messengers, namely AA, could play a significant role in the internal mobilization of calcium during T and B lymphocyte activation.     

Effect of ebselen on Ca2+ transport in mitochondria

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca2+ release from mitochondria. Ebselen stimulates Ca2+ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclosporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca2+ cycling is prevented with ruthenium red, an inhibitor of the Ca2+ uniporter, or by chelation of extramitochondrial Ca2+ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca2+ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca2+ release from intact mitochondria via an NAD+ hydrolysis-dependent mechanism.     

Gliotoxin induces Mg2+ efflux from intact brain mitochondria

Gliotoxin (GT) is a hydrophobic fungal metabolite of the epipolythiodioxopiperazine group which reacts with membrane thiols. When added to a suspension of energized brain mitochondria, it induces matrix swelling of low amplitude, collapse of membrane potential (DeltaPsi), and efflux of endogenous cations such as Ca2+ and Mg2+, typical events of mitochondrial permeability transition (MPT) induction. These effects are due to opening of the membrane transition pore. The addition of cyclosporin A (CsA) or ADP slightly reduces membrane potential collapse, matrix swelling and Ca2+ efflux; Mg2+ efflux is not affected at all. The presence of exogenous Mg2+ or spermine completely preserve mitochondria against DeltaPsi collapse, matrix swelling and Ca2+ release. Instead, Mg2+ efflux is only slightly affected by spermine. Our results demonstrate that, besides inducing MPT, gliotoxin activates a specific Mg2+ efflux system from brain mitochondria.     

Effect of Ca2+, peroxides, SH reagents, phosphate and aging on the permeability of mitochondrial membranes

The mechanism by which a number of agents such as hydroperoxides, inorganic phosphate, azodicarboxylic acid bis(dimethylamide) (diamide), 2-methyl-1,4-naphthoquinone (menadione) and aging, induce Ca2+ release from rat liver mitochondria has been analyzed by following Ca2+ fluxes in parallel with K+ fluxes, matrix swelling and triphenylmethylphosphonium fluxes (as an index of transmembrane potential). Addition of hydroperoxides causes a cycle of Ca2+ efflux and reuptake and an almost parallel cycle of delta psi depression. The hydroperoxide-induced delta psi depression is biphasic. The first phase is rapid and insensitive to ATP and is presumably due to activation of the transhydrogenase reaction during the metabolization of the hydroperoxides. The second phase is slow and markedly inhibited by ATP and presumably linked to the activation of a Ca2+-dependent reaction. The slow phase of delta psi depression is paralleled by matrix K+ release and mitochondrial swelling. Nupercaine and ATP reduce or abolish also K+ release and swelling. Inorganic phosphate, diamide, menadione or aging also cause a process of Ca2+ efflux which is paralleled by a slow delta psi depression, K+ release and swelling. All these processes are reduced or abolished by Nupercaine and ATP. The slow delta psi depression following addition of hydroperoxide and diamide is largely reversible at low Ca2+ concentration but tends to become irreversible at high Ca2+ concentration. The delta psi depression increases with the increase of hydroperoxide, diamide and menadione concentration, but is irreversible only in the latter case. Addition of ruthenium red before the hydroperoxides reduces the extent of the slow but not of the rapid phase of delta psi depression. Addition of ruthenium red after the hydroperoxides results in a slow increase of delta psi. Such an effect differs from the rapid increase of delta psi due to ruthenium-red-induced inhibition of Ca2+ cycling in A23187-supplemented mitochondria. Metabolization of hydroperoxides and diamide is accompanied by a cycle of reversible pyridine nucleotide oxidation. Above certain hydroperoxide and diamide concentrations the pyridine nucleotide oxidation becomes irreversible. Addition of menadione results always in an irreversible nucleotide oxidation. The kinetic correlation between Ca2+ efflux and delta psi decline suggests that hydroperoxides, diamide, menadione, inorganic phosphate and aging cause, in the presence of Ca2+, an increase of the permeability for protons of the inner mitochondrial membrane. This is followed by Ca2+ efflux through a pathway which is not the H+/Ca2+ exchange.     

Ca2+ transport by coupled Trypanosoma cruzi mitochondria in situ

The use of digitonin to permeabilize Trypanosoma cruzi plasma membrane enabled us to study Ca2+ transport and oxidative phosphorylation in mitochondria in situ. Addition of Ca2+ to these preparations evoked a cycle of respiratory stimulation. Ca2+ uptake was partially inhibited by ruthenium red, almost totally inhibited by antimycin A, and stimulated by inorganic phosphate. Addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone to digitonin-permeabilized T. cruzi epimastigotes under steady-state conditions was followed by Ca2+ release. Antimycin A- and carbonyl cyanide p-trifluoromethoxyphenylhydrazonein-sensitive Ca2+ uptake was also detected in digitonin-permeabilized epimastigotes. Accordingly, ATP stimulated Ca2+ uptake by preparations de-energized by oligomycin and antimycin A. In conclusion, in contrast to previous reports indicating that a Ca2+ transport system occurs only in mitochondria from vertebrate tissues, T. cruzi epimastigotes also possess a similar system. In addition, these protozoan mitochondria have an extremely high resistance to the deleterious effects of massive Ca2+ loads in comparison with most types of mammalian mitochondria.     

Quinone imine-induced Ca2+ release from isolated rat liver mitochondria

Incubation of Ca2(+)-loaded rat liver mitochondria with N-acetyl-p-benzoquinone imine (NAPQI) or its two dimethylated analogues resulted in a concentration dependent Ca2+ release, with the following order of potency: 2,6-(Me)2-NAPQI greater than NAPQI greater than 3,5-(Me)2-NAPQI. The quinone imine-induced Ca2+ release was associated with NAD(P)H oxidation and was prevented when NAD(P)+ reduction was stimulated by the addition of 3-hydroxybutyrate. Mitochondrial glutathione was completely depleted within 0.5 min by all three quinone imines, even at low concentrations that did not result in Ca2+ release. Depletion of mitochondrial GSH by pretreatment with 1-chloro-2,4-dinitrobenzene enhanced quinone imine-induced NAD(P)H oxidation and Ca2+ release. However, 3-hydroxybutyrate protected from quinone imine-induced Ca2+ release in GSH-depleted mitochondria. Mitochondrial membrane potential was lost after the addition of quinone imines at concentrations that caused rapid Ca2+ release; however, subsequent addition of EGTA led to the complete recovery of the transmembrane potential. In the absence of Ca2+, the quinone imines caused only a small and transient loss of the transmembrane potential. Taken together, our results suggests that the quinone imine-induced Ca2+ release from mitochondria is a consequence of NAD(P)H oxidation rather than GSH depletion, GSSG formation, or mitochondrial inner membrane damage.     

Stabilising action of carnitine on energy linked processes in rat liver mitochondria

Rat liver mitochondria exposed to stressing conditions - ageing at room temperature, incubation in the presence of t-butyl hydroperoxide or damaging concentrations of Ca2+ and phosphate- undergo a rapid fall in their membrane potential (delta psi) with a concomitant release of endogenous Mg2+ and accumulated Ca2+. Addition of L-carnitine to the incubation medium considerably delays mitochondrial deenergization. A similar, though lower, protection has also been observed in L-carnitine pretreated and subsequently washed rat liver mitochondria. Furthermore mitochondria isolated from livers of starved rats, treated with L-carnitine 30 minutes before death and exposed to the same stressing conditions show similar delay in the decrease of delta psi and concurrent energy linked processes as compared with untreated animals. Both the in vitro and in vivo results strongly indicate that the stabilising action of L-carnitine on liver mitochondria is due to the removal of membrane bound long chain acyl CoA.     

Role of mitochondria in calcium regulation of spontaneously contracting cardiac muscle cells.

Mitochondrial involvement in the regulation of cytosolic calcium concentration ([Ca2+]i) in cardiac myocytes has been largely discounted by many authors. However, recent evidence, including the results of this study, has forced a reappraisal of this role. [Ca2+]i and Ca2+ in the mitochondria ([Ca2+]m) were measured in this study with specific fluorescent probes, fluo-3 and di-hydro-rhod-2, respectively; mitochondrial membrane potential (DeltaPsim) was monitored with JC-1. Addition of uncouplers or inhibitors of the mitochondrial respiratory chain was found to cause a twofold decrease in the rate of removal of Ca2+ from the cytosol after a spontaneously generated Ca2+ wave. These agents also caused a progressive elevation of [Ca2+]i, an increase in the number of hotspots of Ca2+ release (Ca2+ sparks), and depression of mitochondrial potential. The Ca2+-indicative fluorophore dihydro-rhod-2 has a net positive charge that contributes to selective accumulation by mitochondria, as supported by its co-localization with other mitochondrial-specific probes (MitoTracker Green). Treatment of dihydro-rhod-2-loaded cells with NaCN resulted in rapid formation of "black holes" in the otherwise uniformly banded pattern. These are likely to represent individual or small groups of mitochondria that have depressed mitochondrial potential, or have lost accumulated rhod-2 and/or Ca2+; all of these eventualities are possible upon onset of the mitochondrial permeability transition. Release of Ca2+ from the sarcoplasmic reticulum and the resultant spontaneous contractility of cardiac muscle are proposed to be triggered by the induction of the mitochondrial permeability transition and the subsequent loss of [Ca2+]m.     

Mechanism of alloxan-induced calcium release from rat liver mitochondria

The objective of the present work was to investigate the mechanism of alloxan-induced Ca2+ release from rat liver mitochondria. Transport of Ca2+, oxidation and hydrolysis of mitochondrial pyridine nucleotides, changes in the mitochondrial membrane potential, and oxygen consumption by mitochondria were investigated. Alloxan does not inhibit the uptake of Ca2+ but stimulates the release of Ca2+ from liver mitochondria, which is accompanied by oxidation and hydrolysis of pyridine nucleotides. Oxidation of mitochondrial pyridine nucleotides by alloxan is not mediated by glutathione peroxidase and glutathione reductase and may occur largely nonenzymatically. Measurements of the mitochondrial membrane potential in combination with inhibitors of Ca2+ reuptake indicate that Ca2+ release takes place from intact liver mitochondria via a distinct pathway. Limited redox cycling of alloxan by mitochondria is indicated by measurements of the membrane potential and O2 consumption in the presence of cyanide. It is concluded that alloxan can cause Ca2+ release from intact rat liver mitochondria. Redox cycling of alloxan is not significantly involved in the Ca2+ release mechanism. Oxidation and hydrolysis of pyridine nucleotides, possibly in conjunction with oxidation of critical sulfhydryl groups, seem to be key events in the alloxan-induced Ca2+ release. Disturbance of cellular Ca2+ homeostasis may partly explain alloxan toxicity.