4-Hydroxytamoxifen is a potent inhibitor of the mitochondrial permeability transition

The effects of 4-hydroxytamoxifen (OHTAM), the major active metabolite of the antiestrogen tamoxifen used in the breast cancer therapy, were studied on the mitochondrial permeability transition (MPT) and bioenergetic functions of mitochondria to evaluate the mechanisms underlying the cell death and toxic effects. The MPT was induced in vitro by incubating rat liver mitochondria with 1 mM inorganic phosphate plus Ca2+ and with tert-butyl hydroperoxide. OHTAM provides protection against the Ca2+-induced mitochondrial swelling, depolarization of the mitochondrial membrane potential (deltapsi), loss of electrophoretic Ca2+ uptake capacity and uncoupling of respiration, similarly to cyclosporine A. The concentrations of OHTAM used do not significantly affect deltapsi, respiratory control and adenosine diphosphate/oxygen ratios and induce repolarization and Ca2+ re-uptake, suggesting that such inhibitory effects of OHTAM were due to the prevention of the MPT induction and not due to the inhibition of the mitochondrial Ca2+ uniporter. Since the MPT induction has been linked to an oxidized shift in the mitochondrial redox state and/or increase in the generation of reactive oxygen species, the MPT prevention by OHTAM may be related to its high antioxidant capacity.     

Effects of equisetin on rat liver mitochondria: Evidence for inhibition of substrate anion carriers of the inner membrane

The effect of equisetin, an antibiotic produced byFusarium equiseti, has been studied on mitochondrial functions (respiration, ATPase, ion transport). Equisetin inhibits the DNP-stimulated ATPase activity of rat liver mitochondria and mitoplasts in a concentration-dependent manner; 50% inhibition is caused by about 8 nmol equisetin/mg protein. The antibiotic is without effect either on the ATPase activity of submitochondrial particles or on the purified F₁-ATPase. It inhibits both the ADP- or DNP-activated oxygen uptake by mitochondria in the presence of glutamate + malate or succinate as substrates, but only the ADP-stimulated respiration is inhibited if the electron donors are TMPD + ascorbate. It does not affect the NADH or succinate oxidation of submitochondrial particles. Equisetin inhibits in a concentration-dependent manner the active Ca²⁺-uptake of mitochondria energized both by ATP or succinate without affecting the Ca²⁺-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the P_i and dicarboxylate carrier. It does not lower the membrane potential at least up to 200 nmol/mg protein concentration. The data presented in this paper indicate that equisetin specifically inhibits the substrate anion carriers of the mitochondrial inner membrane.Abbreviations EGTA ethyleneglycol bis/-aminoethylether/-N, N-tetraacetic acid - DNP 2, 4-dinitrophenol - TMPD N,N,N,N,tetramethyl-p-phenylenediamine - CCP carbonylcyanide-m-chlorophenyl hydrazone - TPP tetraphenyl-phosphonium - Hepes /4,(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid/     

Oxidative Stress Underlies the Mechanism for Ca2+-induced Permeability Transition of Mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca²⁺ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca²⁺ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca²⁺ increased the generation of H²O² by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca²⁺ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca²⁺-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca²⁺ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

Cardiac subsarcolemmal and interfibrillar mitochondria display distinct responsiveness to protection by diazoxide

Objective

Cardiac subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial subpopulations possess distinct biochemical properties and differ with respect to their protein and lipid compositions, capacities for respiration and protein synthesis, and sensitivity to metabolic challenge, yet their responsiveness to mitochondrially active cardioprotective therapeutics has not been characterized. This study assessed the differential responsiveness of the two mitochondrial subpopulations to diazoxide, a cardioprotective agent targeting mitochondria.

Methods

Mitochondrial subpopulations were freshly isolated from rat ventricles and their morphologies assessed by electron microscopy and enzymatic activities determined using standard biochemical protocols with a plate reader. Oxidative phosphorylation was assessed from State 3 respiration using succinate as a substrate. Calcium dynamics and the status of Ca²⁺-dependent mitochondrial permeability transition (MPT) pore and mitochondrial membrane potential were assessed using standard Ca²⁺ and TPP⁺ ion-selective electrodes.

Results

Compared to IFM, isolated SSM exhibited a higher sensitivity to Ca²⁺ overload-mediated inhibition of adenosine triphosphate (ATP) synthesis with decreased ATP production (from 375±25 to 83±15 nmol ATP/min/mg protein in SSM, and from 875±39 to 583±45 nmol ATP/min/mg protein in IFM). In addition, SSM exhibited reduced Ca²⁺-accumulating capacity as compared to IFM (230±13 vs. 450±46 nmol Ca²⁺/mg protein in SSM and IFM, respectively), suggestive of increased Ca²⁺ sensitivity of MPT pore opening. Despite enhanced susceptibility to stress, SSM were more responsive to the protective effect of diazoxide (100 μM) against Ca²⁺ overload-mediated inhibition of ATP synthesis (67% vs. 2% in SSM and IFM, respectively).

Conclusion

These results provide evidence for the distinct sensitivity of cardiac SSM and IFM toward Ca²⁺-dependent metabolic stress and the protective effect of diazoxide on mitochondrial energetics.     

Induction of calcium-dependent nonspecific permeability of the inner membrane in liver mitochondria of mammals and birds: A comparative study

The kinetics of the processes accompanying the induction of Ca²⁺-dependent permeability (pore opening) of the inner membrane—swelling of organelles and Ca²⁺ release from the matrix—was studied in isolated liver mitochondria of mammals (mice, rats, and rabbits) and birds (pigeons and guinea fowls). It was found that the mitochondria of rats, pigeons, and guinea fowls of the gray-speckled population (GSP) are similar in terms of respiration and oxidative ATP synthesis, whereas mitochondria of rabbits exhibit a greater degree of coupling of respiration and ATP synthesis, and mitochondria of mice and Zagorskaya White breed (ZWB) guinea fowls, a lower degree of coupling. It was established that mammalian mitochondria energized by succinate oxidation and incubated with 1 mM of inorganic phosphate are able to swell upon the addition of 125 nmol of CaCl₂ per 1 mg protein. Under these conditions, mitochondria of GSP and ZWB guinea fowls and pigeons are capable of swelling upon addition of at least 875, 875 and 1000 nmol of CaCl₂ per 1 mg protein, respectively. Cyclosporin A (CsA, 1 μM) inhibits mitochondrial swelling. It was shown that mitochondria of mammalians and guinea fowls but not of pigeons are able to effectively absorb and retain Ca²⁺ in the matrix. Calcium retention capacity of mitochondria from rats, mice, rabbits, GSP, and ZWB guinea fowls were, respectively, 70, 57, 38, 844 and 793 nmol of CaCl₂ per 1 mg of protein. In the presence of an oxidizing agent tert-butylhydroperoxide (TBH), the induction of the Ca²⁺-dependent pore in the mitochondria was observed upon addition of CaCl₂ in substantially smaller quantities. TBH was most effective in the case of rabbit mitochondria and had the lowest efficiency in the case of guinea fowl and pigeon mitochondria.     

The role of a membrane-bound glutathione transferase in the peroxynitrite-induced mitochondrial permeability transition pore: formation of a disulfide-linked protein complex

We have previously shown that the mitochondrial membrane-bound glutathione transferase (mtMGST1) is activated via thiol modifications and contributes to the mitochondrial permeability transition (MPT) pore. In the present study we aimed to confirm the role of mtMGST1 in the oxidant peroxynitrite (PON)-induced MPT pore opening. PON induced the swelling of mitoplasts (inner membranes including the matrix) as well as of the mitochondria. The swelling was markedly suppressed by ADP [an adenine nucleotide translocator (ANT) ligand] and partially suppressed by cyclosporin A or by GST inhibitors (tannic acid, S-hexylglutathione). Dithiothreitol (DTT), a disulfide bond-reducing reagent, prevented the swelling. Western blot analyses of mitoplast proteins after PON-induced swelling positively identified the high molecular weight protein (HMP) including mtMGST1 (monomer), ANT (48 kDa), and cyclophilin D (CypD, 30 kDa). The HMP level was decreased according to suppression of the swelling and undetectable after DTT treatment. The HMP formation and swelling were also suppressed by a Ca²⁺ chelating agent and antioxidants. These results suggest that the HMP is a disulfide-linked protein complex involving mtMGST1, ANT, CypD and function as a MPT pore in PON-induced swelling, in which the Ca²⁺ released by PON might play an important role in the complex formation.     

Vascular smooth muscle mitochondria: Magnesium content and transport

Endogenous magnesium content and magnesium transport of isolated bovine vascular smooth muscle mitochondria were studied. Mitochondria isolated from atherosclerotic bovine arteries contained two to three times as much magnesium (178 nmol/mg of mitochondrial protein) as those isolated from normal arteries (67 nmol/mg of mitochondrial protein). Electron-opaque granules were visible in the unstained unfixed mitochondria and could be shown with electron probe analysis to consist of magnesium, calcium, and phosphorus. At concentrations of external Mg²⁺ from 0 to 6 mm, the vascular smooth muscle mitochondria exhibited respiratory substrate-supported release of Mg²⁺ as studied with metallochromic indicator, eriochrome blue, using dual-wavelength spectrophotometry. The maximal velocity of energized release (3 nmol of Mg²⁺/s/mg of mitochondrial protein) was observed at 4 mm external Mg²⁺ and the half-maximal transport occurred at 0.5 mm.     

Interaction between Connexin 43 and nitric oxide synthase in mice heart mitochondria

Connexin 43 (Cx43), which is highly expressed in the heart and especially in cardiomyocytes, interferes with the expression of nitric oxide synthase (NOS) isoforms. Conversely, Cx43 gene expression is down‐regulated by nitric oxide derived from the inducible NOS. Thus, a complex interplay between Cx43 and NOS expression appears to exist. As cardiac mitochondria are supposed to contain a NOS, we now investigated the expression of NOS isoforms and the nitric oxide production rate in isolated mitochondria of wild‐type and Cx43‐deficient (Cx43^{Cre‐ER(T)/fl}) mice hearts. Mitochondria were isolated from hearts using differential centrifugation and purified via Percoll gradient ultracentrifugation. Isolated mitochondria were stained with an antibody against the mitochondrial marker protein adenine‐nucleotide‐translocator (ANT) in combination with either a neuronal NOS (nNOS) or an inducible NOS (iNOS) antibody and analysed using confocal laser scanning microscopy. The nitric oxide formation was quantified in purified mitochondria using the oxyhaemoglobin assay. Co‐localization of predominantly nNOS (nNOS: 93 ± 4.1%; iNOS: 24.6 ± 7.5%) with ANT was detected in isolated mitochondria of wild‐type mice. In contrast, iNOS expression was increased in Cx43^{Cre‐ER(T)/fl} mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43^{Cre‐ER(T)/fl} mitochondria (0.14 ± 0.02 nmol/min./mg protein) in comparison to wild‐type mitochondria (0.24 ± 0.02 nmol/min./mg). These are the first data demonstrating, that a reduced mitochondrial Cx43 content is associated with a switch of the mitochondrial NOS isoform and the respective mitochondrial rate of nitric oxide formation.     

Alloxan-induced mitochondrial permeability transition triggered by calcium, thiol oxidation, and matrix ATP

In addition to their critical function in energy metabolism, mitochondria contain a permeability transition pore, which is regulated by adenine nucleotides. We investigated conditions required for ATP to induce a permeability transition in mammalian mitochondria. Mitochondrial swelling associated with mitochondria permeability transition (MPT) was initiated by adding succinate to a rat liver mitochondrial suspension containing alloxan, a diabetogenic agent. If alloxan was added immediately with or 5 min after adding succinate, MPT was strikingly decreased. MPT induced by alloxan was inhibited by EGTA and several agents causing thiol oxidation, suggesting that alloxan leads to permeability transition through a mechanism dependent on Ca(2+) uptake and sulfhydryl oxidation. Antimycin A and cyanide, inhibitors of electron transfer, carbonyl cyanide m-chlorophenylhydrazone, and oligomycin all inhibited MPT. During incubation with succinate, alloxan depleted ATP in mitochondria after an initial transient increase. However, in a mitochondrial suspension containing EGTA, ATP significantly increased in the presence of alloxan to a level greater than that of the control. These results suggest the involvement of energized transport of Ca(2+) in the MPT initiation. Addition of exogenous ATP, however, did not trigger MPT in the presence of alloxan and had no effect on MPT induced by alloxan. We conclude that alloxan-induced MPT requires mitochondrial energization, oxidation of protein thiols, and matrix ATP to promote energized uptake of Ca(2+).     

Propofol Prevents Oxidative Stress by Decreasing the Ischemic Accumulation of Succinate in Focal Cerebral Ischemia–Reperfusion Injury

Oxidative stress caused by mitochondrial dysfunction during reperfusion is a key pathogenic mechanism in cerebral ischemia–reperfusion (IR) injury. Propofol (2,6-diisopropylphenol) has been proven to attenuate mitochondrial dysfunction and reperfusion injury. The current study reveals that propofol decreases oxidative stress injury by preventing succinate accumulation in focal cerebral IR injury. We evaluated whether propofol could attenuate ischemic accumulation of succinate in transient middle cerebral artery occlusion in vivo. By isolating mitochondria from cortical tissue, we also examined the in vitro effects of propofol on succinate dehydrogenase (SDH) activity and various mitochondrial bioenergetic parameters related to oxidative stress injury, such as the production of reactive oxidative species, membrane potential, Ca²⁺-induced mitochondrial swelling, and morphology via electron microscopy. Propofol significantly decreased the ischemic accumulation of succinate by inhibiting SDH activity and inhibited the oxidation of succinate in mitochondria. Propofol can decrease membrane potential in normal mitochondria but not in ischemic mitochondria. Propofol prevents Ca²⁺-induced mitochondrial swelling and ultrastructural changes to mitochondria. The protective effect of propofol appears to act, at least in part, by limiting oxidative stress injury by preventing the ischemic accumulation of succinate.     

Intramitochondrial and extramitochondrial free calcium ion concentrations of suspensions of heart mitochondria with very low,plausibly physiological,contents of total calcium

The 2-oxoglutarate dehydrogenase of intact rat heart mitochondria is activated by Ca²⁺, with 50% activation at approximately 0.5 nmol of total Ca/mg of mitochondrial protein, in the presence of P_i and Mg²⁺. Mitochondrial Ca contents in excess of 2 nmol/mg of protein result in 100% activation of the enzyme. Investigation of Ca²⁺ release from the mitochondria using the metallochromic indicator Arsenazo III defines aS _0.5 of 5.4±0.4 nmol of Ca/mg of protein, when the endogenous Ca content of the mitochondria is progressively depleted with EGTA, prior to the initiation of the release process being studied. The subsequent determination of matrix free Ca²⁺ concentration by the null-point technique has allowed expression of these results in terms of free concentration rather than Ca content, with an activity coefficient of approximately 0.001 for matrix Ca²⁺. From the above, Ca²⁺ efflux from heart mitochondria is not saturated at the mitochondrial Ca contents or Ca²⁺ concentrations which give effective regulation of dehydrogenase activity. A consequence is that heart mitochondria do not buffer the pCa of the extramitochondrial medium at these Ca contents (<2 nmol/mg of protein), and this is shown in direct measurements of extramitochondrial pCa. This is taken to question the physiological significance of mitochondrial buffering of cytosolic free Ca²⁺ in normal heart.     

Agmatine prevents the Ca2+-dependent induction of permeability transition in rat brain mitochondria

The arginine metabolite agmatine is able to protect brain mitochondria against the drop in energy capacity by the Ca²⁺-dependent induction of permeability transition (MPT) in rat brain mitochondria. At normal levels, the amine maintains the respiratory control index and ADP/O ratio and prevents mitochondrial colloid-osmotic swelling and any electrical potential (ΔΨ) drop. MPT is due to oxidative stress induced by the interaction of Ca²⁺ with the mitochondrial membrane, leading to the production of hydrogen peroxide and, subsequently, other reactive oxygen species (ROS) such as hydroxyl radicals. This production of ROS induces oxidation of sulfhydryl groups, in particular those of two critical cysteines, most probably located on adenine nucleotide translocase, and also oxidation of pyridine nucleotides, resulting in transition pore opening. The protective effect of agmatine is attributable to a scavenging effect on the most toxic ROS, i.e., the hydroxyl radical, thus preventing oxidative stress and consequent bioenergetic collapse.     

Effect of adenine nucleotide pool size in mitochondria on intramitochondrial ATP levels.

Net adenine nucleotide transport into and out of the mitochondrial matrix via the ATP-Mg/Pi carrier is activated by micromolar calcium concentrations in rat liver mitochondria. The purpose of this study was to induce net adenine nucleotide transport by varying the substrate supply and/or extramitochondrial ATP consumption in order to evaluate the effect of the mitochondrial adenine nucleotide pool size on intramitochondrial adenine nucleotide patterns under phosphorylating conditions. Above 12 nmol/mg protein, intramitochondrial ATP/ADP increased with an increase in the mitochondrial adenine nucleotide pool. The relationship between the rate of respiration and the mitochondrial ADP concentration did not depend on the mitochondrial adenine nucleotide pool size up to 9 nmol ADP/mg mitochondrial protein. The results are compatible with the notion that net uptake of adenine nucleotides at low energy states supports intramitochondrial ATP consuming processes and energized mitochondria may lose adenine nucleotides. The decrease of the mitochondrial adenine nucleotide content below 9 nmol/mg protein inhibits oxidative phosphorylation. In particular, this could be the case within the postischemic phase which is characterized by low cytosolic adenine nucleotide concentrations and energized mitochondria.     

Mitochondrial calcium and the permeability transition in cell death

Dysregulation of Ca²⁺ has long been implicated to be important in cell injury. A Ca²⁺-linked process important in necrosis and apoptosis (or necrapoptosis) is the mitochondrial permeability transition (MPT). In the MPT, large conductance permeability transition (PT) pores open that make the mitochondrial inner membrane abruptly permeable to solutes up to 1500 Da. The importance of Ca²⁺ in MPT induction varies with circumstance. Ca²⁺ overload is sufficient to induce the MPT. By contrast after ischemia-reperfusion to cardiac myocytes, Ca²⁺ overload is the consequence of bioenergetic failure after the MPT rather than its cause. In other models, such as cytotoxicity from Reye-related agents and storage-reperfusion injury to liver grafts, Ca²⁺ appears to be permissive to MPT onset. Lastly in oxidative stress, increased mitochondrial Ca²⁺ and ROS generation act synergistically to produce the MPT and cell death. Thus, the exact role of Ca²⁺ for inducing the MPT and cell death depends on the particular biologic setting.     

Regulation of Ca2+-induced permeability transition by Bcl-2 is antagonized by Drp1 and hFis1

The regulation of mitochondrial permeability transition (MPT) is essential for cell survival. Un-controlled opening of the MPT pore is often associated with cell death. Anti-death protein Bcl-2 can block MPT as assessed by the enhanced capacity of mitochondria to accumulate and retain Ca²⁺. We report here that two proteins of the mitochondrial fission machinery, dynamin-related protein (Drp1) and human mitochondrial fission protein (hFis1), have an antagonistic effect on Bcl-2. Drp1, with the assistance of hFis1, sensitizes cells to MPT by reducing the mitochondrial Ca²⁺ retention capacity (CRC). While the reduction of CRC by Drp1/hFis1 is linked to mitochondrial fission, the antagonism between Bcl-2 and Drp1 appears to be mediated by mutually exclusive interactions of the two proteins with hFis1. The complexity of protein–protein interactions demonstrated in the present study suggests that in addition to the previously described role of Bcl-2 in the control of apoptosis, Bcl-2 may also participate directly or indirectly in the regulation of mitochondrial fission.     

The isolation of coupled mitochondria from Physarum polycephalum and their response to Ca2+

A method for the isolation of coupled mitochondria from the acellular slime mould Physarum polycephalum is described. The mitochondria oxidize respiratory substrates at rates comparable to those of mitochondria from other micro-organisms and show similar responses to respiratory inhibitors. ADP/O values approach similar values to those obtained with mitochondria from higher organisms: 3 with NAD-linked substrates, 2 with succinate, and 1 with ascorbate-TMPD.Mitochondria actively take up low concentrations of Ca²⁺ with stimulation of their respiration. With succinate or pyruvate-malate as substrates respiratory responses are depressed by Ca²⁺ concentrations in excess of 200 μM in the presence or absence of phosphate.Exogenous NADH is unique in supporting the uptake of large amounts of Ca²⁺ in the presence of phosphate and in showing an unusual ‘uncoupled’ response in the absence of phosphate.A sigmoidal relationship occurs between initial velocity of Ca²⁺ uptake and Ca²⁺ concentration with a maximum velocity of approx. 15 nmol/s per mg protein and half maximum velocity occurring at approx. 50 μM Ca²⁺.     

Heterogeneity of chondrocyte mitochondria: A study of the Ca2+ concentration and density banding characteristics of normal and rachitic cartilage

Previous morphological studies of the mineralizing epiphysis suggested that some mitochondria were concerned with Ca²⁺ accumulation while others were associated with cellular energetics and metabolism. To determine if there was mitochondrial heterogeneity in chondrocytes of the epiphyseal growth plate, mitochondria were isolated from four different regions of the plate and subjected to continuous sucrose gradient centrifugation. Centrifugation of the organelles in a narrow density sucrose gradient (1.5–2.0 M) in the presence of inhibitors of Ca²⁺ transport (ruthenium red and 5,5′-dithiobis-(2-nitrobenzoic acid)) revealed that considerable heterogeneity existed. In the least calcified zone 20% of the mitochondria formed a low density band of low Ca²⁺ concentration (309 nmol/mg protein). Organelles isolated from more calcified tissue zones showed a concomitant increase in Ca²⁺ concentration (up to 5700 nmol/mg protein) as well as an increase in the total percentage of mitochondria sedimenting in 2.0 M sucrose. The banding patterns of mitochondria isolated from rachitic and hypertrophic cartilage were similar. In addition, similarities were also noted in the Ca²⁺ concentration and the cytochrome oxidase activities of mitochondria of these tissues. During recovery from the rachitic condition, there was a change in the density centrifugation characteristics of this tissue and a substantial increase was noted in the proportion of mitochondria sedimenting in 2.0 M sucrose. The Ca²⁺ concentration of mitochondria of this rapidly calcifying tissue suggested that the critical Ca²⁺ concentration necessary for initiation of the calcification mechanism was 4 μmol/mg protein.     

Cyclosporin a Increases Mitochondrial Calcium Uptake Capacity in Cortical Astrocytes but not Cerebellar Granule Neurons

Isolated brain mitochondria are a heterogeneous mixture from different cell types and these subsets may have differing sensitivities to Ca²⁺-induced membrane permeability transition (MPT) and to inhibition of the MPT by cyclosporin A (CsA). This study tested the hypothesis that mitochondria within primary cultures of astrocytes and neurons exhibit different energy-dependent Ca²⁺ uptake capacities and different degrees to which CsA increases their uptake capacity. Astrocytes and neurons were suspended in a cytosol-like medium containing respiratory substrates, ATP, and Mg²⁺ in the presence of digitonin to selectively permeabilize the plasma membrane. Uptake of added Ca²⁺ by mitochondria within the cells was measured by Calcium Green 5N fluorescent monitoring of the medium [Ca²⁺]. Permeabilized astrocytes had a fourfold higher Ca²⁺ uptake capacity, relative to neurons and a twofold higher content based on relative contents of mitochondria assessed by measurements of mitochondrial DNA and cytochrome oxidase subunit 1 protein. In astrocytes the Ca²⁺ uptake capacity was increased twofold by preincubation with 2–5 μM CsA, while in neurons CsA had no effect. Similar results were obtained using measurements of the effects of added Ca²⁺ on mitochondrial membrane potential. FK506, a drug similar to CsA but without MPT inhibitory activity, had no effect on either cell type. These results are consistent with the presence of a calcium-induced MPT in astrocytes, even in the presence of ATP, and indicate that the MPT in cerebellar granule neurons is resistant to CsA inhibition. Some of the protective effects of CsA in vivo may therefore be mediated by preservation of mitochondrial functional integrity within astrocytes.     

Bile Acid-Induced Ca<Superscript>2+</Superscript> Efflux from Liver Mitochondria as a Factor Preventing the Formation of Mitochondrial Pores

The effect of bile acids as inducers of Ca²⁺ efflux from the matrix was studied on isolated rat liver mitochondria. Mitochondria in the presence of cyclosporin A (CsA) were energized by succinate, then loaded with Ca²⁺ and after the addition of the calcium uniporter inhibitor ruthenium red were de-energized by malonate. It was shown that under these conditions hydrophobic bile acids lithocholic and chenodeoxycholic at concentrations of 10 and 30 μM respectively and hydrophilic bile acids ursodeoxycholic and cholic at a concentration of 400 μM induce Ca²⁺ efflux from the mitochondrial matrix. It is noted that the efflux of these ions is not associated with damage of the inner mitochondrial membrane by bile acids, since it is accompanied by the generation of Δψ, i.e., the formation of the diffusion potential. It is assumed that along with induction of calcium efflux from the matrix, bile acids are also capable of transporting hydrogen and potassium ions in the opposite direction, i.e., perform H⁺/Ca²⁺ and K⁺/Ca²⁺ exchange. It was found that ruthenium red added to Ca²⁺-loaded energized mitochondria prevents the return of these ions to the matrix and weakens the effect of chenodeoxycholic acid as an inducer of the CsA-sensitive mitochondrial pore and the effect of ursodeoxycholic acid as an inducer of CsA-insensitive permeability of the inner mitochondrial membrane. We conclude that in the conditions of the calcium uniporter activity decrease, Ca²⁺ efflux from the matrix induced by bile acids can be considered as one of the mechanisms reducing their effectiveness as inducers of the Ca²⁺-dependent CsA-sensitive pore and CsA-insensitive permeability transition in mitochondria.     

Ca binding to c-state of adenine nucleotide translocase (ANT)-surrounding cardiolipins enhances (ANT)-Cys relative mobility: A computational-based mitochondrial permeability transition study

The oxidation of critical cysteines/related thiols of adenine nucleotide translocase (ANT) is believed to be an important event of the Ca²⁺-induced mitochondrial permeability transition (MPT), a process mediated by a cyclosporine A/ADP-sensitive permeability transition pores (PTP) opening. We addressed the ANT-Cys⁵⁶ relative mobility status resulting from the interaction of ANT/surrounding cardiolipins with Ca²⁺ and/or ADP by means of computational chemistry analysis (Molecular Interaction Fields and Molecular Dynamics studies), supported by classic mitochondrial swelling assays. The following events were predicted: (i) Ca²⁺ interacts preferentially with the ANT surrounding cardiolipins bound to the H4 helix of translocase, (ii) weakens the cardiolipins/ANT interactions and (iii) destabilizes the initial ANT-Cys⁵⁶ residue increasing its relative mobility. The binding of ADP that stabilizes the conformation “m” of ANT and/or cardiolipin, respectively to H5 and H4 helices, could stabilize their contacts with the short helix h56 that includes Cys⁵⁶, accounting for reducing its relative mobility. The results suggest that Ca²⁺ binding to adenine nucleotide translocase (ANT)-surrounding cardiolipins in c-state of the translocase enhances (ANT)-Cys⁵⁶ relative mobility and that this may constitute a potential critical step of Ca²⁺-induced PTP opening.