Phosphorylation/dephosphorylation of maize mitochondrial proteins with different localization

Phosphorylation and dephosphorylation of the proteins residing in the outer mitochondrial membrane, mitoplasts and whole mitochondria of maize (Zea mays L.) were investigated in order to reveal the possible participation of these processes in mitochondrial signaling. A mitochondrial protein of around 57 kD was identified by immunocytochemistry as α-subunit of the F₁-ATPase complex. In isolated mitochondria of maize, phosphorylation of this protein could be visualized only after treating mitochondria with endotholl, an inhibitor of the PP1a and PP2A protein phosphatases. A phosphorylated protein of 46.6 kD was identified as β-subunit of the F₁-ATPase complex. Ca²⁺ is the most common second messenger participating in mitochondrial signaling. We conclude that the transmission of the Ca²⁺ signal to the plant mitochondria occurs via proteins of the outer mitochondrial membrane. The systems perceiving this signal could include the protein phosphatases residing in the outer mitochondrial membrane, which preferentially dephosphorylate the proteins in the inner membrane.     

Inhibitor analysis of protein phosphorylation/dephosphorylation in maize mitochondria in relation to redox conditions

The role of protein phosphorylation/ dephosphorylation in the redox regulation of mitochondrial functioning was investigated. Incubation of isolated mitochondria of maize (Zea mays L.) in the presence of γ-³²P-ATP revealed phosphorylation of polypeptides with mol wt of 66, 60, 55, 48/50 doublet, 45, 29, 22, and 19 kD. The presence in the incubation medium of oxidized glutathione significantly reduced the level of protein phosphorylation. The addition of reduced glutathione diminished phosphorylation of proteins with mol wt of 60 and 48/50 kD and slightly increased phosphorylation of proteins with mol wt of 66, 55, and 45 kD. The reducing agent, sodium dithionite decreased phosphorylation of proteins with mol wt of 60, 45, 29, 22, and 19 kD but increased phosphorylation of 55 kD protein. The inhibitors of protein kinases and protein phosphatases significantly modified the effects of redox agents. For example, simultaneous action of an oxidant K₃[Fe(CN)₆] and NaF enhanced phosphorylation level compared to separate treatments with these agents. The combined application of sodium dithionite and NaF elevated phosphorylation level of 55 kD protein. Phosphoprotein with mol wt of about 66 kD was identified immunochemically as a heat shock protein (HSP 60). The results indicate the presence in mitochondria of redox-sensitive protein kinases and protein phosphatases. Differential changes in the pattern of mitochondrial phosphoproteins under the action of various redox agents suggest that phosphorylation is probably involved in the transduction of redox signal in plant mitochondria.     

Regulation of Phosphorylation of Wheat Mitochondrial Proteins by Divalent Cations

Mitochondria isolated from 4-day-old dark-grown wheat seedlings were purified by self-generating Percoll gradient. Phosphorylation reaction was carried out in vitro with the addition of [ c-³²P]ATP and polypeptides resolved by 50S-PAGE were subjected to autoradiography. Amongst endogenous polypeptides phosphorylated, four polypeptides of 120, 66, 43 and 21 kD were prominent. Addition of Mg²⁺ (5 mM) caused dephosphorylation of 120 and 66 kO polypeptides but, simultaneously, induced/enhanced the phosphorylation of some polypeptides, with the effect being more pronounced on a 67 kD species. The phosphorylation of 120 kD species and a few other polypeptides was also down-regulated and that of a 18 kD polypeptide was up-regulated by Ca²⁺. The present study provides evidence that phosphorylation status of mitochondrial proteins is regulated by Mg²⁺ and/or Ca²⁺-dependent phosphatase(s) and protein kinase(s).     

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

Control of mitochondrial membrane potential and ROS formation by reversible phosphorylation of cytochrome c oxidase

Phosphorylation of isolated cytochrome c oxidase from bovine kidney and heart, and of the reconstituted heart enzyme, with protein kinase A, cAMP and ATP turns on the allosteric ATP-inhibition at high ATP/ADP ratios. Also incubation of isolated bovine liver mitochondria only with cAMP and ATP turns on, and subsequent incubation with Ca²⁺ turns off the allosteric ATP-inhibition of cytochrome c oxidase. In the bovine heart enzyme occur only three consensus sequences for cAMP-dependent phosphorylation (in subunits I, III and Vb). The evolutionary conservation of RRYS⁴⁴¹ at the cytosolic side of subunit I, together with the above results, suggest that phosphorylation of Ser⁴⁴¹ turns on the allosteric ATP-inhibition of cytochrome c oxidase. The results support the 'molecular-physiological hypothesis' [29], which proposes a low mitochondrial membrane potential through the allosteric ATP-inhibition. A hormone- or agonist-stimulated increase of cellular [Ca²⁺]] is suggested to activate a mitochondrial protein phosphatase which dephosphorylates cytochrome c oxidase, turns off the allosteric ATP-inhibition and results in increase of mitochondrial membrane potential and ROS formation.     

Diacylglycerols Activate Mitochondrial Cationic Channel(s) and Release Sequestered Ca<Superscript>2+</Superscript>

Mitochondria contribute to cytosolic Ca²⁺ homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAGs) induce Ca²⁺ release from Ca²⁺-loaded mammalian mitochondria. Release is not mediated by the uniporter or the Na⁺/Ca²⁺ exchanger, nor is it attributed to putative catabolites. DAGs-induced Ca²⁺ efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca²⁺ and relatively slow reuptake, a secondary progressive release of Ca²⁺ occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAGs-induced Ca²⁺ efflux is abolished by La³⁺ (1 mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca²⁺ efflux. Ca²⁺-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAGs-induced Ca²⁺ release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brain mitoplasts reveal DAGs-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La³⁺. The presence of a second messenger-sensitive Ca²⁺ release mechanism in mitochondria could have an important impact on intracellular Ca²⁺ homeostasis.     

Mitochondrial Ca<Superscript>2+</Superscript> cycle mediated by the palmitate-activated cyclosporin a-insensitive pore

Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca²⁺ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca²⁺, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca²⁺ and the swelling of mitochondria. Inhibitors of the Ca²⁺ uniporter, ruthenium red and La³⁺, as well as EGTA added in 10 min after the Pal/Ca²⁺-activated pore opening, prevent the release of Ca²⁺ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr²⁺], which leads to the activation of phospholipase A₂ and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca²⁺ cycle, in which Ca²⁺ uptake is mediated by the Ca²⁺ uniporter and Ca²⁺ efflux occurs via a short-living Pal/Ca²⁺-activated pore.     

Distinctive characteristics and functions of multiple mitochondrial Ca<Superscript>2+</Superscript> influx mechanisms

Intracellular Ca²⁺ is vital for cell physiology. Disruption of Ca²⁺ homeostasis contributes to human diseases such as heart failure, neuron-degeneration, and diabetes. To ensure an effective intracellular Ca²⁺ dynamics, various Ca²⁺ transport proteins localized in different cellular regions have to work in coordination. The central role of mitochondrial Ca²⁺ transport mechanisms in responding to physiological Ca²⁺ pulses in cytosol is to take up Ca²⁺ for regulating energy production and shaping the amplitude and duration of Ca²⁺ transients in various micro-domains. Since the discovery that isolated mitochondria can take up large quantities of Ca²⁺ approximately 5 decades ago, extensive studies have been focused on the functional characterization and implication of ion channels that dictate Ca²⁺ transport across the inner mitochondrial membrane. The mitochondrial Ca²⁺ uptake sensitive to non-specific inhibitors ruthenium red and Ru360 has long been considered as the activity of mitochondrial Ca²⁺ uniporter (MCU). The general consensus is that MCU is dominantly or exclusively responsible for the mitochondrial Ca²⁺ influx. Since multiple Ca²⁺ influx mechanisms (e.g. L-, T-, and N-type Ca²⁺ channel) have their unique functions in the plasma membrane, it is plausible that mitochondrial inner membrane has more than just MCU to decode complex intracellular Ca²⁺ signaling in various cell types. During the last decade, four molecular identities related to mitochondrial Ca²⁺ influx mechanisms have been identified. These are mitochondrial ryanodine receptor, mitochondrial uncoupling proteins, LETM1 (Ca²⁺/H⁺ exchanger), and MCU and its Ca²⁺ sensing regulatory subunit MICU1. Here, we briefly review recent progress in these and other reported mitochondrial Ca²⁺ influx pathways and their differences in kinetics, Ca²⁺ dependence, and pharmacological characteristics. Their potential physiological and pathological implications are also discussed.     

Cooperative Interactions in Energy-dependent Accumulation of Ca<Superscript>2+</Superscript> by Isolated Rat Liver Mitochondria

THE energy-dependent accumulation of Ca²⁺ by isolated rat liver mitochondria is intimately associated with oxidative phosphorylation¹. Available evidence supports the idea that, like the permeases postulated for some mitochondrial metabolites², this active accumulation of Ca²⁺ may involve a “carrier” in the mitochondrial membrane specific for Ca²⁺ (ref. 3). Several studies have shown that the energy-independent “binding” of Ca²⁺ to sites on the (inner membrane of), intact mitochondria and of submitochondrial particles exhibits hyperbolic saturation curves as a function of Ca²⁺ concentration^{4, 5}.     

Mitochondrial Ca<Superscript>2+</Superscript> uptake pathways

Calcium (Ca²⁺) plays diverse roles in all living organisms ranging from bacteria to humans. It is a structural element for bones, an essential mediator of excitation-contraction coupling, and a universal second messenger in the regulation of ion channel, enzyme and gene expression activities. In mitochondria, Ca²⁺ is crucial for the control of energy production and cellular responses to metabolic stress. Ca²⁺ uptake by the mitochondria occurs by the uniporter mechanism. The Mitochondrial Ca2+ Uniporter (MCU) protein has recently been identified as a core component responsible for mitochondrial Ca²⁺ uptake. MCU knockout (MCU KO) studies have identified a number of important roles played by this high capacity uptake pathway. Interestingly, this work has also shown that MCU-mediated Ca²⁺ uptake is not essential for vital cell functions such as muscle contraction, energy metabolism and neurotransmission. Although mitochondrial Ca²⁺ uptake was markedly reduced, MCU KO mitochondria still contained low but detectable levels of Ca²⁺. In view of the fundamental importance of Ca²⁺ for basic cell signalling, this finding suggests the existence of other currently unrecognized pathways for Ca²⁺ entry. We review the experimental evidence for the existence of alternative Ca²⁺ influx mechanisms and propose how these mechanisms may play an integral role in mitochondrial Ca²⁺ signalling.     

Mitochondrial Ca<Superscript>2+</Superscript> and regulation of the permeability transition pore

The mitochondrial permeability transition pore was originally described in the 1970’s as a Ca²⁺ activated pore and has since been attributed to the pathogenesis of many diseases. Here we evaluate how each of the current models of the pore complex fit to what is known about how Ca²⁺ regulates the pore, and any insight that provides into the molecular identity of the pore complex. We also discuss the central role of Ca²⁺ in modulating the pore’s open probability by directly regulating processes, such as ATP/ADP balance through the tricarboxylic acid cycle, electron transport chain, and mitochondrial membrane potential. We review how Ca²⁺ influences second messengers such as reactive oxygen/nitrogen species production and polyphosphate formation. We discuss the evidence for how Ca²⁺ regulates post-translational modification of cyclophilin D including phosphorylation by glycogen synthase kinase 3 beta, deacetylation by sirtuins, and oxidation/ nitrosylation of key residues. Lastly we introduce a novel view into how Ca²⁺ activated proteolysis through calpains in the mitochondria may be a driver of sustained pore opening during pathologies such as ischemia reperfusion injury.     

Protein phosphorylation in plant mitochondria

Protein kinase activity was detected in osmotically lysed mitochondria isolated from etiolated seedlings of corn, pea, soybean, and wheat, as well as from potato tubers. Ther kinase(s) phosphorylated both endogenous polypeptides and exogenous, nonmitochondrial proteins when supplied with ATP and Mg²⁺. Eight to fifteen endogenous mitochondrial polypeptides were phosphorylated. The major mitochondrial polypeptide labeled in all species migrated during denaturing electrophoresis with an apparent monomeric molecular weight of 47,000. Incorporation of phosphate into endogenous proteins appeared to be biphasic, being most rapid during the first 1 to 2 minutes but slower thereafter. The kinase activity was greatest at neutral and alkaline pH values and utilized ATP with a K_m of approximately 200 micromolar. The kinase was markedly inhibited by CaCl₂ but was essentially unaffected by NaF, calmodulin, oligomycin, or cAMP. These data suggest that plant mitochondrial protein phosphorylation may be similar to protein phosphorylation in animal mitochondria.     

Phosphorylation of triton X-100 soluble and insoluble protein substrates in a demembranated/reactivated human sperm model

Sperm motility is a process which involves a cascade of events mediated by cAMP and Ca²⁺, cAMP in the initiation of flagellar movement, and Ca²⁺ in the regulation of beat asymmetry, and it has been suggested that these two messengers act through phosphorylation/dephosphorylation of axonemal proteins. Only a few studies on human sperm protein phosphorylation have been reported and no relation of this process with motility or other function has been established. In the present study, phosphorylation of human sperm proteins was performed using detergent-demembranated spermatozoa, in which motility is reactivated by the addition of ATP. This system allows direct accessibility of intracellular kinases to [³²P]-γATP and allows some relation between protein phosphorylation and flagellar movements. After electrophoresis and autoradiography, numerous phosphoproteins were detected. Phosphorylation of 2 proteins (36 and 51 kDa) was stimulated by cAMP in a concentration-dependent manner, and this increase was prevented by inhibitors of cAMP-dependent protein kinase. In order to characterize phosphoproteins originating from the cytoskeleton or axoneme, detergent extracted spermatozoa were also subjected to phosphorylation. Three major phosphorylated proteins (14.8, 15.3, and 16.2 kDa) were detected, the first two expressing cAMP-dependency according to their cAMP concentration-dependent increase in phosphorylation and the reversal of this effect by inhibitors of cAMP-dependent protein kinase. Proteins phosphorylation during the reactivation of demembranated spermatozoa previously immobilized H₂O₂, xanthine + xanthine oxidase-generated reactive oxygen species, or the oxidative phosphorylation uncoupler rotenone, revealed increases in cAMP-independent phosphorylation of proteins of 16.2, 46, and 93 kDa. These results documenting human sperm phosphoproteins form a base for further studies on the role of protein phosphorylation in sperm functions. © 1996 Wiley-Liss, Inc.     

Effect of peripheral benzodiazepine receptor (PBR/TSPO) ligands on opening of Ca<Superscript>2+</Superscript>-induced pore and phosphorylation of 3.5-kDa polypeptide in rat brain mitochondria

The effect of nanomolar concentrations of PBR/TSPO ligands—Ro 5-4864, PK11195, and PPIX—on Ca²⁺-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria was investigated. PBR/TSPO agonist Ro 5-4864 (100 nM) and endogenous ligand PPIX (1 μM) were shown to stimulate PTP opening, while antagonist PK11195 (100 nM) suppressed this process. Correlation between PBR ligand action on PTP opening and phosphorylation of a 3.5 kDa polypeptide was investigated. In intact brain mitochondria, incorporation of [γ-³²P]ATP into 3.5 kDa peptide was decreased in the presence of Ro 5-4864 and PPIX and increased in the presence of PK11195. At threshold Ca²⁺ concentrations leading to PTP opening, PBR/TSPO ligands were found to stimulate dephosphorylation of the 3.5 kDa peptide. Specific anti-PBR/TSPO antibody prevented both PTP opening and dephosphorylation of the 3.5-kDa peptide. The peptide was identified as subunit c of F_oF₁-ATPase by Western blot using specific anti-subunit c antibody. The results suggest that subunit c of F_oF₁-ATPase could be an additional target for PBR/TSPO ligands action, is subjected to Ca²⁺- and TSPO-dependent phosphorylation/dephosphorylation, and is involved in PTP operation in mitochondria.     

Life after the birth of the mitochondrial Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger,NCLX

Powered by the mitochondrial membrane potential, Ca²⁺ permeates the mitochondria via a Ca²⁺ channel termed Ca²⁺ uniporter and is pumped out by a Na⁺/Ca²⁺ exchanger, both of which are located on the inner mitochondrial membrane. Mitochondrial Ca²⁺ transients are critical for metabolic activity and regulating global Ca²⁺ responses. On the other hand, failure to control mitochondrial Ca²⁺ is a hallmark of ischemic and neurodegenerative diseases. Despite their importance, identifying the uniporter and exchanger remains elusive and their inhibitors are non-specific. This review will focus on the mitochondrial exchanger, initially describing how it was molecularly identified and linked to a novel member of the Na⁺/Ca²⁺ exchanger superfamily termed NCLX. Molecular control of NCLX expression provides a selective tool to determine its physiological role in a variety of cell types. In lymphocytes, NCLX is essential for refilling the endoplasmic reticulum Ca²⁺ stores required for antigendependent signaling. Communication of NCLX with the store-operated channel in astroglia controls Ca²⁺ influx and thereby neuro-transmitter release and cell proliferation. The refilling of the Ca²⁺ stores in the sarcoplasmic reticulum, which is controlled by NCLX, determines the frequency of action potential and Ca²⁺ transients in cardiomyocytes. NCLX is emerging as a hub for integrating glucose-dependent Na⁺ and Ca²⁺ signaling in pancreatic β cells, and the specific molecular control of NCLX expression resolved the controversy regarding its role in neurons and β cells. Future studies on an NCLX knockdown mouse model and identification of human NCLX mutations are expected to determine the role of mitochondrial Ca²⁺ efflux in organ activity and whether NCLX inactivation is linked to ischemic and/or neurodegenerative syndromes. Structure-function analysis and protein analysis will identify the NCLX mode of regulation and its partners in the inner membrane of the mitochondria.     

Role of yessotoxin in calcium and cAMP‐crosstalks in primary and K‐562 human lymphocytes: The effect is mediated by Anchor kinase a mitochondrial proteins

Yessotoxin (YTX) is a marine polyether toxin previously described as a phosphodiesterase (PDE) activator in fresh human lymphocytes. This toxin induces a decrease of adenosine 3′,5′‐cyclic monophosphate (cAMP) levels in fresh human lymphocytes in a medium with calcium (Ca²⁺), whereas the contrary effect has been observed in a Ca²⁺‐free medium. In the present article, the effect of YTX in K‐562 lymphocytes cell line has been analysed. Surprisingly, results obtained in K‐562 cell line are completely opposite than in fresh human lymphocytes, since in K‐562 cells YTX induces an increase of cAMP levels. YTX cytotoxicity was also studied in both K‐562 cell line and fresh human lymphocytes. Results demonstrate that YTX does not modify fresh human lymphocytes viability, whereas in K‐562 cells, YTX has a highly cytotoxic effect. It has been described in a previous study that YTX induces a small cytosolic Ca²⁺ increase in fresh human lymphocytes but no effect was observed on Ca²⁺ pools depletion in these cells. However, our results show that, in K‐562 cells, YTX has no effect on cytosolic Ca²⁺ levels in a medium with Ca²⁺ and induces an increase on Ca²⁺ pools depletion followed by a Ca²⁺ influx. As far as Ca²⁺ modulation is concerned these results demonstrate that YTX has a clear opposite effect in tumoural and fresh human lymphocytes. In addition, intracellular Ca²⁺ reservoirs affected by YTX are different than thapsigargin‐sensible pools. Furthermore, YTX‐dependent Ca²⁺ pools depletion was abolished by cAMP analogue (dibutyryl cAMP), phosphodiesterase‐4 (PDE4) inhibitor (rolipram), protein kinase A inhibitor (H89) and oxidative phosphorylation uncoupler carbonyl cyanide p‐(trifluoromethoxy) (FCCP) treatments. This evidences the crosstalks between Ca²⁺, YTX and cAMP pathways. Also, results obtain demonstrate that YTX‐dependent Ca²⁺ influx was only abolished by FCCP pre‐treatment, which indicates a link between YTX and mitochondria in K‐562 cell line. Cytosolic expression of A‐kinase anchor proteins (AKAPs), the proteins which integrates phosphodiesterases (PDEs) and PKA to the mitochondria, was determined in both cell models. On the one hand, in human fresh lymphocytes, YTX increases AKAP149 cytosolic expression. This fact is accompanied with a decrease in cAMP levels, and therefore PDEs activation, which finally leads to cell survival. On the other hand, in tumoural lymphocytes, YTX has an opposite effect since decreases AKAP149 cytosolic expression and increase cAMP levels which leads to cell death. This is the first time that YTX and mitochondrial AKAPs proteins relationship is characterised. J. Cell. Biochem. 113: 3752–3761, 2012. © 2012 Wiley Periodicals, Inc.     

Minocycline chelates Ca<Superscript>2+</Superscript>, binds to membranes,and depolarizes mitochondria by formation of Ca<Superscript>2+</Superscript>-dependent ion channels

Minocycline (an anti-inflammatory drug approved by the FDA) has been reported to be effective in mouse models of amyotrophic lateral sclerosis and Huntington disease. It has been suggested that the beneficial effects of minocycline are related to its ability to influence mitochondrial functioning. We tested the hypothesis that minocycline directly inhibits the Ca²⁺-induced permeability transition in rat liver mitochondria. Our data show that minocycline does not directly inhibit the mitochondrial permeability transition. However, minocycline has multiple effects on mitochondrial functioning. First, this drug chelates Ca²⁺ ions. Secondly, minocycline, in a Ca²⁺-dependent manner, binds to mitochondrial membranes. Thirdly, minocycline decreases the proton-motive force by forming ion channels in the inner mitochondrial membrane. Channel formation was confirmed with two bilayer lipid membrane models. We show that minocycline, in the presence of Ca²⁺, induces selective permeability for small ions. We suggest that the beneficial action of minocycline is related to the Ca²⁺-dependent partial uncoupling of mitochondria, which indirectly prevents induction of the mitochondrial permeability transition.     

Regulation of mitochondrial Ca<Superscript>2+</Superscript> and its effects on energetics and redox balance in normal and failing heart

Ca²⁺ has been well accepted as a signal that coordinates changes in cytosolic workload with mitochondrial energy metabolism in cardiomyocytes. During increased work, Ca²⁺ is accumulated in mitochondria and stimulates ATP production to match energy supply and demand. The kinetics of mitochondrial Ca²⁺ ([Ca²⁺]_m) uptake remains unclear, and we review the debate on this subject in this article. [Ca²⁺]_m has multiple targets in oxidative phosphorylation including the F1/FO ATPase, the adenine nucleotide translocase, and Ca²⁺-sensitive dehydrogenases (CaDH) of the tricarboxylic acid (TCA) cycle. The well established effect of [Ca²⁺]_m is to activate CaDHs of the TCA cycle to increase NADH production. Maintaining NADH level is not only critical to keep a high oxidative phosphorylation rate during increased cardiac work, but is also necessary for the reducing system of the cell to maintain its reactive oxygen species (ROS) —scavenging capacity. Further, we review recent data demonstrating the deleterious effects of elevated Na⁺ in cardiac pathology by blunting [Ca²⁺]_m accumulation.     

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.