Inhibition of oxidative phosphorylation by a Ca2+-induced diminution of the adenine nucleotide translocator

The mechanism through which internal Ca²⁺ inhibits oxidative phosphorylation of rat heart mitochondria has been explored. In parallel to a Ca²⁺-induced diminution of the activity of the adenine nucleotide translocator, an efflux of internal adenine nucleotides is observed. The efflux of adenine nucleotides depends on the amount of Ca²⁺ accumulated by the mitochondria and on the time that Ca²⁺ remains in the mitochondria; this efflux is atractyloside insensitive. These results suggest that internal Ca²⁺, by inducing a lowering of the internal concentration of adenine nucleotides, diminishes the rate of exchange of adenine nucleotides via the translocase, and in consequence of oxidative phosphorylation. Under conditions in which the Ca²⁺-induced release of adenine nucleotides takes place, no gross changes of the permeability properties of the membrane are observed. As revealed by studies with arsenate, respiratory activity and the function of the ATPase in the direction of ATP synthesis are not affected by internal Ca²⁺.     

Modulation of matrix Ca2+ content by the ADP/ATP carrier in brown adipose tissue mitochondria. Influence of membrane lipid composition

The role of the adenine nucleotide translocase on Ca²⁺ homeostasis in mitochondria from brown adipose tissue was examined. It was found that in mitochondria incubated with 50 M Ca²⁺, ADP was not needed to retain the cation, but it was required for strengthening the inhibitory effect of cyclosporin on membrane permeability transition as induced by menadione. In addition, carboxyatractyloside was unable to promote matrix Ca²⁺ release, even though it inhibits the ADP exchange reaction. However, when the Ca²⁺ concentration was increased to 150 M, carboxyatractyloside did induce Ca²⁺ release, and ADP favored Ca²⁺ retention. Determination of cardiolipin content in the inner membrane vesicles showed a greater concentration in brown adipose tissue mitochondria than that found in kidney mitochondria. It is suggested that the failure of the adenine nucleotide translocase to influence membrane permeability transition depends on the lipid composition of the inner membrane.     

Calcium Induces Mitochondrial Oxidative Stress Because of its Binding to Adenine Nucleotide Translocase

Several studies have demonstrated that the mitochondrial membrane switches from selective to non-selective permeability because of its improved matrix Ca²⁺ accumulation and oxidative stress. This process, known as permeability transition, evokes severe dysfunction in mitochondria through the opening of a non-specific pore, whose chemical nature is still under discussion. There are some proposals regarding the components of the pore structure, e.g., the adenine nucleotide translocase and dimers of the F1 Fo-ATP synthase. Our results reveal that Ca²⁺ induces oxidative stress, which not only increases lipid peroxidation and ROS generation but also brings about both the collapse of the transmembrane potential and the membrane release of cytochrome c. Additionally, it is shown that Ca²⁺ increases the binding of the probe eosin-5-maleimide to adenine nucleotide translocase. Interestingly, these effects are diminished after the addition of ADP. It is suggested that pore opening is caused by the binding of Ca²⁺ to the adenine nucleotide translocase.     

Control of the Mitochondrial Permeability Transition Pore by High-Affinity ADP Binding at the ADP/ATP Translocase in Permeabilized Mitochondria

Low levels of ADP binding at the ADP/ATP translocase caused inhibition of the Ca²⁺-inducedpermeability transition of the mitochondrial inner membrane, when measured using the shrinkage assay on mitochondria, which have already undergone a transition. Inhibition was preventedby carboxyatractyloside, but potentiated by bongkrekic acid, which increased the affinity forinhibition by ADP. This suggests that inhibition was related to the conformation of thetranslocase. Ca²⁺ addition was calculated to remove most of the free ADP. Ca²⁺ added after ADPinduced a slow decay of the inhibition, which probably reflected the dissociation of ADP fromthe translocator. We conclude that the probability of forming a permeability transition pore(PTP) is much greater when the translocase is in the CAT conformation than in the BKAconformation, and, in the absence of CAT and BKA, the translocator is shifted between theBKA and CAT conformations by ADP binding and removal, even in deenergized mitochondria with no nucleotide gradients.     

Effects of adenine nucleotide translocase inhibitors on dinitrophenol-induced Ca2+ efflux from pig heart mitochondria

Bongkrekic acid and atractyloside, inhibitors of adenine nucleotide translocase, do not inhibit Ca²⁺ uptake and H⁺ production by pig heart mitochondria. However, bongkrekic acid, but not atractyloside, inhibits dinitrophenol-induced Ca²⁺ efflux and H⁺ uptake. Conversely, ruthenium red blocks Ca²⁺ uptake and H⁺ production but does not prevent dinitrophenol-induced Ca²⁺ efflux and H⁺ uptake by mitochondria. These results suggest that mitochondrial Ca²⁺ uptake and release exist as two independent pathways. The efflux of Ca²⁺ from mitochondria is mediated by a bongkrekic acid sensitive component which is apparently not identical to the ruthenium red sensitive Ca²⁺ uptake carrier.     

Distinct characteristics of Ca-induced depolarization of isolated brain and liver mitochondria

Ca²⁺-induced mitochondrial depolarization was studied in single isolated rat brain and liver mitochondria. Digital imaging techniques and rhodamine 123 were used for mitochondrial membrane potential measurements. Low Ca²⁺ concentrations (about 30-100 nM) initiated oscillations of the membrane potential followed by complete depolarization in brain mitochondria. In contrast, liver mitochondria were less sensitive to Ca²⁺; 20 μM Ca²⁺ was required to depolarize liver mitochondria. Ca²⁺ did not initiate oscillatory depolarizations in liver mitochondria, where each individual mitochondrion depolarized abruptly and irreversibly. Adenine nucleotides dramatically reduced the oscillatory depolarization in brain mitochondria and delayed the onset of the depolarization in liver mitochondria. In both type of mitochondria, the stabilizing effect of adenine nucleotides completely abolished by an inhibition of adenine nucleotide translocator function with carboxyatractyloside, but was not sensitive to bongkrekic acid. Inhibitors of mitochondrial permeability transition cyclosporine A and bongkrekic acid also delayed Ca²⁺-depolarization. We hypothesize that the oscillatory depolarization in brain mitochondria is associated with the transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore, whereas the non-oscillatory depolarization in liver mitochondria is caused by the irreversible opening of the pore.     

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.     

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 μM, GE prevents oxidative stress and MPT induced by supraphysiological Ca²⁺ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe-S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca²⁺. GE also inhibits Ca²⁺ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca²⁺ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe-S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.     

Stable enhancement of calcium retention in mitochondria isolated from rat liver after the administration of glucagon to the intact animal

1. Mitochondria isolated from rat liver by centrifugation of the homogenate in buffered iso-osmotic sucrose at between 4000 and 8000g-min, 1h after the administration in vivo of 30μg of glucagon/100g body wt., retain Ca²⁺ for over 45min after its addition at 100nmol/mg of mitochondrial protein in the presence of 2mm-P_i. In similar experiments, but after the administration of saline (0.9% NaCl) in place of glucagon, Ca²⁺ is retained for 6–8min. The ability of glucagon to enhance Ca²⁺ retention is completely prevented by co-administration of 4.2mg of puromycin/100g body wt. 2. The resting rate of respiration after Ca²⁺ accumulation by mitochondria from glucagon-treated rats remains low by contrast with that from saline-treated rats. Respiration in the latter mitochondria increased markedly after the Ca²⁺ accumulation, reflecting the uncoupling action of the ion. 3. Concomitant with the enhanced retention of Ca²⁺ and low rates of resting respiration by mitochondria from glucagon-treated rats was an increased ability to retain endogenous adenine nucleotides. 4. An investigation of properties of mitochondria known to influence Ca²⁺ transport revealed a significantly higher concentration of adenine nucleotides but not of P_i in those from glucagon-treated rats. The membrane potential remained unchanged, but the transmembrane pH gradient increased by approx. 10mV, indicating increased alkalinity of the matrix space. 5. Depletion of endogenous adenine nucleotides by P_i treatment in mitochondria from both glucagon-treated and saline-treated rats led to a marked diminution in ability to retain Ca²⁺. The activity of the adenine nucleotide translocase was unaffected by glucagon treatment of rats in vivo. 6. Although the data are consistent with the argument that the Ca²⁺-translocation cycle in rat liver mitochondria is a target for glucagon action in vivo, they do not permit conclusions to be drawn about the molecular mechanisms involved in the glucagon-induced alteration to this cycle.     

Adenine nucleotide translocation in plant mitochondria

The rapid translocation of external ADP-[^14C]by corn mitochondria is inhibited by high concentrations of atractyloside with enhanced inhibition occurring in the presence of Mg²⁺. This translocation is also inhibited by AMP or ATP but CDP, GDP, IDP or UDP have little effect. Backward exchange of internal ADP-[¹⁴C] occurs in the presence of AMP, ADP or ATP but is not promoted by other nucleoside diphosphates. It is suggested that the adenine nucleotide (AdN) carrier is specific for ADP and ATP and that apparent translocation of AMP is a result of adenylate kinase activity. The translocated ADP can be separated into 3 components: (1) atractyloside-insensitive binding; (2) carrier-bound ADP saturated at ca 30 μM external ADP; and (3) exchanged ADP saturated as ca 5 μM external ADP. It is suggested that the adenine nucleotide carrier of plant mitochondria possesses similar properties to the classical carrier of vertebrate mitochondria.     

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.     

Adenine nucleotide pool size,adenine nucleotide translocase activity,and respiratory activity in newborn rabbit liver mitochondria

The adenine nucleotide content (ATP+ADP+AMP) of newborn rabbit liver mitochondria was 6.0±0.5 nmol/mg mitochondrial protein at birth, increased rapidly to 14.5±1.7 nmol/mg protein by 2 h postnatal, peaked at 6 h, then decreased gradually to 7.8±0.6 nmol/mg protein by 4 days postnatal. There was a strong positive correlation (r=0.82) between the total adenine nucleotide pool size and adenine nucleotide translocase activity in these mitochondria. In contrast, glutamate + malate-supported State 3 respiratory rates remained constant from birth through the first week of life. State 4 rates also remained constant, as did the respiratory control index and uncoupled respiratory rates. The following conclusions are suggested: (1) The maximum rate of translocase activity is limited by the intramitochondrial adenine nucleotide pool size. (2) In newborn rabbit liver mitochondria, the State 3 respiratory rate is not limited by either the adenine pool size or the maximum capacity for translocase-mediated adenine exchange. (3) In contrast to rat, rabbit liver mitochondria are fully functional at birth with regard to respiratory rates and oxidative phosphorylation. (4) The rapid postnatal accumulation of adenine nucleotides by liver mitochondria, now documented in two species, may be a general characteristic of normal metabolic adjustment in neonatal mammals.     

Adenine nucleotide transport in hepatoma mitochondria. Characterization of factors influencing the kinetics of ADP and ATP uptake

Initial velocity measurements of [³H]ADP and [³H]ATP uptake have been made with mitochondria isolated from Morris hepatomas of differing growth rates, and factors known to influence the rates of nucleotide exchange have been examined in an effort to determine whether the elevated rates of aerobic glycolysis in these tumors can be attributed to altered carrier activity. These studies included the determination of the apparent kinetic constants for nucleotide uptake as a function of the mitochondrial energy state and the dependence of transport rates on temperature. Also included in these studies were measurements of the mitochondrial levels of endogenous inhibitors, divalent cations and internal adenine nucleotides. Results obtained showed that with mitochondria isolated from the various tumor lines, the apparent kinetic constants for nucleotide uptake are different from those of control rat or regenerating liver mitochondria; the apparent V_max values for both ADP and ATP uptake are significantly lower. Furthermore, under conditions of a high-energy state, the K_m and V_max values for ATP uptake are greater than the K_m and V_max value for ADP uptake but that under uncoupled conditions, the opposite is observed. Comparison of the levels of mitochondrial Ca²⁺, Mg²⁺, long-chain acyl-CoA ester and adenine nucleotide from the various mitochondria showed that important differences exist between liver and hepatoma mitochondria in the levels of Ca²⁺, long-chain acyl-CoA ester and AMP. Mitochondrial Ca²⁺ levels are elevated 3–5-fold in all tumor lines, and for Morris 7777 hepatoma (a rapidly growing tumor) by a remarkable 70-fold; whereas the levels of acyl-CoA ester and AMP are significantly lower in the more rapidly growing tumors. Arrhenius plots for nucleotide uptake in mitochondria from liver and hepatoma are characterized as being biphasic, having similar activation energies above and below the break point temperature (28–38 and 6–16 kcal/mol, respectively). However, the transition temperature for mitochondria from the various hepatomas is uniformly 4–5°C lower than mitochondria from control liver. The latter difference may reflect a variation in membrane composition, most probably lipid components. It is concluded that the presence of elevated levels of Ca²⁺ and lower levels of AMP in hepatoma mitochondria and difference of membrane compositions may play an important role in limiting adenine nucleotide transport activity in vivo and that the impaired carrier activity may contribute to higher rates of aerobic glycolysis observed in these tumors.     

Inhibition by Sr2+ of specific mitochondrial Ca2+-efflux pathways

The effect of Sr²⁺ on the set point for external Ca²⁺ was studied in rat heart and liver mitochondria with the aid of a Ca²⁺-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca²⁺ influx on the Ca²⁺ uniporter and efflux by various mechanisms. We studied the Ca²⁺-Na⁺ exchange pathway in heart mitochondria and the Δψ-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr²⁺ was found to shift the set points towards lower external Ca²⁺ both in heart mitochondria under conditions of Ca²⁺-Na⁺ exchange and in liver mitochondria under conditions that should promote opening of the Δψ-modulated pathway. The effect on the set point was found to be due to inhibition of Ca²⁺ efflux by Sr²⁺ taken up by the mitochondria, while Sr²⁺ efflux was too slow to be measurable.     

Relationship between the systems of activation of fatty acids and ademine nucleotide translocase in the mitochondria]

Experiments were conducted on freshly isolated rat liver mitochondria and mitochondria subjected to ageing by two different methods. It was shown that the work of the mitochondrial system of fatty acid activation could lead to inhibition of the adenine nucleotide transport through the internal mitochondrial membrane. Inhibition of adenine nucleotide translocase was eliminated by preincubation of mitochondria with carnitine. The presence in the mitochondrial preparations of fatty acids in the concentration adequate for induction of inhibition of addition of CoA and ATP served as a preculiarity of adenine nucleotide translocase inhibition of the ageing mitochondria. The data obtained permitted to make a supposition on the participation of acyl-CoA formed by the mitochondrial acyl-CoA-synthetase in the regulation of adenine nucleotide transport into the mitochondria.     

Effects of hypothermia on canine kidney mitochondria

The effect of hypothermia on the function of isolated dog kidney cortex mitochondria was determined with an FAD- and NAD⁺-linked substrate. In dog kidney mitochondria, temperatures of 10 °C or less suppress ADP stimulation of respiration but have little or no effect upon uncoupler, Ca²⁺ or valinomycin-K⁺ stimulation of respiration. This suggests that the adenine nucleotide translocase which catalyses the transport of ADP into the mitochondria limits the rate of respiration and generation of ATP at 10 °C in kidneys undergoing preservation. The coupling of oxidation to phosphylation, as determined by measuring the amount of ATP formed at low temperatures, indicates, however, that mitochondria are fully coupled at both 10 and 5 °C. The respiratory control index at 15 °C is greater (with pyruvate plus malate) than at 30 or 10 °C and suggests that 15 °C may be the optimum perfusion temperature for maintaining adenine nucleotide levels in the perfused kidney.     

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.     

The interaction of Ga2+ with mitochondria from human myometrium.

Mitochondria prepared from human myometrium contain large amounts of endogenous Ca²⁺ (up to 200 nmol/mg of protein) even if isolated in media containing ethylene glycol-bis(β-aminoethylether)-N,N′-tetraacetic acid. The endogenous Ca⁺², however, is not irreversibly sequestered, since it can be rapidly and quantitatively discharged by uncouplers. Human myometrial mitochondria are capable of efficient energy-linked Ca²⁺ transport. In the absence of phosphate, the amount of Ca²⁺ accumulated is reduced to insignificant levels. Mg²⁺ has a strong inhibitory effect, which has been exploited to develop an inhibitor-stop method which has permitted the determination of the affinity of myometrial mitochondria for Ca²⁺ (K_m, ~5 μM) and of the maximal velocity of uptake (0.55 nmol/mg of protein/s). The respiration of human myometrial mitochondria is stimulated by Ca²⁺, with respiratory control indexes of the order of 4–5. In contrast, ADP induces an insignificant stimulation, or no stimulation at all. The response of respiration to ADP is somewhat improved if mitochondria are preincubated under conditions which decrease their endogenous Ca²⁺ content. The adenine nucleotide exchange in human myometrial mitochondria is deficient with respect to liver mitochondria.     

A kinetic assay of mitochondrial ADP-ATP exchange rate in permeabilized cells

We previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg²⁺] reported by an Mg²⁺-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg²⁺. In the current article, we describe a modification of this method suited for following ADP-ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity, and myosin ATPase activity. Here we report that the addition of BeF₃⁻ and sodium orthovanadate (Na₃VO₄) to medium containing digitonin-permeabilized cells inhibits all ADP-ATP-using reactions except the adenine nucleotide translocase (ANT)-mediated mitochondrial ADP-ATP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F₁F_o-ATPase due to its sensitivity to BeF₃⁻ and Na₃VO₄. With this assay, ADP-ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler and expressed as a function of citrate synthase activity per total amount of protein.     

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.