Interaction of Sr2+ with Ca2+-induced Ca2+ release in mitochondria

Respiring rat liver mitochondria are known to spontaneously release the Ca²⁺ taken up when they have accumulated Ca²⁺ over a certain threshold, while Sr²⁺ and Mn²⁺ are well tolerated and retained. We have studied the interaction of Sr²⁺ with Ca²⁺ release. When Sr²⁺ was added to respiring mitochondria simultaneously with or soon after the addition of Ca²⁺, the release was potently inhibited or reversed. On the other hand, when Sr²⁺ was added before Ca²⁺, the release was stimulated. Ca²⁺-induced mitochondrial damage and release of accumulated Ca²⁺ is generally believed to be due to activation of mitochondrial phospholipase A (EC 3.1.1.4.) by Ca²⁺. However, isolated mitochondrial phospholipase A activity was little if at all inhibited by Sr²⁺. The Ca²⁺ -release may thus be triggered by some Ca²⁺ -dependent function other than phospholipase.     

Mn2+ prevents the Ca2+-induced inhibition of ATP synthesis in brain mitochondria

The differential scanning microcalorimetry and fluorescence methods, using probes ANS and pyrene, have been employed to study thermotropic behaviour of rat liver microsomes in the presence and absence of Mg2+. Addition of Mg2+ yields three partially reversible phase transitions at 18, 27 and 32 degrees C, respectively. A character of Mg2+-induced rearrangements in a membrane and their relation to a catalytic function of a cytochrome P-450-dependent enzymatic system is discussed.     

Physiological Ca2+ level and Ca2+-induced Permeability Transition Pore control protein phosphorylation in rat brain mitochondria

Phosphorylation of several low molecular mass proteins (3.5, 17, 23 and 29kDa) was observed in rat brain mitochondria (RBM) at ATP concentration close to that in the mitochondrial matrix. Furthermore, regulatory effects of Ca2+ on phosphorylation of these proteins were investigated. Protein phosphorylation was found to be modulated by Ca2+ in the physiological concentration range (10(-8) to 10(-6)M free Ca2+). Incorporation of 32P from [gamma-32P]ATP into the 17kDa protein was dramatically increased within the 10(-7) to 10(-6)M free Ca2+ range, whereas an opposite effect was observed for the 3.5kDa polypeptide. Strong de-phosphorylation of the 3.5kDa polypeptide and enhanced 32P-incorporation into the 17 and 23kDa proteins were found with supra-threshold Ca2+ loads and these effects were eliminated or reduced in the presence of cyclosporin A, an inhibitor of Permeability Transition Pore (PTP) opening. In the presence of calmidazolium (Cmz), a calmodulin antagonist, enhanced levels of phosphorylation of the 17 and 3.5kDa polypeptides were observed and the 17kDa protein phosphorylation was suppressed by H-8, a protein kinase A inhibitor. It is concluded that Ca2+ in physiological concentrations, as a second messenger, can control phosphorylation of the low molecular mass phospoproteins in RBM, in addition to well known regulation of some Krebs cycle dehydrogenases by Ca2+. The protein phosphorylation was strongly dependent on the Ca2+-induced PTP opening.     

Kinetic model for Ca2+-induced permeability transition in energized liver mitochondria discriminates between inhibitor mechanisms

Cytotoxicity associated with pathophysiological Ca(2+) overload (e.g. in stroke) appears mediated by an event termed the mitochondrial permeability transition (mPT). We built and solved a kinetic model of the mPT in populations of isolated rat liver mitochondria that quantitatively describes Ca(2+)-induced mPT as a two-step sequence of pre-swelling induction followed by Ca(2+)-driven, positive feedback, autocatalytic propagation. The model was formulated as two differential equations, each directly related to experimental parameters (Ca(2+) flux/mitochondrial swelling). These parameters were simultaneously assessed using a spectroscopic approach to monitor multiple mitochondrial properties. The derived kinetic model correctly identifies a correlation between initial Ca(2+) concentration and delay interval prior to mPT induction. Within the model's framework, Ru-360 (a ruthenium complex) and Mg(2+) were shown to compete with the Ca(2+)-stimulated initiation phase of mPT induction, consistent with known inhibition at the phenomenological level of the Ca(2+) uniporter. The model further reveals that Mg(2+), but not Ru-360, inhibits Ca(2+)-induced effects on a downstream stage of mPT induction at a site distinct from the uniporter. The analytical approach was then applied to promethazine, an FDA-approved drug previously shown to inhibit both mPT and ischemia-reperfusion injury. Kinetic analysis revealed that promethazine delayed mPT induction in a manner qualitatively distinct from that of lower concentrations of Mg(2+). In summary, we have developed a kinetic model to aid in the quantitative characterization of mPT induction. This model is consistent with/informative about the biochemistry of several mPT inhibitors, and its success suggests that this kinetic approach can aid in the classification of agents or targets that modulate mPT induction.     

Doxorubicin increases the susceptibility of brain mitochondria to Ca(2+)-induced permeability transition and oxidative damage

This study was aimed at investigating the effects of subchronic administration of doxorubicin (DOX) on brain mitochondrial bioenergetics and oxidative status. Rats were treated with seven weekly injections of vehicle (sc, saline solution) or DOX (sc, 2 mg kg(-1)), and 1 week after the last administration of the drug the animals were sacrificed and brain mitochondrial fractions were obtained. Several parameters were analyzed: respiratory chain, phosphorylation system, induction of the permeability transition pore (PTP), mitochondrial aconitase activity, lipid peroxidation markers, and nonenzymatic antioxidant defenses. DOX treatment induced an increase in thiobarbituric acid-reactive substances and vitamin E levels and a decrease in reduced glutathione content and aconitase activity. Furthermore, DOX potentiated PTP induced by Ca2+. No statistical differences were observed in the other parameters analyzed. Altogether our results show that DOX treatment increases the susceptibility of brain mitochondria to Ca(2+)-induced PTP opening and oxidative stress, predisposing brain cells to degeneration and death.     

Exercise training induces respiratory substrate-specific decrease in Ca2+-induced permeability transition pore opening in heart mitochondria

The purpose of this study was to determine whether regular exercise (treadmill running, 10 wk) alters the susceptibility of rat isolated heart mitochondria to Ca(2+)-induced permeability transition pore (PTP) opening and whether this could be associated with changes in the modulation of PTP opening by selected physiological effectors. Basal leak-driven and ADP-stimulated respiration in the presence of substrates for complex I, II, and IV were not affected by training. Fluorimetric studies revealed that in the control and exercise-trained groups, the amount of Ca(2+) required to trigger PTP opening was greater in the presence of complex II vs. I substrates (230 +/- 12 vs. 134 +/- 7 nmol Ca(2+)/mg protein, P < 0.01; pooled average of control and trained groups). In addition, with a substrate feeding the complex II, training increased by 45% (P < 0.01) the amount of Ca(2+) required to trigger PTP opening both in the presence and absence of the PTP inhibitor cyclosporin A. However, membrane potential, reactive oxygen species production, NAD(P)H ratio, and Ca(2+) uptake kinetics were not different in mitochondria from both groups. Together, these results suggest the existence of a substrate-specific regulation of the PTP in heart mitochondria and suggest that regular exercise results in a reduced sensitivity to Ca(2+)-induced PTP opening in presence of complex II substrates.     

Ca2+-induced permeabilization promotes free radical release from rat brain mitochondria with partially inhibited complex I

Mitochondrial complex I dysfunction has been implicated in a number of brain pathologies, putatively owing to an increased rate of reactive oxygen species (ROS) release. However, the mechanisms regulating the ROS burden are poorly understood. In this study we investigated the effect of Ca2+ loads on ROS release from rat brain mitochondria with complex I partially inhibited by rotenone. The addition of 20 nm rotenone to brain mitochondria increased ROS release. Ca2+ (100 microm) alone had no effect on ROS release, but greatly potentiated the effects of rotenone. The effect of Ca2+ was decreased by ruthenium red. Ca2+-challenged mitochondria lose about 88% of their glutathione and 46% of their cytochrome c under these conditions, although this depends only on Ca2+ loading and not complex I inhibition. ADP in combination with oligomycin decreased the loss of glutathione and cytochrome c and free radical generation. Cyclosporin A alone was ineffective in preventing these effects, but augmented the protection provided by ADP and oligomycin. Non-specific permeabilization of mitochondria with alamethicin also increased the ROS signal, but only when combined with partial inhibition of complex I. These results demonstrate that Ca2+ can greatly increase ROS release by brain mitochondria when complex I is impaired.     

The Ca2+-induced membrane transition in mitochondria. III. Transitional Ca2+ release.

The efflux of Ca²⁺ from mitochondria respiring at steady state, and much of uncoupler-induced Ca²⁺ efflux, is shown to be a consequence of the Ca²⁺-induced membrane transition (the Ca²⁺-induced transition is the Ca²⁺-dependent sudden increase in the nonspecific permeability of the mitochondrial inner membrane which occurs spontaneously when mitochondria are incubated under a variety of conditions (D. R. Hunter, R. A. Haworth, and J. H. Southard, 1976, J. Biol. Chem.251, 5069–5077)). Ca²⁺ release from mitochondria respiring at steady state is shown to be transitional by four criteria: (1) Ca²⁺ release is inhibited by Mg²⁺, ADP, and bovine serum albumin (BSA), all inhibitors of the transition; (2) release is selective for Ca²⁺ over Sr²⁺, a selectivity also found for the transition; (3) the time course of Ca²⁺ release is identical to the time course of the change in the mitochondrial population from the aggregated to the orthodox configuration; and (4) from kinetics, Ca²⁺ release from individual mitochondria is shown to occur suddenly, following a lag period during which no release occurs. Ca²⁺ release induced by uncoupler is shown to be mostly by a transitional mechanism, as judged by four criteria: (1) release of Ca²⁺ is ruthenium red-insensitive and is an order of magnitude faster than Sr²⁺ release which is ruthenium red-sensitive; (2) release of Ca²⁺ is strongly inhibited by keeping the mitochondrial NAD⁺ reduced; (3) the kinetics of Ca²⁺ release indicates a transitional release mechanism; and (4) uncoupler addition triggers the aggregated to orthodox configurational transition which, at higher levels of Ca²⁺ uptake, occurs in the whole mitochondrial population at a rate equal to the rate of Ca²⁺ release. Na²⁺-induced Ca²⁺ release was not accompanied by a configurational change; we therefore conclude that it is not mediated by the Ca²⁺-induced transition.     

Copper deficiency inhibits Ca2+-induced swelling in rat cardiac mitochondria

Cu deficiency disrupts the architecture of mitochondria, impairs respiration, and inhibits the activity of cytochrome c oxidase - the terminal, Cu-dependent respiratory complex (Complex IV) of the electron transport chain. This suggests that perturbations in the respiratory chain may contribute to the changes in mitochondrial structure caused by Cu deficiency. This study investigates the effect of Cu deficiency on Ca2+-induced mitochondrial swelling as it relates to changes in respiratory complex activities in cardiac mitochondria of rats. Male weanling rats were fed diets containing either no added Cu (Cu0), 1.5 mg Cu/kg (Cu1.5), 3 mg Cu/kg (Cu3) or 6 mg Cu/kg (Cu6). The rate of Ca2+-induced mitochondrial swelling in the presence of succinate and oligomycin was reduced, and the time to reach maximal swelling was increased only in the rats consuming Cu0 diet. Cytochrome c oxidase activity was reduced 60% and 30% in rats fed Cu0 and Cu1.5, respectively, while NADH:cytochrome c reductase (Complex I+ComplexIII) activity was reduced 30% in rats consuming both Cu0 and Cu1.5. Mitochondrial swelling is representative of mitochondrial permeability transition pore (MPTP) formation and the results suggest that Ca2+-induced MPTP formation occurs in cardiac mitochondria of Cu-deficient rats only when cytochrome c oxidase activity falls below 30% of normal. Decreased respiratory complex activities caused by severe Cu deficiency may inhibit MPTP formation by increasing matrix ADP concentration or promoting oxidative modifications that reduce the sensitivity of the calcium trigger for MPTP formation.     

Acrolein induces oxidative stress in brain mitochondria

Acrolein, a byproduct of lipid peroxidation, has been shown to inflict significant structural and functional damage to isolated guinea pig spinal cord. Reactive oxygen species (ROS) are thought to mediate such detrimental effects. The current study demonstrates that acrolein can directly stimulate mitochondrial oxidative stress. Specifically, exposure of purified brain mitochondria to acrolein resulted in a dose-dependent increase of ROS and decreases in glutathione content and aconitase activity. This effect was not accompanied by significant intramitochondrial calcium influx or mitochondrial permeability transition, but rather by impaired function of the mitochondrial electron transport system. As well, we detected a significant inhibition of mitochondrial adenine nucleotide translocase (ANT) in the presence of acrolein. This inhibition of ANT likely contributes to acrolein-induced ROS elevation since application of atractyloside, a specific ANT inhibitor, induced significant increase of ROS. We hypothesize that inhibition of ANT may mediate, in part, the acrolein-induced ROS increase in mitochondria.     

Resistance to Ca2+-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxidative muscles

This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (Sol) and superficial white gastrocnemius (WG). Threshold for Ca2+-induced mitochondrial Ca2+ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124+/-47 vs. 30.4+/-6.8 pmol Ca2+/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121+/-60 vs. 40+/-10 nmol Ca2+/mg protein in WG and Sol), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative protein modulators (cyclophilin D, adenylate nucleotide translocator-1, and voltage-dependent anion channels) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the Sol. However, endogenous matrix Ca2+ measured in mitochondria isolated under resting baseline conditions was approximately twofold lower in the WG than in the Sol (56+/-4 vs. 111+/-11 nmol/mg protein), which significantly accounted for the resistance of WG. Together, these results reveal fiber type differences in the sensitivity to Ca2+-induced PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca2+ dynamics between fiber types.     

Mitochondrial oxidative stress induced by Ca2+ and monoamines: different behaviour of liver and brain mitochondria in undergoing permeability transition

Mitochondrial permeability transition (MPT) is correlated with the opening of a nonspecific pore, the so-called transition pore, that triggers bidirectional traffic of inorganic solutes and metabolites across the mitochondrial membrane. This phenomenon is caused by supraphysiological Ca(2+) concentrations and by other compounds leading to oxidative stress, while cyclosporin A, ADP, bongkrekic acid, antioxidant agents and naturally occurring polyamines strongly inhibit it. The effects of polyamines, including the diamine agmatine, have been widely studied in several types of mitochondria. The effects of monoamines on MPT have to date, been less well-studied, even if they are involved in a variety of neurological and neuroendocrine processes. This study shows that in rat liver mitochondria (RLM), monoamines such as tyramine, serotonin and dopamine amplify the swelling induced by calcium, and increase the oxidation of thiol groups and the production of hydrogen peroxide, effects that are counteracted by the above-mentioned inhibitors. In rat brain mitochondria (RBM), the monoamines do not amplify calcium-induced swelling, even if they demonstrate increases in the extent of oxidation of thiol groups and hydrogen peroxide production. In these mitochondria, the antioxidants are not at all or scarcely effective in suppressing mitochondrial swelling. In conclusion, we hypothesize that different mechanisms induce the MPT in the two different types of mitochondria evaluated. Calcium and monoamines induce oxidative stress in RLM, which in turn appears to induce and amplify MPT. This process is not apparent in RBM, where MPT seems resistant to oxidative stress.     

The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site.

The permeability of isolated mitochondria which have undergone the Ca²⁺-induced transition can be modulated over a wide range simply by adjusting the concentration of free Ca²⁺ in the medium. The effect varies sigmoidally with respect to Ca²⁺ concentration, with an apparent K_m of 16 μm at pH 7.0. It is concluded that the trigger site (by “trigger site” we mean the site of binding of Ca²⁺ which, when Ca²⁺ is bound, will allow the transition in permeability to occur) is possibly also the site for high-affinity Ca²⁺ uptake. Added ADP, NADH and Mg²⁺ inhibit the Ca²⁺-induced permeability of mitochondria which have undergone the Ca²⁺-induced transition. Mg²⁺ and other ions, including H⁺, act like competitive inhibitors of the Ca²⁺ effect. In the presence of Ca²⁺, both neutral and charged molecules of molecular weight <1000 pass readily through the membrane. This response to Ca²⁺ is interpreted as a gating effect at the internal end of hydrophilic channels which span the inner membrane.     

A possible role for calmodulin in Ca2+-induced swelling of mitochondria

Ca2+-induced mitochondrial swelling was inhibited by a low concentration of calmodulin antagonists. Two affinities of Ca2+ to mitochondrial swelling were observed: high (2-5 microM) and low (more than 100 microM) systems. The high-affinity change was inhibited by micromolar level of trifluoperazine and W-7, but not by W-5. The possible mechanism of this inhibition and the role of CaM in mitochondria are discussed.     

Enhancement of Ca2+-induced Ca2+ release in calpain treated rabbit skinned muscle fibers.

Calpain treatment of rabbit skinned muscle fibers resulted in proteolysis of junctional foot protein or Ca2+ release channel of the sarcoplasmic reticulum. Electrophoretic and immunoblot analyses indicate that calpain cleaves off approximately 130 kDa peptide from the N-terminus. After such treatment, Ca2+ capacity of the sarcoplasmic reticulum remained normal and both Ca2+ and adenine nucleotide dependence of Ca2+-induced Ca2+ release mechanism were retained. However, the Ca2+-activated Ca2+ release rate was increased by two fold after the proteolysis. The results suggest the presence of functional domains in the junctional foot protein, and the N-terminus domain controls the activity of the Ca2+ channel without changing Ca2+ and nucleotide sensitivities.     

The reversible Ca2+-induced permeabilization of rat liver mitochondria.

Rat liver mitochondria became permeabilized to sucrose according to an apparent first-order process after accumulating 35 nmol of Ca2+/mg of protein in the presence of 2.5 mM-Pi, but not in its absence. A fraction (24-32%) of the internal space remains sucrose-inaccessible. The rate constant for permeabilization to sucrose decreases slightly when the pH is decreased from 7.5 to 6.5, whereas the rate of inner-membrane potential (delta psi) dissipation is markedly increased, which indicates that H+ permeation precedes sucrose permeation. Permeabilization does not release mitochondrial proteins. [14C]Sucrose appears to enter permeabilized mitochondria instantaneously. Chelation of Ca2+ with EGTA restores delta psi and entraps sucrose in the matrix space. With 20 mM-sucrose at the instant of resealing, about 21 nmol of sucrose/mg of protein becomes entrapped. The amount of sucrose entrapped is proportional to the degree of permeabilization. Entrapped sucrose is not removed by dilution of the mitochondrial suspension. Resealed mitochondria washed three times retain about 74% of the entrapped sucrose. In the presence of Ruthenium Red and Ca2+ buffers permeabilized mitochondria reseal only partially with free [Ca2+] greater than 3 microM. [14C]Sucrose enters partially resealed mitochondria continuously with time, despite maintenance of delta psi, in accordance with continued interconversion of permeable and impermeable forms. Kinetic analyses of [14C]sucrose entry indicate two Ca2+-sensitive reactions in permeabilization. This conclusion is supported by the biphasic time courses of resealing and repolarization of permeabilized mitochondria and the acute dependence of the rapid repolarization on the free [Ca2+]. A hypothetical model of permeabilization and resealing is suggested and the potential of the procedure for matrix entrapment of substances is discussed.     

Complex contribution of cyclophilin D to Ca2+-induced permeability transition in brain mitochondria, with relation to the bioenergetic state

Cyclophilin D (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca(2+), and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca(2+) caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca(2+) in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca(2+) deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.     

Ca2+-induced accumulation of pyrophosphate in mitochondria during acetate metabolism.

The mechanism of pyrophosphate (PPi) accumulation in rat liver during acetate metabolism was investigated. Perfusion of the liver with acetate in the presence of noradrenaline and glucagon induced marked accumulation of PPi (2 mumol/g of liver, 200 times that of control). In contrast, perfusion with glutamine, which generates PPi only in the cytosol, caused little accumulation of PPi, even in the presence of the two hormones. The site of PPi accumulation was shown to be the mitochondria by the finding that isolated mitochondria from the liver perfused with acetate and the hormones contained 50 nmol of PPi/mg of protein. The addition of an uncoupler to mitochondria with accumulated PPi caused gradual decrease in their PPi content, with concomitant release of a stoichiometric amount of Ca2+. Similar accumulation of PPi was observed when isolated mitochondria were incubated with acetate and Ca2+. These results show that an increase in cytosolic Ca2+ caused by the co-administration of the two hormones induced uptake of the ion into mitochondria, and that PPi accumulated in mitochondria only when it was generated in the organelles with an elevated concentration of Ca2+. High mitochondrial concentrations of Ca2+ are considered to inhibit inorganic pyrophosphatase through the formation of a stable complex, CaPPi-. Mitochondria with accumulated PPi had normal respiratory activities, and their adenine nucleotide concentrations were increased 2-fold rather than being decreased, the increases also being considered to be caused by their high concentration of Ca2+.