Changes in calcium-dependent membrane permeability properties in mitochondria of livers from arthritic rats

The involvement of the mitochondrial permeability transition pore (PTP) in the responses of mitochondria from adjuvant-induced arthritic rats to Ca(2+) addition was investigated. The respiratory activity, the Ca(2+)-induced osmotic swelling and the electrophoretic (45)Ca(2+) uptake were evaluated in the absence and in the presence of cyclosporin A (CsA), a well-known inhibitor of the mitochondrial PTP. The Ca(2+)-induced mitochondrial permeability transition (MPT) process occurred in mitochondria from arthritic rats even in the presence of a low Ca(2+) concentration. Whereas in the normal condition, the Ca(2+)-induced uncoupling of oxidative phosphorylation and osmotic swelling was observed in the presence of 10 or 20 microM Ca(2+) concentration, in the arthritic condition, these events occurred at 1.0 microM concentration. In addition, mitochondria from arthritic rats presented an impaired ability to accumulate (45)Ca(2+). All these effects were completely prevented by the administration of CsA. The results of the present study suggest that the higher sensitivity of mitochondria from arthritic rats to Ca(2+)-induced MPT may be an important factor in the pathogenesis of the arthritis disease.     

Transport and oxidation of choline by liver mitochondria

1. Rapid choline oxidation and the onset of P(i)-induced swelling by liver mitochondria, incubated in a sucrose medium at or above pH7.0, required the addition of both P(i) and an uncoupling agent. Below pH7.0, P(i) alone was required for rapid choline oxidation and swelling. 2. Choline oxidation was inhibited by each of several reagents that also inhibited P(i)-induced swelling under similar conditions of incubation, including EGTA, mersalyl, Mg(2+), the Ca(2+)-ionophore A23187, rotenone and nupercaine. None of these reagents had any significant effect on the rate of choline oxidation by sonicated mitochondria. There was therefore a close correlation between the conditions required for rapid choline oxidation and for P(i)-induced swelling to occur, suggesting that in the absence of mitochondrial swelling the rate of choline oxidation is regulated by the rate of choline transport across the mitochondrial membrane. 3. Respiratory-chain inhibitors, uncoupling agents (at pH6.5) and ionophore A23187 caused a loss of endogenous Ca(2+) from mitochondria, whereas nupercaine and Mg(2+) had no significant effect on the Ca(2+) content. Inhibition of choline oxidation and mitochondrial swelling by ionophore A23187 was reversed by adding Ca(2+), but not by Mg(2+). It is concluded that added P(i) promotes the Ca(2+)-dependent activation of mitochondrial membrane phospholipase activity in respiring mitochondria, causing an increase in the permeability of the mitochondrial inner membrane to choline and therefore enabling rapid choline oxidation to occur. Nupercaine and Mg(2+) appear to block choline oxidation and swelling by inhibiting phospholipase activity. 4. Choline was oxidized slowly by tightly coupled mitochondria largely depleted of their endogenous adenine nucleotides, suggesting that these compounds are not directly concerned in the regulation of choline oxidation. 5. The results are discussed in relation to the possible mechanism of choline transport across the mitochondrial membrane in vivo and the influence of this process on the pathways of choline metabolism in the liver.     

Elevation of mitochondrial calcium by ryanodine-sensitive calcium-induced calcium release

Calcium is an important regulator of mitochondrial function. Since there can be tight coupling between inositol 1,4, 5-trisphosphate-sensitive Ca(2+) release and elevation of mitochondrial calcium concentration, we have investigated whether a similar relationship exists between the release of Ca(2+) from the ryanodine receptor and the elevation of mitochondrial Ca(2+). Perfusion of permeabilized A10 cells with inositol 1,4, 5-trisphosphate resulted in a large transient elevation of mitochondrial Ca(2+) to about 8 microm. The response was inhibited by heparin but not ryanodine. Perfusion of the cells with Ca(2+) buffers in excess of 1 microm leads to large increases in mitochondrial Ca(2+) that are much greater than the perfused Ca(2+). These increases, which average around 10 microm, are enhanced by caffeine and inhibited by ryanodine and depletion of the intracellular stores with either orthovanadate or thapsigargin. We conclude that Ca(2+)-induced Ca(2+) release at the ryanodine receptor generates microdomains of elevated Ca(2+) that are sensed by adjacent mitochondria. In addition to ryanodine-sensitive stores acting as a source of Ca(2+), Ca(2+)-induced Ca(2+) release is required to generate efficient elevation of mitochondrial Ca(2+).     

Copper induces permeability transition through its interaction with the adenine nucleotide translocase

In this work we examined the effect of low concentrations of Cu(2+) on the opening of the mitochondrial non-specific pore. The purpose was addressed to further contribute to the knowledge of the mechanisms that regulate the open/closed cycles of the permeability transition pore. Membrane leakage was established by measuring matrix Ca(2+) efflux and mitochondrial swelling. The experimental results indicate that Cu(2+) at very low concentrations promoted the release of accumulated Ca(2+), as well as mitochondrial swelling, provided 1,10-phenanthroline has been added. Carboxyatractyloside and Cu(2+) exhibited additive effects on these parameters. After Cu(2+) titration of membrane thiols, it might be assumed that the blockage of 5.9nmol of SH/mg protein suffices to open the non-specific pore. Taking into account the reinforcing effect of carboxyatractyloside, the increasing ADP concentrations, and that N-ethylmaleimide inhibited the Cu(2+)-induced Ca(2+) efflux, it is proposed that the target site for Cu(2+) is located in the ADP/ATP carrier.     

Lysosomal involvement in hepatocyte cytotoxicity induced by Cu(2+) but not Cd(2+)

Previously we showed that the redox active Cu(2+) was much more effective than Cd(2+) at inducing reactive oxygen species ("ROS") formation in hepatocytes and furthermore "ROS" scavengers prevented Cu(2+)-induced hepatocyte cytotoxicity (Pourahmad and O'Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu(2+), but not Cd(2+), was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, "ROS" formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu(2+), but not Cd(2+), as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu(2+)-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu(2+)-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu(2+). Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu(2+)-induced hepatocyte cytotoxicity. It is proposed that Cu(2+)-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by "ROS" generated by lysosomal Cu(2+) redox cycling.     

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.     

Lead and calcium produce rod photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore

Calcium overload is suggested to play a fundamental role in the process of rod apoptosis in chemical-induced and inherited retinal degenerations. However, this hypothesis has not been tested directly. We developed an in vitro model utilizing isolated rat retinas to determine the mechanisms underlying Ca(2+)- and/or Pb(2+)-induced retinal degeneration. Confocal microscopy, histological, and biochemical studies established that the elevated [Ca(2+)] and/or [Pb(2+)] were localized to photoreceptors and produced rod-selective apoptosis. Ca(2+) and/or Pb(2+) induced mitochondrial depolarization, swelling, and cytochrome c release. Subsequently caspase-9 and caspase-3 were sequentially activated. Caspase-7 and caspase-8 were not activated. The effects of Ca(2+) and Pb(2+) were additive and blocked completely by the mitochondrial permeability transition pore (PTP) inhibitor cyclosporin A, whereas the calcineurin inhibitor FK506 had no effect. The caspase inhibitors carbobenzoxy-Leu-Glu-His-Asp-CH(2)F and carbobenzoxy-Asp-Glu-Val-Asp-CH(2)F, but not carbobenzoxy-Ile-Glu-Thr-Asp-CH(2)F, differentially blocked post-mitochondrial events. The levels of reduced and oxidized glutathione and pyridine nucleotides in rods were unchanged. Our results demonstrate that rod mitochondria are the target site for Ca(2+) and Pb(2+). Moreover, they suggest that Ca(2+) and Pb(2+) bind to the internal metal (Me(2+)) binding site of the PTP and subsequently open the PTP, which initiates the cytochrome c-caspase cascade of apoptosis in rods.     

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.     

Impaired regulation of brain mitochondria by extramitochondrial Ca2+ in transgenic Huntington disease rats

Huntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt(51Q)), modeling the adult form of HD. Ca(free)(2+) up to 2 mum activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. Ca(free)(2+) above 2 mum inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca(2+) uniporter, did not affect the Ca(2+)-dependent activation of respiration but reduced Ca(2+)-induced inhibition. Thus, Ca(2+) activation was mediated exclusively by extramitochondrial Ca(2+), whereas inhibition was promoted also by intramitochondrial Ca(2+). In contrast, htt(51Q) mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca(2+) activation, and a higher susceptibility to Ca(2+)-dependent inhibition. Furthermore htt(51Q) mitochondria exhibited a diminished membrane potential stability in response to Ca(2+), lower capacities and rates of Ca(2+) accumulation, and a decreased Ca(2+) threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca(2+)-induced inhibition of respiration of htt(51Q) mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca(2+). In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca(2+). We suggest that specific regulatory Ca(2+) binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt(51Q) and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD.     

Occurrence and characteristics of the mitochondrial permeability transition in plants.

The behavior of purified potato mitochondria toward the main effectors of the animal mitochondrial permeability transition has been studied by light scattering, fluorescence, SDS-polyacrylamide gel electrophoresis, and immunoblotting techniques. The addition of Ca(2+) induces a phosphate-dependent swelling that is fully inhibited by cyclosporin A if dithioerythritol is present. Mg(2+) cannot be substituted for Ca(2+) but competes with it. Disruption of the outer membrane and release of several proteins, including cytochrome c, occur upon completion of swelling. Ca(2+)-induced swelling is delayed and its rate is decreased when pH is shifted from 7.4 to 6.6. It is accelerated by diamide, phenylarsine oxide, and linolenic acid. In the absence of Ca(2+), however, linolenic acid (< or =20 microm) rapidly dissipates the succinate-driven membrane potential while having no effect on mitochondrial volume. Anoxic conditions favor in vitro swelling and the concomitant release of cytochrome c and of other proteins in a pH-dependent way. These data indicate that the classical mitochondrial permeability transition occurs also in plants. This may have important implications for our understanding of cell stress and death processes.     

Brain Region-Specific, Age-Related, Alterations in Mitochondrial Responses to Elevated Calcium

An age-related Ca(2+) dysregulation and increased production of reactive oxygen species (ROS) may contribute to late-onset neurodegenerative disorders. These alterations are often attributed to impaired mitochondrial function yet few studies have directly examined mitochondria isolated from various regions of the aged brain. The purpose of this study was to examine Ca(2+)-buffering and ROS production in mitochondria isolated from Fischer 344 rats ranging in age from 4 to 25 months. Mitchondria isolated from the cortex of the 25 month rat brain exhibited greater rates of ROS production and mitochondrial swelling in response to increasing Ca(2+) loads as compared to mitochondria isolated from younger (4, 13 month) animals. The increased swelling is indicative of opening of the mitochondrial permeability transition pore indicating impaired Ca(2+) buffering/cycling in aged animals. These age-related differences were not observed in mitochondria isolated from cerebellum. Together, these results demonstrate region specific, age-related, alterations in mitochondrial responses to Ca(2+).     

Ca(2+)-induced high amplitude swelling and cytochrome c release from wheat (Triticum aestivum L.) mitochondria under anoxic stress

Under stress conditions, mitochondria sense metabolic changes, e.g. in pH, cytoplasmic Ca(2+), energy status, and reactive oxygen species (ROS), and respond by induction of the permeability transition pore (PTP) and by releasing cytochrome c, thus initiating the programmed cell death (PCD) cascade in animal cells. In plant cells, the presence of all the components of the cascade has not yet been shown. In wheat (Triticum aestivum L.) root mitochondria, the onset of anoxia caused rapid dissipation of the inner membrane potential, initial shrinkage of the mitochondrial matrix and the release of previously accumulated Ca(2+). Ca(2+) uptake by mitochondria was dependent on the presence of inorganic phosphate. Treatment of mitochondria with high micromolar and millimolar Ca(2+) (but not Mg(2+)) concentrations induced high amplitude swelling, indicative of PTP opening. Alterations in mitochondrial volume were confirmed by transmission electron microscopy. Mitochondrial swelling was not sensitive to cyclosporin A (CsA)-an inhibitor of mammalian PTP. The release of cytochrome c was monitored under lack of oxygen. Anoxia alone failed to induce cytochrome c release from mitochondria. Oxygen deprivation and Ca(2+) ions together caused cytochrome c release in a CsA-insensitive manner. This process correlated positively with Ca(2+) concentration and required Ca(2+) localization in the mitochondrial matrix. Functional characteristics of wheat root mitochondria, such as membrane potential, Ca(2+) transport, swelling, and cytochrome c release under lack of oxygen are discussed in relation to PCD.     

Exposure of developing oligodendrocytes to cadmium causes HSP72 induction, free radical generation, reduction in glutathione levels, and cell death

Primary cultures of oligodendrocytes were used to study the toxic effects of cadmium chloride. Cell viability was evaluated by the mitochondrial dehydrogenase activity and confirmed by propidium iodide (PI) fluorescence staining. The expression of the 72 kDa stress protein, HSP72, was assayed by Western blot analysis. The results showed that Cd(2+)-induced toxicity was dependent on the time and dose of exposure, as well as on the developmental stage of the cultures. Oligodendrocyte progenitors were more vulnerable to Cd(2+) toxicity than were mature oligodendrocytes. Mature oligodendrocytes accumulated relatively higher levels of Cd(2+) than did progenitors, as determined by (109)CdCl(2) uptake; treatment with the metal ion caused a more pronounced reduction in intracellular glutathione levels and significantly higher free radical accumulation in progenitors. The latter could explain the observed differences in Cd(2+) susceptibility. HSP72 protein expression was increased both in progenitors and in mature cells exposed to Cd(2+). Pretreatment with N-acetylcysteine, a thiocompound with antioxidant activity and a precursor of glutathione, prevented Cd(2+)-induced (i) reduction in glutathione levels and (ii) induction of HSP72 and diminished (i) Cd(2+) uptake and (ii) Cd(2+)-evoked cell death. In contrast, buthionine sulfoximine, an inhibitor of gamma-glutamyl-cysteine synthetase, depleted glutathione, and potentiated the toxic effect of Cd(2+). These results strongly suggest that Cd(2+)-induced cytotoxicity in oligodendrocytes is mediated by reactive oxygen species and is modulated by glutathione levels.     

Effect of magnesium on calcium-induced depolarisation of mitochondrial transmembrane potential

An effect of magnesium on calcium-induced depolarisation of mitochondrial transmembrane potential (DeltaPsi(m)) was investigated. Depending on the presence of Mg(2+), addition of Ca(2+) to suspension of isolated rat heart mitochondria induced either reversible depolarisation or irreversible collapse of succinate-driven DeltaPsi(m). Irreversible collapse of DeltaPsi(m), observed in the absence of Mg(2+), was insensitive to Ca(2+) chelation, inhibition of Ca(2+) uptake and increased efflux of Ca(2+) from mitochondrial matrix. Based on these data, opening of mPTP in a high-conductance mode is considered to be a major cause of the Ca(2+)-induced irreversible collapse of DeltaPsi(m) in the absence of Mg(2+). Involvement of mPTP in the process of Ca(2+)-induced collapse of DeltaPsi(m) was further supported by protective effect of both CsA and ADP. Reversible collapse of DeltaPsi(m), observed in the presence of Mg(2+), was sensitive to EGTA, ADP; and inhibition of Ca(2+) uptake and increased efflux of Ca(2+) from mitochondrial matrix. This may represent selective induction of a low-conductance permeability pathway. Presented results indicate important role of Mg(2+) in the process of Ca(2+)-induced depolarisation of DeltaPsi(m) mainly through discrimination between low- and high-conductance modes of mPTP. Minor effect of Mg(2+) on Ca(2+)-induced depolarisation of DeltaPsi(m) was observed at the level of stimulation of DeltaPsi(m) generation and inhibition of mitochondrial Ca(2+) uptake.     

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

Interrelationships in trace-element metabolism in metal toxicities in a cobalt-resistant strain of Neurospora crassa

A strain of Neurospora crassa was isolated by training the mould to grow on media containing high concentrations of Co(2+). This strain, the Co(R) strain, exhibited approximately tenfold the resistance of the parent strain to Co(2+) and Ni(2+) but not to Zn(2+) or Cu(2+). Co(2+) toxicity in the Co(R) strain was reversed by Mg(2+) but not by Fe(3+). Also, Co(2+) did not affect iron metabolism in this strain. It is suggested that the mechanism of resistance in the Co(R) strain involves an alteration in the pattern of iron metabolism such that the latter is no longer adversely affected by toxic concentrations of Co(2+). The Co(R) strain is genetically stable and is most probably a result of a resistance mutation in N. crassa induced by Co(2+).     

Cyclosporin A-sensitive permeability transition pore is involved in Cd(2+)-induced dysfunction of isolated rat liver mitochondria: doubts no more

There is dose-dependent Cd(2+)-evoked swelling of isolated rat liver mitochondria energized by complex I, II, or IV respiratory substrates in sucrose medium in the absence of added Ca(2+) and P(i), which is prevented by Sr(2+). Permeability transition effectors (ADP, CsA, EGTA, RR, DTT, ATR, P(i), and Ca(2+)) affect in a corresponding way Cd(2+)-promoted membrane permeabilization in NH(4)NO(3), KCl, and sucrose media. Maximal depression of Cd(2+)-induced swelling is achieved by simultaneous addition of ADP, Mg(2+), and CsA that produces either synergistic (NH(4)NO(3)) or additive (KCl and sucrose media) action. Sustained activation by low [Cd(2+)] of mitochondrial basal respiration in KCl medium is observed both in the absence and in the presence of rotenone and/or oligomycin but only in the latter case (rotenone+oligomycin) CsA inhibits completely Cd(2+) activation of St 4 respiration and partially reverses DNP-uncoupled respiration depressed by cadmium. Cd(2+) effects are discussed in terms of comparison with those of Zn(2+) and PhAsO.     

In Vitro Effect of Copper Chloride Exposure on Reactive Oxygen Species Generation and Respiratory Chain Complex Activities of Mitochondria Isolated from Broiler Liver

This study is to examine if Cu(2+) can act directly on mitochondria or indirectly by producing reactive oxygen species (ROS), isolated broiler hepatic mitochondria were exposed to different concentrations of Cu(2+) (10, 30, 50?μM). Respiratory chain complex activities, ROS generation, respiratory control ratio (RCR) and mitochondrial membrane potential were investigated. Dose-dependent inhibition of respiratory chain complexes and induction of ROS were observed, which coincided with decreasing RCR both with glutamate?+?malate or succinate. Further investigation indicated that the membrane potential determined by rhodamine 123 release decreased after CuCl(2) exposure at 30 and 50?μM. In addition, the effects of the antioxidants NAC (200?μM) and GSH (200?μM) were studied at 50?μM Cu(2+). The results indicate that Cu can induce mitochondrial dysfunction in excessive dose and the effect of Cu(2+) exposure on respiratory chain is not site-specific, and antioxidants can protect the mitochondrial function by reducing the formation of free radicals.     

Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening

Inhibition of Na(+)/H(+) exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by ～60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl(2) to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl(2)-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 μM) decreased mitochondrial Ca(2+)-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.     

Alteration in mitochondrial thiol enhances calcium ion dependent membrane permeability transition and dysfunction in vitro: a cross-talk between mtThiol, Ca2+, and ROS

Mitochondrial permeability transition (MPT) and dysfunctions play a pivotal role in many patho-physiological and toxicological conditions. The interplay of mitochondrial thiol (mtThiol), MPT, Ca(2+) homeostasis, and resulting dysfunctions still remains controversial despite studies by several research groups. Present study was undertaken to ascertain the correlation between Ca(2+) homeostasis, mtThiol alteration and reactive oxygen species (ROS) in causing MPT leading to mitochondrial dysfunction. mtThiol depletion significantly enhanced Ca(2+) dependent MPT (swelling) and depolarization of mitochondria resulting in release of pro-apoptotic proteins like Cyt c, AIF, and EndoG. mtThiol alteration and Ca(2+) overload caused reduced mitochondrial electron flow, oxidation of pyridine nucleotides (NAD(P)H) and significantly enhanced ROS generation (DHE and DCFH-DA fluorescence). Studies with MPT inhibitor (Cyclosporin A), Ca(2+) uniport blocker (ruthenium red) and Ca(2+) chelator (BAPTA) indicated that mitochondrial dysfunction was more pronounced under dual stress of altered mtThiol and Ca(2+) overload in comparison with single stress of excessive Ca(2+). Transmission electron microscopy confirmed the changes in mitochondrial integrity under stress. Our findings suggest that the Ca(2+) overload itself is not solely responsible for structural and functional impairment of mitochondria. A multi-factorial cross-talk between mtThiol, Ca(2+) and ROS is responsible for mitochondrial dysfunction. Furthermore, minor depletion of mtThiol was found to be an important factor along with Ca(2+) overload in triggering MPT in isolated mitochondria, tilting the balance towards disturbed functionality.