Oxidative stress is involved in the permeabilization of the inner membrane of brain mitochondria exposed to hypoxia/reoxygenation and low micromolar Ca2+

From in vivo models of stroke it is known that ischemia/reperfusion induces oxidative stress that is accompanied by deterioration of brain mitochondria. Previously, we reported that the increase in Ca2+ induces functional breakdown and morphological disintegration in brain mitochondria subjected to hypoxia/reoxygenation (H/R). Protection by ADP indicated the involvement of the mitochondrial permeability transition pore in the mechanism of membrane permeabilization. Until now it has been unclear how reactive oxygen species (ROS) contribute to this process. We now report that brain mitochondria which had been subjected to H/R in the presence of low micromolar Ca2+ display low state 3 respiration (20% of control), loss of cytochrome c, and reduced glutathione levels (75% of control). During reoxygenation, significant mitochondrial generation of hydrogen peroxide (H2O2) was detected. The addition of the membrane permeant superoxide anion scavenger TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) suppressed the production of H2O2 by brain mitochondria metabolizing glutamate plus malate by 80% under normoxic conditions. TEMPOL partially protected brain mitochondria exposed to H/R and low micromolar Ca2+ from decrease in state 3 respiration (from 25% of control to 60% of control with TEMPOL) and permeabilization of the inner membrane. Membrane permeabilization was obvious, because state 3 respiration could be stimulated by extramitochondrial NADH. Our data suggest that ROS and Ca2+ synergistically induce permeabilization of the inner membrane of brain mitochondria exposed to H/R. However, permeabilization can only partially be prevented by suppressing mitochondrial generation of ROS. We conclude that transient deprivation of oxygen and glucose during temporary ischemia coupled with elevation in cytosolic Ca2+ concentration triggers ROS generation and mitochondrial permeabilization, resulting in neural cell death.     

Adenine nucleotide and phosphate transport systems of mitochondria. Relative location of sulfhydryl groups based on the use of the novel fluorescent probe eosin-5-maleimide

Eosin-5-maleimide is impermeable to the inner mitochondrial membrane, exhibiting essentially no reactivity with matrix glutathione or with beta-hydroxybutyrate dehydrogenase located on the matrix surface of the inner membrane. In intact mitochondria, eosin-5-maleimide is unreactive with the ADP/ATP antiporter even under conditions which promote maximal labeling by N-[3H]ethylmaleimide (i.e., ADP present). However, eosin-5-maleimide readily labels the ADP/ATP antiporter in "inverted" inner membrane vesicles even in the presence of N-ethylmaleimide. Labeling is prevented if the vesicles are prepared from mitochondria pretreated with carboxyatractyloside. In contrast to the ADP/ATP antiporter, essential sulfhydryl groups of the Pi/H+ symporter are accessible to eosin-5-maleimide in intact mitochondria with optimal inhibition of phosphate transport being observed at 25 degrees C. Eosin-5-maleimide also prevents labeling of the Pi/H+ symporter by N-[3H]ethylmaleimide. These results show that essential sulfhydryl groups of the ADP/ATP antiporter and the Pi/H+ symporter have differing reactivities and locations in functionally intact mitochondria. With respect to eosin-5-maleimide, sulfhydryl groups of the ADP/ATP carrier occur in two distinct classes, both of which are inaccessible in intact mitochondria. Only one class, depending on conditions, can be exposed in submitochondrial particles. In contrast, sulfhydryl group(s) of the Pi/H+ symporter behave as a single reactive class which is readily accessible in mitochondria at 25 degrees C.     

Effects of extramitochondrial ADP on permeability transition of mouse liver mitochondria

Carboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 microM CAT and 75 microM Ca2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca2+ but the release of Ca2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 microM CAT did not change the respiration, but increased the mitochondrial capacity for Ca2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 microM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP. We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression.     

Nature of endogenous proton conductance of the inner mitochondrial membrane. Role of Ca2+ transport system in proton transfer

In order to elucidate the nature of endogenous proton conductance of rat liver inner mitochondrial membrane, the dependence of the rate of Ca2+ transport on pH was studied. It was found that the inhibiting effect of H+ is independent of protonation of functional groups of hypothetical Ca2+ carrier, but results from electrogenic transfer of H+ across the membrane, which is highly permeable for the proton. The adsorption of H+ by mitochondria is inhibited by ruthenium red and other specific inhibitors of Ca2+ transport. It is concluded that endogenous proton conductance of the inner mitochondrial membrane depends on the functioning of the same transport system essential for membrane permeability for Ca2+ and other bivalent cations. The correlation observed between the rates of H+ and Ca2+ transport in mitochondria and the ratio of cation mobilities in aqueous solutions is in favour of a "porous" mechanism of cation transport across the mitochondrial membrane.     

Detection and functional role of local gradients of H+-ions on the intracellular mitochondrial membrane with covalently linked pH-probe

The study is devoted to the registration of local H+ gradients on the inner membrane of mitochondria under conditions of H+ pump functioning were recorded. By using a covalently linked pH probe (fluorescein isothiocyanate), a local increase in the activity of hydrogen ions on the outer face of the inner mitochondrial membrane in the presence of the respiration substrate at increased permeability of the membrane for K+ was registered. It was also found that the buffer capacity of medium affects the respiration rate of completely uncoupled mitochondria; a change in respiration rate strictly correlates with changes in local H+ gradients on the mitochondrial membrane. It was concluded that local gradients of H+ activity can control the rate of functioning of H+ pumps. It was shown that, under certain conditions, the system of H+ pumps incorporated into succinate oxidase of mitochondria functions as a nonliner system.     

Reconstitution and partial purification of the glibenclamide-sensitive, ATP-dependent K+ channel from rat liver and beef heart mitochondria.

The transport properties of mitochondria are such that net potassium flux across the inner membrane determines mitochondrial volume. It has been known that K+ uptake is mediated by diffusive leak driven by the high electrical membrane potential maintained by redox-driven, electrogenic proton ejection and that regulated K+ efflux is mediated by an 82-kDa inner membrane K+/H+ antiporter. There is also long-standing suggestive evidence for the existence of an inner membrane protein designed to catalyze electrophoretic K+ uptake into mitochondria. We report reconstitution of a highly purified inner membrane protein fraction from rat liver and beef heart mitochondria that catalyzes electrophoretic K+ flux in liposomes and channel activity in planar lipid bilayers. The unit conductance of the channel at saturating [K+] is about 30 pS. Reconstituted K+ flux is inhibited with high affinity by ATP and ADP in the presence of divalent cations and by glibenclamide in the absence of divalent cations. The mitochondrial ATP-dependent K+ channel is selective for K+, with a Km of 32 mM, and does not transport Na+. K+ transport depends on voltage in a manner consistent with a channel activity that is not voltage-regulated. Thus, the mitochondrial ATP-dependent K+ channel exhibits properties that are remarkably similar to those of the ATP-dependent K+ channels of plasma membranes.     

Mechanism of alterations in isolated rat liver mitochondrial function induced by gold complexes of bidentate phosphines

Au(DPPE)+2 (bis[1,2-bis(diphenylphosphino)ethane] gold(I] is an organo-gold antineoplastic agent that has anti-tumor activity in a variety of in vitro cell lines and in vivo rodent tumor models. Preliminary studies suggested that this compound represented a novel class of inhibitors of mitochondrial function. The purpose of this study was, therefore, to determine the mechanism of mitochondrial dysfunction induced by Au(DPPE)+2. Au(DPPE)+2 induced a rapid, dose-related collapse of the inner mitochondrial membrane potential (EC50 = 28.0 microM) that was not potentiated by Ca2+ preloading. Au(DPPE)+2-induced dissipation of mitochondrial membrane potential was accompanied by an efflux of Ca2+ from mitochondria upon exposure to Au(DPPE)+2. Ca2+ efflux in these experiments was via a reversal of the Ca2+ uniporter as efflux could be inhibited with ruthenium red. Au(DPPE)+2 did not increase the permeability of mitochondria to oxalacetate, indicating that the collapse of membrane potential may not be a result of gross increased inner membrane permeability. However, Au(DPPE)+2 may mediate an increased permeability of the inner membrane to cations and protons. Au(DPPE)+2 caused passive swelling in potassium acetate buffer in the absence of valinomycin, suggesting Au(DPPE)+2 facilitated the exchange of H+ and K+. Ca2+ cycling was not extensive and did not contribute to the decrease in membrane potential. These data suggest that one possible mechanism of Au(DPPE+2-induced uncoupling of mitochondrial oxidative phosphorylation is via increased permeability of the inner mitochondrial membrane to cations. The disruption of mitochondrial function may be a key process leading to hepatocyte cell injury by this drug.     

Ca2+ Induces a Cyclosporin A-Insensitive Permeability Transition Pore in Isolated Potato Tuber Mitochondria Mediated by Reactive Oxygen Species

Oxidative damage of mammalian mitochondria induced by Ca²⁺ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca²⁺ uniport, undergo inner membrane permeabilization when treated with Ca²⁺ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg²⁺ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis–trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca²⁺ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.     

Calcium-Dependent Nonspecific Permeability of the Inner Mitochondrial Membrane Is Not Induced in Mitochondria of the Yeast Endomyces magnusii

Mitochondria of the yeast Endomyces magnusii were examined for the presence of a Ca2+- and phosphate-induced permeability of the inner mitochondrial membrane (pore). For this purpose, coupled mitochondria were incubated under conditions known to induce the permeability transition pore in animal mitochondria, i.e., in the presence of high concentrations of Ca2+ and P(i), prooxidants (t-butylhydroperoxide), oxaloacetate, atractyloside (an inhibitor of ADP/ATP translocator), SH-reagents, by depletion of adenine nucleotide pools, and deenergization of the mitochondria. Large amplitude swelling, collapse of the membrane potential, and efflux of the accumulated Ca2+ were used as parameters for demonstrating pore induction. E. magnusii mitochondria were highly resistant to the above-mentioned substances. Deenergization of mitochondria or depletion of adenine nucleotide pools have no effect on low-amplitude swelling or the other parameters. Cyclosporin A, a specific inhibitor of the nonspecific permeability transition in animal mitochondria, did not affect the parameters measured. It is thus evident that E. magnusii mitochondria lack a functional Ca2+-dependent pore, or possess a pore differently regulated as compared to that of mammalian mitochondria.     

Local coupling of respiration processes and phosphorylation in rat liver mitochondria

One variant of the model of the local coupling of phosphorylation and respiration in intact mitochondria was experimentally verified. The model is based on the following postulates: (1). Upon the functioning of H+ pumps, hydrogen ions bound to the outer membrane surface do not enter the aqueous phase but are utilized for ATP synthesis in the membrane supercomplex respiratory H+ pump--ATP synthetase. (2). During the functioning of H+ pumps, an appreciable part of the energy of oxidation reactions can be stored in the form of the thermodynamic (solvation) potential of H+ ions bound to the outer membrane surface. According to the model, the hydration of hydrogen ions during the transition from the outer face of the inner membrane to the aqueous phase should lead to a decrease in the efficiency of the system of the coupling of respiration and phosphorylation. The model takes into account the ability of the nonpermeating buffer to catalyze the detachment of hydrogen ions from the membrane surface to the aqueous phase and provide their complete solvation. A preparation of phosphorylating mitochondria with the covalently bound pH probe was obtained. This made it possible to register for the first time the presence of a local H+ gradient on the outer side of the inner mitochondrial membrane during the stable functioning of the oxidative phosphorylation system. It was shown on these mitochondrial preparations that a decrease in the outer local H+ gradient by the action of increased concentrations of buffer is accompanied by a significant decrease in the ADP/O parameter and a partial dissociation of oxidative phosphorylation. Conditions were determined under which increased concentrations of buffer in the incubation medium cause a partial dissociation and a decrease in the ADP/O value from 20% to twofold (depending on the quality of mitochondrial preparations). The results obtained are in full agreement with the predictions of the model.     

Effect of succinate on mitochondrial lipid peroxidation. 1. Comparative studies on ferrous ion and ADP · Fe/NADPH-induced peroxidation

Lipid peroxidation in isolated rat liver mitochondria, mitoplast, and mitochondrial inner membrane fragments was induced either by ferrous ions, or in an NADPH-dependent process by complexing with adenine nucleotides (ADP or ATP) iron. The Fe²⁺-induced lipid peroxidation is nonenzymic when inner membrane fragments are used, while the differences in the inhibitory effect of Mn²⁺ ions and the stimulatory effect of the ionophore A-23187 in mitochondria and inner membrane fragments suggest an enzymic mechanism for ferrous ion-induced lipid peroxidation in intact mitochondria. Contrary to this the ADP/Fe/NADPH-dependent lipid peroxidation is an enzymic process both in mitochondria and inner membrane preparations. We have shown that cytochrome P₄₅₀ is involved in the ADP/Fe/NADPH-induced lipid peroxidation. Succinate, a known inhibitor of NADPH-dependent lipid peroxidation, inhibited the Fe²⁺-induced process also, and there was no difference in this effect when inner membrane preparations, mitochondria, or mitoplasts were used.     

The maturation of the inner membrane of foetal rat liver mitochondria.

A new method was devised for the isolation of foetal and neonatal rat lvier mitochondria, giving higher yields than conventional methods. 2. During development from the perinatal period to the mature adult, the ratio of cytochrome oxidase/succinate-cytochrome c reductase changes. 3. The inner mitochondrial membrane of foetal liver mitochondria possesses virtually no osmotic activity; the permeability to sucrose decreases with increasing developmental age. 4. Foetal rat liver mitochondria possess only marginal respiratory control and do not maintain Ca2+-induced respiration; they also swell in respiratory-control medium in the absence of substrate. ATP enhances respiratory control and prevents swelling, adenylyl imidodiphosphate, ATP+atractyloside enhance the R.C.I. (respiratory control index), Ca2+-induced respiratory control and prevent swelling, whereas GTP and low concentrations of ADP have none of these actions. It is concluded that the effect of ATP depends on steric interaction with the inner mitochondrial membrane. 5. When 1-day pre-partum foetuses are obtained by Caesarean section and maintained in a Humidicrib for 90 min, mitochondrial maturation is "triggered", so that their R.C.I. is enhanced and no ATP is required to support Ca2+-dependent respiratory control or to inhibit mitochondrial swelling. 6. It is concluded that foetal rat liver mitochondria in utero do not respire, although they are capable of oxidative phosphorylation in spite of their low R.C.I. The different environmental conditions which the neonatal rat encounters ex utero enable the hepatic mitochondria to produce ATP, which interacts with the inner mitochondrial membrane to enhance oxidative phosphorylation by an autocatalytic mechanism.     

Studies on the interactions of Ca2+ and pyruvate in the regulation of rat heart pyruvate dehydrogenase activity. Effects of starvation and diabetes.

1. Previous studies showed that the activation of pyruvate dehydrogenase within intact rat heart mitochondria of pyruvate is much diminished in mitochondria from starved or diabetic animals [see Kerbey, Randle, Cooper, Whitehouse, Pask & Denton (1976) Biochem. J. 154, 327-348]. In the present study, diminished responses to added Ca2+ and ADP were also found in these mitochondria. 2. Starvation or diabetes did not affect the mitochondrial respiratory control ratio of the ATP content. Moreover, starvation and diabetes did not alter the response of the intramitochondrial Ca2+-sensitive enzyme, 2-oxoglutarate dehydrogenase, to changes in the extramitochondrial concentration of Ca2+ and 2-oxoglutarate, thus indicating that there were no appreciable changes in the distribution of Ca2+ and H+ across the mitochondrial inner membrane. 3. Pyruvate, Ca2+ and ADP were found to have synergistic effects on pyruvate dehydrogenase activity, particularly in mitochondria from starved and diabetic rats. 4. The results suggest that the effects of diabetes and starvation on pyruvate dehydrogenase are not brought about by changes in the distribution of these effectors across the mitochondrial inner membrane or by changes in the intrinsic sensitivity of the kinase or phosphatase of the pyruvate dehydrogenase system to pyruvate, Ca2+ or ADP; rather it is probably that there is an increase in the maximum activity of kinase relative to that of the phosphatase. 6. The results also lend further support to the hypothesis that adrenaline may bring about the activation of pyruvate dehydrogenase in the rat heart by an increase in the intramitochondrial concentration of Ca2+.     

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.     

Tetrandrine concentrations not affecting oxidative phosphorylation protect rat liver mitochondria from oxidative stress

The effects of tetrandrine (6,6', 7,12-tetramethoxy-2, 2'-dimethyl-berbaman) on the mitochondrial function were assessed on oxidative stress, mitochondrial permeability transition (MPT), and bioenergetics of rat liver mitochondria. At concentrations lower than 100nmol/mg protein, tetrandrine decreased the hydrogen peroxide formation, the extent of lipid peroxidation, the susceptibility to Ca(2+)-induced opening of MPT pore, and inhibited the inner membrane anion channel activity, not significantly affecting the mitochondrial bioenergetics. High tetrandrine concentrations (100-300nmol/mg protein) stimulated succinate-dependent state 4 respiration, while some inhibition was observed for state 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations. The respiratory control ratio and the transmembrane potential were depressed but the adenosine diphosphate to oxygen (ADP/O) ratio was less affected. A slight increase of the inner mitochondrial membrane permeability to H(+) and K(+) by tetrandrine was also observed. It was concluded that low concentrations of tetrandrine afford protection against liver mitochondria injury promoted by oxidative-stress events, such as hydrogen peroxide production, lipid peroxidation, and induction of MPT. Conversely, high tetrandrine concentrations revealed toxicological effects expressed by interference with mitochondrial bioenergetics, as a consequence of some inner membrane permeability to H(+) and K(+) and inhibition of the electron flux in the respiratory chain. The direct immediate protective role of tetrandrine against mitochondrial oxidative stress may be relevant to clarify the mechanisms responsible for its multiple pharmacological actions.     

Effect of oxaloacetic acid on hydrogen-calcium metabolism in mitochondria

It has been shown by using o-phthalic acid--a concurrent inhibitor of the transport of oxalacetic acid in mitochondria that the effect of the latter on the mechanism of 2H+/Ca2+-metabolism is realized at the inner side of the inner mitochondrial membrane. Oxalacetic acid was shown to induce not only the release of Ca2+ ions but also those of strontium and manganese accumulated in the mitochondria (100-150 nmol/mg of protein). Mechanism of the effect of oxalacetic acid on permeability of the mitochondria membranes are discussed.     

Localization of the ATP/ADP translocator in the inner membrane and regulation of contact sites between mitochondrial envelope membranes by ADP. A study on freeze-fractured isolated liver mitochondria.

The frequency of contacts between the mitochondrial envelope membranes was determined in freeze-fractured samples of isolated mitochondria by means of quantifying the frequency of fracture plane deflections between the two membranes. It was observed that the formation of contacts correlated with the concentration of free ADP despite of inhibition of electron transport by antimycin A. The activity of ATPase partially inhibited by oligomycin or depletion of membrane potential by K+ and valinomycin had no effect on the induction of the contacts by ADP. ATP was ineffective in creating contacts irrespective of the presence or absence of a membrane potential, whereas carboxyatractyloside induced the contacts under all conditions in a manner similar to ADP. These results suggest the involvement of the ATP/ADP translocator in regulation of contact sites. As a consequence, we analyzed its distribution in the inner membrane of kidney and liver mitochondria by binding of [3H]atractyloside to subfractions of this membrane. The experiments demonstrated that the translocator was located in the peripheral part of the inner membrane as well as in the portion which formed the cristae.     

Effect of cadmium ions on the respiration, cation transport and morphology of the liver mitochondria in the rat and the lamprey

The influence of Cd2+ on the function and structure of liver mitochondria of rats and lamprey (Lampetra fluviatilis L.) has been studied in vitro. It is shown that Cd2+ can penetrate into the mitochondrial matrix due to Ca2+-transport mechanism. Being stored in the mitochondria, Cd2+ inhibits respiration and an energy dependent transport of penetrating cations (Cs+-valinomycin), and disturbs passive permeability of the inner mitochondrial membrane for monovalent cations and H+. The effect of Cd2+ on the lamprey liver mitochondria is more pronounced than in the case of rats.     

Adenine nucleotide translocase as a site of regulation by ADP of the rat liver mitochondria permeability to H+ and K+ lons

In the presence of oligomycin ADP inhibits the osmotic swelling of the nonenergized rat liver mitochondria in the NH₄NO₃ medium. With the energized mitochondria ADP enhances contraction of the mitochondria swollen in the NH₄NO₃ medium. Carboxyatractyloside and atractyloside abolish or prevent the effects of ADP. The direct measurements of the proton conductance of rat liver mitochondria shows that the inhibitory action of ADP + oligomycin on the H⁺ permeability does not depend on the energization of mitochondria. In these experiments the local anesthetic nupercaine and ADP additively inhibit the inner membrane conductance for protons, but carboxyatractyloside abolishes only the effect of ADP. In the presence of oligomycin ADP also inhibits the osmotic swelling of the nonenergized liver mitochondria in the KNO₃ medium, and the energy-dependent swelling of rat liver mitochondria in the medium with K⁺ ions and P_i. The inhibition by ADP of the membrane passive permeability for K⁺ is also sensitive to carboxyatractyloside. It is concluded that rat liver mitochondria possess an ADP-regulated channel for H⁺ and K⁺. The properties of this pathway for protons and potassium ions favor the idea that ADP regulates the mitochondrial permeability via adenine nucleotide translocase. It is assumed that the adenine nucleotides carrier should operate according to the “gated pore” mechanism.     

Heterogeneity of the calcium-induced permeability transition in isolated non-synaptic brain mitochondria

Calcium overload of neural cell mitochondria plays a key role in excitotoxic and ischemic brain injury. This study tested the hypothesis that brain mitochondria consist of subpopulations with differential sensitivity to calcium-induced inner membrane permeability transition, and that this sensitivity is greatly reduced by physiological levels of adenine nucleotides. Isolated non-synaptosomal rat brain mitochondria were incubated in a potassium-based medium in the absence or presence of ATP or ADP. Measurements were made of medium and intramitochondrial free calcium, light scattering, mitochondrial ultrastructure, and the elemental composition of electron-opaque deposits within mitochondria treated with calcium. In the absence of adenine nucleotides, calcium induced a partial decrease in light scattering, accompanied by three distinct ultrastructural morphologies, including large-amplitude swelling, matrix vacuolization and a normal appearance. In the presence of ATP or ADP the mitochondrial calcium uptake capacity was greatly enhanced and calcium induced an increase rather than a decrease in mitochondrial light scattering. Approximately 10% of the mitochondria appeared damaged and the rest contained electron-dense precipitates that contained calcium, as determined by electron-energy loss spectroscopy. These results indicate that brain mitochondria are heterogeneous in their response to calcium. In the absence of adenine nucleotides, approximately 20% of the mitochondrial population exhibit morphological alterations consistent with activation of the permeability transition, but less than 10% exhibit evidence of osmotic swelling and membrane disruption in the presence of ATP or ADP.