Sodium inhibits permeability transition by decreasing potassium matrix content in rat kidney mitochondria

Inner membrane mitochondria undergo a permeability increase elicited after the opening of a nonspecific pore due to supraphysiological matrix Ca²⁺ load, and the presence of an inducer. Multiple inducers have been used to promote the transition in permeability; among them are carboxyatractyloside (CAT) and reactive oxygen-derived species. In contrast, inhibitors such as ADP and cyclosporin A have been commonly used. In this work, we show that the opening or closure of the nonspecific pore depends on the cationic composition of the incubation medium. It was found that when mitochondria were incubated in either 125 mM KCl or 125 mM LiCl, ADP was essential to maintain selective membrane permeability. Interestingly, the nucleotide was not required when the medium contained 125 mM NaCl. Furthermore, it was established that CAT promotes membrane leakage in K⁺- or Li⁺-incubated mitochondria, while it failed to do so in Na⁺-incubated mitochondria. Evidence is also presented on the ability of Na⁺ to induce resistance in mitochondria against membrane damage by oxidative stress. Mitochondrial Ca²⁺ discharge, swelling, and transmembrane electric gradient were analyzed to establish permeability transition. It is concluded that the protection provided by Na⁺ was accomplished by inducing matrix K⁺ depletion, which, in turn, diminished the free fraction of matrix Ca²⁺.     

The redox state of pyridine nucleotides controls permeability of uncoupled mitochondria to K+

Heart mitochondria respiring in the presence of P_i release endogenous K⁺ to a sucrose medium when an uncoupler is added. The uncoupled mitochondria retain K⁺, however, if the oxidation of NAD(P)H is prevented by the addition of rotenone or antimycin. Addition of rotenone, once the uncoupler-dependent K⁺-efflux has been initiated, results in a rapid reduction of NAD(P) and a simultaneous decrease in permeability to K⁺. These changes are independent of respiration. The results suggest that a latent pathway for K⁺-permeability is present in the membrane, that it can be opened and closed reversibly, and that it reflects, either directly or indirectly, the redox status of mitochondrial pyridine nucleotides. The possible relationship of this putative pathway to those available for Ca²⁺ uptake and release is considered.     

Proton permeability and the regulation of potassium permeability in mitochondria by uncoupling agents

Summary The addition of agents that uncouple electron transfer from energy conservation (uncouplers) to state 4 mitochondria causes the following ion movements: K⁺ is extruded from the mitochondria in association with phosphate and possibly other anions, but not H⁺. Endogenous Ca⁺⁺ is extruded from the mitochondria, and H⁺ moves in to counter-balance the Ca⁺⁺ movement; some phosphate movement may be associated with Ca⁺⁺ extrusion. The rate and extent of K⁺ extrusion induced by uncoupler is dependent on the concentrations of external phosphate and divalent ions. Phosphate induces K⁺ extrusion, while Mg⁺⁺ and Mn⁺⁺ inhibit it. TheV _max of K⁺ transport is 300 moles K⁺/g protein per min. The K _m for FCCP-induced potassium extrusion is 0.25 M at pH 7.4. The inhibitory effect of Mg⁺⁺ is noncompetitive with respect to uncoupler concentration but competitive with respect to phosphate concentration. The experimental evidence does not support the existence of high H⁺ permeability in the presence of uncoupler. A correlation is observed between the rate of K⁺ extrusion and the energy reserves supplied from the high energy intermediate. The action of uncoupler in inducing K⁺ permeability is considered to arise through its action in depleting the energy reserves of mitochondria rather than through a specific activating effect of permeability by the uncoupler itself. The relationship of membrane potential to regulation of K⁺ permeability is discussed.     

Induction of Ca2+-dependent cyclosporin a-insensitive nonspecific permeability of the inner membrane of liver mitochondria and cytochrome c release by α,ω-hexadecanedioic acid in media of varying ionic strength

In liver mitochondria loaded with Ca²⁺ or Sr²⁺, α,ω-hexadecanedioic acid (HDA) can induce nonspecific permeability of the inner membrane (mitochondrial pore) by the mechanism insensitive to cyclosporin A (CsA). In this work we studied the effect of ionic strength of the incubation medium on the kinetics of the processes that accompany Ca²⁺-dependent induction of the mitochondrial pore by fatty acid: organelle swelling, Ca²⁺ release from the matrix, changes in transmembrane potential (Δψ) and rate of oxygen consumption, and the release of cytochrome c from the intermembrane space. Two basic incubation media were used: sucrose medium and isotonic ionic medium containing KCl without sucrose. We found that 200 μM Ca²⁺ and 20 μM HDA in the presence of CsA effectively induce high-amplitude swelling of mitochondria both in the case of sucrose and in the ionic incubation medium. In the presence of CsA, mitochondria can rapidly absorb Ca²⁺ and retain it in the matrix for a while without reducing Δψ. Upon incubation in the ionic medium, mitochondria retain most of the added Ca²⁺ in the matrix for a short time without reducing the Δψ. In both cases the addition of HDA to the mitochondria 2 min after the introduction of Ca²⁺ leads to the rapid release of these ions from the matrix and total drop in Δψ. The mitochondrial swelling induced by Ca²⁺ and HDA in non-ionic medium is accompanied by almost maximal stimulation of respiration. Under the same conditions, but during incubation of mitochondria in the ionic medium, it is necessary to add cytochrome c for significant stimulation of respiration. The mitochondrial swelling induced by Ca²⁺ and HDA leads to the release of cytochrome c in a larger amount in the case of ionic medium than for the sucrose medium. We conclude that high ionic strength of the incubation medium determines the massive release of cytochrome c from mitochondria and liberates it from the respiratory chain, which leads to blockade of electron transport along the respiratory chain and consequently to disruption of the energy functions of the organelles.     

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.     

Induction of permeability of the inner membrane of yeast mitochondria

The current view on apoptosis is given, with a special emphasis placed on apoptosis in yeasts. Induction of a non-specific permeability transition pore (mPTP) in mammalian and yeast mitochondria is described, particularly in mitochon-dria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, which are aerobes possessing the fully competent respiratory chain with all three points of energy conservation and well-structured mitochondria. They were examined for their ability to induce an elevated permeability transition of the inner mitochondrial membrane, being subjected to virtually all conditions known to induce the mPTP in animal mitochondria. Yeast mitochondria do not form Ca²⁺-dependent pores, neither the classical Ca²⁺/P_i-dependent, cyclosporin A-sensitive pore even under deenergization of mitochondria or depletion of the intramitochondrial nucleotide pools, nor a pore induced in mammalian mitochondria upon concerted action of moderate Ca²⁺ concentrations (in the presence of the Ca²⁺ ionophore ETH129) and saturated fatty acids. No pore formation was found in yeast mitochondria in the presence of elevated phosphate concentrations at acidic pH values. It is concluded that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Bivalent Metal Ions Modulate Cd2+ Effects on Isolated Rat Liver Mitochondria

We have studied Cd²⁺-induced effects on mitochondrial respiration and swelling in various media as a function of the [Cd²⁺] in the presence or absence of different bivalent metal ions or ruthenium red (RR). It was confirmed by monitoring oxygen consumption by isolated rat liver mitochondria that, beginning from 5 M, Cd²⁺ decreased both ADP and uncoupler-stimulated respiration and increased their basal respiration when succinate was used as respiratory substrate. At concentrations higher than 5 M, Cd²⁺ stimulated ion permeability of the inner mitochondrial membrane, which was monitored in this study by swelling of both nonenergized mitochondria in 125 mM KNO₃ or NH₄NO₃ medium and succinate-energized mitochondria incubated in a medium containing 25 mM K-acetate and 100 mM sucrose. We have found substantial changes in the above-mentioned Cd²⁺ effects on mitochondria treated in sequence with 100 M of Ca²⁺, Sr²⁺, Mn²⁺ or Ba²⁺(Me²⁺) and 7.5 M RR, as well as the alterations in Cd²⁺ action on the uptake of ¹³⁷Cs⁺ by succinate-energized mitochondria in the presence or absence of valinomycin in acetate medium (50 mM Tris-acetate and 140 mM sucrose) with or without Ca²⁺ or RR. The evidence obtained indicate that Ca²⁺ exhibits a synergestic action on all Cd²⁺ effects examined, whereas Sr²⁺ and Mn²⁺, conversely, are antagonistic. In the presence of RR, the Cd²⁺ effects on respiration [stimulation of State 4 respiration and inhibition of 2,4-dinitrophenol (DNP)-uncoupled respiration] still exist, but are observed at concentrations of cadmium more than one order higher; the inhibition of State 3 respiration by Cd²⁺, conversely, takes place under even lower cadmium concentrations than those determined without RR in the medium. In addition, RR added simultaneously with cadmium in the incubation medium prevents any swelling in the nitrate media, but induces an increment both in Cd²⁺-stimulated swelling and ¹³⁷Cs⁺ (analog of K⁺) uptake in the acetate media. For the first time, we have shown that Cd²⁺-induced swelling in all media under study is susceptible to cyclosporin A (CSA), a high-potency inhibitor of the mitochondrial permeability transition (PT) pore. The observations are interpreted in terms of a dual effect of cadmium on respiratory chain activity and permeability transition.     

Ascorbate and low concentrations of FeSO4 induce Ca2+-dependent pore in rat liver mitochondria

Oxidative stress is one of the most frequent causes of tissue and cell injury in various pathologies. The molecular mechanism of mitochondrial damage under conditions of oxidative stress induced in vitro with low concentrations of FeSO₄ and ascorbate (vitamin C) was studied. FeSO₄ (1-4 M) added to rat liver mitochondria that were incubated in the presence of 2.3 mM ascorbate induced (with a certain delay) a decrease in membrane potential and high-amplitude swelling. It also significantly decreased the ability of mitochondria to accumulate exogenous Ca²⁺. All the effects of FeSO₄ + ascorbate were essentially prevented by cyclosporin A, a specific inhibitor of the mitochondrial Ca²⁺-dependent pore (also known as the mitochondrial permeability transition). EGTA restored the membrane potential of mitochondria de-energized with FeSO₄ + ascorbate. We hypothesize that oxidative stress induced in vitro with FeSO₄ and millimolar concentrations of ascorbate damages mitochondria by inducing the cyclosporin A-sensitive Ca²⁺-dependent pore in the inner mitochondrial membrane.     

Reactive oxygen species and permeability transition pore in rat liver and kidney mitoplasts

Mitochondrial permeability transition is typically characterized by Ca²⁺ and oxidative stress-induced opening of a nonselective proteinaceous membrane pore sensitive to cyclosporin A, known as the permeability transition pore (PTP). Data from our laboratory provide evidence that the PTP is formed when inner membrane proteins aggregate as a result of disulfide cross-linking caused by thiol oxidation. Here we compared the redox properties between PTP in intact mitochondria and mitoplasts. The rat liver mitoplasts retained less than 5% and 10% of the original outer membrane markers monoamine oxidase and VDAC, respectively. Kidney mitoplasts also showed a partial depletion of hexokinase. In line with the redox nature of the PTP, mitoplasts that were more susceptible to PTP opening than intact mitochondria showed higher rates of H₂O₂ generation and decreased matrix NADPH-dependent antioxidant activity. Mitoplast PTP was also sensitive to the permeability transition inducer tert-butyl hydroperoxide and to the inhibitors cyclosporin A, EGTA, ADP, dithiothreitol and catalase. Taken together, these data indicate that, in mitoplasts, PTP exhibits redox regulatory characteristics similar to those described for intact mitochondria.     

On the role of K+ on succinic dehydrogenase activity

The effect of potassium ions on succinic dehydrogenase activity of mitochondria was studied. The results showed that in these organelles K⁺ induces inhibition of the respiratory control; moreover, in submitochondrial particles potassium inhibits the rate of oxidation of succinate. The results showed also that K⁺ does not changes theK _m for succinate but diminishes theV _max. In addition, the data provide evidence that mitochondria oxidizing glutamatemalate in a sucrose medium show a higher activity of succinate dehydrogenase than mitrochondria incubated in KCl.     

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.     

The permeability transition pore as a mitochondrial calcium release channel: A critical appraisal

Mitochondria from a variety of sources possess an inner membrane channel, the permeability transition pore. The pore is a voltage-dependent channel, activated by matrix Ca²⁺ and inhibited by matrix H⁺, which can be blocked by cyclosporin A, presumably after binding to mitochondrial cyclophilin. The physiological function of the permeability transition pore remains unknown. Here we evaluate its potential role as a fast Ca²⁺ release channel involved in mitochondrial and cellular Ca²⁺ homeostasis. We (i) discuss the theoretical and experimental reasons why mitochondria need a fast, inducible Ca²⁺ release channel; (ii) analyze the striking analogies between the mitochondrial permeability transition pore and the sarcoplasmic reticulum ryanodine receptor-Ca²⁺ release channel; (iii) argue that the permeability transition pore can act as a selective release channel for Ca²⁺ despite its apparent lack of selectivity for the transported speciesin vitro; and (iv) discuss the importance of mitochondria in cellular Ca²⁺ homeostasis, and how disruption of this function could impinge upon cell viability, particularly under conditions of oxidative stress.     

Conditions for activity of glutaminase in kidney mitochondria

1. Rat kidney mitochondria oxidize glutamate very slowly. Addition of glutamine stimulates this respiration two- to three-fold. Addition of glutamate also stimulates respiration in the presence of glutamine. 2. By measuring mitochondrial swelling in iso-osmotic solutions of glutamine or of ammonium glutamate it was shown that glutamine penetrates the mitochondrial membrane rapidly whereas ammonium glutamate penetrates very slowly. 3. Experiments in which reduction of NAD(P)⁺ was measured in preparations of intact and broken mitochondria indicated that glutamate dehydrogenase shows the phenomenon of `latency'. On the addition of glutamine rapid reduction of nicotinamide nucleotides in intact mitochondria was obtained. 4. During the action of glutaminase there is an accumulation of glutamate inside the mitochondria. 5. When the mitochondria were suspended in a medium containing glutamine, P_i and rotenone the rate of production of ammonia was stimulated by the addition of a substrate, e.g. succinate. Addition of an uncoupler or antimycin A abolished this stimulation. 6. The effects of succinate and uncoupler were especially pronounced in the presence of glutamate, which is an inhibitor of glutaminase activity by competition with P_i. 7. Determination of the enzyme activity in media at different pH values showed that the optimum pH for glutaminase activity in the preparation of broken mitochondria was 8, whereas for intact mitochondria it was dependent on the energy state. In the presence of succinate as an energy source it was pH 8.5, but in the presence of uncoupler or antimycin A it was 9. This displacement of the pH optimum to a higher value was especially pronounced in the presence of both glutamate and uncoupler. 8. If nigericin was present in potassium chloride medium the pH optimum for enzyme activity in intact non-respiring mitochondria was nearly the same as in the preparation of broken mitochondria; however, its presence in K⁺-free medium displaced the pH optimum for glutaminase activity to a very high value. 9. It is postulated that because of low permeability of the kidney mitochondrial membrane to glutamate the latter accumulates inside the mitochondria, and that this leads to the inhibition of the enzyme by competition with P_i and also by lowering the pH of the intramitochondrial space. With succinate as substrate for respiration there is an outward translocation of H⁺ ions, which together with accumulation of P_i increases glutaminase activity. Translocation of K⁺ ions inward increases the enzyme activity, perhaps by increasing the pH of the internal spaces and causing an accumulation of P_i. 10. The importance of the location of the enzyme in the mitochondria in relation to its biological function and conditions for activity is discussed.     

Modulation of the Permeability Transition Pore by Inhibition of the Mitochondrial KATP Channel in Liver vs. Brain Mitochondria

Inhibition of the mitochondrial K_ATP (mitoK_ATP) channel abrogates the beneficial effects of preconditioning induced by a brief episode of sublethal ischemia. We studied the effect of 5-hydroxydecanoate, a well-known inhibitor of the mitoK_ATP channel, on swelling of isolated liver and brain mitochondria. Volume changes were determined by measurement of light absorbance at 540 nm. Mitochondrial swelling induced by adding Ca²⁺ ions correlated with opening of the permeability transition pore as shown by modulation by 1 μM cyclosporin A. In brain mitochondria, 5-hydroxydecanoate did not significantly affect Ca²⁺-induced swelling. In contrast, 50 or 500 μM 5-hydroxydecanoate increased swelling of liver mitochondria by 9.7 ± 5.1% (n = 6, P = 0.057) and 29.4 ± 1.4% (n = 5, P < 0.0001), respectively. The effect of 5-hydroxydecanoate was blocked by cyclosporin A and was dependent on the presence of potassium in the medium. In medium containing 200 μM ATP to inhibit the mitoK_ATP channel, 5–hydroxydecanoate did not further increase Ca²⁺-induced swelling. We conclude that inhibition of the mitoK_ATP channel exerts its detrimental effect by facilitation of permeability transition pore opening.     

The features of activation of free oxidation by α,ω-tetradecanedioic acid in liver mitochondria

It was found that α,ω-tetradecanedioic acid (TDA) at the concentration of 0–500 μM doubles the rate of nonphosphorylating respiration (free oxidation) of liver mitochondria in a dose-dependent manner. This effect of TDA is observed in the presence of the excess of EGTA, which eliminates the induction of the Ca²⁺-dependent nonspecific permeability of the mitochondrial inner membrane (pore opening). An unusually high concentration of cyclosporin A (10 mM) completely eliminates this effect when added to the mitochondria before or after TDA. The stimulatory effect of TDA is not accompanied by inhibition of oxidative ATP synthesis and decrease in the ADP/O ratio, in contrast to the effects of other activators of free oxidation, such as protonophore uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone and palmitic acid. It was shown that neither oligomycin, an inhibitor of H⁺-ATP synthase, nor ADP, ATP and P_i affected the activity of TDA. This is seen as an evidence that the effect of TDA is not associated with the influence on H⁺-ATP synthase and it differs from the action of membranotropic uncouplers. In the presence of the lipophilic cation tetraphenylphosphonium (TPP⁺) cyclosporin A does not affect the TDA-stimulated respiration of mitochondria, but carboxyatractylate and glutamate added after TDA do inhibit the respiration. In addition, under these conditions TDA decreases the rate of oxidative ATP synthesis and reduces the ADP/O ratio. It is assumed that the mechanism of the TDA-induced activation of free oxidation in liver mitochondria in the absence of TPP⁺ is similar to that of the so-called decouplers and is associated with the switching of the respiratory chain complexes to the idle mode (inner uncoupling).     

Mitochondrial Effects of Triarylmethane Dyes

The mitochondrial effects of submicromolar concentrations of six triarylmethane dyes, withpotential applications in antioncotic photodynamic therapy, were studied. All dyes promotedan inhibition of glutamate or succinate-supported respiration in uncoupled mitochondria, in amanner stimulated photodynamically. No inhibition of N,N,N,N-tetramethyl-p-phenylenediamine(TMPD) supported respiration was observed, indicating that these dyes do not affectmitochondrial complex IV. When mitochondria were energized with TMPD in the absence ofan uncoupler, treatment with victoria blue R, B, or BO, promoted a dissipation of mitochondrialmembrane potential and increase of respiratory rates, compatible with mitochondrialuncoupling. This effect was observed even in the dark, and was not prevented by EGTA, Mg²⁺ orcyclosporin A, suggesting that it is promoted by a direct effect of the dye on inner mitochondrialmembrane permeability to protons. Indeed, victoria blue R, B, and BO promoted swellingof valinomycin-treated mitochondria incubated in a hyposmotic K⁺-acetate-based medium,confirming that these dyes act as classic protonophores such as FCCP. On the other hand, ethylviolet, crystal violet, and malachite green promoted a dissipation of mitochondrial membranepotential, accompanied by mitochondrial swelling, which was prevented by EGTA, Mg²⁺, andcyclosporin A, demonstrating that these drugs induce mitochondrial permeability transition.This mitochondrial permeabilization was followed by respiratory inhibition, attributable tocytochrome c release, and was caused by the oxidation of NAD(P)H promoted by these drugs.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

Induction of the non-selective mitochondrial pore in lymphoid cells. 2. Intact rat thymocytes.

Increase of Ca2+ concentration in the cytosol of thymocytes to 400-600 nM causes slow accumulation of Ca2+ in mitochondria. Release of Ca2+ from mitochondria into the cytosol is induced by an uncoupler (FCCP) or by a dithiol cross-linking agent (phenylarsine oxide) and is inhibited by cyclosporin A--a specific inhibitor of the permeability transition pore in the inner mitochondrial membrane. In the presence of oxidizing agents (tert-butyl hydroperoxide and diamide), sub-optimal concentrations of uncoupler induce rapid cyclosporin-sensitive release of Ca2+. 6-Ketocholestanol, a recoupler under these conditions, causes redistribution of Ca2+ from the cytosol into mitochondria. These data indicate that partial uncoupling under conditions of oxidative stress causes opening of the permeability transition pore in a fraction of the mitochondria in intact lymphocytes. This mechanism mediates rapid release of Ca2+ from mitochondria into the cytosol.     

A general characteristic of tumor mitochondria: Leakage of endogenous Mg2+ on incubation with uncoupler and resultant reduction of uncoupler-stimulated ATPase activity

Previous studies showed that stimulation of mouse mitochondrial ATPase activity of tumor cells, fetal liver, and adult brain by the uncoupler 2,4-dinitrophenol was markedly suppressed during incubation of the mitochondria with the uncoupler (J.-I. Hayashi et al., 1980, Biochem. Biophys. Res. Commun.92, 261–267). The present work showed the reason for this suppression. More than half the endogenous Mg²⁺ leaked from mitochondria of all tumor cells tested, and of fetal liver and adult brain during incubation with the uncoupler, while only about 30% of the endogenous Mg²⁺ leaked from mitochondria of other normal tissues. The effect of the uncoupler on Mg²⁺ leakage from liver mitochondria changed from the fetal to the adult type within about 30 min after birth. In hypotonic medium, normal liver mitochondria also lost more than half their total Mg²⁺ and concomitantly stimulation of their ATPase activity by uncoupler was considerably reduced. Exogenously added Mg²⁺ could reverse this reduced effect of the uncoupler on ATPase activity of mitochondria from normal tissues and tumor cells. These results show that the endogenous Mg²⁺ content of mitochondria directly affects the stimulation by uncoupler of ATPase activity of mitochondria from both normal tissues and tumor cells. Thus, mitochondria of all tumor cells tested, and of fetal liver and adult brain are leaky to Mg²⁺ during incubation with uncoupler and as a result of the leakage, the stimulatory effect of the uncoupler on their ATPase activity is greatly reduced.     

Tl+ induces both cationic and transition pore permeability in the inner membrane of rat heart mitochondria

Effects of Tl⁺ were studied in experiments with isolated rat heart mitochondria (RHM) injected into 400 mOsm medium containing TlNO₃ and a nitrate salt (KNO₃ or NH₄NO₃) or TlNO₃ and sucrose. Tl⁺ increased permeability of the inner membrane of the RHM to K⁺ and H⁺. This manifested as an increase of the non-energized RHM swelling, in the order of sucrose < K⁺ < NH₄ ⁺, respectively. After succinate administration, the swollen RHM contracted. The Tl⁺-induced opening of the mitochondrial permeability pore (MPTP) in Ca²⁺-loaded rat heart mitochondria increased both the swelling and the inner membrane potential dissipation, as well as decreased basal state and 2,4-dinitrophenol-stimulated respiration. These effects of Tl⁺ were suppressed by the MPTP inhibitors (cyclosporine A, ADP, bongkrekic acid, and n-ethylmaleimide), activated in the presence of the MPTP inducer (carboxyatractyloside) or mitoK_ATP inhibitor (5-hydroxydecanoate), but were not altered in the presence of mitoK_ATP agonists (diazoxide or pinacidil). We suggest that the greater sensitivity of heart and striated muscles, versus liver, to thallium salts in vivo can result in more vigorous Tl⁺ effects on muscle cell mitochondria.