The nonspecific inner membrane pore of liver mitochondria: modulation of cyclosporin sensitivity by ADP at carboxyatractyloside-sensitive and insensitive sites

Cyclosporin A prevents the opening of a nonspecific pore in the inner membrane of liver mitochondria when added prior to Ca2+. In the presence of 10 microM Ca2+ cyclosporin is unable to close the pore and restore the original permeability unless ADP is also added. ADP acts at a high-affinity site (Km 5 microM), corresponding to the adenine nucleotide transporter. This effect of ADP is prevented and reversed by carboxyatractyloside. In the presence of carboxyatractyloside, cyclosporin added with higher concentrations of ADP (Km 70 microM) also can close the pore. This suggests that a lower-affinity ADP-binding component as well as cyclophilin and the adenine nucleotide transporter can modulate the sensitivity of the pore to cyclosporin.     

Protective action of tamoxifen on carboxyatractyloside-induced mitochondrial permeability transition

AimsMitochondrial permeability transition is established after massive Ca²⁺ accumulation inside the matrix, in addition to an inducer. The closure of the pore can be accomplished by adenosine diphosphate and the immunosuppressant cyclosporin A. Recently, the estrogen antagonist, tamoxifen, has been introduced as an inhibitor of the opening of the permeability transition pore. However, the mechanism by which this drug inhibits pore opening is still under discussion. This work was performed with the purpose of establishing the membrane system involved in tamoxifen-induced pore closure. For this purpose, permeability transition was induced after the addition of carboxyatractyloside, which is a specific reagent that interacts with the adenine nucleotide translocase.Main methodsPermeability transition was assessed by analyzing matrix Ca²⁺ release, transmembrane electric gradient, and mitochondrial swelling in aged, as well as in freshly prepared mitochondria. Also, cytochrome c content was analyzed in membrane mitochondria as well as in the supernatant.Key findingsIn freshly prepared mitochondria, tamoxifen, at the concentration of 10 μM, totally inhibited nonspecific membrane permeability induced by 1 μM carboxyatractyloside. In addition, tamoxifen inhibited non-specific permeability in aged mitochondria and diminished membrane fluidity.SignificancePlausibly, the inhibitory effect of tamoxifen on nonspecific membrane permeability, as induced by carboxyatractyloside, should be ascribed to a diminution, of membrane fluidity by this drug.     

Carboxyatractyloside increases the effect of oleate on mitochondrial permeability transition

Addition of a low concentration of carboxyatractyloside (0.075 microM) renders mitochondria susceptible to the opening of the non-specific pore by 5 microM oleate, in a cyclosporin A-sensitive fashion. Matrix Ca2+ efflux as well as collapse of the transmembrane potential reveal permeability transition. The effect of oleate is reached after the titration, by carboxyatractyloside, of 38 pmol of adenine nucleotide translocase per mg mitochondrial protein. We propose that permeability transition may result from an additive action of carboxyatractyloside plus oleate on the ADP/ATP carrier.     

The permeability transition in heart mitochondria is regulated synergistically by ADP and cyclosporin A.

Heart mitochondria respiring in a sucrose medium containing P(i) show a permeability transition when challenged with Ca2+ and an oxidant such as cumene hydroperoxide. The transition results from the opening of a Ca(2+)-dependent pore and is evidenced by loss of membrane potential (delta psi) and osmotic swelling due to uptake of sucrose and other solutes. In the absence of oxidant, high concentrations of Ca2+ (100-150 microM) are necessary to induce loss of delta psi and initiate swelling. Cyclosporin A delays the loss of delta psi but enhances swelling under these conditions, apparently by promoting better retention of accumulated Ca2+. Cyclosporin A and ADP together restore delta psi in respiring mitochondria that have undergone the permeability transition at levels that are not effective when either is added alone. When the state of the Ca(2+)-dependent pore is assessed using passive osmotic contraction in response to polyethylene glycol (Haworth, R. A., and Hunter, D. R. (1979) Arch. Biochem. Biophys. 195, 460-467), cyclosporin A is found to be a partial inhibitor of solute flow through the open pore. Cyclosporin A decreases the Vmax of passive contraction and increases the Km for Ca2+ without affecting the Hill slope. ADP in the presence of carboxyatractyloside closes the pore almost completely even in the presence of 40 microM Ca2+. ADP shows mixed type inhibition of the Ca(2+)-dependent pore, and cyclosporin A increases the affinity of the pore for ADP. It is concluded that cyclosporin A and ADP act synergistically to close the Ca(2+)-dependent pore of the mitochondrion and that the pore is probably not formed directly from the adenine nucleotide transporter.     

Inhibition of Ca2(+)-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase.

1. Isolated rat liver and heart mitochondria incubated in 150 mM-KSCN or sucrose medium in the presence of respiratory-chain inhibitors showed a large increase in swelling when exposed to 250 microM-Ca2+. Swelling was inhibited by bongkrekic acid and cyclosporin A in both media and by ADP in KSCN medium; the effect of ADP was reversed by carboxyatractyloside. These results demonstrate that this is a suitable technique with which to study the opening of the Ca2(+)-induced non-specific pore of the mitochondrial inner membrane and implicate the adenine nucleotide carrier in this process. 2. Titration of the rate of swelling with increasing concentrations of cyclosporin showed the number of cyclosporin-binding sites (+/- S.E.M.) in liver and heart mitochondria to be respectively 113.7 +/- 5.0 (n = 9) and 124.3 +/- 11.2 (n = 10) pmol/mg of protein, with a Ki of about 5 nM. 3. Liver and heart mitochondrial-matrix fractions were prepared free of membrane and cytosolic contamination and shown to contain cyclosporin-sensitive peptidyl-prolyl cis-trans isomerase (cyclophilin) activity. Titration of isomerase activity with cyclosporin gave values (+/- S.E.M.) of 110.6 +/- 10.1 (n = 5) and 165.4 +/- 15.0 (n = 3) pmol of enzyme/mg of liver and heart mitochondrial protein respectively, with a Ki of 2.5 nM. The similarity of these results to those from the swelling experiments suggest that the isomerase may be involved in the Ca2(+)-induced swelling. 4. The rapid light-scattering change induced in energized heart mitochondria exposed to submicromolar Ca2+ [Halestrap (1987) Biochem. J. 244, 159-164] was inhibited by ADP and bongkrekic acid, the former effect being reversed by carboxyatractyloside. These results suggest an interaction of Ca2+ with the adenine nucleotide carrier when the 'c' conformation. 5. A model is proposed in which mitochondrial peptidyl-prolyl cis-trans isomerase interacts with the adenine nucleotide carrier in the presence of Ca2+ to cause non-specific pore opening. The model also explains the involvement of the adenine nucleotide translocase in the PPi-mediated cyclosporin-insensitive increase in K+ permeability described in the preceding paper [Davidson & Halestrap (1990) Biochem. J. 268, 147-152]. 6. The physiological and pathological implications of the model are discussed in relation to reperfusion injury and cyclosporin toxicity.     

The flavonoid quercetin induces changes in mitochondrial permeability by inhibiting adenine nucleotide translocase

This study shows the effects of the flavonoid quercetin on diverse mitochondrial functions, among them membrane permeability. Our findings indicate that the addition of 50 μM quercetin did not produce reactive oxygen derived species; however, it inhibited the oxidative stress induced after the addition of Fe²/H₂O₂ by about 38%. At this concentration, quercetin also promoted a fast calcium release, inhibited oxidative phosphorylation, stimulated oxygen consumption, and decreased membrane potential. In addition 50 μM quercetin inhibited the adenine nucleotide translocase (ANT) by 46%. These effects induced the opening of the permeability transition pore and release of cytochrome c, by its interaction with a component of the non-specific pore complex, fixed to the carrier in the conformation c, as carboxyatractyloside does. Quercetin-induced permeability transition pore opening was inhibited by 0.5 μM cyclosporin A, but, interestingly, the release of cytochrome c was not inhibited by the immunosuppressor, as quercetin was found to disrupt the outer membrane.     

The participation of pyridine nucleotides redox state and reactive oxygen in the fatty acid-induced permeability transition in rat liver mitochondria

The ability of low concentrations (5-15 microM) of long-chain fatty acids to open the permeability transition pore (PTP) in Ca(2+)-loaded mitochondria has been ascribed to their protonophoric effect mediated by mitochondrial anion carriers, as well as to a direct interaction with the pore assembly [M.R. Wieckowski and L. Wojtczak, FEBS Lett. 423 (1998) 339-342]. Here, we have compared the PTP opening ability of arachidonic acid (AA) with that of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) at concentrations that cause similar quantitative dissipation of the membrane potential (DeltaPsi) in Ca(2+)-loaded rat liver mitochondria respiring on succinate. The initial protonophoric effects of AA and FCCP were only slightly modified by carboxyatractyloside and were followed by PTP opening, as indicated by a second phase of DeltaPsi disruption sensitive to EGTA, ADP, dithiothreitol and cyclosporin A. This second phase of DeltaPsi dissipation could also be prevented by rotenone or NAD(P)H-linked substrates which decrease the pyridine nucleotide (PN) oxidation that follows the stimulation of oxygen consumption induced by AA or FCCP. These results suggest that, under the experimental conditions used here, the PTP opening induced by AA or FCCP was a consequence of PN oxidation. Exogenous catalase also inhibited both AA- and FCCP-induced PTP opening. These results indicate that a condition of oxidative stress associated with the oxidized state of PN underlies membrane protein thiol oxidation and PTP opening.     

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⁺.     

On the Protection by Inorganic Phosphate of Calcium-Induced Membrane Permeability Transition

The role of inorganic phosphate as inhibitor of mitochondrial membrane permeability transition was studied. It is shown that in mitochondria containing a high phosphate concentration, i.e., 68 nmol/mg, Ca²⁺ did not activate the pore opening. Conversely, at lower levels of matrix phosphate, i.e., 38 nmol/mg, Ca²⁺ was able to induce subsequent pore opening. The inhibitory effect of phosphate was apparent in sucrose-based media, but it was not achieved in KCl media. The matrix free Ca²⁺ concentration and matrix pH were lowered by phosphate, but they were always higher in K⁺-media. In the absence of ADP, phosphate strengthened the inhibitory effect of cyclosporin A on carboxyatractyloside-induced Ca²⁺ efflux. Acetate was unable to replace phosphate in the induction of the aforementioned effects. It is concluded that phosphate preserves selective membrane permeability by diminishing the matrix free Ca²⁺ concentration.     

Mitochondria and reperfusion injury of the heart—A holey death but not beyond salvation

The combination of calcium overload and oxidative stress opens a non-specific pore in the inner mitochondrial membrane known as the mitochondrial permeability transition pore (MPTP). This uncouples oxidative phosphorylation and compromises intracellular ATP levels eventually leading to necrotic cell death. In cardiac ischemia and reperfusion, as during treatment of a coronary thrombosis or cardiac surgery, the extent of MPTP opening determines the amount of irreversible damage (infarct size). Furthermore, cardioprotection can be achieved by inhibiting MPTP opening either directly with cyclosporin A analogues, or indirectly by reducing oxidative stress. The detailed molecular mechanism of the MPTP remains uncertain. Knockout studies have confirmed important regulatory roles for cyclophilin-D (CyP-D) and the adenine nucleotide translocase (ANT) but not the voltage dependent anion channel. Our own studies have implicated a calcium-triggered conformational change of the mitochondrial phosphate carrier that is facilitated by CyP-D and modulated by the conformation of the ANT.     

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.     

Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress.

The capacity of cyclosporin A to inhibit opening of a Ca2+-dependent pore in the inner membrane of heart mitochondria was investigated. Whereas in the presence of 25 nmol of Ca2+/mg of mitochondrial protein and 5 mM-Pi mitochondria were unable to maintain accumulated Ca2+, inner-membrane potential and sucrose impermeability, all three parameters were preserved when cyclosporin was included. Pore opening was assayed directly by [14C]sucrose entry and entrapment in the matrix space. [14C]Sucrose entry induced by both Ca2+ plus Pi and Ca2+ plus t-butyl hydroperoxide was almost completely inhibited by 60 pmol of cyclosporin/mg of mitochondrial protein. It is concluded that cyclosporin A is a potent inhibitor of the pore.     

The composition of the incubation medium influences the sensitivity of mitochondrial permeability transition to cyclosporin A

The aim of this work was to study permeability transition, and the influence of the composition of the incubation medium, on the inhibitory action of cyclosporin A. It was found that cyclosporin inhibited the opening of a nonspecific pore, as induced by the uncoupler carbonyl cyanide m-chlorophenylhydrazone, provided K⁺ was present in the incubation medium, but failed to do so if mitochondria are incubated in sucrose or Na⁺-based medium. It was also found that the sensitivity of mitochondria to the uncoupler depended on the incubation mixture, being more sensitive when sucrose was the osmotic support. Matrix Ca²⁺ release, large amplitude swelling, and drop in transmembrane electric gradient revealed permeability transition. The titration of membrane thiol groups shows them to be increased in mitochondria incubated in sucrose medium, in comparison with the values found in mitochondria incubated in KCl or NaCl medium. Our proposal is that the incubation in sucrose medium propitiated a conformational change of membrane proteins in such a way that cyclosporin was unable to bind to its target site.     

Long-chain fatty acids promote opening of the reconstituted mitochondrial permeability transition pore

Adenine nucleotide translocase-porin-hexokinase complex isolated from rat brain, when reconstituted into phospholipid-cholesterol vesicles, exhibits all properties of the mitochondrial permeability transition pore [Beutner, G., Rück, A., Riede, B., Welte, W. and Brdiczka, D. (1996) FEBS Lett. 396, 189-195]. In the present work, the effect of long-chain fatty acids on such reconstituted pore was examined. Opening of the pore was measured by leakage of either malate or fluorescein sulphonate entrapped inside the vesicles. It was found that myristate and oleate in the presence of 50 or 100 microM Ca(2+) produced a partial release of the probes in a dose-dependent way. A dicarboxylic fatty acid analogue, that appeared inactive as protonophore in intact mitochondria, exerted no effect on pore opening in the reconstituted system. 100 microM Ca(2+) alone was without effect. Pore opening by fatty acids in the reconstituted system was partly prevented by cyclosporin A. The pore opening also occurred when the vesicles were incubated in the presence of pancreatic phospholipase A(2). In this case, the opening was decreased by cyclosporin A or serum albumin. These results indicate that long-chain fatty acids elicit opening of the permeability transition pore reconstituted in phospholipid vesicles in a similar way as in intact mitochondria [Wi&ecedil;ckowski, M.R. and Wojtczak, L. (1998) FEBS Lett. 423, 339-342].     

Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane

The mitochondrial permeability transition pore allows solutes with a m.w. 1500 to equilibrate across the inner membrane. A closed pore is favored by cyclosporin A acting at a high-affinity site, which may be the matrix space cylophilin isozyme. Early results obtained with cyclosporin A analogs and metabolites support this hypothesis. Inhibition by cyclosporin does not appear to require inhibition of calcineurin activity; however, it may relate to inhibition of cyclophilin peptide bond isomerase activity. The permeability transition pore is strongly regulated by both the membrane potential () and pH components of the mitochondrial protonmotive force. A voltage sensor which is influenced by the disulfide/sulhydryl state of vicinal sulfhydryls is proposed to render pore opening sensitive to . Early results indicate that this sensor is also responsive to membrane surface potential and/or to surface potential gradients. Histidine residues located on the matrix side of the inner membrane render the pore responsive to pH. The pore is also regulated by several ions and metabolites which act at sites that are interactive. There are many analogies between the systems which regulate the permeability transition pore and the NMDA receptor channel. These suggest structural similarities and that the permeability transition pore belongs to the family of ligand gated ion channels.     

Possible Mechanism for Formation and Regulation of the Palmitate-Induced Cyclosporin A-Insensitive Mitochondrial Pore

The mechanism of the palmitate-induced opening of the mitochondrial Ca2+-dependent cyclosporin A (CsA)-insensitive pore was studied, as well as the influence on this process of well-known modulators of the CsA-sensitive Ca2+-dependent pore. Palmitic acid, which can bind Ca2+ with high affinity, induced the cyclosporin A-insensitive swelling of mitochondria, whereas palmitoleic and 2-bromopalmitic acids, which have no such affinity for Ca2+, failed to induce the pore opening. The palmitate-induced Ca2+-dependent swelling of mitochondria was not affected by a well-known inhibitor of the CsA-sensitive pore (ADP) and an activator of this pore (inorganic phosphate, P(i)). However, this swelling was inhibited by physiological concentrations of ATP ([I]50 = 1.3 mM), but 100 microM ATP increased by 30% the rate of mitochondria swelling if Ca2+ had been added earlier. The effects of ATP (inhibition and activation) manifested themselves from different sides of the inner mitochondrial membrane. Mg2+ inhibited the palmitate-induced Ca2+-dependent swelling of mitochondria with [I]50 = 0.8 mM. It is concluded that palmitic acid induces the opening of the CsA-insensitive pore due to its ability for complexing with Ca2+. A possible mechanism of the pore formation and the influence of some modulators on this process are discussed.     

Copper sensitizes the mitochondrial permeability transition to carboxyatractyloside and oleate

Addition of 5 M copper to rat kidney mitochondria enhances the effect of carboxyatractyloside and oleate on pore opening, in a cyclosporin A-sensitive fashion. The effects of the pair copper-carboxyatractyloside were observed on matrix Ca²⁺ efflux, mitochondrial swelling and on the transmembrane electric gradient. The effect of Cu²⁺ emphasizes the importance of membrane thiol groups located, probably, in the ADP/ATP translocase (ANT), on permeability transition. It was also found that Cu²⁺ does not block the fluorescent label of ANT by eosin 5-maleimide, but abolishes the inhibition by CAT on the labeling. This suggests that the binding of Cu²⁺ to cysteine residues of ANT promotes a conformational change in the carrier, strengthening the effect of CAT and oleate on membrane leakage.     

Nitric oxide donors, nitrosothiols and mitochondrial respiration inhibitors induce caspase activation by different mechanisms

We investigated to what extent different types of NO donors induce caspase activation by opening of the mitochondrial permeability transition pore (PTP) or inhibition of mitochondrial respiration. We found that nitrosothiols can directly open the PTP in isolated mitochondria and cause cytochrome c release, whereas NONOate donors can not. In macrophages nitrosothiols cause caspase activation that is blocked by cyclosporin A or calcium chelation, both of which prevent PTP opening, whereas caspase activation caused by NONOates is much less sensitive to these agents. Inhibitors of mitochondrial respiration did not promote PTP opening in isolated mitochondria, and although they cause caspase activation in macrophages, this activation was slower than that caused by NO donors, and was relatively insensitive to cyclosporin and calcium chelators suggesting that PTP opening was not involved.     

A study on permeability transition pore opening and cytochrome c release from mitochondria, induced by caspase-3 in vitro.

We recently described that there is a feedback amplification of cytochrome c release from mitochondria by caspases. Here we investigated how caspases impact on mitochondria to induce cytochrome c release and found that recombinant caspase-3 induced opening of permeability transition pore and reduction of membrane potential in vitro. These events were inhibited by Bcl-xL, cyclosporin A and z-VAD.fmk. Moreover, caspase-3 stimulated the rate of mitochondrial state 4 respiration, superoxide production and NAD(P)H oxidation in a Bcl-xL- and cyclosporin A-inhibitable manner. These results suggest that caspase-3 induces cytochrome c release by inducing permeability transition pore opening which is associated with changes in mitochondrial respiration and redox potential.