Báo cáo Y học: Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin ppt

Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin Yasuo Shinohara 1 , Mohamad Radwan Almofti 1 , Takenori Yamamoto 1 , Taro Ishida 1 , Fumiyo Kita 1 , Hideki Kanzaki 1 , Masakatsu Ohnishi 1 , Kikuji Yamashita 2 , Shigeomi Shimizu 3,4 and Hiroshi Terada 5 1 Faculty of Pharmaceutical Sciences, University of Tokushima, Japan; 2 School of Dentistry, University of Tokushima, Japan; 3 Department of Medical Genetics, Osaka University Graduate School of Medicine, Japan; 4 CREST of Japan Science and Technology Corp., Osaka, Japan; 5 Faculty of Pharmaceutical Sciences, Tokyo University of Science, Tokyo, Japan To examine whether valinomycin induces a mitochondrial permeability transition (PT), we investigated its effects on mitochondrial functions under various conditions. The acceleration of mitochondrial respiration and swelling, induced by valinomycin, were found to be insensitive to inhibitors of the ordinary PT, indicating that valinomycin does not induce the ordinary PT. Results of experiments using mitochondria isolated from transgenic mice expressing human bcl-2 also supported this conclusion. Furthermore, evidence for induction of PT pores by valinomycin was not obtained by either electron microscopic analysis of mito- chondrial configurations or by measurement of the per- meability of the inner mitochondrial membrane by use of polyethylene glycol. However, valinomycin did induce a significant release of cytochrome c, and thus it may be a nice tool to study the processes of mitochondrial cytochrome c release. Keywords: mitochondria; valinomycin; permeability trans- ition; apoptosis; cytochrome c. The inner mitochondrial membrane is highly impermeable even to tiny solutes and ions in order to enable efficient energy conversion. However, in the presence of certain triggers such as Ca 2+ , the inner mitochondrial membrane is known to become highly permeable to such molecules. Nowadays, this transition is referred to as the mitochondrial permeability transition (PT), and it is believed to reflect the opening of proteinaceous pores (for reviews see [1–3]). Although the molecular features of the PT pores are still uncertain, cyclosporin A (CsA) and bongkrekic acid (BKA) are known to be effective inhibitors of the PT, and thus they have been used to judge induction of the mitochondrial PT. However, recent studies indicated that a PT insensitive to these inhibitors could also be induced [4–7]. In this paper, to distinguish PT’s sensitive and insensitive to these inhibitors, werefertothePTsensitivetoCsAandBKAasthe Ôordinary PTÕ. The physiological roles of mitochondrial PT are still obscure; however, recent studies revealed that PT is a key event during the process of programmed cell death, also known as apoptosis (for reviews see [8–11]). PT causes the release of apoptosis-inducible mitochondrial proteins such as cytochrome c into the cytosol, and these proteins trigger the subsequent reactions that execute apoptosis. The action of valinomycin, a K + selective ionophore [12], serves to dissipate the membrane potential of respiring mitochondria. Its effect as an apoptosis inducer was reported in the late 1980s to early 1990s [13–15], but the mechanism of apoptosis induction by it is not well understood at present. Recently, two discrepant papers on the effect of valinomycin as an apoptosis inducer have appeared. Inai et al. [16] reported that the valinomycin- induced apoptosis of malignant tumor cells was due to the dissipation of the mitochondrial membrane potential, which was independent of the PT and the actions of members of the proapoptotic and antiapoptotic Bcl-2 protein families. In contrast, Furlong et al. [17] reported that the apoptosis of a pre-B cell line caused by valinomycin was due to the induction of a PT, which was prevented by the PT inhibitor BKA. The reason why such discrepant results were obtained is uncertain. Furthermore, the question of whether the release of cytochrome c from mitochondria is inducible by valino- mycin has not yet been clearly answered. For clear answers to these questions, in addition to the studies using whole cells, investigations on the direct actions of valinomycin on isolated mitochondria would seem to be very important. Thus, in this study, we examined the effects of valinomycin on isolated mitochondria under various conditions. MATERIALS AND METHODS Materials Cytochrome c (code C-2037) and valinomycin (code V-0627) were purchased from Sigma. Maleimide-activated keyhole limpet hemocyanin (KLH, code 1-376-438) was obtained from Roche Diagnostics K.K. (Tokyo). Cyclo- sporin A (CsA) was kindly provided by Novartis Pharma Inc. (Tokyo). Poly(ethylene glycol) 6000 was purchased from Nacalai Tesque (Kyoto). Correspondence to Y. Shinohara, Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan. Fax: + 81 88 633 9512, Tel.: + 81 88 633 7278, E-mail: yasuo@ph.tokushima-u.ac.jp Abbreviations: BKA, bongkrekic acid; CsA, cyclosporin A; KLH, keyhole limpet hemocyanin; PT, permeability transition; SF6847, 3,5-di-tert-butyl-4-hydroxybenzylidene malononitrile. (Received 8 July 2002, revised 1 September 2002, accepted 5 September 2002) Eur. J. Biochem. 269, 5224–5230 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03229.x Preparation of mitochondria Liver mitochondria were isolated from normal male Wistar rats, mice or transgenic mice expressing human bcl-2 in their livers [18] according to the method described previously [19]. Brain mitochondria were prepared essentially by the same procedure used for the preparation of liver mitochondria but using a medium containing 300 m M mannitol, 5 m M Hepes buffer, and 0.5 mgÆmL )1 bovine serum albumin (fatty acid free, Sigma, code A-6003) at pH 7.4. Measurements of mitochondrial oxygen consumption and swelling Mitochondrial function and absorbance change were examined essentially as described previously [19]. Briefly, mitochondria (0.7 mg proteinÆmL )1 ) suspended in +P i medium (200 m M sucrose and 10 m M potassium phosphate buffer, pH 7.4) were energized with 10 m M succinate in the presence of rotenone (0.5 lg rotenoneÆmg protein )1 ), and their respiration was measured at 25 °CwithaClark oxygen-electrode (Yellow Springs, code 5331) in a total volume of 2.5 mL. Mitochondrial swelling in the same medium was monitored by measuring the absorbance at 540 nm with a Shimadzu spectrophotometer, model UV- 3000. Transmission electron microscopic analysis of mitochondrial configuration Mitochondrial configurations were analyzed by transmis- sion electron microscopy according to the method previ- ously published [19]. Preparation of antiserum against cytochrome c and Western blotting Antiserum against cytochrome c was prepared as follows: A peptide with the amino acid sequence of HTVEKGGKHKTGPNLHGLFC, which is completely conserved in mouse, rat, human and bovine cytochrome c’s except for the last cysteine residue, was synthesized by means of a Shimadzu peptide synthesizer, model PSSM-8. One milligram of this peptide was conjugated to 1 mg of maleimide-activated KLH. The peptide-KLH conjugate (500 lg) was dissolved in 500 lLofsterileNaCl/P i [–] and then emulsified with 500 lL of Freund’s complete adjuvant. This mixture was injected intramuscularly into an adult New Zealand White rabbit. Boosters were given twice at 3-week intervals. Total blood was obtained 10 days after the final booster shot, and allowed to stand at room tempera- ture for 1 h and then overnight at 4 °C. The blood clot was removed by centrifugation at 5000 g for 10 min at 4 °C, and the resulting supernatant was used as antiserum without further purification. The antiserum thus obtained recog- nized mouse, rat, human and bovine cytochrome c’s to the same degree. For an assay of cytochrome c, a standard solution was prepared by dissolving powdered cytochrome c in distilled water to a concentration of 10 mgÆmL )1 ,anditsexact concentration was determined from an absorbance meas- urement at 550 nm assuming e (m M )tobe27.8[20]after reduction with sodium hydrosulfite. To determine the amount of cytochrome c released from the respiring mito- chondria, we placed an aliquot (100 lL) of mitochondrial suspension in an Eppendorf centrifuge tube, promptly centrifuged it, and separated the supernatant from the pellet. The pelleted mitochondria were resuspended in 100 lL of the incubation medium, and 2 lLofthis suspension and 5 lL of supernatant were individually subjected to SDS/PAGE. Western blotting was carried out essentially as described earlier [21]. Determination of the amount of cytochrome c was performed as follows: first, to make a calibration curve, we subjected different but known amounts of cytochrome c to SDS/PAGE and then transferred them to a nitrocellulose membrane. Intensities of the immunodetected protein band of cytochrome c were determined by using an ATTO image analyzer, model AE-6900. Quantification of the released cytochrome c was performed in the range in which inten- sities of the immunodetected protein band and amount of cytochrome c showed a linear relationship. RESULTS Effects of valinomycin on mitochondrial respiration and volume and their sensitivity to PT inhibitors When a mitochondrial PT is induced, acceleration of mitochondrial respiration and swelling of mitochondria are commonly observed, and these changes are often used as a simple means to judge whether or not a PT is induced. Thus, we first examined the effects of valinomycin on mitochondrial respiration and volume change. As Ca 2+ is a well-known PT inducer, the effect of Ca 2+ on the mito- chondrial membrane was used as a positive control of PT induction. The protonophore SF6847, which dissipates the mitochondrial membrane potential but does not induce a PT, was used as a negative control for the PT induction. In addition, the induction of the ordinary PT was judged by the effects of the typical PT inhibitors CsA and BKA. As shown in Fig. 1A, Ca 2+ gradually accelerated the mito- chondrial respiration with succinate as a substrate (upper trace b), as well as mitochondrial swelling (lower trace b), and both were prevented in the presence of either CsA or BKA (upper and lower traces c and d, respectively). It is noteworthy that different from the relatively moderate inhibitory effect of BKA, the inhibitory effect of CsA on Ca 2+ -induced mitochondrial changes was very strong; and CsA inhibited Ca 2+ -induced PT even when PT was induced by higher concentrations of Ca 2+ such as 500 l M (data not shown). SF6847 increased mitochondrial respiration with no accompanying swelling, and CsA and BKA were ineffective in preventing accelerated respiration (Fig. 1B). In contrast, as shown in Fig. 1C, valinomycin accelerated the rate of oxygen consumption, and the accelerated respiration continued until all of the oxygen had been exhausted (upper trace b). On the addition of valinomycin, the turbidity of the mitochondrial suspension also decreased instantly, and after this change, showed a nearly constant value until all oxygen had been depleted (lower trace b). However, the PT inhibitors CsA and BKA had no effect on these changes (upper and lower traces c and d, respectively), showing that valinomycin did not induce the ordinary PT. Carboxyatractyloside, known to be a specific inhibitor of the mitochondrial ADP/ATP carrier, as is BKA [22], was Ó FEBS 2002 Cytochrome c release induced by valinomycin (Eur. J. Biochem. 269) 5225 shown earlier to have the opposite (i.e. stimulatory) effect on the Ca 2+ -induced PT [23]. The effects of valinomycin on mitochondrial structure and function were not influenced at all by carboxyatractyloside (data not shown), like in the case of BKA. Effects of valinomycin on the mitochondria prepared from rat brain and liver of transgenic mice expressing bcl-2 The above experiments were carried out by using mito- chondria prepared from rat liver. To know whether the observed effects of valinomycin are particular to mitochon- dria prepared from liver, we examined the effects of the drug on mitochondria prepared from rat brain. By employing the procedure described in Materials and methods, tightly coupled mitochondria showing a high respiratory control ratio (v state3 /v state4 ) of 5–6 were successfully prepared from rat brain; although they showed slower respiration at state 4 (v state4 , 8–10 natoms OÆmg protein )1 Æmin )1 ) than the liver mitochondria (data not shown). When valinomycin was added to the brain mitochondria, CsA-insensitive acceler- ation of respiration was observed (data not shown), indicating that the effects of valinomycin were not specific to the liver mitochondria. Furthermore, we examined the effect of valinomycin on the mitochondria isolated from livers of transgenic mice expressing human bcl-2, as the ordinary PT was reported to be sensitive to bcl-2 [24,25]. As a result, valinomycin caused acceleration of mitochondrial respiration in a similar manner to that observed with liver mitochondria of nontransgenic mice (data not shown). This result also supports the above conclusion that valinomycin is not an inducer of the ordinary PT. Permeability of inner mitochondrial membrane treated with valinomycin The preceding experiments clearly demonstrated that vali- nomycin is not an inducer of the ordinary PT, which is sensitive to CsA or BKA. However, as described above, recent studies showed the presence of another type of PT, one that is CsA insensitive; and thus it is difficult to conclude from these data that valinomycin is not a PT inducer. Thus, we used the following 2 strategies to examine the permeability of the inner mitochondrial membrane treated with valinomycin. First, when a PT is induced, changes in the configuration of the inner mitochondrial membrane take place, causing the disappearance of the inner membrane structure [19,26– 28], possibly as a result of a change in its permeability to solutes and ions. Thus, we examined the configuration of mitochondria that had been treated with valinomycin. As shown in Fig. 2A,B, the addition of Ca 2+ caused the disappearance of the inner mitochondrial membrane struc- ture, as is commonly observed during the induction of a PT by Ca 2+ [19,26–28]. Valinomycin caused a significant level of swelling of the mitochondrial matrix (Fig. 2C), but, different from the case of Ca 2+ -treated mitochondria, the structure of the mitochondrial inner membrane was retained. Thus, transmission electron microscopic analysis clearly showed that valinomycin did not induce the ordinary PT of inner mitochondrial membrane like that induced by Ca 2+ . Fig. 1. Effects of CsA and BKA on mitochondrial respiration and swelling in the presence of Ca 2+ , SF6847, and valinomycin. Panels A, B, and C represent effects of Ca 2+ , SF6847, and valinomycin, respectively, on the mitochondrial structure and function. Upper and lower traces show mitochondrial respiration and the optical absorbance of mitochondrial suspensions at 540 nm, respectively. In each panel, trace ÔaÕ represents the controlwithnoaddedCa 2+ , SF6847 or valinomycin; and trace ÔbÕ,theeffectof50l M Ca 2+ (A), 50 n M SF6847 (B) or 2.2 lgÆmL )1 valinomycin (C). Traces ÔcÕ and ÔdÕ show the effects of 600 n M CsA and 10 l M BKA, respectively.Rat liver mitochondria (RLM, 1.75 mg) were suspended in 2.5 mL of +P i medium (200 m M sucrose and 10 m M potassium phosphate buffer, pH 7.4) at 25 °C and energized by the addition of 10 m M succinate (+0.5 lgrotenoneÆmg mitochondrial protein )1 ) as a respiratory substrate. Time-dependent changes in mitochondrial oxygen con- sumption and turbidity of the mitochondrial suspension (absorbance at 540 nm) were monitored at 25 °C. 5226 Y. Shinohara et al. (Eur. J. Biochem. 269) Ó FEBS 2002 We next directly evaluated the permeability of the inner mitochondrial membrane by using poly(ethylene glycol). As reported previously [4,7] and shown in Fig. 3, when PT was induced by Ca 2+ , the addition of poly(ethylene glycol) 6000 caused mitochondrial shrinkage, reflecting the extrusion of matrix water via the formed PT pores. The effect of poly(ethylene glycol) 6000 was negligible when the pore was closed by the addition of CsA. Contrary to these findings, when poly(ethylene glycol) 6000 was added to the mito- chondria preswollen by valinomycin, no shrinkage was observed. Thus, we concluded that a PT is not induced at least at this stage. Release of mitochondrial cytochrome c induced by valinomycin Finally, we examined whether cytochrome c is released from mitochondria when they are treated with valinomycin. For this, mitochondria were first treated with Ca 2+ or valinomycin and then pelleted by centrifugation. Proteins released from and retained in mitochondria were then subjected to electrophoresis. After transfer of the proteins to a nitrocellulose membrane, cytochrome c was immunolo- gically detected by using anti cytochrome c antibody. The amounts of cytochrome c in various samples were deter- mined with reference to a calibration curve that had been generated from known amounts of standard cytochrome c. By this method, the amount of cytochrome c in the untreated rat liver mitochondria was determined to be 200 pmolÆmg )1 mitochondrial protein, which is in good agreement with previously reported values [29–31]. Ca 2+ caused a significant release of cytochrome c from the mitochondria (Fig. 4, Ca 2+ ), and the amount released was 34% of the total mitochondrial cytochrome c,whichis consistent with a previously reported value [32]. CsA and BKA were effective in inhibiting cytochrome c release induced by Ca 2+ however, SF6847 had little effect on the release (Fig. 4, SF). In contrast, valinomycin caused the release of cytochrome c at levels as high as about 60% of the total (Fig. 4, Val), whereas this release was insensitive to Fig. 3. Different effects of poly (ethylene glycol) 6000 on mitochondria preswollenbyCa 2+ or valinomycin. Mitochondria were first treated with Ca 2+ or valinomycin as shown in Fig. 1. Then, 1.1 mL of 300 mOsM solution of poly(ethylene glycol) 6000 was added. Time- dependent changes in the turbidity of mitochondrial suspensions were monitored as in Fig. 1. Effects of preaddition of CsA were also examined. Fig. 2. Transmission electron micrographs of mitochondria. Mitochondria were energized with succinate (plus rotenone) in +P i medium at 25 °C and treated with 100 l M Ca 2+ (B) or 0.8 lgÆmL )1 valinomycin (C) for 5 min. As a reference, mitochondria with no added Ca 2+ or valinomycin were also analyzed (A). Of approximately 30 randomly selected sections observed for each condition, typical images are shown. Ó FEBS 2002 Cytochrome c release induced by valinomycin (Eur. J. Biochem. 269) 5227 CsA and BKA. Accordingly, we concluded that valino- mycin caused the release of cytochrome c in an ordinary PT-independent manner. DISCUSSION It is well known that mitochondrial dysfunction and the subsequent release of mitochondrial proteins are tightly linked to the processes of programmed cell death, also called apoptosis. Valinomycin, a known K + -ionophore, has often been used to dissipate the membrane potential of cells and mitochondria. Although its activity as an apoptosis inducer is well known [13–15], the mechanism of its action is controversial; as it has been reported to function as either a non-PT-inducer [16] or PT-inducer [17]. As direct actions of valinomycin on isolated mitochondria had never been previously examined, in this study we examined the effects of the drug on isolated mitochondria to understand the reason for such discrepancies. Addition of valinomycin caused acceleration of mito- chondrial respiration and swelling, but these effects were insensitive to BKA or CsA, showing that valinomycin is not an inducer of the ordinary PT. As ordinary PT was reported to be sensitive to bcl-2 [24,25], we also examined the effects of valinomycin on the mitochondria of transgenic mice expressing human bcl-2. Our results clearly showed that effects of valinomycin were independent of the action of bcl-2. As described above, Furlong et al. [17], using whole pre-B cells, reported that valinomycin induced BKA-sensitive apoptosis, suggesting that valinomycin-induced apoptosis was due to the induction of PT. Contrary to their results, our results using isolated mitochondria clearly demonstra- ted that the effects of valinomycin were not related to the ordinary PT. The specific action site of BKA was estab- lished to be the ADP/ATP carrier existing at the inner mitochondrial membrane, and thus this protein is believed to be a component of the PT pore. However, to understand why such discrepant results could be observed, we should pay close attention to the unidentified side-effect(s) of BKA. We further examined possible changes in the permeability of the inner mitochondrial membrane by transmission electron microscopic analysis and by measuring the osmotic response of the inner membrane. Both of these experimental results supported our proposition that the actions of valinomycin were not effected via the ordinary PT. The status of the membrane structure under these conditions is still uncertain and currently under investigation. Recently, Salvioli et al. examined whether cytochrome c could be released from mitochondria when U937 cells were treated with valinomycin and concluded that valinomycin did not induce the release of cytochrome c from these organelles [33]. However, their findings were based only on the result of a confocal analysis, and the question of whether valinomycin had an inducing effect on cytochrome c release from mitochondria was not clearly answered. Thus, in this study, we examined the release of cytochrome c from isolated mitochondria by valinomycin. As a result, we found valinomycin to induce a significant release of mitochondrial cytochrome c; and this release was insensitive to ordinary PT inhibitors. Based on these results, we conclude that valinomycin induced the release of cytochrome c from mitochondria in an ordinary PT-independent manner. As released cytochrome c triggers the subsequent effector steps resulting in apoptosis, it is very important to under- stand how mitochondrial cytochrome c is released from mitochondria. Two types of mechanisms have been pro- posed for the release of cytochrome c across the mitocho- ndrial outer membrane that accompanies the induction of the mitochondrial PT, namely: (a) release via the rupture of the outer mitochondrial membrane [8,34,35], and (b) release from channels formed by proteins such as Bax or mitoch- ondrial porin (also referred to as the voltage-dependent anion channel, VDAC) [36,37]. At present, the question as to which of these mechanisms is used for the valinomycin- induced release of mitochondrial cytochrome c is still unanswered. However, our present results show that the release of mitochondrial cytochrome c, which is present on the outer surface of the inner membrane, could occur even under the condition in which a PT at the inner mitochondrial membrane was not induced. Furthermore, it is also noteworthy that the amount of cytochrome c Fig. 4. Effects of CsA and BKA on the release of cytochrome c from mitochondria induced by Ca 2+ and by valinomycin. During the meas- urement of respiration, aliquots of a mitochondrial suspension were placed in an Eppendorf centrifuge tube, and the mitochondria were separated by prompt centrifugation. Both the pellet (P), corresponding to 1.4 lg of mitochondria, and supernatant (S) containing cyto- chrome c released from 3.5 lg of mitochondria were subjected to Western analysis using the specific antibody against cytochrome c.A typical result obtained with three independent mitochondrial prepa- rations is shown. Numerical values shown under the immunostained bands represent the amounts of released cytochrome c relative to total mitochondrial cytochrome c (in percentage), determined from the intensities of immunoreactive protein bands for each set of experiments (mean values for three separate experiments). Lane M represents total mitochondrial proteins (1.4 lg). 5228 Y. Shinohara et al. (Eur. J. Biochem. 269) Ó FEBS 2002 released by valinomycin was larger than that induced by Ca 2+ . As the amount of the latter accorded well with the reported value, we can conclude that the effect of valino- mycin on the release of mitochondrial cytochrome c is much stronger than that of the ordinary PT inducer Ca 2+ , possibly reflecting the differences of their action mecha- nisms. Furthermore, even when we varied the concentration of valinomycin from 0.1 lgÆmL )1 to 5 lgÆmL )1 ,we observed essentially the same results (data not shown). 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