Microcin J25 induces the opening of the mitochondrial transition pore and cytochrome c release through superoxide generation Marı ´ a Niklison Chirou, Augusto Bellomio, Fernando Dupuy, Beatriz Arcuri, Carlos Minahk and Roberto Morero Departamento de Bioquı ´ mica de la Nutricio ´ n, Instituto Superior de Investigaciones Biolo ´ gicas (Consejo Nacional de Investigaciones Cientı ´ ficas y Te ´ cnicas—Universidad Nacional de Tucuma ´ n), Instituto de Quı ´ mica Biolo ´ gica ‘‘Dr. Bernabe Bloj,’’ San Miguel de Tucuma ´ n, Argentina Microcin J25 (MccJ25), a 21-amino acid antimicrobial peptide that is active against certain human pathogens such as Salmonella and Shigella [1], has an unusual lasso distinctive structure [2–4] and a dual mechanism of action. Microcin J25 inhibits transcription by obstructing the RNA polymerase secondary channel [5] and affects, independently, the cytoplasmic membrane of Escherichia coli and Salmonella enterica serovars [6]. In this regard, it was shown that MccJ25 disrupts the membrane integrity of S. enterica and therefore causes dissipation of its membrane electrical potential. In addition, MccJ25 inhibits respiratory enzymes such as NADH, succinate dehydrogenase and lactate dehydrogenase and alters the oxygen consumption rate in vivo and in vitro [7]. The fact that MccJ25 is a membrane-active peptide is also supported by studies carried out on liposomes [8]. Recently, the effect of MccJ25 on intact rat heart mitochondria was explored [9]. The peptide modifies the membrane permeability, displays a potent effect as inhibitor of thecomplex III and diminishes drastically the internal ATP level. Mitochondria play a vital role in the regulation of energy metabolism and cell death by apoptosis and necrosis. Mitochondrial function requires a continuous transmembrane potential, which depends on the generation of an electrochemical pro- ton gradient across the inner membrane. The loss of the mitochondrial membrane integrity induces the release of pro-apoptotic proteins [10–13]. On the one hand, the apoptotic cascade can be triggered by a Keywords antibiotics; Ca 2+ ; cytochrome c; microcin; mitochondria Correspondence R. Morero, Chacabuco 461, S.M. de Tucuma ´ n 4000, Argentina Fax: +54 0381 4248025 Tel: +54 0381 4248921 E-mail: rdmorero@fbqf.unt.edu.ar (Received 23 April 2008, revised 21 May 2008, accepted 12 June 2008) doi:10.1111/j.1742-4658.2008.06550.x Microcin J25, an antimicrobial lasso-structure peptide, induces the opening of mitochondrial permeability transition pores and the subsequent loss of cytochrome c. The microcin J25 effect is mediated by the stimulation of superoxide anion overproduction. An increased uptake of calcium is also involved in this process. Additional studies with superoxide dismutase, ascorbic acid and different specific inhibitors, such as ruthenium red, cyclo- sporin A and Mn 2+ , allowed us to establish a time sequence of events starting with the binding of microcin J25, followed by superoxide anion overproduction, opening of mitochondrial permeability transition pores, mitochondrial swelling and the concomitant leakage of cytochrome c. Abbreviations carboxy-DCF, 5-(-6)-carboxy-2¢,7¢-dichlorofluorescein; carboxy-H 2 DCFDA, 5-(-6)-carboxy-2¢,7¢-dichloro-dihydrofluorescein diacetate; carboxy- H 2 DCFH, 5-(-6)-carboxy-2¢,7¢-dichloro-dihydrofluorescein; CsA, cyclosporin A; DNP, 2,4-dinitrophenol; FITC, fluorescein isothiocyanate; MccJ25, microcin J25; MccJ25F*, fluorescent derivative of microcin J25; MTP, mitochondrial transition pore; ROS, reactive oxygen species; RR, ruthenium red; SOD, manganese-superoxide dismutase. 4088 FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS Ca 2+ -independent mechanism that involves the Bcl-2 protein family, but this is not necessarily associated with mitochondrial volume changes [14]. On the other hand, the opening of MTP could be activated by Ca +2 , resulting in mitochondrial swelling with the con- sequent loss of the electrochemical gradient and the uncoupling of oxidative phosphorylation [15]. As a result, cytochrome c and other proteins are released into the cytosol. The leakage of cytochrome c from mitochondria is considered to be an early critical event in apoptotic cascade induction, which ultimately leads to programmed cell death [16–19]. However, additional evidence from studies with intact cells and isolated mitochondria suggests that mitochondrial membrane permeability changes may also occur by some other mechanism [20]. In the present study we analysed the effect of MccJ25 on isolated heart mitochondria. Our results indicated that MccJ25 induces the overproduction of superoxide anions, thus increasing the mitochondrial inner membrane permeability and activation of the mitochondrial transition pore (MTP), resulting in swelling and cytochrome c release. Results Mitochondrial uptake of MccJ25 Mitochondrial uptake of MccJ25 was examined using the peptide fluorescent derivative MccJ25F*, which showed antibiotic activity and membrane gradient dis- sipater capability similar to the native peptide (data not shown). Addition of MccJ25F* to energized mito- chondria (in the presence of 10 mm succinate) resulted in the immediate uptake of the microcin analogue (Fig. 1). The uptake was rapid, with a maximal level reached within 20 min. To ensure that the uptake of MccJ25F* was not an artifact, we confirmed the results by determining binding displacement with native MccJ25 (see Fig. 1) and fluorescence in the mitochondrial pellet (results not shown). Studies car- ried out with nonenergized mitochondria (i.e. in the absence of succinate) showed a marked decrease in the uptake capability of MccJ25F * . Pretreatment of ener- gized mitochondria with either 200 lm vanadate (an ATPase inhibitor) or 100 lm 2,4-dinitrophenol (2,-DNP; anelectrochemical gradient dissipater) reduced the uptake of MccJ25F* by approximately 85%, suggesting that the uptake of MccJ25F* depends on both the energy of mitochondria and the mitochondrial proton membrane gradient, mainly determined by ATP level and provided by succinate oxidation, respectively. Effect of MccJ25 on the mitochondrial transition pore of energized mitochondria The addition of 20 lm MccJ25 induced the swelling of energized mitochondria (Fig. 2). The kinetics of swell- ing during a period of 5 h had a linear pattern charac- teristic of MTP induction, showing an initial rate of 7.02 4 light scattering ⁄ min · 10 4 . The initial rate of swelling increased as a function of MccJ25 concentra- tion (inset in Fig. 2) and became saturated at about 60 lm (results not shown). Similar results were obtained, although with reduced swelling, when the buffer was devoid of succinate. Furthermore, the pres- ence of 10 lm antimycin A inhibited completely the swelling, suggesting that the electronic flow through the respiratory chain stimulates the MccJ25 effect (results not shown). We next determined whether MccJ25 was able to induce the permeation of solutes with a relatively low molecular mass. To study this we loaded mitochondria with calcein-AM, which is con- verted into calcein by endogenous esterases. Loaded mitochondria were treated with increasing concentra- tions of MccJ25 and the release of calcein was studied. A marked calcein release, parallel to the increased swelling, was observed following the addition of 20 lm MccJ25 (Fig. 2). 0 5 10 15 20 25 30 35 Uptake (%) Time (min) 0 5 10 15 20 25 30 35 Fig. 1. Uptake of MccJ25 by isolated mitochondria. Energized mitochondria without pre-incubation ( ) or pre-incubated with 200 l M vanadate ( ), 100 lM 2,4-DNP (d)or5lM native MccJ25 (h), were suspended in 10 m M Tris–sodium phosphate buffer (pH 7.4), 230 m M mannitol, 70 mM sucrose, 3 mM HEPES, supple- mented with 10 m M succinate and 1 lM rotenone. Nonenergized mitochondria (.) were suspended in the same buffer but in the absence of succinate. Then, 1.4 l M MccJ25F* (final concentration) was added to the suspensions, which were incubated for different periods of time at 25 °C. The fluorescence incorporated by mito- chondria was plotted as a function of the incubation time. Results are expressed as mean ± SD of five independent experiments. M. Niklison Chirou et al. MccJ25 induces the opening of mitochondrial transition pore FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS 4089 In an attempt to identify the role played by the MTP in the swelling and permeability changes of mito- chondria induced by MccJ25, we tested the influence of the specific pore inhibitor cyclosporin A (CsA), a calcium chelator (EDTA), a calcium competitor (Mn 2+ ) and the calcium uniporter inhibitor ruthenium red (RR). As shown in Table 1, swelling and calcein release induced by MccJ25 were almost completely inhibited by 1 lm CsA. Additionally, 50 lm EDTA, 125 lm Mn 2+ and 2.5 lm RR also almost completely inhibited the swelling induced by MccJ25, strongly suggesting that the peptide was able to induce mito- chondrial swelling through the opening of the MTP mediated by the uptake of calcium through the uniport of calcium. On the other hand, KCN and antimycin A, both inhibitors of electron transport, also inhibited the swelling induced by MccJ25. The inhibition of swelling induced by ascorbic acid will be discussed later. As a positive control, the swelling and calcein release induced by calcium, and the inhibitory effect of CsA and RR, are also shown in Table 1, confirming previ- ous results [21,22]. Cytochrome c release An initial aim of this work was to study the influence of MccJ25 on the release of cytochrome c. Therefore, we investigated whether the indirect opening of MTP by MccJ25 was followed by the release of cyto- chrome c. The experimental results (inset of Fig. 3) showed that increasing amounts of cytochrome c were released, showing a positive linear correlation with the 0 50 100 150 250200 300 –0.4 –0.3 –0.2 –0.1 0.0 0.1 0 10203040 0 2 4 6 8 10 12 14 2 4 Light scattering (540 nm) Time (min) Δ Light scattering ·min –1 x 10 4 MccJ25 (µM) Fluorescence (AU) Fig. 2. Effect of MccJ25 on mitochondrial swelling. The time course of swelling (h) and calcein release (D) of energized mito- chondria induced by 20 l M MccJ25 was assessed by measuring the change of light scattering at 540 nm or of calcein fluorescence, respectively, of mitochondria suspended in buffer, as described in the Materials and methods. Controls of swelling ( ) and calcein release ( ) were performed by incubation in the absence of MccJ25. The data shown are representative of at least five sepa- rate studies. The inset shows mitochondrial swelling as a function of MccJ25 concentration. Table 1. Effect of different drugs on the swelling and calcein release from mitochondria induced by MccJ25. Swelling and calcein release of energized mitochondria were induced by 20 l M MccJ25 and 50 l M Ca +2 in the absence or presence of different inhibitors. ND, not determined. Mitochondrial swelling DLight scattering ⁄ min · 10 4 Calcein release (%) MccJ25 (20 l M) 7.02 ± 0.01 a 52.00 ± 1.00 +EDTA (50 l M) 1.19 ± 0.01 ND +Mn 2+ (125 lM) 0.01 ± 0.02 0.10 ± 0.10 +RR (2.5 l M) 0.28 ± 0.01 ND +CsA (1 l M) 0.30 ± 0.01 4.80 ± 1.00 +Ascorbic acid (0.5 m M) 0.09 ± 0.02 ND +Antimycin A (10 l M) 0.50 ± 0.02 ND +KCN (2 m M) 0.43 ± 0.10 ND Calcium (50 l M) 15.20 ± 0.02 54.50 ± 2.00 +CsA (1 l M) 0.10 ± 0.01 0.30 ± 0.01 +RR (2.5 l M) 0.25 ± 0.01 ND Control 0.03 ± 0.01 0.30 ± 0.10 a Results are expressed as means ± SD of three separate experiments. 0 50 100 150 200 0 4 8 12 16 0 10203040 0 2 4 6 8 10 12 Cyt c release (pmol·mg –1 protein) Cyt c release (pmol·mg –1 protein) Time (min) MccJ25 (µM) Fig. 3. Effect of MccJ25 on cytochrome c release from mitochon- dria. Isolated mitochondria were suspended in 100 m M potassium phosphate buffer (pH 7.4), 10 m M succinate, 1 lM rotenone, and incubated for different periods of time at 37 °C in the absence (h) and in the presence of 20 l M MccJ25 ( ). Cytochrome c was determined chromatographically, as described in the Materials and methods. Data are the mean ± SD of three independent experi- ments. The inset shows cytochrome c release as a function of the MccJ25 concentration after 2 h of incubation at 37 °C. MccJ25 induces the opening of mitochondrial transition pore M. Niklison Chirou et al. 4090 FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS concentration of MccJ25. This result agrees with the swelling effect induced by MccJ25. It should be noted that at 20 lm MccJ25 the release effect was time dependent. After a lag phase of approximately 50 min, the release of cytochrome c increased abruptly, reach- ing a plateau at about 3 h (Fig. 3). Effect of MccJ25 on the production rate of superoxide anions It was recently found in our laboratory that the effect of MccJ25 on the E. coli respiratory chain enzymes is mediated by increased superoxide production [6]. As it is widely accepted that the mitochondrial electron transport chain is the main source of superoxide anions, we decided to determine the effect of MccJ25 on the mitochondrial electron transport chain and the possible implications on the swelling and permeability changes. As shown in Fig. 4, the rate of O À 2 generation by the submitochondrial particles increased as a func- tion of time, reaching a plateau at just about 45 min (data not shown in Fig. 4). The effect was dependent on the MccJ25 concentration (inset of Fig. 4) and was almost completely inhibited by superoxide dismutase (SOD), ensuring the superoxide anion determination. To demonstrate and better understand the mechanism of the reactive oxygen species (ROS) overproduction induced by the antimicrobial peptide, MccJ25, we examined the effect on isolated mitochondria. The ROS production was monitored using a ROS-sensitive fluorescent probe [5-(-6)-carboxy-2¢,7¢-dichloro-dihy- drofluorescein diacetate (carboxy-H 2 DCFDA)]. The results shown in Fig. 5 indicate that 20 lm MccJ25 induced a clear increment of ROS production com- pared with the control experiment performed in the absence of the peptide. Indeed, the rate of ROS pro- duction in the presence of MccJ25 was three times higher than in the absence of this peptide. The maxi- mum ROS production rate was obtained after 30 min of exposure to antibiotic. Pretreatment of isolated mitochondria with antioxidants such as 0.5 mm ascor- bic acid or 0.5 mm a-tocoferol acetate almost com- pletely suppressed the ROS production induced by MccJ25. By contrast, pretreatment with 2.5 lm RR was unable to prevent the ROS overproduction induced by MccJ25. Effect of MccJ25 on NADPH oxidation Reactive oxygen species produced in mitochondria are inactivated by a set of protective enzymes, including SOD, glutathione peroxidase and glutathione reductase [23]. Glutathione reductase recycles oxidized glutathi- one to its reduced form, using electrons from the NADPH. As a consequence, the overproduction of superoxide anions induced by MccJ25 would implicate 0 5 10 20 3015 25 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 102030405060 0.0 0.4 0.8 1.2 O 2 – Production (nmoles) O 2 – Production (nmoles) Time (min) MccJ25 (µM) Fig. 4. Rate of O À 2 generation induced by MccJ25 in rat heart sub- mitochondrial particles. The rate of superoxide generation in the presence of 20 l M MccJ25 (d) or in the presence of 20 lM MccJ25 and manganese-SOD (300 unitsÆmL )1 )( ) was determined as described in the Materials and methods. Data are mean ± SD of three independent experiments. The rate of superoxide generation as a function of MccJ25 concentration is shown in the inset. 0 500 1000 1500 2000 2500 Fluorescence (AU) 0 5 10 15 20 25 30 35 Time (min) Fig. 5. Effects of MccJ25 on the generation of ROS in isolated mitochondria. A preparation of mitochondria, preloaded with the flu- orescence probe carboxy-H 2 DCFDA, was suspended in 10 mM Tris–potassium phosphate buffer (pH 7.4), 150 mM sucrose, 50 mM KCl, 1 lM rotenone (h), or in buffer containing 20 lM MccJ25 ( ). Pretreatment with 2.5 l M RR (s), 0.5 mM a-tocopherol acetate (d) or 0.5 m M ascorbic acid (Ñ) was also performed. The time course of 5-(-6)-carboxy-2¢,7¢-dichlorofluorescein (carboxy-DCF) fluores- cence (k ex , 485 nm; k em , 525 nm) was monitored after the addition of 10 m M succinate. The data shown are representative of at least five separate studies. M. Niklison Chirou et al. MccJ25 induces the opening of mitochondrial transition pore FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS 4091 oxidation of NADPH. As shown in Fig. 6, 20 lm MccJ25 markedly increased the oxidation of NADPH compared with a control in the absence of the peptide. EDTA, RR and ascorbic acid completely inhibited the effect of MccJ25. Surprisingly, CsA, a specific inhibitor of the pore, was unable to inhibit the effect of MccJ25, which strengthens the hypothesis that MccJ25 gener- ates ROS, regardless of the presence of calcium. Discussion Understanding the mechanisms of action of some types of proteins and peptides on the structural and func- tional state of mitochondria seems to be important in the development of apoptosis-regulating technologies, particularly for anticancer therapy, and for the design of new classes of antibiotics, taking into account a cer- tain similarity between mitochondria and bacteria. In this work we observed that mitochondrial uptake of MccJ25 proceeds very rapidly and in a concentration- dependent manner. The peptide inserts into the mem- brane, modifying its permeability and provoking conse- quently an electrical potential dissipation, as described previously [9]. Such an effect promotes the uncoupling of electron transport with a concomitant loss of the efficiency to reduce oxygen and an increase in superox- ide species and subsequently ROS generation. Interest- ingly, this MccJ25 effect is similar to that described recently for E. coli [6]. The membrane perturbations would induce a small increase in the internal calcium concentration, which in turn activates the uniporter of calcium, increasing even more the calcium influx and ROS production. Both the increase of intramitochond- rial calcium concentration and the opening of MTP trigger mitochondrial swelling, with the concomitant release of the apoptotic inducer cytochrome c [24]. The sequence of these events is schematically represented in Fig. 7 and ordered on a timescale according to the 0 20 40 60 80 100 –40 –35 –30 –25 –20 –15 –10 –5 0 5 Fluorescence (AU) Time (min) Fig. 6. Effect of MccJ25 on the mitochondrial NADPH level. Mito- chondria suspended (0.5 mgÆmL )1 ) in Tris–potassium phosphate buffer (pH 7.4), 10 m M succinate, 1 lM rotenone, 150 mM sucrose, 50 m M KCl were incubated in the absence (d) or in the presence of 20 l M MccJ25 without any pretreatment (D), or pretreated for 1 min with 1 l M CsA ( ), 2.5 lM RR (s), 0.1 mM EDTA (.)or 0.5 m M ascorbic acid (h). At different time-points NADPH intrinsic fluorescence was monitored at excitation and emission wave- lengths of 366 and 450 nm respectively. 60 min Cyt. r c elease Seg 15 min 45 min 8 min 5 min MccJ25 Fluidity increase Gradient dissipation Uncoupling O 2 – production ROS increase SOD Vit. C Vit. E NADPH Ca 2+ Calcium uniporter MTP opening Swelling RR EDTA CsA ADP, Mn 2+ Timescale events Uptake Fig. 7. Schematic representation of the time sequence steps triggered by MccJ25 acting on isolated heart mitochondria. MccJ25 induces the opening of mitochondrial transition pore M. Niklison Chirou et al. 4092 FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS kinetics of each process. The interaction between MccJ25 and the membrane is followed by membrane perturbation, superoxide and ROS overproduction, stimulation of the calcium uniporter, opening of the MTP and, finally, mitochondrial swelling with cyto- chrome c leakage. This sequence is supported by the effect of several specific inhibitors. The overproduction of superoxide and ROS is the first event induced by MccJ25 and is responsible for triggering the subsequent effects because the presence of ascorbic acid was suffi- cient to inhibit mitochondrial swelling (see Table 1). The kinetic of superoxide and ROS overproduction clearly demonstrates that the generation of this reactive species precedes MTP opening and mitochondrial swelling. The inability of RR and CsA, specific inhibi- tors of the calcium uniport and MTP respectively, to inhibit the increase of ROS strongly supports the above-mentioned hypothesis. The NADPH oxidation occurs prior to the opening of the MTP pore and is coupled to the transport of calcium from the intermem- brane space to the matrix because it is inhibited by RR but not by CsA. The mitochondrial swelling induced by MccJ25 was also inhibited by EDTA, Mn 2+ and RR, indicating that the influx of Ca 2+ to the mitochondrial matrix was necessary for activation of the MTP. These results indicate that the effect of MccJ25 on the respira- tory chain forces the opening of the MTP pore, which is mediated by an increase of the matrix calcium con- centration, with the concomitant release of cyto- chrome c. The fact that the mitochondrial swelling induced by MccJ25 was inhibited by antimycin A allowed us to presume that the peptide effect takes place only when the electron flow through the respiratory chain is oper- ating. In addition, and supporting this result, the swell- ing effect is highly elevated in activated mitochondria and completely inhibited by rotenone (complex I inhib- itor) in ‘not activated’ mitochondria. Alternatively, we could consider that in the presence of antimycin A the mitochondrial membrane potential cannot be built up and therefore mitochondria would not accumulate calcium, which is required for MTP induction. In conclusion, we have shown that MccJ25 has a mitochondrial deleterious effect that is associated with the induction of the MTP. Microcin J25 would target the site of anion superoxide generation, increasing the production of ROS. However, the results presented here do not clarify the mechanism by which MccJ25 induces ROS production. Superoxide can be produced at complex I and ⁄ or at complex III [25]. Our results clearly indicate that complex III is essentially impli- cated in the mechanism of superoxide production because the peptide effect was obtained in the presence of rotenone, a specific inhibitor of complex I. We dem- onstrated that ROS play a major role in mediating mitochondrial dysfunction induced by MccJ25. Taking into account the induced swelling and the cytochrome c release, we could hypothesize that MccJ25 behaves as an apoptotic agent. However, any disruption in the electrochemical gradient and ⁄ or oxidative phosphory- lation, resulting in a decrease of ATP production, could compromise the progression of this form of cell death because an energy requirement is clearly needed for apoptosome formation. Moreover, there is contro- versy about whether or not the mitochondria indeed swell during apoptosis. Some studies have reported observing mitochondrial swelling [26], whereas others have reported that swelling never occurred [27,28]. Recent investigations suggest that cytochrome c release in apoptosis was not caused by mitochondrial swelling [29]. Additional studies will be helpful to understand, in greater detail, the molecular basis of this effect, its biological significance and the possible peptide bio-applicability mainly in whole human cells. These studies are currently underway in our laboratory. Materials and methods Chemicals and reagents Calcein-AM, CsA, fluorescein isothiocyanate (FITC) and ATP were purchased from Sigma Chemical Co. (St Louis, MO, USA). Carboxy-H 2 DCFDA-SE was obtained from Molecular Probes Inc. (Eugene, OR, USA). All other reagents were of analytical grade or the purest available commercial form. Peptides synthesis and purification Microcin J25 was obtained from the supernatant of E. coli AB259 harboring pTUC200 and was purified according to the procedure previously reported [7]. This procedure yielded a preparation that appeared homogeneous in two different systems of analytical RP-HPLC [30,31]. A fluores- cent analog containing FITC (MccJ25F*) was prepared for mitochondrial uptake studies. A mutated peptide in which isoleucine of position thirteen was replaced with lysine was obtained and purified from the supernatant of E. coli DH5a harboring pI13K. The strain was generously provided by P. Vincent (INSIBIO, CONICET ⁄ UNT, S. M. Tucuma ´ n). The fluorescent peptide was obtained by incubation of the mutated peptide with FITC (1 : 3, w ⁄ w) for 2 h in alkaline medium and in darkness at room tem- perature (25 °C). The labeled peptide was purified by chro- matography on a hydrophobic C8 cartridge. The fraction eluted with 100% methanol was reduced under vacuum, resuspended in water and chromatographed on a C18 M. Niklison Chirou et al. MccJ25 induces the opening of mitochondrial transition pore FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS 4093 column using a Gilson HPLC system, being finally eluted with a 50 mm sodium phosphate buffer (pH 6.5) ⁄ methanol gradient. Peptide concentrations were determined by mea- surement of the absorbance at 278 nm [32]. Isolation of heart mitochondrial Winstar rats (250–300 g) were killed by CO 2 inhalation, in accordance with the European directive for protection of vertebrate animals for scientific research. Hearts were rap- idly removed and placed in 10 mL of ice-cold 5 mm HEPES buffer (pH 7.4), 200 mm mannitol, 10 m m sucrose, 1mm EDTA, 0.1% BSA. The tissue was finely minced with scissors and then homogenized using an Omni-Mixer (Sor- vall, Norwalk, CT, USA). The homogenate was centrifuged at 900 · g for 10 min, the pellet was discarded and the supernatant centrifuged again at 17 000 · g for 10 min. The pellet containing the mitochondria was resuspended in the isolation buffer without EDTA. To prepare submito- chondrial particles, the mitochondrial pellet was resus- pended (20 mgÆmL )1 )in50mm Tris–HCl (pH 7.6), 230 mm mannitol, 70 mm sucrose, and sonicated three times (each consisting of a 30-s pulse burst) at 1-min intervals, at 4 °C. The sonicated mitochondria were centrifuged at 8500 · g for 10 min to remove the unbroken organelles. The super- natant was centrifuged again at 100 000 · g for 60 min, and the resulting pellet was washed and resuspended in the same buffer [33]. The protein concentration was determined by the method of Lowry et al., with bovine albumin as the standard [34]. Mitochondrial uptake studies For mitochondrial uptake of MccJ25F*, mitochondria (1 mgÆmL )1 ) were suspended in 10 mm Tris–sodium phos- phate buffer (pH 7.4), 230 mm mannitol, 70 mm sucrose, 3mm HEPES, supplemented with 10 mm succinate and 1 lm rotenone, at 25 °C, then 1.4 lm MccJ25F* was added and the solution was incubated at 25 °C. Uptake was stopped at different time-points by centrifugation (12 000 · g, 5 min, 4 °C) and fluorescence of tyrosine (k ex : 277 nm, k em : 305 nm) and FITC (k ex : 490 nm, k em : 520 nm) in the supernatant was measured using an ISS (Champaign, IL, USA) PC1 spectrofluorometer at 25 °C. Uptake experiments were also determined in mitochondrial samples incubated previously with 200 mm vanadate, or 100 mm DNP, for 5 min at 37 °C. Mitochondrial swelling assay Isolated mitochondria (1 mgÆmL )1 ) were incubated in 10 mm Tris–sodium phosphate buffer (pH 7.4), 230 mm mannitol, 70 mm sucrose, 3 mm HEPES, supplemented with 10 mm succinate and 1 lm rotenone at 25 °C [27]. Different quantities of MccJ25 were added to the incuba- tion buffer. In additional experiments, inhibitors such as EDTA, Mn 2+ , CsA, KCN, antimycin A, ascorbic acid and RR were added before the addition of MccJ25. Calcium (50 lm) was used as a positive control. A control experiment in the absence of MccJ25 and Ca 2+ was also performed. Swelling was estimated from the changes of light scattering at 540 nm in a DU7500 spectrophotometer (Beckman, Fullerton, CA, USA) equipped with a peltier constant temperature chamber. The rate of swelling (Dlight scattering ⁄ min) was calculated from the slope of the initial linear portion of the curve. Calcein release from mitochondria Mitochondria isolated from rat heart were incubated at 25 °C for 30 min in the suspension buffer containing 2 lm calcein-AM and then washed and resuspended in the same buffer. To assess calcein release, loaded mitochondria (0.1 mgÆmL )1 ) were added to the assay buffer [5 mm Tris– HEPES buffer (pH 7.4), 250 mm sucrose, 0.1% BSA, 10 lm CoCl 2 ] and variations in the fluorescence were mea- sured using an ISS PC1 spectrofluorometer [22]. The excita- tion and emission wavelengths were 488 and 530 nm respectively. The buffer contained 10 mm CoCl 2 to quench the fluorescence of calcein released from mitochondria. The fluorescence value obtained in the presence of 0.2% Triton X-100 was considered as 100% leakage. Cytochrome c release Quantification of cytochrome c release from rat heart mito- chondria was performed as described by Crouser et al. [24], with minor modifications. Essentially, intact mitochondria (1 mgÆmL )1 ) suspended in 100 mm potassium phosphate buffer (pH 7.4), 10 mm succinate and 1 lm rotenone were incubated at 37 °C with MccJ25. At different time-points, aliquots were centrifuged (30 000 g, 10 min, 4 °C) and the supernatant was analyzed using HPLC (Gilson HPLC equipped with a UV-VIS detector) through a C8 reverse- phase analytical column (Waters XTerra MS C8 5 l m, 4.6 · 250 mm) preceded by a guard column. A linear gradi- ent, increasing from 20 to 60% acetonitrile in water, was employed. Both the 20% and the 60% acetonitrile solutions also contained 100 mm KCl and 0.1% trifluoroacetic acid (v ⁄ v). The eluted cytochrome c was detected at 393 nm. Cytochrome c concentrations were calculated from a standard measurement. Superoxide anion radical generation The rate of O À 2 generation by submitochondrial particles was measured as reduction of acetylated ferricytochrome c, which is an excellent quantitative trap for O À 2 [35]. The MccJ25 induces the opening of mitochondrial transition pore M. Niklison Chirou et al. 4094 FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS reduction was followed spectrophotometrically at 550 nm in a Beckman DU 7500 at 25 °C. The reaction mixture contained 100 mm potassium phosphate buffer (pH 7.4), 10 lm acetylated ferricytochrome c,1lm rotenone, small mitochondrial particles (0.5 mg of proteinÆmL )1 ) and 20 lm MccJ25. The reaction was started by the addition of 10 mm succinate. We also performed experiments in which the effect of MccJ25 was studied after pre-incubation for 5 min with SOD. Reactive oxygen species production Reactive oxygen species production was monitored using the ROS-sensitive fluorescent probe, 5-(-6)-carboxy-2¢, 7¢-dichloro-dihydrofluorescein diacetate (carboxy-H 2 DCFDA). Once in the mitochondria, the acetate groups are cleaved by nonspecific esterases, hence the nonfluorescent 5-(-6)- carboxy-2¢,7¢-dichloro-dihydrofluorescein (carboxy-H 2 DCFH) is trapped inside. This probe was selected because is well retained not only in cells but also in mitochondria [36]. Oxidation of carboxy-H 2 DCFH by ROS yields carboxy- DCF. This fluorescent product indirectly measures the O À 2 produced that has dismutated to H 2 O 2 through the action of endogenous Mn 2+ -dependent SOD. The suspension of mitochondria (10 mgÆmL )1 ) was incubated with carboxy- H 2 DCFDA for 30 min at 30 °C and then washed twice with 10 mm Tris–sodium phosphate buffer (pH 7.4), 150 mm sucrose and 50 mm KCl to eliminate nonincorpo- rated probe. To monitor ROS production, carboxy- H 2 DCFH-loaded mitochondria were suspended (0.1 mgÆmL )1 )in10mm Tris–sodium phosphate buffer (pH 7.4), 150 mm sucrose, 50 mm KCl, 10 mm succinate, 1 lm rotenone, and the variation in fluorescence was followed using an ISS PC1 spectrofluorometer at 25 °C after the addition of MccJ25. The excitation and emission wavelengths were 490 and 520 nm respectively. Determination of NADPH oxidation Mitochondrial pyridine nucleotides were monitored, in an ISS PC1 spectrofluorometer at 25 °C, by measuring their intrinsic fluorescence at 450 nm after exciting at 340 nm [37]. Mitochondria were suspended (0.1 mgÆmL )1 )in10mm Tris–potassium phosphate buffer (pH 7.4), 10 mm succi- nate, 1 lm rotenone, 150 mm sucrose and 50 mm KCl. The suspension was pre-incubated for 1 min, and the oxidation of NADPH was started by adding 20 lm MccJ25 or 50 lm Ca 2+ . 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FITC, fluorescein isothiocyanate; MccJ25, microcin J25; MccJ25F*, fluorescent derivative of microcin J25; MTP, mitochondrial transition pore; ROS, reactive