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Diamide accelerates opening of the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria
Accepted Manuscript + 2+ Diamide accelerates opening of the Tl -induced permeability transition pore in Ca loaded rat liver mitochondria Sergey M. Korotkov, Svetlana A. Konovalova, Irina V. Brailovskaya PII:
S0006-291X(15)30792-0
DOI:
10.1016/j.bbrc.2015.10.091
Reference:
YBBRC 34775
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 8 October 2015 Accepted Date: 19 October 2015
Please cite this article as: S.M. Korotkov, S.A. Konovalova, I.V. Brailovskaya, Diamide accelerates + 2+ opening of the Tl -induced permeability transition pore in Ca -loaded rat liver mitochondria, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.10.091. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Diamide accelerates opening of the Tl+-induced permeability transition pore in
Ca2+-loaded rat liver mitochondria Sergey M. Korotkov* · Svetlana A. Konovalova · Irina V. Brailovskaya
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Sechenov Institute of Evolutionary Physiology and Biochemistry, the Russian Academy of Sciences, Thorez pr. 44, 194223 St. Petersburg, Russia Keywords: Tl+ · Diamide · Glutathione oxidation · Mitochondrial permeability transition · Adenine nucleotide translocase · Rat liver mitochondria.
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* Corresponding author. E-mail address: [email protected] (S.M. Korotkov).
ACCEPTED MANUSCRIPT Abstract
Opening of the mitochondrial permeability transition pore (MPTP) in the inner membrane is due to matrix Ca2+ overload and matrix glutathione loss. Fixing the ‘m’ conformation of the adenine nucleotide translocase (ANT) by ADP or N-ethylmaleimide (NEM) inhibits opening of the MPTP.
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Oxidants (diamide or tert-butylhydroperoxide (tBHP)) fix the ANT in ‘c’ conformation, and the ability of ADP to inhibit the MPTP is thus attenuated. Earlier we found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria resulted in Tl+-induced MPTP opening, which was
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accompanied by a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration, as well as increased swelling and membrane potential dissipation. These effects, which were increased by
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diamide and tBHP, were visibly reduced in the presence of the MPTP inhibitors (ADP, NEM, and cyclosporine A). Our data suggest that conformational changes of the ANT and matrix glutathione loss may be directly involved in opening the Tl+-induced MPTP in the inner membrane of Ca2+-
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loaded rat liver mitochondria.
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ACCEPTED MANUSCRIPT Introduction
It is known that matrix Ca2+ overload induces opening of the mitochondrial permeability transition pore (MPTP) which in turn leads to a potential membrane (∆Ψmito) drop, mitochondrial swelling, cytochrome c release in the intermembrane space, and loss of matrix solutes, including
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glutathione (GSH), NAD(P)H, pyridine and adenine nucleotides [1,2]. It is now believed that cyclophilin D (CyP-D) and mitochondrial phosphate carrier (PiC) are the main components of the MPTP, whereas adenine nucleotide translocase (ANT) interacting closely with the first two may be
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attributed to the regulatory part of the pore [1,3-5]. ADP fixing of the ANT in ‘m’ conformation decreases the sensitivity of calcium trigger sites to Ca2+ and inhibition of the MPTP consequently
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occurs [1,6]. On the contrary, diamide (GSH oxidant) and tBHP (oxidative stress inducer) fix the ANT in ‘c’ conformation which increases both Cyp-D binding with specific sites of the inner mitochondrial membrane (IMM) and sensitivity of the pore opening to Ca2+ [1,2,6-8]. Arsenite-, tBHP-, or diamide-induced MPTP with a subsequent increase in mitochondrial swelling, ∆Ψmito disruption, and affinity of the calcium binding site to Ca2+ were inhibited by the membrane-penetrant
‘m’ conformation [6-9].
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hydrophobic thiol reagent, N-ethylmaleimide (NEM) which reacts with Cys56 and fixes the ANT in
The swelling of rat liver mitochondria (RLM) in nitrate media demonstrated that the IMM displays low permeability to K+ but a higher permeability to Tl+ [10-12]. Calcium-loaded RLM
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(CaRLM) in medium containing TlNO3 and KNO3 showed that Tl+-induced MPTP opening is accompanied by increased mitochondrial swelling and ∆Ψmito dissipation, as well as decreased state 4,
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state 3 and 2,4-dinitrophenol (DNP)-uncoupled respiration [13]. We recently showed that Tl+ weak interacted with the IMM thiol groups, but it very likely formed complexes with soluble matrix proteins of RLM [14]. Diamide converted selectively GSH to its disulfide form (GSSG) and promoted S-glutathionylation which leads to the formation of protein–glutathione (PSSG) mixed disulfides resulting from the reaction of diamide with SH groups of proteins [15]. However, we are still uncertain about the possible joint effect of the ANT conformation and matrix GSH in the Tl+induced MPTP opening. Our aim was therefore to study the effect of diamide, compared with tBHP, on the ability of ADP and NEM to inhibit Tl+-induced MPTP opening in the inner membrane of CaRLM. We investigated swelling, basal, state 4, state 3 and DNP-stimulated respiration, and ∆Ψmito 3
ACCEPTED MANUSCRIPT dissipation of CaRLM, injected into medium containing TlNO3, KNO3, succinate, rotenone as well as diamide and tBHP (where indicated) and MPTP inhibitors (ADP, cyclosporine A (CsA), and NEM).
Materials and methods
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Animals
Male Wistar rats (250-300 g) were kept at 20–23 ºC under 12-h light/dark cycle with free
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access to water ad libitum and the standard rat diet. All treatment procedures of animals were performed according to the Animal Welfare act and the Institute Guide for Care and Use of
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Laboratory Animals.
Isolation of mitochondria
Liver mitochondria were isolated according to [16] in a buffer containing 250 mM sucrose, 3
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mM Tris-HCl (pH 7.3), and 0.5 mM ethylene glycol tetraacetic acid (EGTA); next mitochondrial pellet was washed out twice by resuspension-centrifugation in a buffer containing 250 mM sucrose and 3 mM Tris-HCl (pH 7.3) and finally suspended in 1 ml of the latter buffer. The mitochondrial
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protein content assayed according to Bradford was within 50-60 mg/ml.
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Swelling of mitochondria
Mitochondrial swelling (Figs. 1 and 2) was measured as a decrease in A540 at 20°C using a SF-
46 spectrophotometer (LOMO, St. Petersburg, Russia). Mitochondria (1.5 mg protein/ml) were administrated into a 1-cm cuvette with 1.5 ml of 400 mOsm medium containing 75 mM TlNO3, 125 mM KNO3, 5 mM succinate, 5 mM Tris-NO3 (pH 7.3), 2 µM rotenone, and 1 µg/ml of oligomycin. Diamide, Ca2+, NEM, ADP, and CsA were added into the medium before or after mitochondria (see Figs. 1 and 2 legends). The swelling, oxygen consumption rates, and ∆Ψmito were researched in 400
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Oxygen consumption assay
Respiration (oxygen consumption rate) was measured polarographically using Expert-001
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analyzer (Econix-Expert Ltd., Moscow, Russia) in a 1.3-ml closed thermostatic chamber with magnetic stirring at 26 °C. Mitochondria (1.5 mg protein/ml) were added to 400 mOsm medium
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containing 25 mM TlNO3, 100 mM sucrose, 3 mM Mg(NO3)2, and 3 mM Tris-Pi (Fig. 3a) or 75 mM TlNO3 and 1 µg/ml of oligomycin (Fig. 3b) as well as 125 mM KNO3, 5 mM Tris-NO3 (pH 7.3), 5 mM succinate, and 2 µM rotenone. Diamide, Ca2+, NEM, ADP, and CsA were added in the medium before or after mitochondria (see Fig. 3 legend). ADP of 130 µM (Fig. 3a) and DNP at 30 µM (Fig. 3)
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were correspondingly administrated into the medium after 2 min recording of state 4 or basal state to induce state 3 and DNP-stimulated respiration.
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Mitochondrial membrane potential
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The ∆Ψmito induced in succinate-energized RLM (Fig. 4) was estimated according to
Waldmeier et al. [17] by the intensity of safranin fluorescence (arbitrary units) in the mitochondrial suspension with magnetic stirring at 20 °C using a Shimadzu RF-1501 spectrofluorimeter (Shimadzu, Japan) at 485/590 nm wavelength (excitation/emission). Mitochondria (0.5 mg protein/ml) were placed into a quartz cuvette of four clear walls with 3 ml of a medium containing 20 mM TlNO3, 125 mM KNO3, 110 mM sucrose, 5 mM Tris-NO3 (pH 7.3), 1 mM Tris-Pi, 3 µM safranin, 2 µM rotenone, and 1 µg/ml of oligomycin. In addition, the next chemicals were injected in the medium before
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mitochondria: diamide, ADP, and CsA (where indicated). Succinate, Ca2+, and DNP were administrated into the medium after mitochondria (see Fig. 7 legend).
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Chemicals
CaCl2, Mg(NO3)2, H3PO4, KNO3, TlNO3, and DNP were of analytical grade from Nevareactiv (St. Petersburg, Russia). Rotenone, oligomycin, diamide, NEM, tris-OH, EGTA, ADP, CsA, and succinate were from Sigma (St. Louis, MO, USA). Sucrose as 1 M solution was refined from cation
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traces on a column filled with a KU-2-8 resin from Azot (Kemerovo, Russia).
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Results
The swelling of mitochondria in media containing KNO3 and TlNO3 permits studying the movement of K+ and Tl+ across the IMM, which is easily permeable to NO3- [10-12]. Succinateenergized RLM free of additions swelled weakly in medium containing TlNO3 and KNO3 (Figs. 1 and
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2). Swelling of succinate-energized mitochondria in the TlNO3 medium increased at 50-100 µM of diamide and especially at 200-500 µM (Fig. 1). The swelling induced by 100-500 µM tBHP (dotted trace) was less than with 100-500 µM diamide (Fig. 1). The swelling of energized CaRLM (control
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trace) in TlNO3 medium increased in series of 25-50 µM diamide < 500 µM tBHP (dotted trace) < 100 µM diamide (Fig. 2A). ADP completely inhibited the Ca2+-induced swelling (control trace) in
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experiments with 25 µM tBHP (dotted trace) and partly at 25 µM diamide (Fig. 2B). Figure 2C illustrates that ADP or NEM, but not CsA, partly inhibited 50 µM diamide-induced swelling of CaRLM. Binary combinations (ADP, CsA, and NEM) caused maximal inhibition of the swelling, which decreased in a lot of CsA > control > ADP > NEM > CsA + NEM > ADP + CsA > ADP + NEM. State 3, state 4, and DNP-stimulated respiration of RLM in medium containing 25 mM TlNO3 and 125 mM KNO3 was not affected by 50-200 µM diamide, except for a slight decrease in DNPstimulated respiration at 200 µM diamide (Fig. 3A). Basal respiration (after a short-term burst) and DNP-stimulated respiration decreased after injection of Ca2+ into medium containing 75 mM TlNO3
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ACCEPTED MANUSCRIPT and 125 mM KNO3 (Fig. 3B). The decrease in respiration was more visible in the presence of 50-100 µM diamide (Fig. 3B). If ADP plus CsA or NEM alone were injected before diamide, the Ca2+induced decrease was markedly eliminated (Fig. 3B). Figure 4 (non Ca2+ trace) shows ∆Ψmito-driven safranine uptake into succinate-energized RLM added to the medium containing 20 mM TlNO3 and 125 mM KNO3. The drop in the ∆Ψmito for some traces immediately after Ca2+ administration is
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caused by the energy-dependent uptake of Ca2+ by the RLM. Subsequent ∆Ψmito dissipation (control trace) after some delay was due to the Tl+-induced MPTP opening [13]. Diamide induced more visible Ca2+-induced ∆Ψmito dissipation, which was completely inhibited by ADP plus CsA (Fig. 4).
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The effects of 50 µM tBHP on mitochondrial respiration and ∆Ψmito dissipation were the same as the
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above effects of diamide (not shown).
Discussion
It has been shown that diamide, in comparison to tBHP, caused more extensive protein-protein and protein-glutathione mixed-disulfide formation which was accompanied by extensive conversion
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of GSH to its disulfide form in experiments with HEK, HeLa, and cultured aortic endothelial cells, as well as neonatal rat heart cell cultures, rat erythrocytes, and human platelets [15,18-21]. The MPTP opening, perturbation of IMM, and decrease of ∆Ψmito caused by Ca2+ plus diamide or Ca2+ plus tBHP were associated with protein polymerization due to thiol cross-linking and the production of high
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molecular mass protein aggregates [9,22,23]. It was previously proposed that the formed ligand complexes of Tl+ with SH groups of soluble matrix mitochondrial proteins may alter the spatially
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structural integrality of IMM, with which these proteins are coupled [14,24]. Tl+ and Tl3+ enhanced the production of ROS and H2O2, stimulated lipid peroxidation, and decreased the intracellular and matrix glutathione in experiments with PC12 cells, isolated hepatocytes, and long-term incubated RLM. However, the biochemical mechanisms of these effects are still unclear [25,26]. We have previously suggested that Tl3+ could be formed by the reaction of Tl+ with ROS generated near mitochondrial respiratory chain complexes [27]. Mitochondrial swelling induced by Zn2+ was stimulated by diamide, tBHP, and monobromobimane [28]. Diamide, more than tBHP, affected Ca2+ retention in rat liver mitoplasts [22]. tBHP has induced swelling of RLM only in the presence of exogenous Ca2+, whereas diamide was effective in its absence [29]. Thus, greater swelling of 7
ACCEPTED MANUSCRIPT energized RLM in experiments with diamide, rather than tBHP, (Figs. 1-2) is suggested to be due to oxidation of GSH by diamide and Tl3+. This oxidation could increase membrane permeabilization resulting from the aggregation of mitochondrial proteins with Tl+ and the formation of disulfide bridges between vicinal protein thiol groups of mitochondrial enzymes, including the ANT [9,27,30]. The calcium overload of mitochondria and cross-linkage of Cys159 and Cys256 by diamide or
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tBHP fixed the ANT in ‘c’ conformation which increased both Cyp-D binding with the IMM-specific sites and the sensitivity of pore opening to Ca2+ [1,6-8]. As a result, a decrease was noted in the ability of ADP to inhibit the diamide- or tBHP-induced MPTP opening, swelling and ∆Ψmito
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dissipation in succinate-energized CaRLM. This has given reason to believe that the ANT is involved in pore formation [1,7,23,31]. We have found that bongkrekic acid (BKA) fixing the ANT in ‘m’
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conformation inhibited Tl+-induced MPTP opening, whereas carboxyatractyloside that fixed the translocase in ‘c’ conformation attenuated the ability of ADP to inhibit the pore formation (not shown here). So, inhibition of swelling by ADP at 25 µM of diamide or tBHP (Fig. 2b) gives us reason to assume that the ANT conformation can be involved in the Tl+-induced MPTP opening. On the other hand, ANT can be transformed from an antiporter to a non-selective porter by increasing CyP-D
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binding with the IMM [6,7]. The swelling of non-energized RLM in medium free of Ca2+ was attenuated by ADP in our experiments with Y3+ and La3+ [32] or Tl+ alone [13] indicating that ANT participates in regulating inner membrane ion permeability. Thus, a second reason for the increase in the diamide-induced swelling (Fig. 1) may be enhancement of the IMM ion permeability, caused by a
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reaction of diamide with SH groups of the ANT [9,23].
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It is known that CsA prevents reaction of Cyp-D with the matrix surface and thus inhibits MPTP opening [1,6]. Diamide enhanced the binding which was partially prevented by CsA [6,7]. ADP, BKA, and CsA inhibited diamide- and tBHP-induced swelling, decreased calcium retention, ∆Ψmito drop, and cytochrome c release from RLM [29,30,34]. Earlier we found [13] that the Tl+induced MPTP opening in CaRLM manifested as increased swelling and decreased ∆Ψmito, as well as decreased mitochondrial respiration. These effects [13] similar to those in experiments with diamide and CaRLM (Figs. 2C, 3, and 4) declined in the series of controls, CsA > ADP >> ADP + CsA. NEM attacking Cys56 at the ANT in ‘m’ conformation inhibited swelling, ∆Ψmito dissipation, and MPTP opening of Ca2+-loaded mitochondria in experiments with thiol reagents (tBHP, diamide, arsenite, menadione, and PAO) [6-9]. We can therefore propose that a decrease by NEM of diamide-induced 8
ACCEPTED MANUSCRIPT swelling in CaRLM (Fig. 2C) and the additive effect of ADP or NEM in the presence of CsA in inhibiting the Tl+-induced MPTP (Figs. 2C, 3, and 4) is possible when the ANT is in ‘m’ conformation [1,6,9]. Diamide enhanced As2O3-induced apoptosis in promyelocytic leukemia NB4 cells [35]. Diamide and tBHP induced cross-linkage of mitochondrial proteins that led to a disorder of the respiratory chain, at the level of the coenzyme Q [36]. A decrease in DNP-uncoupled respiration
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caused by diamide (Fig. 3) indicates a disturbance in the respiratory chain, which can favor MPTP opening [37]. One may therefore suggest that another reason for the more potent swelling of succinate-energized RLM in the medium containing TlNO3 and KNO3 as well as diamide and tBHP
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(Fig. 1) may be due to oxidative stress caused by these oxidizers. Ultimately, our experiments suggest that the stronger action of diamide, compared to tBHP, on mitochondria in TlNO3 media can result in
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diamide-induced matrix glutathione loss, accompanied by aggregation of mitochondrial proteins, and interconnected with the ANT in ‘c’ conformation fixing. The use of K+ surrogate Tl+ in this model of the Tl+-induced MPTP can help in the search for the mitochondrial transition pore of new inhibitors and inducers among different chemical and natural compounds.
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Acknowledgments
Authors are grateful to Ms Terttu Kaustia for correcting the English. Purchase of diamide was financed by a grant from the Russian Foundation for Basic Research to Diana Gutzaeva (#09-04-
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01439); the funding organization does not have control over the resulting publication.
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Conflict of interest
No conflicts of interest, financial or otherwise, are declared by the authors.
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ACCEPTED MANUSCRIPT Figure legends
Fig. 1 The influence of diamide and tert-butylhydroperoxide on swelling of succinate-energized rat liver mitochondria. Mitochondria (1.5 mg/ml of protein) were injected to the medium containing 75 mM TlNO3, 125 mM KNO3, 5 mM Tris-succinate, 5 mM Tris-NO3 (pH 7.3), 2 µM rotenone, and 1
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µg/ml of oligomycin. Additions (µM) of diamide and tert-butylhydroperoxide (dashed trace) before mitochondria are indicated on the right of the traces. Addition of mitochondria (RLM) is shown by arrow. The apparent absorbance change (Figs. 1-2) was within 4% (p < 0.05). Representative traces
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from one of three independent experiments are presented.
Fig. 2 Effects of diamide, tert-butylhydroperoxide, and Ca2+ on Tl+-induced swelling of succinate-
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energized rat liver mitochondria in the presence of ADP. Mitochondria (1.5 mg/ml of protein) were administrated to medium containing 75 mM TlNO3, 125 mM KNO3, 5 mM Tris-succinate, 5 mM Tris-NO3 (pH 7.3), 2 µM rotenone, and 1 µg/ml of oligomycin. Additions (µM) of diamide and tertbutylhydroperoxide (dashed trace) before mitochondria are indicated on the right of the traces. Additions before mitochondria are indicated to right of traces: free of additions (none Ca2+, in bold), 100 µM Ca2+ alone (control, in bold), 500 µM ADP (ADP), 50 µM NEM (NEM), and 1 µM CsA
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(CsA). Additions of mitochondria (RLM) and 100 µM Ca2+ (Ca2+) are shown by arrows. Typical traces from one of three independent different mitochondrial preparations are presented.
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Fig. 3 Influence of diamide and Ca2+ on oxygen consumption rates of rat liver mitochondria. Mitochondria (1.5 mg/ml of protein) were injected in medium containing 25 mM TlNO3, 100 mM
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sucrose, 3 mM Tris-Pi, and 3 mM Mg(NO3)2 (panel A) or 75 mM TlNO3, and 1 µg/ml of oligomycin (panel B) as well as 125 mM KNO3, 5 mM Tris-NO3 (pH 7.3), 5 mM succinate and 2 µM rotenone. Additions of mitochondria (RLM), 0-200 µM diamide (diamide), 100 µM Ca2+ (Ca2+), and 30 µM DNP (DNP) are correspondingly showed by vertical arrows, inclined long arrows, inclined long bold arrows, and inclined bold arrows. Inclined arrows show additions of 130 µM ADP (ADP), 500 µM ADP (ADP(2)), 1 µM CsA (CsA), and 50 µM NEM (NEM). The numbers on the right of the traces show concentrations of diamide (µM). Oxygen consumption rates (ng atom O min/mg of protein) are presented as numbers placed above experimental traces. The rates' deviation was within 5% (p < 0.05). Representative traces from one of three independent experiments are presented. 14
ACCEPTED MANUSCRIPT Fig. 4 Effects of diamide and Ca2+ on the inner membrane potential (∆Ψmito) of rat liver mitochondria in the nitrate medium. Mitochondria (0.5 mg/ml of protein) were added to medium containing 20 mM TlNO3, 125 mM KNO3, 110 mM sucrose, 5 mM Tris-NO3 (pH 7.3), 1 mM Tris-Pi, 3 µM safranin, 2 µM rotenone, and 1 µg/ml of oligomycin. Additions of mitochondria (RLM) and 5 mM succinate
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(Succ) are shown by arrows. Injections of 75 µM Ca2+ and 20 µM DNP are correspondingly marked by up-directed arrows and up-directed bold arrows. Additions before mitochondria are indicated on the right of the traces: 50 µM diamide (diamide), 500 µM ADP (ADP), and 1 µM CsA (CsA). The
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apparent absorbance change was within 8% (p < 0.05). Representative traces from one of three
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• ANT conformation and glutathione loss in opening Tl+-induced pore is proposed. • Thiol oxidants (diamide and tBHP) fixing the ANT in ‘c’ conformation researched. • Ability of ADP to inhibit the pore decreased in the presence of the oxidants. • N-ethylmaleimide (NEM) fixing the ANT in ‘m’ conformation inhibited the pore opening.
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• The pore inhibition by ADP or NEM was considerably stronger in the presence of CsA.
S0006-291X(15)30792-0
DOI:
10.1016/j.bbrc.2015.10.091
Reference:
YBBRC 34775
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 8 October 2015 Accepted Date: 19 October 2015
Please cite this article as: S.M. Korotkov, S.A. Konovalova, I.V. Brailovskaya, Diamide accelerates + 2+ opening of the Tl -induced permeability transition pore in Ca -loaded rat liver mitochondria, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.10.091. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Diamide accelerates opening of the Tl+-induced permeability transition pore in
Ca2+-loaded rat liver mitochondria Sergey M. Korotkov* · Svetlana A. Konovalova · Irina V. Brailovskaya
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Sechenov Institute of Evolutionary Physiology and Biochemistry, the Russian Academy of Sciences, Thorez pr. 44, 194223 St. Petersburg, Russia Keywords: Tl+ · Diamide · Glutathione oxidation · Mitochondrial permeability transition · Adenine nucleotide translocase · Rat liver mitochondria.
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* Corresponding author. E-mail address: [email protected] (S.M. Korotkov).
ACCEPTED MANUSCRIPT Abstract
Opening of the mitochondrial permeability transition pore (MPTP) in the inner membrane is due to matrix Ca2+ overload and matrix glutathione loss. Fixing the ‘m’ conformation of the adenine nucleotide translocase (ANT) by ADP or N-ethylmaleimide (NEM) inhibits opening of the MPTP.
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Oxidants (diamide or tert-butylhydroperoxide (tBHP)) fix the ANT in ‘c’ conformation, and the ability of ADP to inhibit the MPTP is thus attenuated. Earlier we found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria resulted in Tl+-induced MPTP opening, which was
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accompanied by a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration, as well as increased swelling and membrane potential dissipation. These effects, which were increased by
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diamide and tBHP, were visibly reduced in the presence of the MPTP inhibitors (ADP, NEM, and cyclosporine A). Our data suggest that conformational changes of the ANT and matrix glutathione loss may be directly involved in opening the Tl+-induced MPTP in the inner membrane of Ca2+-
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loaded rat liver mitochondria.
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ACCEPTED MANUSCRIPT Introduction
It is known that matrix Ca2+ overload induces opening of the mitochondrial permeability transition pore (MPTP) which in turn leads to a potential membrane (∆Ψmito) drop, mitochondrial swelling, cytochrome c release in the intermembrane space, and loss of matrix solutes, including
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glutathione (GSH), NAD(P)H, pyridine and adenine nucleotides [1,2]. It is now believed that cyclophilin D (CyP-D) and mitochondrial phosphate carrier (PiC) are the main components of the MPTP, whereas adenine nucleotide translocase (ANT) interacting closely with the first two may be
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attributed to the regulatory part of the pore [1,3-5]. ADP fixing of the ANT in ‘m’ conformation decreases the sensitivity of calcium trigger sites to Ca2+ and inhibition of the MPTP consequently
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occurs [1,6]. On the contrary, diamide (GSH oxidant) and tBHP (oxidative stress inducer) fix the ANT in ‘c’ conformation which increases both Cyp-D binding with specific sites of the inner mitochondrial membrane (IMM) and sensitivity of the pore opening to Ca2+ [1,2,6-8]. Arsenite-, tBHP-, or diamide-induced MPTP with a subsequent increase in mitochondrial swelling, ∆Ψmito disruption, and affinity of the calcium binding site to Ca2+ were inhibited by the membrane-penetrant
‘m’ conformation [6-9].
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hydrophobic thiol reagent, N-ethylmaleimide (NEM) which reacts with Cys56 and fixes the ANT in
The swelling of rat liver mitochondria (RLM) in nitrate media demonstrated that the IMM displays low permeability to K+ but a higher permeability to Tl+ [10-12]. Calcium-loaded RLM
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(CaRLM) in medium containing TlNO3 and KNO3 showed that Tl+-induced MPTP opening is accompanied by increased mitochondrial swelling and ∆Ψmito dissipation, as well as decreased state 4,
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state 3 and 2,4-dinitrophenol (DNP)-uncoupled respiration [13]. We recently showed that Tl+ weak interacted with the IMM thiol groups, but it very likely formed complexes with soluble matrix proteins of RLM [14]. Diamide converted selectively GSH to its disulfide form (GSSG) and promoted S-glutathionylation which leads to the formation of protein–glutathione (PSSG) mixed disulfides resulting from the reaction of diamide with SH groups of proteins [15]. However, we are still uncertain about the possible joint effect of the ANT conformation and matrix GSH in the Tl+induced MPTP opening. Our aim was therefore to study the effect of diamide, compared with tBHP, on the ability of ADP and NEM to inhibit Tl+-induced MPTP opening in the inner membrane of CaRLM. We investigated swelling, basal, state 4, state 3 and DNP-stimulated respiration, and ∆Ψmito 3
ACCEPTED MANUSCRIPT dissipation of CaRLM, injected into medium containing TlNO3, KNO3, succinate, rotenone as well as diamide and tBHP (where indicated) and MPTP inhibitors (ADP, cyclosporine A (CsA), and NEM).
Materials and methods
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Animals
Male Wistar rats (250-300 g) were kept at 20–23 ºC under 12-h light/dark cycle with free
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access to water ad libitum and the standard rat diet. All treatment procedures of animals were performed according to the Animal Welfare act and the Institute Guide for Care and Use of
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Laboratory Animals.
Isolation of mitochondria
Liver mitochondria were isolated according to [16] in a buffer containing 250 mM sucrose, 3
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mM Tris-HCl (pH 7.3), and 0.5 mM ethylene glycol tetraacetic acid (EGTA); next mitochondrial pellet was washed out twice by resuspension-centrifugation in a buffer containing 250 mM sucrose and 3 mM Tris-HCl (pH 7.3) and finally suspended in 1 ml of the latter buffer. The mitochondrial
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protein content assayed according to Bradford was within 50-60 mg/ml.
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Swelling of mitochondria
Mitochondrial swelling (Figs. 1 and 2) was measured as a decrease in A540 at 20°C using a SF-
46 spectrophotometer (LOMO, St. Petersburg, Russia). Mitochondria (1.5 mg protein/ml) were administrated into a 1-cm cuvette with 1.5 ml of 400 mOsm medium containing 75 mM TlNO3, 125 mM KNO3, 5 mM succinate, 5 mM Tris-NO3 (pH 7.3), 2 µM rotenone, and 1 µg/ml of oligomycin. Diamide, Ca2+, NEM, ADP, and CsA were added into the medium before or after mitochondria (see Figs. 1 and 2 legends). The swelling, oxygen consumption rates, and ∆Ψmito were researched in 400
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Oxygen consumption assay
Respiration (oxygen consumption rate) was measured polarographically using Expert-001
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analyzer (Econix-Expert Ltd., Moscow, Russia) in a 1.3-ml closed thermostatic chamber with magnetic stirring at 26 °C. Mitochondria (1.5 mg protein/ml) were added to 400 mOsm medium
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containing 25 mM TlNO3, 100 mM sucrose, 3 mM Mg(NO3)2, and 3 mM Tris-Pi (Fig. 3a) or 75 mM TlNO3 and 1 µg/ml of oligomycin (Fig. 3b) as well as 125 mM KNO3, 5 mM Tris-NO3 (pH 7.3), 5 mM succinate, and 2 µM rotenone. Diamide, Ca2+, NEM, ADP, and CsA were added in the medium before or after mitochondria (see Fig. 3 legend). ADP of 130 µM (Fig. 3a) and DNP at 30 µM (Fig. 3)
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were correspondingly administrated into the medium after 2 min recording of state 4 or basal state to induce state 3 and DNP-stimulated respiration.
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Mitochondrial membrane potential
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The ∆Ψmito induced in succinate-energized RLM (Fig. 4) was estimated according to
Waldmeier et al. [17] by the intensity of safranin fluorescence (arbitrary units) in the mitochondrial suspension with magnetic stirring at 20 °C using a Shimadzu RF-1501 spectrofluorimeter (Shimadzu, Japan) at 485/590 nm wavelength (excitation/emission). Mitochondria (0.5 mg protein/ml) were placed into a quartz cuvette of four clear walls with 3 ml of a medium containing 20 mM TlNO3, 125 mM KNO3, 110 mM sucrose, 5 mM Tris-NO3 (pH 7.3), 1 mM Tris-Pi, 3 µM safranin, 2 µM rotenone, and 1 µg/ml of oligomycin. In addition, the next chemicals were injected in the medium before
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mitochondria: diamide, ADP, and CsA (where indicated). Succinate, Ca2+, and DNP were administrated into the medium after mitochondria (see Fig. 7 legend).
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Chemicals
CaCl2, Mg(NO3)2, H3PO4, KNO3, TlNO3, and DNP were of analytical grade from Nevareactiv (St. Petersburg, Russia). Rotenone, oligomycin, diamide, NEM, tris-OH, EGTA, ADP, CsA, and succinate were from Sigma (St. Louis, MO, USA). Sucrose as 1 M solution was refined from cation
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traces on a column filled with a KU-2-8 resin from Azot (Kemerovo, Russia).
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Results
The swelling of mitochondria in media containing KNO3 and TlNO3 permits studying the movement of K+ and Tl+ across the IMM, which is easily permeable to NO3- [10-12]. Succinateenergized RLM free of additions swelled weakly in medium containing TlNO3 and KNO3 (Figs. 1 and
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2). Swelling of succinate-energized mitochondria in the TlNO3 medium increased at 50-100 µM of diamide and especially at 200-500 µM (Fig. 1). The swelling induced by 100-500 µM tBHP (dotted trace) was less than with 100-500 µM diamide (Fig. 1). The swelling of energized CaRLM (control
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trace) in TlNO3 medium increased in series of 25-50 µM diamide < 500 µM tBHP (dotted trace) < 100 µM diamide (Fig. 2A). ADP completely inhibited the Ca2+-induced swelling (control trace) in
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experiments with 25 µM tBHP (dotted trace) and partly at 25 µM diamide (Fig. 2B). Figure 2C illustrates that ADP or NEM, but not CsA, partly inhibited 50 µM diamide-induced swelling of CaRLM. Binary combinations (ADP, CsA, and NEM) caused maximal inhibition of the swelling, which decreased in a lot of CsA > control > ADP > NEM > CsA + NEM > ADP + CsA > ADP + NEM. State 3, state 4, and DNP-stimulated respiration of RLM in medium containing 25 mM TlNO3 and 125 mM KNO3 was not affected by 50-200 µM diamide, except for a slight decrease in DNPstimulated respiration at 200 µM diamide (Fig. 3A). Basal respiration (after a short-term burst) and DNP-stimulated respiration decreased after injection of Ca2+ into medium containing 75 mM TlNO3
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ACCEPTED MANUSCRIPT and 125 mM KNO3 (Fig. 3B). The decrease in respiration was more visible in the presence of 50-100 µM diamide (Fig. 3B). If ADP plus CsA or NEM alone were injected before diamide, the Ca2+induced decrease was markedly eliminated (Fig. 3B). Figure 4 (non Ca2+ trace) shows ∆Ψmito-driven safranine uptake into succinate-energized RLM added to the medium containing 20 mM TlNO3 and 125 mM KNO3. The drop in the ∆Ψmito for some traces immediately after Ca2+ administration is
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caused by the energy-dependent uptake of Ca2+ by the RLM. Subsequent ∆Ψmito dissipation (control trace) after some delay was due to the Tl+-induced MPTP opening [13]. Diamide induced more visible Ca2+-induced ∆Ψmito dissipation, which was completely inhibited by ADP plus CsA (Fig. 4).
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The effects of 50 µM tBHP on mitochondrial respiration and ∆Ψmito dissipation were the same as the
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above effects of diamide (not shown).
Discussion
It has been shown that diamide, in comparison to tBHP, caused more extensive protein-protein and protein-glutathione mixed-disulfide formation which was accompanied by extensive conversion
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of GSH to its disulfide form in experiments with HEK, HeLa, and cultured aortic endothelial cells, as well as neonatal rat heart cell cultures, rat erythrocytes, and human platelets [15,18-21]. The MPTP opening, perturbation of IMM, and decrease of ∆Ψmito caused by Ca2+ plus diamide or Ca2+ plus tBHP were associated with protein polymerization due to thiol cross-linking and the production of high
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molecular mass protein aggregates [9,22,23]. It was previously proposed that the formed ligand complexes of Tl+ with SH groups of soluble matrix mitochondrial proteins may alter the spatially
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structural integrality of IMM, with which these proteins are coupled [14,24]. Tl+ and Tl3+ enhanced the production of ROS and H2O2, stimulated lipid peroxidation, and decreased the intracellular and matrix glutathione in experiments with PC12 cells, isolated hepatocytes, and long-term incubated RLM. However, the biochemical mechanisms of these effects are still unclear [25,26]. We have previously suggested that Tl3+ could be formed by the reaction of Tl+ with ROS generated near mitochondrial respiratory chain complexes [27]. Mitochondrial swelling induced by Zn2+ was stimulated by diamide, tBHP, and monobromobimane [28]. Diamide, more than tBHP, affected Ca2+ retention in rat liver mitoplasts [22]. tBHP has induced swelling of RLM only in the presence of exogenous Ca2+, whereas diamide was effective in its absence [29]. Thus, greater swelling of 7
ACCEPTED MANUSCRIPT energized RLM in experiments with diamide, rather than tBHP, (Figs. 1-2) is suggested to be due to oxidation of GSH by diamide and Tl3+. This oxidation could increase membrane permeabilization resulting from the aggregation of mitochondrial proteins with Tl+ and the formation of disulfide bridges between vicinal protein thiol groups of mitochondrial enzymes, including the ANT [9,27,30]. The calcium overload of mitochondria and cross-linkage of Cys159 and Cys256 by diamide or
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tBHP fixed the ANT in ‘c’ conformation which increased both Cyp-D binding with the IMM-specific sites and the sensitivity of pore opening to Ca2+ [1,6-8]. As a result, a decrease was noted in the ability of ADP to inhibit the diamide- or tBHP-induced MPTP opening, swelling and ∆Ψmito
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dissipation in succinate-energized CaRLM. This has given reason to believe that the ANT is involved in pore formation [1,7,23,31]. We have found that bongkrekic acid (BKA) fixing the ANT in ‘m’
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conformation inhibited Tl+-induced MPTP opening, whereas carboxyatractyloside that fixed the translocase in ‘c’ conformation attenuated the ability of ADP to inhibit the pore formation (not shown here). So, inhibition of swelling by ADP at 25 µM of diamide or tBHP (Fig. 2b) gives us reason to assume that the ANT conformation can be involved in the Tl+-induced MPTP opening. On the other hand, ANT can be transformed from an antiporter to a non-selective porter by increasing CyP-D
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binding with the IMM [6,7]. The swelling of non-energized RLM in medium free of Ca2+ was attenuated by ADP in our experiments with Y3+ and La3+ [32] or Tl+ alone [13] indicating that ANT participates in regulating inner membrane ion permeability. Thus, a second reason for the increase in the diamide-induced swelling (Fig. 1) may be enhancement of the IMM ion permeability, caused by a
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reaction of diamide with SH groups of the ANT [9,23].
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It is known that CsA prevents reaction of Cyp-D with the matrix surface and thus inhibits MPTP opening [1,6]. Diamide enhanced the binding which was partially prevented by CsA [6,7]. ADP, BKA, and CsA inhibited diamide- and tBHP-induced swelling, decreased calcium retention, ∆Ψmito drop, and cytochrome c release from RLM [29,30,34]. Earlier we found [13] that the Tl+induced MPTP opening in CaRLM manifested as increased swelling and decreased ∆Ψmito, as well as decreased mitochondrial respiration. These effects [13] similar to those in experiments with diamide and CaRLM (Figs. 2C, 3, and 4) declined in the series of controls, CsA > ADP >> ADP + CsA. NEM attacking Cys56 at the ANT in ‘m’ conformation inhibited swelling, ∆Ψmito dissipation, and MPTP opening of Ca2+-loaded mitochondria in experiments with thiol reagents (tBHP, diamide, arsenite, menadione, and PAO) [6-9]. We can therefore propose that a decrease by NEM of diamide-induced 8
ACCEPTED MANUSCRIPT swelling in CaRLM (Fig. 2C) and the additive effect of ADP or NEM in the presence of CsA in inhibiting the Tl+-induced MPTP (Figs. 2C, 3, and 4) is possible when the ANT is in ‘m’ conformation [1,6,9]. Diamide enhanced As2O3-induced apoptosis in promyelocytic leukemia NB4 cells [35]. Diamide and tBHP induced cross-linkage of mitochondrial proteins that led to a disorder of the respiratory chain, at the level of the coenzyme Q [36]. A decrease in DNP-uncoupled respiration
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caused by diamide (Fig. 3) indicates a disturbance in the respiratory chain, which can favor MPTP opening [37]. One may therefore suggest that another reason for the more potent swelling of succinate-energized RLM in the medium containing TlNO3 and KNO3 as well as diamide and tBHP
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(Fig. 1) may be due to oxidative stress caused by these oxidizers. Ultimately, our experiments suggest that the stronger action of diamide, compared to tBHP, on mitochondria in TlNO3 media can result in
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diamide-induced matrix glutathione loss, accompanied by aggregation of mitochondrial proteins, and interconnected with the ANT in ‘c’ conformation fixing. The use of K+ surrogate Tl+ in this model of the Tl+-induced MPTP can help in the search for the mitochondrial transition pore of new inhibitors and inducers among different chemical and natural compounds.
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Acknowledgments
Authors are grateful to Ms Terttu Kaustia for correcting the English. Purchase of diamide was financed by a grant from the Russian Foundation for Basic Research to Diana Gutzaeva (#09-04-
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01439); the funding organization does not have control over the resulting publication.
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Conflict of interest
No conflicts of interest, financial or otherwise, are declared by the authors.
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[31] A.P. Halestrap, K.Y. Woodfield, C.P. Connern, Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase, J. Biol. Chem. 272(1997) 3346-3354. [32] S. Korotkov, S. Konovalova, L. Emelyanova, I. Brailovskaya, Y3+, La3+, and some bivalent metals inhibited the opening of the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria, J. Inorg. Biochem. 141 (2014) 1-9. 12
ACCEPTED MANUSCRIPT [33] F. Ricchelli, J. Sileikytė, P. Bernardi, Shedding light on the mitochondrial permeability transition, Biochim. Biophys. Acta 1807 (2011) 482-490. [34] K.K. Lee, M. Shimoji, Q.S. Hossain, H. Sunakawa, Y. Aniya, Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria, Toxicol. Appl. Pharmacol. 232 (2008) 109-118.
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[35] Y. Yu, P. Jia, Y. Huang, X. Cai, G. Chen, Diamide and cyclosporin A enhanced arsenic trioxideinduced apoptosis in NB4 cells, Zhonghua. Xue. Ye. Xue. Za. Zhi. (In Chinese). 23 (2002) 254-257. [36] A.E. Vercesi, A.J. Kowaltowski, M.T. Grijalba, A.R. Meinicke, R.F. Castilho, The role of
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reactive oxygen species in mitochondrial permeability transition, Biosci. Rep. 17 (1997) 43-52. [37] E. Chávez, E. Meléndez, C. Zazueta, H. Reyes-Vivas, S.G. Perales, Membrane permeability
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transition as induced by dysfunction of the electron transport chain, Biochem. Mol. Biol. Int. 41
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Fig. 1 The influence of diamide and tert-butylhydroperoxide on swelling of succinate-energized rat liver mitochondria. Mitochondria (1.5 mg/ml of protein) were injected to the medium containing 75 mM TlNO3, 125 mM KNO3, 5 mM Tris-succinate, 5 mM Tris-NO3 (pH 7.3), 2 µM rotenone, and 1
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µg/ml of oligomycin. Additions (µM) of diamide and tert-butylhydroperoxide (dashed trace) before mitochondria are indicated on the right of the traces. Addition of mitochondria (RLM) is shown by arrow. The apparent absorbance change (Figs. 1-2) was within 4% (p < 0.05). Representative traces
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Fig. 2 Effects of diamide, tert-butylhydroperoxide, and Ca2+ on Tl+-induced swelling of succinate-
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energized rat liver mitochondria in the presence of ADP. Mitochondria (1.5 mg/ml of protein) were administrated to medium containing 75 mM TlNO3, 125 mM KNO3, 5 mM Tris-succinate, 5 mM Tris-NO3 (pH 7.3), 2 µM rotenone, and 1 µg/ml of oligomycin. Additions (µM) of diamide and tertbutylhydroperoxide (dashed trace) before mitochondria are indicated on the right of the traces. Additions before mitochondria are indicated to right of traces: free of additions (none Ca2+, in bold), 100 µM Ca2+ alone (control, in bold), 500 µM ADP (ADP), 50 µM NEM (NEM), and 1 µM CsA
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(CsA). Additions of mitochondria (RLM) and 100 µM Ca2+ (Ca2+) are shown by arrows. Typical traces from one of three independent different mitochondrial preparations are presented.
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Fig. 3 Influence of diamide and Ca2+ on oxygen consumption rates of rat liver mitochondria. Mitochondria (1.5 mg/ml of protein) were injected in medium containing 25 mM TlNO3, 100 mM
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sucrose, 3 mM Tris-Pi, and 3 mM Mg(NO3)2 (panel A) or 75 mM TlNO3, and 1 µg/ml of oligomycin (panel B) as well as 125 mM KNO3, 5 mM Tris-NO3 (pH 7.3), 5 mM succinate and 2 µM rotenone. Additions of mitochondria (RLM), 0-200 µM diamide (diamide), 100 µM Ca2+ (Ca2+), and 30 µM DNP (DNP) are correspondingly showed by vertical arrows, inclined long arrows, inclined long bold arrows, and inclined bold arrows. Inclined arrows show additions of 130 µM ADP (ADP), 500 µM ADP (ADP(2)), 1 µM CsA (CsA), and 50 µM NEM (NEM). The numbers on the right of the traces show concentrations of diamide (µM). Oxygen consumption rates (ng atom O min/mg of protein) are presented as numbers placed above experimental traces. The rates' deviation was within 5% (p < 0.05). Representative traces from one of three independent experiments are presented. 14
ACCEPTED MANUSCRIPT Fig. 4 Effects of diamide and Ca2+ on the inner membrane potential (∆Ψmito) of rat liver mitochondria in the nitrate medium. Mitochondria (0.5 mg/ml of protein) were added to medium containing 20 mM TlNO3, 125 mM KNO3, 110 mM sucrose, 5 mM Tris-NO3 (pH 7.3), 1 mM Tris-Pi, 3 µM safranin, 2 µM rotenone, and 1 µg/ml of oligomycin. Additions of mitochondria (RLM) and 5 mM succinate
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(Succ) are shown by arrows. Injections of 75 µM Ca2+ and 20 µM DNP are correspondingly marked by up-directed arrows and up-directed bold arrows. Additions before mitochondria are indicated on the right of the traces: 50 µM diamide (diamide), 500 µM ADP (ADP), and 1 µM CsA (CsA). The
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• ANT conformation and glutathione loss in opening Tl+-induced pore is proposed. • Thiol oxidants (diamide and tBHP) fixing the ANT in ‘c’ conformation researched. • Ability of ADP to inhibit the pore decreased in the presence of the oxidants. • N-ethylmaleimide (NEM) fixing the ANT in ‘m’ conformation inhibited the pore opening.
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• The pore inhibition by ADP or NEM was considerably stronger in the presence of CsA.