Pyridine nucleotides regulate the superoxide anion

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Pyridine nucleotides regulate the superoxide anion flash upon permeabilization of mitochondrial membranes: an MCLA-based study

Ekaterina S. Kharechkina1, Anna B. Nikiforova1, Alexey G. Kruglov1*

1

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences,

Pushchino, Moscow Region, 142290 Russia

*

Address correspondence to Alexey G. Kruglov: [email protected]

Abstract

The permeabilization of mitochondrial membranes via permeability transition pore opening or treatment with the pore-forming peptide alamethicin causes a flash of superoxide anion (SA) and hydrogen peroxide production and the inhibition of matrix aconitase. It was shown using the SA probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine3-one (MCLA) that the substrates of NAD-dependent dehydrogenases, inhibitors of the respiratory chain, and NAD(P)H at millimolar concentrations suppressed or delayed SA flashes. In the presence of added NADH and NADPH, SA flashes were observed only after considerable oxidation of pyridine nucleotides. The production of SA was maximal at NADPH and NADH redox potentials from -310 to -274 mV and from -330 to -220 mV, respectively, depending on NAD(P)H concentration. SA generation supported by NADPH was several-fold greater than that supported by NADH. In intact mitochondria, NADPH- and NADH-dependent SA generation was negligible. Respiratory substrates at physiological or lower concentrations were incapable of suppressing the NADPH-supported SA flash. These data indicate that, in pathophysiologyrelevant conditions, matrix NADPH oxidoreductases, presumably, an adrenodoxin reductase– adrenodoxin complex, can essentially contribute to SA flashes associated with transient or irreversible permeability transition pore opening.

Keywords: permeability transition pore, inner mitochondrial membrane, superoxide flash, NADH, NADPH, redox potential, adrenodoxin reductase

Abbreviations: Alam, alamethicin; ADx, adrenodoxin; AR, adrenodoxin reductase; DLD, dihydrolypoamide dehydrogenase; IMM, inner mitochondrial membrane; MDCL, MCLAderived chemiluminescence; MCLA, 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2a]pyrazine-3-one; mPT, mitochondrial permeability transition; mPTP, mitochondrial

permeability transition pore; OMM, outer mitochondrial membrane; ROS, reactive oxygen species; SA, superoxide anion; SOD, superoxide dismutase.

Introduction

Mitochondrial permeability transition (mPT) is a Ca2+-dependent permeabilization of the inner mitochondrial membrane (IMM) for ions and solutes of molecular masses less than 1.5 kDa via the creation of an unspecific pore (mPTP) [1]. The molecular composition of mPTP is a matter of debates [2]. However, the regulation of mPTP opening has been well studied. Reactive oxygen species (ROS), oxidants, and thiol reagents facilitate, while cyclosporin A (an inhibitor of peptidyl-prolyl cis-trans isomerase), reduced pyridine nucleotides in the matrix, Mg2+, ATP, and ADP suppress mPTP opening triggered by Ca2+ [1]. The inhibitors of adenine nucleotide translocase, which stabilize the carrier either in the cytosolic or the matrix conformation, facilitate or inhibit mPTP opening, respectively [3]. It is well known that the opening of mPTP by Ca2+ stimulates the generation of ROS in isolated mitochondria [4–6]. A burst of ROS production occurs in cells in pathologic states, which can trigger the mPTP opening [7–9]. The elevation of cytosolic Ca2+ and the induction of mPTP are considered to be the main reasons for the activation of ROS production and cell death upon ischemia/reperfusion [10–12]. The transient mPTP opening associated with the excitotoxicity of glutamate in motor neuron-like cells [13], the suppression of proliferation of neural progenitor cells by beta-amyloid [14], as well as with Ca2+ and oxidative stress [15] also causes a short-term acceleration of SA production (so-called flash) by mitochondria [16]. Several mechanisms were proposed to explain ROS flashes/bursts during mPTP opening and under mPTP-facilitating conditions. It was suggested that Ca2+-activated dehydrogenases, pyruvate dehydrogenase and a-ketoglutarate dehydrogenase, can generate ROS with higher rates than non-activated enzymes [17]. It was also proposed that mPTP induces conformational

changes in complexes I, II and III, which accelerates ROS production [4, 6, 18]. In this case, malic enzyme may generate fuel for ROS production by damaged complex III [4]. An exhaustion of antioxidant systems due to the release of glutathione through the mPTP may also contribute to the acceleration of ROS production [5, 19]. In the heart, the burst of ROS production may be related to the accumulation of serotonin and its oxidation by monoamine oxidase during ischemia and reperfusion, respectively [20]. However, up to now, the mechanisms underlying the mPTP-related ROS burst remain unclear. Here we explored possible mechanisms of SA flash in mitochondria upon the mPTP opening using the chemiluminescent probe MCLA. MCLA seems to be a superior SA probe for many applications [21–25]. It is extremely sensitive to SA (hundreds of times more sensitive than lucigenin) [26], though it can also sense 1О2 [27]. Many SA probes, namely, tetrazolium salts, lucigenin, cytochrome c, and epinephrine are either unspecific or capable of inducing the additional ROS production in biological systems [23, 28–30]. MCLA, presumably, lacks these drawbacks. By contrast to lucigenin [29, 31] and oxidized products of hydroethidine (MitoSOX), ethidium and 2-hydroxyethidium [32], both MCLA and its superoxide adduct are uncharged [27], which makes chemiluminescence independent of the membrane potential. This property seems advantageous in the context of the present study, since the permeabilization of the IMM causes immediate dissipation of the membrane potential. The known drawbacks of MCLA are a rather high spontaneous luminescence in solution [27] and chemiluminescence quenching by sulfur-containing compounds [33]. Both problems can be solved by the selection of appropriate experimental conditions. Using MCLA in a model of isolated mitochondria, we found that the permeabilization of the IMM via mPTP opening and by treatment with alamethicin (Alam) causes an SA flash, which is regulated by the redox state of internal and external NADH and NADPH. External (cytosolic) NADPH and internal NADPH oxidoreductase(s) can make a major contribution to

the SA flash upon the IMM permeabilization. The pathophysiological relevance of our findings was discussed.

2. Materials and Methods

2.1. Materials

Alamethicin, AmplifluTM Red (Amplex Red), bovine serum albumin (BSA), cytochrome c from the equine heart, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), glutamate, αketoglutarate, malate, mannitol, 7- dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2a]pyrazine- 3-one (MCLA), myxothiazol, NADH, NADPH, NAD, NADP, β-oxybutyrate, pig heart isocitrate dehydrogenase, potassium peroxide, pyruvate, rotenone, sucrose, succinate, superoxide dismutase (SOD), and Trizma Base were obtained from the Sigma-Aldrich Corporation (St. Louis, MO; USA). Other chemicals were of analytical grade and were purchased from local suppliers.

2.2. Isolation and purification of rat liver mitochondria

All manipulations with animals before the isolation of the liver were performed in accordance with the Helsinki Declaration of 1975 (revised in 1983) and national requirements for the care and use of laboratory animals. Adult male Wistar rats were killed by cutting the neck after anesthesia with CO2. Rat liver mitochondria were isolated according to a standard differential centrifugation procedure [34]. The homogenization medium contained 220 mM mannitol, 70 mM sucrose, 10 mM HEPES (pH adjusted to 7.4 with Trizma Base), 1 mM EGTA, and 0.05% BSA. The pellet was washed three times with a medium devoid of EGTA and BSA.

Final pellets were resuspended in this medium to yield 80–90 mg protein/ml. Measurements were performed at 30ºC in KCl-based medium (120 mM KCl, 20 mM sucrose, 10 mM HEPES (pH adjusted to 7.3 with Trizma Base), 2 mM KH2PO4, and 2 mM MgCl2), unless otherwise indicated. Other experimental details are given in figures and figure legends. The total mitochondrial protein was determined by the Biuret method using BSA as a standard [35].

2.3. Assessment of the intactness of mitochondrial membranes

The permeabilization of the IMM to ions and solutes causes the immediate swelling of mitochondria and the rupture of the outer mitochondrial membrane (OMM). The intactness of mitochondrial membranes was defined as the ratio of the rates of succinate-supported reduction of exogenous cytochrome c in the absence and in the presence of pore-forming peptide Alam (40 µg/mg protein) expressed in percent [24]. Mitochondria were used in experiments if contained less than 5% of damaged organelles (usually about 3%).

2.4. Recording of the permeabilization of mitochondrial membranes

The permeabilization of mitochondrial membranes for solutes was assessed by highamplitude mitochondrial swelling. Mitochondrial swelling (a decrease in A540) was recorded using a plate reader (Infinite 200 Tecan, Austria) and 96-well plates. Other details are given in figures and figure legends.

2.5. Assessment of ROS production

Hydrogen peroxide. The level of hydrogen peroxide in a mitochondrial suspension was measured in standard KCl-BM supplemented with 500 µM EGTA, 20 µM Amplex Red, and

horseradish peroxidase (HRP) (3 U/ml). Resorufin accumulation was traced using a plate fluorimeter (Infinite 200 Tecan) in 96-well plates at excitation and emission wavelengths of 530 and 595nm. For the quantitative assessment of hydrogen peroxide, fluorescence was calibrated by an excess of hydrogen peroxide at the end of measurements. In order to avoid light-induced resorufin formation, the fluorescence was measured once or two times a minute. SA. The rate of SA production was assessed using the highly sensitive chemiluminescent probe MCLA [27]. The kinetics of MCLA-derived chemiluminescence (MDCL) was recorded using a plate reader (Infinite 200 Tecan). Each value on the curve is the mean ± S.E.M. of three integrations of luminescence for 900 ms expressed in arbitrary units. Aconitase assay. Aconitase activity was measured by fluorescent monitoring of NADP reduction (Ex360/Em465) in a freshly prepared reaction mixture (50 mM Tris-HCl (pH 7.4), 5 mM sodium citrate, 0.6 mM MnCl2, 200 µM NADP, and pig heart isocitrate dehydrogenase (1 unit/ml)) [36] with addition of Alam (20 µg/ml) for the permeabilization of the IMM. The reaction was started by the addition of aliquots (10 µg protein) of MCLA-free samples to 100 µl of assay mixture preequilibrated at 25ºC. The activity of aconitase was expressed in nmol∙min1

∙mg protein-1. Other experimental details are given in figure legends.

2.6. Generation of SA by potassium peroxide

Measurements were performed at 25ºC in KCl-based medium supplemented with 50 mM HEPES (pH adjusted to 7.3 with Trizma Base), 20 µM MCLA, and, where indicated, SOD (100 U/ml), NADH and NADPH at different concentrations. Chunks of potassium peroxide were crashed into 2–3-mg pieces and mixed with an appropriate volume of the medium immediately before measurements. The reaction was initiated by the addition of 500 µM KO2 into the reaction mixture. Since SA production induced by potassium peroxide is transient, the kinetics of MDCL

was recorded in 3–6 wells, and the chemiluminescence integration time was reduced to 500 ms. Each experiment was repeated 10–20 times.

2.7. Media of different osmolarity

Incubation media of different osmolarity (tonicity) were prepared by the addition of 25, 50, 75, 100, 125, 175, and 275 mM KCl (final concentrations) to the basal 50 mOsM solution (20 mM sucrose, 10 mM HEPES (pH 7.3), 3 mM KCl, 2 mM KH2PO4, 2 mM MgCl2).

2.8. Determination of the redox state of pyridine nucleotides in a mitochondrial suspension

Concentration of pyridine nucleotides in solutions. The concentrations of oxidized (NAD+/NADP+) and reduced (NADH/NADPH) forms of pyridine nucleotides in solution were calculated taking the ε259 and ε339 values to be 17.8 and 6.22 mM-1∙cm-1, respectively. In order to determine ε259 for reduced forms under the incubation conditions used, we recorded 30 scans of absorbance at 200–500 nm (1 scan/min) upon the reduction of NAD or NADP by glutamate dehydrogenase in standard KCl-based medium in the presence of 10 mM glutamate. This allowed us to establish that the reduction of NADP and NAD led to a decrease in absorbance at 259 nm by 0.00286 ± 0.00012 and 0.00311 ± 0.00008 per µM of NADPH and NADH, respectively. Thus, ε259 for NADPH and NADH was 14.94 and 14.49, respectively. With the use of generated A259/A339 ratio curves, the percentage of the reduced forms of NADH and NADPH in our matrix solution was determined to be 98.6 and 96.2%, respectively. Redox state of pyridine nucleotides in mitochondrial suspension. The kinetics of oxidation of added NADH and NADPH was recorded as a decrease in fluorescence (Ex 360/Em 465 nm) using a plate reader (Infinite 200 Tecan). The dependence of fluorescence on the

NAD(P)H concentration was not linear in the near-millimolar range (see Figs. 4 and 5). Therefore, the concentration of NAD(P)H in solution at each time point was determined using the equations of sigmoid curves generated for each series by the Boltzmann method (Sigmoidal Fit, Origin 7.0) (see insert in Fig. 5B). The dynamics of the redox state of NADH and NADPH in a mitochondrial suspension, ENAD(H) and ENADP(H), was calculated using the Nernst equation assuming that (1) E0 for NADH and NADPH is equal to -0.32 V; (2) the concentrations of endogenous NAD(H) and NADP(H) in the mitochondrial matrix are 2 and 3 mM, respectively; (3) in permeabilized mitochondria, NAD(P)H is totally oxidized in the absence of respiratory substrates; and (4) the volume of the mitochondrial matrix is equal to 1 µl/mg protein.

2.9. Statistical analysis

The data shown represent the means ± standard error of means (S.E.M.) or are the means of at least three experiments. Statistical probability (P) values were derived by the Student’s ttest, unless otherwise indicated.

3. Results

1. Activation of ROS production by the permeabilization of the mitochondrial membranes

Figure 1 shows that the permeabilization of mitochondrial membranes by the pore-forming peptide Alam or as a consequence of mPTP opening by Ca2+ causes the mitochondrial swelling (B), which is accompanied by the production of SA (A) and hydrogen peroxide (D), and the inhibition of the ROS-sensitive matrix enzyme aconitase (C). The prevention of mPTP opening by CsA and/or EGTA suppressed both ROS production and aconitase inhibition. Thus, the

permeabilization of the IMM can cause a drastic increase in ROS production irrespective of the mechanism of permeabilization. However, ROS production and swelling in mitochondria permeabilized by Alam were much stronger than in Ca2+-treated organelles.

2. Effect of osmotic swelling of intact mitochondria on chemiluminescence of MCLA

The high-amplitude swelling of mitochondria caused by the permeabilization of the IMM by Alam or Ca2+ considerably decreases the light absorbance in the sample. Therefore, we checked whether the increase in MDCL in permeabilized mitochondria can be explained by an increase in the transparency of the suspension. Figure 2 shows that the decrease in tonicity from 600 to 50 mOsM caused a strong decrease in light absorption at 540 nm in a mitochondrial suspension, which was comparable with that induced by Alam (B). However, SOD-sensitive MDCL changed not so dramatically (C). The decrease in tonicity from 400 to 100 mOsM resulted in a decrease in absorbance from ~0.8 to ~0.5 without any effect on the apparent SA level (B–D). The acceleration of SA generation was observed only at extreme values of tonicity (50 and 600 mOsM) after prolonged incubation, and, therefore, was related to the damage to mitochondrial membranes. These data indicate that (a) MDCL reflects changes in the level of SA but not in the transparency of mitochondrial suspension; and (b) when IMM remained intact, mitochondrial contraction, but not swelling, stimulated SA production.

3. Effect of respiratory substrates and inhibitors of respiratory chain on the superoxide production in permeabilized mitochondria

Alam-dependent SA flash was much higher than the Ca2+-induced one (see Fig. 1A). This could be due to the IMM permeabilization throughout the mitochondrial population (see Fig. 1B) and/or the creation of Alam pores of larger size than mPTP (see Supplementary Fig. 1). On the

other hand, mPTP opening requires the oxidation of matrix NAD(P)H [5], while the creation of Alam pores does not. Therefore, it was logical to suggest that respiratory substrates and inhibitors of NAD(P)H oxidation will support SA production in permeabilized mitochondria. Figure 3 shows that Ca2+ (A) and Alam (B) caused a fast activation of SA generation in the absence of added substrates (-S). However, the substrates of NAD- and NADP-dependent dehydrogenases (malate plus glutamate, malate plus pyruvate, and hydroxybutyrate) at high concentrations (5 mM) decreased and/or delayed the maximal SA production caused by Alam (B). The inhibitors of the respiratory chain (rotenone, myxothiazol, and cyanide) also strongly delayed the SA overproduction (C). Since NADH donates electrons to the respiratory complex I, one of the main sites of ROS production in mitochondria, and NADPH is a substrate of glutathione reductase, a key enzyme in the mitochondrial antioxidant system, we examined the effect of NADH and NADPH at high concentrations on SA generation in permeabilized mitochondria (D). As it follows from Fig. 3D, both NADH and NADPH caused a relatively weak stimulation of SA production but completely suppressed the SA flash. In mitochondria permeabilized due to mPTP opening, the effect of respiratory substrates was less pronounced. However, again, malate plus pyruvate, which inhibited the oxidation of endogenous NAD(P)H (insert in panel A), delayed the activation of SA production. These data imply that keeping the mitochondrial NAD(P)H-binding redox centers in a fully reduced state prevents a SA flash.

4. Superoxide flash in permeabilized mitochondria in the presence of NADPH

Figure 4 shows that the addition of NADPH to mitochondria preliminarily permeabilized by Alam caused an immediate strong activation of SA production (flash) if the initial concentration of NADPH was low (A). An increase in NADPH concentration gradually postponed the initiation of SA production and completely prevented it at 660 µM NADPH during the recording. SA production was activated only when a major part of NADPH was

oxidized (B). Rotenone had a weak effect on the rate of NADPH oxidation and, as a consequence, on the time of activation of SA production. The superimposing of the dynamics of MDCL and NADP/NADPH redox potential (ENADP(H)) in the suspension (C) made it possible to determine the value of ENADP(H), at which SA production was maximal (ENADP(H)Max). An increase in the total NADP(H) concentration in the suspension shifted the ENADP(H)Max to more positive values (D). These data show that a decrease in the NADPH/NADP ratio below a certain limit can increase the probability of electron leakage from the redox centers to oxygen.

5. Generation of superoxide in permeabilized mitochondria in the presence of NADH

Figure 5 shows that the activation of SA production in permeabilized mitochondria in the presence of NADH occurred only after a considerable oxidation of the latter, as this happened in the presence of NADPH (see Fig. 4). Rotenone (B) and 1.5 mM cyanide (not shown) strongly inhibited NADH oxidation and delayed the activation of SA generation (A and C). The values of NADH redox potential at which the SA production was maximal (ENADP(H)Max) were much higher than that of NADPH (see Fig. 4) and also increased with increasing NADH+NAD concentration (D). Rotenone and NaCN shifted the ENADP(H)Max to more negative values at NADH+NAD concentrations higher than 50 µM. Presumably, NADH and NADPH at high concentrations (low redox potential) prevented the SA flash due to interactions with enzymatic redox centers, but not with the MCLA molecule, since neither NADH nor NADPH quenched the SOD-sensitive MDCL in the presence of the chemical SA donor, potassium peroxide (see Supplementary Figure 2S). Also, the tenfold acceleration of hydrogen peroxide production (measured using Amplex Red) occurred only after a partial oxidation of NADH in a suspension of permeabilized mitochondria (see Supplementary Fig. 3S). These data indicate that, at physiologic concentrations of cytosolic NAD(P) and NAD(P)H, the flash of SA in permeabilized mitochondria would occur only if the NAD(P)H

redox state is considerably shifted to positive values (considerable NAD(P)H oxidation). Systems responsible for NADH- and NADPH-dependent SA generation may be different.

6. Comparison of NADH- and NADPH-dependent stimulation of superoxide production in intact and permeabilized mitochondria

Because the outer leaflet of the IMM and the OMM contain NAD(P)H-binding enzymes capable of generating ROS under certain conditions (apoptosis-inducing factor 1, cytochrome b5 reductase isoform 3, NADPH oxidase 4), we compared the effect of added NADH and NADPH on the SA production in permeabilized and intact mitochondria. Figure 6 shows that, in intact mitochondria, NADPH at different concentrations caused an about 15-fold lesser stimulation of SA production (B) than in organelles permeabilized by Alam (A). Moreover, in intact mitochondria, the NADPH-supported SA production occurred without a concentrationdependent delay. NADH in intact mitochondria caused no stimulation of the SA production at all concentrations tested (D). In permeabilized mitochondria, NADH supported SA generation several fold weaker than NADPH (A and C). Similar results were obtained for mitochondria permeabilized due to the mPTP opening (see Supplementary Fig. 4S). These data indicate that internal redox systems make a major contribution to both NADHand NADPH-dependent SA production in permeabilized mitochondria. In turn, NADPHdependent systems are the main source of SA in permeabilized mitochondria in the presence of external NADPH.

7. Effect of respiratory substrates on the ability of NAD(P)H to support superoxide flashes in permeabilized mitochondria

An increase in the concentration of cytosolic Ca2+ during the ischemia or extensive workloads can be accompanied by a decrease in the concentration of respiratory substrates below the normal physiological values of 100–300 µM. Therefore, we examined whether mitochondrial dehydrogenases can prevent the oxidation of exogenous and endogenous NAD(P)H, the increase in ENAD(P)H to ENAD(P)HMax values, and the SA flash in permeabilized mitochondria in the presence of respiratory substrates at physiological and subphysiological concentrations. Figure 7 shows that, in mitochondria permeabilized by Ca2+, malate plus pyruvate at low (75 plus 25 µM), “physiological” (300 plus 100 µM), and high concentrations (1 mM plus 500 µM) considerably delayed the oxidation of endogenous NAD(P)H but only slightly suppressed the initial SA production. The dose-dependent effects of substrates on the SA level were most pronounced after prolonged incubation when the difference in the redox state of matrix NAD(P)H was undetectable. At the same time, substrates inhibited the oxidation of exogenous NADPH, delayed SA flashes, and reduced their intensity in a concentration-dependent manner. Thus, the conditions that favor the SA flash in mitochondria are (1) the permeabilization of the inner membrane by any mechanism, (2) the availability of cytosolic NADPH, and (3) a decrease in the concentration of NAD(P)H-regenerating substrates.

Discussion

For the goals of the present study, MCLA seems a superior SA probe. Its relatively high spontaneous luminescence in suspension (SOD-insensitive MDCL) [27, 37] can be subtracted from the total signal. Chemiluminescence quenching by sulfur-containing compounds cannot remarkably affect the MDCL under our experimental conditions (low concentrations of a mitochondrial protein) [33]. Slow penetration through membranes also was not an essential drawback, since we studied the effect of the permeabilization of membranes on the SA release. Moreover, the high-amplitude mitochondrial swelling had a minor effect on the SOD-sensitive

MDCL if membranes remained intact (Fig. 2). In addition, NADH and NADPH neither interfered with the reaction of SA with MCLA nor quenched the MDCL (Supplementary Fig. 2S). Taken together, this means that SOD-sensitive MDCL can be a semi-quantitative measure of the SA level in a suspension of permeabilized mitochondria in the presence of substrates of different dehydrogenases. Indeed, the data on the strong increase in the SA production upon the permeabilization of mitochondrial membranes, obtained using MCLA, are in good agreement with the data obtained by other direct and indirect approaches (Fig. 1, Supplementary Fig. 2S). Presumably, the mechanism of the acceleration of SA production is not related to the earlier proposed changes in the structure of the respiratory complex upon the mPTP opening [4, 6, 18]. Indeed, the pore-forming peptide Alam, which permeabilizes membranes in the whole mitochondrial population via an mPTP-independent mechanism, caused a much stronger SA production than Ca2+. The Ca2+-dependent activation of matrix dehydrogenases [4, 17 and references therein] must be excluded for the same reason. The permeabilization of the IMM by Alam or via mPTP opening facilitates the release of SA, hydrogen peroxide [38], endogenous pyridine nucleotides, and GSH, but not matrix antioxidant enzymes (MnSOD, GSH-peroxidase, GSSG reductase) [5, 19]. This must decrease the capability of mitochondria to utilize ROS. At the same time, the oxidation/reduction of matrix NAD(P)(H) should be mediated not only by internal (Complex I, adrenodoxin reductase (AR), dihydrolypoamide dehydrogenase (DLD)) but also by external dehydrogenases (apoptosisinducing factor 1, cytochrome b5 reductase isoform 3, NADPH oxidase 4), which would support short-term ROS production (ROS burst). Therefore, it was logical to suggest that the addition of respiratory substrates and NAD(P)H would enhance the level of ROS in permeabilized mitochondria and extend the time of ROS production. Our experimental results support this suggestion only in part. First, the substrates of NAD- and NADP-dependent dehydrogenases and the inhibitors of the respiratory chain, which prevent NAD(P)H oxidation, considerably suppressed and/or delayed the SA flash

(Fig. 3). Second, NADH and NADPH at high concentrations weakly stimulated SA production. However, the oxidation of NADH and NADPH to certain, rather low E(NAD(P)H) values was accompanied by a flash in SA generation (Figs. 4 and 5). Third, NADH- and NADPH-dependent SA flashes were mediated predominantly by internal redox system(s) (Fig. 6). Some facts indicate that the NADPH-dependent SA flash is mediated by the system AR– adrenodoxin (ADx) [39; 40], a stable complex in the matrix (Kdiss ≤ 1 nM) [41]. Standard redox potentials for flavin of AR and the iron sulfur cluster of ADx were determined to be -295 and 331 mV, respectively [41, 42]. The reduction of AR by NADPH (Km = 1.82 µM) results in the formation of a charge transfer complex (ARH2-NADP+) (Kdiss ~ 10 nM), which can be oxidized by oxygen to form SA and the “blue” or neutral form of flavin semiquinone [42, 43]. The redox potential of ADx is lower than that of AR and much lower than that of the AR–NADP+ complex (-198 mV), which decreases the probability of ADx reduction (≤ 20 %) upon the equimolar reduction of the AR–ADx complex by NADPH [41, 43]. It is important that NADPH can bind to reduced AR (Kdiss was not determined) and decrease the lifetime of the charge transfer complex, thereby increasing the reduction of ADx [42, 43]. Reduced ADx is only slowly oxidized by oxygen [43]. NADP+ can bind to oxidized and one-electron reduced AR (Kdiss = 14 and 24 µM, respectively) and competitively inhibit the activity of the AR–ADx complex (Ki ~ 25 µM) [42, 43]. Therefore, the competition of NADPH and NADP+ for binding to the ARH2–NADP+ charge transfer complex can explain the appearance of an SA flash upon a decrease in the NADPH/NADP+ ratio (Fig. 4). Moreover, the difference between Kdiss values for NADPH and NADP+ explains the observed decline in ENADP(H)Max caused by a rise in NADP(H) concentration. The nature of the system responsible for the NADH-dependent SA flash is even less clear. On the one hand, one may suggest that NADH-supported SA flashes are mediated by Complex I or DLD. Complex I and DLD are considered to be the main generators of SA and hydrogen peroxide in mitochondria [44–47]. Rotenone significantly affected ENAD(H)Max but had a minor

effect on ENADP(H)Max at different NAD(P)H concentrations (Figs 4 and 5). On the other hand, the maximal rates of ROS production by Complex I were observed at much more negative potentials (from -400 to -380 mV) [46, 48, 49] than SA flashes that occurred in our experiments (in the range from -330 to -220 mV depending on NAD(H) concentration) (Fig. 5). In the case of DLD, the maximal rates of hydrogen peroxide production were also observed at minimal NAD+/NADH ratios [50]. This discrepancy could be explained by the shift of apparent ENAD(H)Max values to more positive area due to the different affinity of oxidized and reduced enzymes to oxidized and reduced forms of nucleotides [51]. (In the present study, we applied relatively high NADH concentrations (20–750 µM)). Also, SA generation may be mediated by a redox center with a much more positive potential than FMN [45]. The existence of two centers of SA production in Complex I has been postulated earlier [52]. However, the question is complicated by the fact that NADH can also reduce AR (Km = 5.56 mM) [42], which, in turn, can form a charge transfer complex with endogenous NADP+ [41]. In addition, the binding of NAD+ to AR is extremely weak [41]. Taken together, this can explain the low ENAD(H)Max values obtained in our experiments. Nevertheless, the question concerning the system(s) responsible for NAD(P)H-dependent SA flashes in permeabilized mitochondria remains to be solved. Presumably, the results of the present study are relevant to pathophysiologic states. In fact, ischemia [10–12], toxin- and hypometabolism-induced hyperactivation of neuronal cells [13, 14, 53, 54], oxidative stress [15], as well as the exposure to environmental and technogenic toxins can cause a rise in cytosolic Ca2+. The removal of Ca2+ and the detoxication of toxic metabolites must activate the utilization of respiratory substrates and NADPH and decrease their concentration below the normal physiologic values [53, 55, 56]. Together, this can increase the probability of sustainable or transient mPTP opening [10–16] and create optimal conditions for SA flashes (Fig. 7). Thus, the data obtained can explain the mechanism of SA flash, which

accompanies the IMM permeabilization in isolated mitochondria and in cells and tissues in pathologic states.

Acknowledgement

This work was supported by a grant to Nikiforova AB from the Russian Science Foundation (project no. 17-75-10122). We cordially thank Professor A.D. Vinogradov from the Moscow State University for useful discussion.

Conflict of interest/disclosures

None

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Figure legends

Fig. 1. Permeabilization of mitochondrial membranes causes the production of ROS and inhibition of aconitase. A and B. Simultaneous registration of SA-dependent MDCL and Ca2+- and Alam-induced swelling of isolated mitochondria. Mitochondria (0.5 mg/ml) were placed in standard KCl-based medium supplemented with 5 mM malate, 5 mM pyruvate, 10 µM EGTA, and 20 µM MCLA (A). The suspension was placed in wells, which contained, where shown, 40 µg/ml Alam, 1 µM CsA, 1mM EGTA, 50 µM CaCl2, and SOD (100 units/ml). The arrow shows when samples (10 µg protein) were taken from wells without MCLA for the aconitase assay. C. Aconitase activity in intact and swollen mitochondria. Aconitase activity was measured as described in Materials and methods. Insert. Original traces of NADP reduction in the assay mixture. D. H2O2 production in rat liver mitochondria upon permeabilization of

membranes by Ca2+ and Alam, measured using Amplex Red. Mitochondria (0.5 mg protein/ml) were placed in KCl-Based medium (without substrates but supplemented with 10 µM EGTA, 20 µM Amplex Red, and HRP 3 U/ml) and dispensed into wells, which contained 5 mM pyruvate, 5 mM malate, and, where indicated, 50 µM Ca2+, 40 µg/ml of Alam, 5 µM menadione (Men). The line marked “-S” shows the ROS production in the absence of added respiratory substrates. Numbers at traces indicate the maximal rates of hydrogen peroxide production (pmol∙min-1∙mg protein-1). The data of one representative experiment of at least four similar are shown. The values on traces are means ± S.E.M. (n = 3). Fig. 2. Effect of mitochondrial swelling not related to the inner membrane permeabilization on SOD-sensitive MDCL in a mitochondrial suspension. Mitochondria (0.5 mg/ml) were placed in KCl-based medium of different tonicity (50–600 mOsM) supplemented with 5 mM malate, 5 mM pyruvate, 10 µM EGTA, and 20 µM MCLA. The suspension was placed into wells, which contained, where shown, Alam (40 µg/ml) and SOD (100 units/ml), and parallel recording of absorbance and luminescence was started. A. Original traces of MDCL in standard (300 mOsM) medium. B. Absorbance at 540 nm in a mitochondrial suspension at different tonicity. C. The SOD-sensitive part of MDCL at different tonicity. D. Relation of osmotic swelling after 60 min of incubation (B) and SOD-sensitive MDCL integrated for 60 min (C). The values at zero tonicity correspond to Alam-containing samples. The data of one representative experiment of at least four similar are shown. Values on traces are means ± S.E.M. (n = 3) (A and B) or means (n = 3) (C and D). Fig. 3. Effect of respiratory substrates, inhibitors of respiratory chain, NADH, and NADPH on the generation of SA in permeabilized mitochondria. Mitochondria (0.6 mg protein/ml) were placed in standard incubation medium supplemented with 10 µM EGTA, 20 µM MCLA, and, where indicated, respiratory substrates (Panels A and B), 5 mM malate (M), 5 mM pyruvate (P), 5 mM glutamate (G), and 5 mM hydroxylbutyrate (β-Oxy); the inhibitors of respiratory chain (Panel C), rotenone (2 µg/ml) (Rot), 2 mM myxothiazol (Myx), and 1 mM

NaCN. The lines marked “-S” show SA production in the absence of added respiratory substrates. Reference wells also contained SOD (200 U/ml). Arrows show the additions of 200 µM Ca2+ (A), Alam (30 µg/ml) (B–D), 2 mM NADH, and 2 mM NADPH (D). The SODsensitive part of a MDCL signal is presented. The data of representative experiments of at least four similar are shown. Values on traces are means ± S.E.M. (n = 3). The insert in panel A shows the effect of malate plus pyruvate on the redox state of endogenous NAD(P)H in mitochondria permeabilized by Ca2+ recorded in parallel experiments. Fig. 4. A decrease in the NADPH redox state causes the activation of SA production in permeabilized mitochondria. Mitochondria (0.5 mg protein/ml) were incubated in standard medium without respiratory substrates in the presence of Alam (40 µg/mg prot.) for 15 min (insert in panel B). Where indicated, medium also contained rotenone (2 µg/ml) and SOD (200 U/ml). Then, the suspension was transferred to wells that contained NADPH at different concentrations and 20 µM MCLA (A) or NADPH only (B), and luminescence and fluorescence were recorded in parallel. C. Superposition of the dynamics of SA production and NADPH redox state. D. Effect of NADPH+NADP concentration on the apparent E(NADP/NADPH)Max. Panels A–C show the data of one representative experiment of three identical. Values on traces are means ± S.E.M. (n = 3). Data on panel D are means ± S.E.M. for three independent experiments (n = 9). Fig. 5. Effect of the redox state of NADH on the production of SA in permeabilized mitochondria. Experimental conditions were as in Fig. 4. At 0 time, mitochondria were added to wells containing NADH at indicated concentrations. A and B. The data of parallel recording of SA production kinetics in mitochondrial suspension, measured as SOD-sensitive MDCL (A) and NADH oxidation (B). The insert in panel B shows the generation of an equation for the determination of NADH concentration from the fluorescence data by the Boltzmann method (Origin 7.0 software). C. Superposition of the dynamics of SA production and NADH redox state. D. Effect of NADH+NAD concentration on the apparent E(NAD/NADH)Max. Panels A–C show the data of one representative experiment of three identical. Values on traces are means ±

S.E.M. (n = 3). Data on panel D are the means ± S.E.M. for two (+ 1.5 mM NaCN) and three (Control, +Rotenone) independent experiments (n = 6 and 9, respectively). Fig. 6. Comparison of the effects of NADH and NADPH on SA production in intact and permeabilized mitochondria. Mitochondria (0.5 mg/ml) were incubated in standard KClbased medium without respiratory substrates but supplemented with 500 µM EGTA, 20 µM MCLA (B and D), and Alam (25 μg/ml) (A and C) for 10 min. Then, the suspension was transferred to the wells with NADPH (A and B) and NADH (C and D) at indicated concentrations. Inserts show the data of parallel recording of mitochondrial swelling. Representative traces of one of three similar experiments are shown. Values on traces are the means ± S.E.M. (n = 3). Fig. 7. Effect of respiratory substrates at near-physiological concentrations on the SA flash and oxidation of endogenous and added NAD(P)H in mitochondria permeabilized by Ca2+. Mitochondria were placed in standard incubation medium supplemented with 20 µM MCLA, 10 µM EGTA (-S/NADPH), and, where indicated, 75 µM NADPH (panel B), and malate plus pyruvate at low (L, 75 + 25 µM), “physiological” (P, 300 + 100 µM), and high (H, 1 + 0.5 mM) concentrations. Incubation medium for NAD(P)H measurements (inserts) was without MCLA. Arrows show the additions of 200 µM Ca2+. Representative traces of one of four similar experiments are shown. Values on the traces are the means ± S.E.M. (n = 3). Supplementary Fig. 1S. Comparison of sizes of mPT and Alam-created pores in the inner mitochondrial membrane by the solute size exclusion test. Traces show the percent of the recovery of mitochondrial volume after addition of polyethylene glycol of different molecular weight (PEG) to RLM permeabilized by Alam (40 µg/mg protein) and via Ca2+dependent mPTP opening (250 nmol CaCl2/mg protein). The data are the means ± S.E.M. (n = 3). The size of pores created by Alam and Ca2+ (mPTP) was assessed by the method of Pfeiffer (Pfeiffer, D.R., Gudz, T.I., Novgorodov, S.A., Erdahl, W.L., 1995. The peptide

mastoparan is a potent facilitator of the mitochondrial permeability transition. J. Biol. Chem. 270, 4923–4932.) with our modifications (Kruglov AG, Teplova VV, Saris NE. The effect of the lipophilic cation lucigenin on mitochondria depends on the site of its reduction. Biochem Pharmacol. 2007 74(4):545-56.). RLM (1 mg/ml) were exposed to Ca2+ (250 nmol/mg protein) or Alam (40 µg/ml) and allowed to swell for 10 min in KCl-based medium. After the termination of high-amplitude swelling, 10 % (v/v) solutions of PEGs of different molecular masses were added to samples to create a 40% increase in osmotic pressure. The shrinkage of mitochondrial matrix was recorded as an increase in absorbance at 540 nm. Complete matrix volume recovery was assumed to be caused by PEGs totally incapable of penetrating through the IMM. Supplementary Fig. 2S. Effect of pyridine nucleotides on the level of KO2-induced SA in the incubation medium. Experimental conditions are described in Materials and methods. Where it is shown, the medium contained SOD (100 U/ml), NADH, and NADPH at indicated concentrations. Arrows show the addition of 500 µM KO2. Representative traces of 10–20 similar experiments are presented. Supplementary Fig. 3S. Kinetics of NADH-dependent production of hydrogen peroxide in permeabilized mitochondria. Numbers at traces show the rates of hydrogen peroxide production in pmoles∙min-1∙mg protein-1. Mitochondria (0.5 mg protein/ml) were incubated in standard medium without respiratory substrates in the presence of Alam (40 µg/mg prot.) for 15 min. Then, the suspension was supplemented with 20 µM AmplifluTM Red and horseradish peroxidase (3 U/ml) and transferred to wells containing NADH at indicated concentrations, and fluorescence was recorded. The data of one representative experiment of three identical are shown. Values on traces are the means ± S.E.M. (n = 3). Supplementary Fig. 4S. Comparison of the effects of NADH and NADPH on SA production in intact and permeabilized mitochondria. Mitochondria (0.5 mg/ml) were incubated in standard KCl-based medium without respiratory substrates but supplemented with 10 µM EGTA, 20 µM MCLA, NADH (A and C), and NADPH (B and D) at indicated

concentrations. Arrows show the addition of 200 µM Ca2+ (C and D). Inserts in panels A and C show the data of the parallel recording of mitochondrial swelling. The inserts in panels B present a low-scale picture of the same data. Representative traces of one of three similar experiments are shown. Values on traces are the means ± S.E.M. (n = 3).

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SOD-sensitive MDCL, AU

5000

1000

30

Endogenous substrates

10000

SOD-sensitive MDCL, AU

Hydroxybutirate

+ NaCN

20

Time, min

Alam

2000

-Oxy

M+P

7000

0

50

B

Alam

10000

SOD-sensitive MDCL, AU

SOD-sensitive MDCL, AU

6000

NAD(P)H fluorescence, AU

Figure 3ABCD

2000 0

+NADH +NADPH 0

5

10

15

Time, min

20

25

30

Figure 4ABCD

30000

70+Rot

18

175 175+Rot

25000 20000 15000 10000

330

5000

660 0

0 -20

36+SOD 0

20

40

60

80

100 120 140 160

12000

0,4

660

10000

330

0,1

8000

0

4

6

8

10 12 14 16

175 6000 4000

70

2000

36 18

0

0

C

-20

175+Rot

36+SOD

70+Rot 0

20

40

60

80

100 120 140 160

D

-270

-0,26

30000

-0,30

[NADPH]0 = 69.6M

-0,32

10000

[NADP]0 = 7.47 M [NADPH]max = 16.1M -0,34

5000

-0,36

0

-0,38

ENADP(H), V

-0,28

E= 292 mV

ENADP(H)Max, mV

35000

MDCL, AU

2

Time, min

-0,24

15000

Alam, NADPH 0-250 M

Time, min

40000

20000

0,3 0,2

Time, min

25000

Control

0,5

A540

70

Fluorescence 360/465, AU

36

35000

MDCL, AU

B

A

40000

-280

+Rotenone

-290 -300 -310

-Rotenone

-320 0

20

40

60

80

100

Time, min

Figure 4ABCD

120

140

160

0

25

50

75

100

125

150

[NADP(H)], M

175

200

Figure 5ABCD

375+Rot

75+Rot 375

6000 75 4000 2000 0

0

25

50

75

100

125

B

14000

12000

750

12000

10000

375 10000

150

8000

C

1200

E~-259 mV

-0,27

1000

-0,30

800

-0,33

600

NADH0 = 750 M

400

-0,39 0

25

50

Time, min

Figure 5ABCD

Chi^2/DoF = 262053.77584 R^2 = 0.99428

4000

A1 A2 x0 dx

2000

-138212.31828 ±1167702.7076 12758.38355 ±671.23464 -419.07044 ±1632.68983 171.9112 ±83.51711

0 0

100

200

300

400

500

600

75

100

200

700

+Rot

4000

75 +Rot

+Rot

2000 Endo 0 -25 0

25

50

75

D

-200 -220

ENAD(H)Max, mV

ENAD(H), V

+Rotenone

-0,36

6000

100

125

150

Time, min

SOD-sensitive MDCL, AU

-0,24

Data: Data1_E Model: Boltzmann Equation: y = A2 + (A1-A2)/(1 + exp((x-x0)/dx)) Weighting: y No weighting

8000

[NADH], mM

6000

Time, min -0,21

Boltzmann Fit of experimental data

14000

Fluorescence 360/465, AU

MDCL, AU

750

Fluorescence 360/465, AU

10000 8000

A

750+Rot

12000

Control

-240 -260

+NaCN +Rotenone

-280 -300 -320 -340 0

100

200

300

400

500

600

[NAD(H)], mM

700

800

800

Figure 6ABCD

100M

12000

25M 10000

0,4

Control 25-1000 M

0,2 0

40

80

120

Time, min

8000

250M

6000 4000 2000 0

500M 1000M

Control 0

20

40

60

80

100

120

0,6

14000 12000 10000

0

100M

Control 50, 250, 500 M 40

120

500M

250M

25M

3000

80

Time, min

4000

2000 1000 0

Control

1000M 0

20

40

60

80

Time, min Figure 6ABCD

120

4000

251000 M

2000

Control

0 20

40

60

80

100

120

8000

100

120

140

Contro

D

0,6

7000 A540

0,4

0

80

Intact mitochondria (EGTA)

SOD-sensitive MDCL, AU

A540

SOD-sensitive MDCL, AU

5000

40

Time, min

0

C

Intact mitochondria

0,2

0,2

Time, min

0,6

6000 50M

Control 25-1000 M

6000

140

Permeabilized mitochondria (Alam)

7000

0,4

8000

Time, min 8000

B

16000

A540

50M

14000

Intact mitochondria

Intact mitochondria (EGTA)

SOD-sensitive MDCL, AU

A

0,6

A540

SOD-sensitive MDCL, AU

Permeabilized mitochondria (Alam) 16000

6000

0,4

Control 50, 250, 500 M

0,2

5000

0

4000

40

80

120

Time, min

3000 2000 1000

Control 50, 250, 500 M

0 0

20

40

60

80

Time, min

100

120

4000

5000 4000 H

3000

P

2000

H

L

1000 0

P

-S/NADPH 0

20

40

60

80

Time, min

3000

Ca

L

2+

2000

-S/NADPH

1000 0

0

20

40

60

Time, min

Figure 7AB

80

100

B

2+

25000 20000 15000

Fluorescence 360/465

5000

A

2+

Ca

6000

SOD-sensitive MDCL, AU

SOD-sensitive MDCL, AU

6000

Fluorescence 360/465

Figure 7AB

Ca

50000 40000 30000

+NADPH

20000

H

10000 0

P L

+NADPH

0

20

40

60

80

100

L

120

P

Time, min

H

10000 5000 0

Ca

2+

-S/NADPH 0

20

40

60

Time, min

80

100

120