The Role of Reactive Oxygen Species in ... - Semantic Scholar

Bioscience Reports, Vol. 17, No. 1, 1997

REVIEW

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition Anibal E. Vercesi,1,2 Alicia J. Kowaltowski,1 Mercedes T. Grijalba,1 Andre R. Meinicke,1 and Roger F. Castilho1 Received September 5, 1996

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as r-butyl hydroperoxide and diamide is caused by a Ca^-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca2* binding. Studies with submitochondrial particles have demonstrated that the binding of Ca2+ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation. KEY WORDS: Calcium; cyclosporin A; lipid peroxidation; mitochondria; mitochondrial membrane permeability transition; protein oxidation; reactive oxygen species.

1. MITOCHONDRIAL MEMBRANE PERMEABILITY TRANSITION It has long been known that mitochondrial Ca2+ overload leads to nonspecific inner mitochondrial membrane permeabilization (for review, see refs. 1 and 2). In 1976 Hunter et al. (3) suggested that this increased membrane permeability was reversible and named it "Ca2+-induced membrane permeability transition". Further studies evidenced various potentiators of the Ca2+-induced permeability transition, such as inorganic phosphate (Pi), arsenate, fatty acids and thiol oxidants, referred to as "Ca2+ releasing agents or inducers" (for review, see refs. 1, 2, 4, 5). On the other hand, agents that protect mitochondria against this Ca2+ effect, such as ADP, ATP, Mg 2+ , local anaesthetics and disulfide reductants (1-5) were identified. An interesting observation made by one of us in Professor Lehninger's laboratory (6,7) suggested that mitochondrial membrane permeabilization by Ca2+ ions could be stimulated by the oxidized state of mitochondrial pyridine nucleotides and reversed by the reduction of this 'The Departamento de Bioquimica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP 13083-970, Brazil. 2 To whom correspondence should be addressed. 43 0144-8463/97/0200-0043$12.50/0 © 1997 Plenum Publishing Corporation

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Vercesi, Kowaltowski, Grijalba, Meinicke and Castilho

coenzyme. This finding stimulated many groups to work on the topic, using isolated mitochondria (2), intact cells (8) and even perfused organs (9). These studies established an important relationship between mitochondrial reducing power and mitochondrial membrane permeability transition (MPT) (see ref. 10). Recent literature suggests that MPT is a condition triggered by Ca 2 ^ through the opening of a regulated cyclosporin A-sensitive protein channel (the mitochondrial membrane permeability transition pore—PTP) (2,4,5,11,12) that permits an equilibration of solutes with molecular weights of 1500 Da or less (3). The closed conformation of this pore is favored by high mitochondrial membrane potential (13) and matrix acidification (14). It has been proposed that the PTP may operate under some pathologic conditions such as ischemia/reperfusion (15,16), or even under physiologic conditions (17,18). Recent reviews on electrophysiology (5) and regulation (4,5,18) characteristics of the PTP are available, but no emphasis has been given on the role of reactive oxygen species (ROS) on MPT or the sequence of events that lead to irreversible mitochondrial permeabilization. These are the aim of the present review. 2. MITOCHONDRIAL GENERATION OF REACTIVE OXYGEN SPECIES The generation of ROS by mitochondria is a continuous and physiological event under aerobic conditions. It has been calculated that up to 2% of the total mitochondrial oxygen consumption results in the generation of the superoxide anion (O-7) at the level of NADH-coenzyme Q, succinate-coenzyme Q and coenzyme QH2-cytochrome c reductases (complexes I, II and III) (19, 20), due to the monoelectronic reduction of O2. Despite the moderate chemical reactivity of O^ in aqueous solutions, it can generate a highly oxidative and cytotoxic ROS, the hydroxyl radical (HO'), through the reductive homolytic cleavage of H2O2, a dismutation product of O^ (21). It is probable that most of the HO' generated in vivo comes from the iron-dependent breakdown of H2O2, via Fenton's reaction (H2O2 + Fe2+ -+ Fe3+ + HO' + HCr) (22, 23). Mitochondria possesses an efficient antioxidant defense system, represented by the enzymes superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, NAD(P) transhydrogenase, and other compounds such as glutathione (GSH), NADPH, vitamins E and C (23,24). Although catalase constitutes the main cytosolic defense against H2O2, it has only been detected in rat heart mitochondria (25,26). Under physiologic conditions, the oxidant and antioxidant systems of the organelle are in balance, but under conditions in which an excess of ROS is genrated and/or the antioxidant defense system is exhausted, a state of oxidative stress is created. Under increased ROS production, many oxidative alterations of mitochondrial membrane components may occur, such as lipid peroxidation or protein thiol oxidation, leading to mitochondrial permeabilization and dysfunction (24). Our group demonstrated that MPT caused by Ca2+ alone or Ca2+ in the presence of different inducers is related to an increased mitochondrial ROS production and characterized by oxidation of membrane protein thiols (see topic below).

Mitochondrial Membrane Permeability Transition

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Fig. 1. Pictorial image of the lipid phase separation process. A monolayer membrane model is exemplified, in which Ca2+-induced cardiolipin patches can be visualized. Below, the typical 5-doxylstearic acid EPR spectra of labeled, nonrespiring submitochondrial particles in the absence (left) or presence (right) of Ca2* are shown. The broadening observed in the spectrum in the presence of Ca2* is consistent with the formation of lipid domains.

In 1980, Cadenas and Boveris (27) demonstrated that the mitochondrial O^/H2O2 production is increased by ionofores and high Ca2+ levels. Bovine heart submitochondrial particles (SMP) are also effective sources of Oj/H2O2. These particles are devoid of auxiliary dehydrogenases, so the H2O2 formation can be directly related to the componeonts of the respiratory chain, characteristics that make SMP very useful and interesting models to study the effect of Ca2+ on ROS production. In this regard, we have demonstrated that Ca2+ binding to SMP, probably to cardiolipin (CL—the only anionic lipid present in the mitochondria), causes important alterations in lipid organization, as attested by means of electron paramagnetic resonance (EPR) spectroscopy of lipophylic spin labels (28). These alterations are characterized by an increased lipid packing and lipid lateral phase separation (or lipid domain formation) (see Fig. 1), conditions that compromise the conformation and consequently the functionality of all membrane proteins/enzymes (28). In these conditions, the free radical production at the electron transport system could be optimized. This is consistent with the increased production of H2O2 by mitochondria in the presence of Ca2+ (28,29) and subsequent oxidative alterations of the mitochondrial membrane (initiated by HO' radicals). 3. MITOCHONDRIAL MEMBRANE PERMEABILITY TRANSITION IS INDUCED BY OXIDATIVE STRESS CONDITIONS It was first observed in Professor Lehninger's laboratory (6,7) that Ca2+ efflux from isolated mitochondria could be stimulated by the presence of

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acetoacetate or oxaloacetate. These compounds can mimic a situation of oxidative stress in vitro, because they can exhaust mitochondrial GSH and NADPH (30-33), substrates of the antioxidant enzymes glutathione peroxidase and glutathione reductase, respectively, thus favoring accumulation of H2O2 (see Fig. 2). Further studies using these and other prooxidants (diamide and r-butyl hydroperoxide) (29,32-34) demonstrated that this Ca2+ efflux was related to MPT. Literature data show that MPT may also be induced by exogenous ROS-generating systems (5), such as menadione (35), nitrofurantoin (36), 5-aminolevulinic acid (37, 38) and xantine/xantine oxidase (39, 40), in the presence of Ca z+ . The similarity between the membrane alterations caused by exogenous ROS-genrating systems, prooxidants and other MPT inducers led us to propose that Ca2+-induced mitochondrial membrane permeabilization is a situation related to mitochondrial oxidative stress. This proposal is supported by the fact that mitochondrial Ca2+ overload itself is capable of increasing mitochondrial ROS generation (27-29). The participation of mitochondrial-generated ROS in the mechanism of inner mitochondrial membrane permeabilization by Ca2+ was demonstrated by experiments showing that exogenous catalase prevents the disruption of membrane potential and mitochondrial swelling caused by the cation alone or in the presence of f-butyl hydroperoxide (29,34), and by latter experiments (29) showing that no mitochondrial permeabilization occurs in the absence of molecular oxygen or in the presence of o-phenanthroline, a Fe2+ chelator. Fe2+ is necessary for the production of the highly reactive HO' radical via the superoxide-driven Fenton reaction (22,23) (Fig. 2), and is present in rat liver mitochondria in concentrations of about 1.7 nmol of Fe/mg of protein, not including iron-sulfur or heme proteins (41). Intramitochondrial Fe2+ may be mobilized by Ca2+ (29,42), possibly via Ca2+-induced stimulation of the production of the superoxide radical, known to promote Fe2+ mobilization (43). This effect increases HO' production and favors MPT. Regarding the mitochondrial site of electron leakage (ROS production) in Ca2+-induced MPT, our group (44) has shown that when rotenone plus antimycin A-poisoned mitochondria are energized by N,N,A^W-tetramethyl-pphenylenediamine (TMPD), which reduces respiratory chain complex IV, mitochondrial permeabilization does not occur, unless succinate, which reduces coenzyme Q, is also added (44,45). This suggests an important role of reduced and semiquinone forms of coenzyme Q as univalent oxygen reductants. One of the earliest MPT inducing agents described is inorganic phosphate (Pi) (2,5). Although many studies have been conducted on P r induced MPT, the available data do not provide knowledge about the mechanism by which this agent induces membrane permeabilization. Recently, however, our group demonstrated that (i) Pi increases ROS generation in Ca2+-loaded mitochondria (46,47) and that (ii) the permeabilization induced by P( and Ca2+ is inhibited by catalase or the absence of molecular oxygen (46,47), demonstrating that ROS participate in the mechanism. In the presence of high (>4mM) P; concentrations mitochondrial permeabilization is caused by both MPT and mitochondrial


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Fig. 2. Scheme summarizing the proposed molecular processes involved in Ca 2+ /ROS-induced mitochondria! membrane permeabilization. Matrix Ca2* interacts with inner mitochondrial membrane (IMM) components (proteins, lipids or both) increasing ROS production that attack membrane protein thiols leading to the opening of nonspecific pores. HO' radicals can also attack mtDNA, resulting in breakage of the DNA strands, and polyunsaturated fatty acids (PUFA). According to our data, the increased rate of matrix HO' radical production depends on: (i) Ca2* stimulation of electron-leakage from the respiratory chain producing OJ and hence H2O2; (ii) stimulation of the Fenton reaction of H2O2 through matrix Fe2* mobilization by Ca2*; and (iii) lack of NAD(P)H and GSH to permit H2O2 consumption by the enzymes GSH-dependent glutathione peroxidase (GP) and NAD(P)H-dependent glutathione reductase (GR). In contrast, H2O2 diffusion to the medium where it can be consumed by added catalase (CAT) protects mitochondria against Ca2*/ROS-induced oxidative damage. In the presence of high phosphate concentration and cytochrome c (cyt Fe3*), aldehydes (Cn, lipid peroxidation products) can produce lower homologous aldehydes in the triplet state (C n _i), which are able to reinitiate the lipid peroxidation cycle.

membrane lipid peroxidation (47). We proposed that P, stimulates membrane lipid peroxidation by catalyzing the tautomerization of aldehydes (lipid peroxidation products) (48), favoring the oxidation of enol forms by hemoproteins, such as cytocrome c. This oxidation produces triplet state products (49) (see Fig. 2),

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which increase the propagation step of the peroxidative process (49-51). The participation of triplet species is supported by the inhibitory effect of sorbate (47), a quencher of triplet carbonyls (52). Bernardi's group proposed that FTP opening can be inhibited by a high membrane potential, and can be induced by membrane depolarization (13,18). In this regard, we have demonstrated that when membrane depolarization is induced, an important increase in mitochondrial ROS generation occurs, followed by a membrane permeabilization sensitive to both exogenous catalase and the absence of molecular oxygen (46). Consequently, membrane depolarization may act via two concerted mechanisms: (i) increase in ROS production, which leads to protein alterations that generate the PTP, and (ii) increase in the PTP opening probability. Indeed, in 1987 Vercesi (53) demonstrated that at low membrane potentials the NAD(P)H transhydrogenase cannot sustain high levels of mitochondrial reducing power (NADPH and GSH), favoring oxidative stress.

4. MITOCHONDRIAL TARGETS FOR ROS The complete protection against Ca2+/ROS-induced mitochondrial permeabilization conferred by dithiothreitol (34,38,47), a disulfide reductant, is a strong indication that protein thiol groups are important targets for mitochondrial-generated ROS. In this regard, Fagian et al. (54) presented evidence that the membrane permeability increase of heart submitochondrial particles and liver mitoplasts incubated in the presence of Ca2+ and diamide was associated with protein polymerization due to thiol cross-linking. Protein thiol attack by HO' generates high molecular weight protein aggregates via disulfide cross-linkage reactions (29,34,38). These protein alterations are accompanied by a decrease in the mitochondrial membrane thiol content (55). Reactive oxygen species (mainly the hydroxyl radical) most probably promote the oxidation of protein thiol groups buried in the lipid phase of mitochondrial membrane, close to their production sites (56). One of the possible membrane protein targets for ROS oxidation is the ADP/ATP carrier, which possesses four cystein residues (57,58). The carboxyatractyloside-sensitive inhibition by ADP of MPT supports the participation of the ADP/ATP carrier in membrane permeabilization. This carrier is the most abundant integral protein of the inner mitochondrial membrane and its interaction with various ligands (ADP, ATP, bongkrekate, carboxyatractyloside) induces conformation changes in the carrier itself (m or c conformation) (57) and structural modification of the mitochondria (59). It might be possible that binding of ADP to the carrier could grant protection against protein thiol oxidation by (i) promoting alterations of the ADP/ATP carrier conformation that change the position of its thiol groups (57, 58, 60), rendering them not accessible to oxidation by ROS or (ii) changing mitochondria from the orthodox to condensed configuration, an alteration that may protect thiol groups of other membrane proteins against oxidation. The second hypothesis is supported by results of


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Novgorodov et al. (61), showing indirect involvement of the ADP/ATP carrier in the mechanism of PTP opening. Many of the MPT inducers are thiol cross-linking reagents (5). These compounds promote a Ca2+-dependent mitochondrial permeabilization sensitive to cyclosporin A, but not to catalase or the lack of molecular oxygen (29). This suggests that protein thiol cross-linkage that leads to MPT may be caused by ROS or directly by thiol cross-linking reagents. MPT promoted by thiol cross-linking reagents provides an interesting experimental condition for the study of the participation of Ca2+ in MPT, because this permeabilization is independent of Ca2+-induced stimulation of ROS production and HO' generation. We have focused our studies on MPT induced by the bifunctional hydrophotib thiol cross-linker phenylarsine oxide (13) and the bifunctional hydrophilic thiol cross-linker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) (62), in comparison with ROS-induced MPT (in the presence of r-butyl hydroperoxide). We observed that the monofunctional hydrophobic thiol reagent Nethylmaleimide inhibits the permeabilization effects of /-butyl hydroperoxide, DIDS and phenylarsine oxide, while the monofunctional hydrophilic thiol reagent mersalyl inhibits only the effect of DIDS (56). These results show that different MPT inducers may react with distinct mitochondrial membrane protein thiol groups. It is important to stress that although MPT seems to be induced mainly by membrane protein thiol cross-linkage, in the presence of ROS other oxidative alterations of mitochondria may occur, such as lipid peroxidation (see above) and mtDNA fragmentation (63; and Almeida, A. M. and Vercesi, A. E., unpublished results). 5. REVERSIBILITY VS. IRREVERSIBILITY OF Ca2+/ROS INDUCED MITOCHONDRIAL PERMEABILITY It is known that mitochondrial membrane potential (Ai/») drop due to PTP opening is reversed by pyridine nucleotide reductants (64), EGTA (38, 55, 62, 65, 66), dithiothreitol (34), or cyclosporin A in the presence of Mg2+ or ADP (61). Interestingly, we have observed that when EGTA is added long after (>10min) the completion of mitochondrial membrane permeabilization (swelling or Ai// elimination), no membrane resealing occurs and there is a large decrease in the content of membrane protein thiols and increase in protein aggregation due to thiol cross-linkage (55). This suggests that when membrane protein oxidation and aggregation is extensive, due to a high degree of oxidative stress, membrane permeabilization becomes irreversible. We propose that different phases of the permeabilization process occur. Initially, intrapeptide bond formation would confer permeability to small ions and molecules. Progress of protein thiol cross-linking could create a protein assembly that would lead to membrane permeabilization to sucrose. Finally, generalized protein aggregation, producing protein clusters, may cause a large and irreversible degree of permeabilization in which even matrix proteins can cross the membrane.

so


Irreversible mitochondrial membrane permeabilization is also obtained in the presence of high Pj concentrations (>4 mM) due to the concomitant occurrence of membrane lipid peroxidation and PTP opening (see discussion above, Ref. 47). In addition, lipid peroxidation is the main form of irreversible mitochondrial membrane permeabilization caused by Ca2+ in the presence of other oxidants such as Fe2+-citrate (67) or Fe2+-ATP (68).

6. THE ROLE OF Ca2+ IN MPT The presence of intramitochondrial Ca2+ is known to be required in MPT due to the inhibitory effect of ruthenium red, which blocks the Ca2+ uniport (69). According to our and literature data, Ca2+ ions can act in four steps of MPT: (i) stimulation of the production of O^ and hence H2O2 by the respiratory chain (27-29) (Figs 1 and 2); (ii) stimulation of the Fen ton reaction through matrix Fe2"1" mobilization (29) (Fig. 2); (iii) unmasking of membrane protein thiol groups, permitting MPT in the presence of hydrophilic thiol reagents (56); (iv) directly regulated PTP opening by binding to specific mitochondrial membrane proteins (2, 4, 5, 18, 38, 62). The competitive inhibition of Ca z+ -induced MPT by dibucaine, first shown by Vercesi el al. (70), and later confirmed by Bernardi et al. (71) also suggests that Ca2+ binding to membrane lipids is essential for MPT.

7. PATHOLOGIC AND/OR PHYSIOLOGICAL ROLE OF THE MPT Despite extensive studies, the role of the MPT still remains unknown. It has been proposed that MPT may participate in physiological mitochondrial functions (17, 18, 72) such as eliminating excess matrix Ca 2+ , protein transport or even heat-generating mechanisms. On the other hand, nonspecific characteristics of MPT, leading to colloidosmotic swelling of the mitochondrial matrix (for review see Ref. 2) and, finally, loss of matrix proteins (73), are incompatible with the maintenance of mitochondrial integrity. In this regard, evidence has been accumulated that PTP may play a role in cell damage and death in situations of oxidative stress such as ischemia/reperfusion (15, 16, 74).

ACKNOWLEDGMENTS This work was partially supported by grants from the Brazilian Agencies Funda9ao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientifico e Technologico (CNPq) e Programa de Apoio ao Desenvolvimento Cientifico e Technologico (PADCT).


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