Prooxidants open both the mitochondrial permeability transition pore and a low-conductance channel in the inner mitochondrial membrane

Mitochondria can be induced by a variety of agents/conditions to undergo a permeability transition (MPT), which nonselectively increases the permeability of the inner membrane (i.m.) to small (<1500 Da) solutes. Prooxidants are generally considered to trigger the MPT, but some investigators suggest instead that prooxidants open a Ca(2+)-selective channel in the inner mitochondrial membrane and that the opening of this channel, when coupled with Ca(2+) cycling mediated by the Ca(2+) uniporter, leads ultimately to the observed increase in mitochondrial permeability [see, e.g., Schlegel et al. (1992) Biochem. J. 285, 65]. S. A. Novgorodov and T. I. Gudz [J. Bioenerg. Biomembr. (1996) 28, 139] propose that the i.m. contains a pore that, upon exposure to prooxidants, can open to two states, one of which conducts only H(+) and one of which is the classic MPT pore. Given the current interest in increased mitochondrial permeability as a factor in apoptotic cell death, it is important to determine whether i.m. permeability is regulated in one or multiple ways and, in the latter event, to characterize each regulatory mechanism in detail. This study examined the effects of the prooxidants diamide and t-butylhydroperoxide (t-BuOOH) on the permeability of isolated rat liver mitochondria. Under the experimental conditions used, t-BuOOH induced mitochondrial swelling only in the presence of exogenous Ca(2+) (>2 microM), whereas diamide was effective in its absence. In the absence of exogenous inorganic phosphate (P(i)), (1) both prooxidants caused a collapse of the membrane potential (DeltaPsi) that preceded the onset of mitochondrial swelling; (2) cyclosporin A eliminated the swelling induced by diamide and dramatically slowed that elicited by t-BuOOH, without altering prooxidant-induced depolarization; (3) collapse of DeltaPsi was associated with Ca(2+) efflux but not with efflux of glutathione; (4) neither Ca(2+) efflux nor DeltaPsi collapse was sensitive to ruthenium red; (5) collapse of DeltaPsi was accompanied by an increase in matrix pH; no stimulation of respiration was observed; (6) Sr(2+) was able to substitute for Ca(2+) in supporting t-BuOOH-induced i.m. depolarization, but not swelling; (7) in addition to being insensitive to CsA, the collapse of DeltaPsi was also resistant to trifluoperazine, spermine, and Mg(2+), all of which block the MPT; and (8) DeltaPsi was restored (and its collapse was inhibited) upon addition of dithiothreitol, ADP, ATP or EGTA. We suggest that these results indicate that prooxidants open two channels in the i.m.: the classic MPT and a low-conductance channel with clearly distinct properties. Opening of the low-conductance channel requires sulfhydryl group oxidation and the presence of a divalent cation; both Ca(2+) and Sr(2+) are effective. The channel permits the passage of cations, including Ca(2+), but not of protons. It is insensitive to inhibitors of the classic MPT.     

Menadione induces a low conductance state of the mitochondrial inner membrane sensitive to bongkrekic acid

When rat liver mitochondria are allowed to cycle Ca(2+) and are incubated in the presence of the pro-oxidant menadione, they undergo swelling, membrane potential (DeltaPsi) collapse, and ion release. These effects, which are inhibited by cyclosporin A (CsA), are fully consistent with the opening of the so-called permeability transition pore. However, when Ca(2+) cycling is abolished by EGTA, the mitochondria remain energized (DeltaPsi collapse and swelling are avoided), but Ca(2+) efflux, promoted by the chelating agent, is stimulated by menadione. This stimulation goes together with the release of Mg(2+), K(+), and adenine nucleotides (AdN) and is inhibited by bongkrekic acid (BKA). The effect of menadione is also characterized by biphasic NAD(P)H oxidation which becomes monophasic in the presence of BKA, CsA, or EGTA and by the oxidation of thiol groups not restrained by the above-mentioned inhibitors. These results suggest that BKA acts indirectly by preserving in the matrix a critical amount of AdN without modifying the monophasic oxidation of pyridine nucleotides by menadione. A critical number of thiol groups also seems to be involved in the phenomenon. Their oxidation most probably causes a conformational change on adenine nucleotide translocase with the opening of the "low-conductance state" of the mitochondrial permeability transition, resulting in ion permeability without DeltaPsi disruption and mitochondrial swelling.     

Effect of magnesium on calcium-induced depolarisation of mitochondrial transmembrane potential

An effect of magnesium on calcium-induced depolarisation of mitochondrial transmembrane potential (DeltaPsi(m)) was investigated. Depending on the presence of Mg(2+), addition of Ca(2+) to suspension of isolated rat heart mitochondria induced either reversible depolarisation or irreversible collapse of succinate-driven DeltaPsi(m). Irreversible collapse of DeltaPsi(m), observed in the absence of Mg(2+), was insensitive to Ca(2+) chelation, inhibition of Ca(2+) uptake and increased efflux of Ca(2+) from mitochondrial matrix. Based on these data, opening of mPTP in a high-conductance mode is considered to be a major cause of the Ca(2+)-induced irreversible collapse of DeltaPsi(m) in the absence of Mg(2+). Involvement of mPTP in the process of Ca(2+)-induced collapse of DeltaPsi(m) was further supported by protective effect of both CsA and ADP. Reversible collapse of DeltaPsi(m), observed in the presence of Mg(2+), was sensitive to EGTA, ADP; and inhibition of Ca(2+) uptake and increased efflux of Ca(2+) from mitochondrial matrix. This may represent selective induction of a low-conductance permeability pathway. Presented results indicate important role of Mg(2+) in the process of Ca(2+)-induced depolarisation of DeltaPsi(m) mainly through discrimination between low- and high-conductance modes of mPTP. Minor effect of Mg(2+) on Ca(2+)-induced depolarisation of DeltaPsi(m) was observed at the level of stimulation of DeltaPsi(m) generation and inhibition of mitochondrial Ca(2+) uptake.     

The oat mitochondrial permeability transition and its implication in victorin binding and induced cell death

The mitochondrion has emerged as a key regulator of apoptosis, a form of animal programmed cell death (PCD). The mitochondrial permeability transition (MPT), facilitated by a pore-mediated, rapid permeability increase in the inner membrane, has been implicated as an early and critical step of apoptosis. Victorin, the host-selective toxin produced by Cochliobolus victoriae, the causal agent of victoria blight of oats, has been demonstrated to bind to the mitochondrial P-protein and also induces a form of PCD. Previous results suggest that a MPT may facilitate victorin's access to the mitochondrial matrix and binding to the P-protein: (i) victorin-induced cell death displays features similar to apoptosis; (ii) in vivo, victorin binds to the mitochondrial P-protein only in toxin-sensitive genotypes whereas victorin binds equally well to P-protein isolated from toxin-sensitive and insensitive oats; (iii) isolated, untreated mitochondria are impermeable to victorin. The data implicate an in vivo change in mitochondrial permeability in response to victorin. This study focused on whether oat mitochondria can undergo a MPT. Isolated oat mitochondria demonstrated high-amplitude swelling when treated with spermine or Ca2+ in the presence of the Ca2+-ionophore A23187, and when treated with mastoparan, an inducer of the MPT in rat liver mitochondria. In all cases, swelling demonstrated size exclusion in the range 0.9-1.7 kDa, similar to that found in animal mitochondria. Further, MPT-inducing conditions permitted victorin access to the mitochondrial matrix and binding to the P-protein. In vivo, victorin treatment induced the collapse of mitochondrial transmembrane potential within 2 h, indicating a MPT. Also, the victorin-induced collapse of membrane potential was clearly distinct from that induced by uncoupling respiration, as the latter event prevented the victorin-induced PCD response and binding to P-protein. These results demonstrate that a MPT can occur in oat mitochondria in vitro, and are consistent with the hypothesis that an MPT, which allows victorin access to the mitochondrial matrix and binding to the P-protein, occurs in vivo during victorin-induced PCD.     

Interaction of peroxidized cardiolipin with rat-heart mitochondrial membranes: induction of permeability transition and cytochrome c release

Cardiolipin peroxidation plays a critical role in mitochondrial cytochrome c release and subsequent apoptotic process. Mitochondrial pore transition (MPT) is considered as an important step in this process. In this work, the effect of peroxidized cardiolipin on MPT induction and cytochrome c release in rat heart mitochondria was investigated. Treatment of mitochondria with micromolar concentrations of cardiolipin hydroperoxide (CLOOH) resulted in a dose-dependent matrix swelling, DeltaPsi collapse, release of preaccumulated Ca2+ and release of cytochrome c. All these events were inhibited by cyclosporin A and bongkrekic acid, indicating that peroxidized cardiolipin behaves as an inducer of MPT. Ca2+ accumulation by mitochondria was required for this effect. ANT (ADP/ATP translocator) appears to be involved in the CLOOH-dependent MPT induction, as suggested by the modulation by ligands and inhibitors of adenine nucleotide translocator (ANT). Together, these results indicate that peroxidized cardiolipin lowers the threshold of Ca2+ for MPT induction and cytochrome c release. This synergistic effect of Ca2+ and peroxidized cardiolipin on MPT induction and cytochrome c release in mitochondria, might be important in regulating the initial phase of apoptosis and also may have important implications in those physiopathological situations, characterized by both Ca2+ and peroxidized cardiolipin accumulation in mitochondria, such as aging, ischemia/reperfusion and other degenerative diseases.     

Tyramine and monoamine oxidase inhibitors as modulators of the mitochondrial membrane permeability transition

Incubation of rat liver mitochondria with 100-500 mM tyramine, a substrate for monoamine oxidases A and B (MAOs), in the presence of 30 mM Ca2+ induces matrix swelling, accompanied by collapse of membrane potential, efflux of endogenous Mg2+ and accumulated Ca2+ and oxidation of endogenous pyridine nucleotides. These effects are completely abolished in the presence of cyclosporin A, ADP, dithioerythritol and N-ethylmaleimide, thus confirming the induction of the mitochondrial membrane permeability transition (MPT). The observed partial protective effect exerted by catalase indicates the involvement of both MAO-derived hydrogen peroxide and aldehyde. Higher concentrations of tyramine (1-2 mM) are less effective or even completely ineffective. At these high concentrations tyramine has an inhibitory effect when the MPT is induced by 100 mM Ca2+. The MAO inhibitors clorgyline (50 mM) and pargyline (500 mM) completely protect against MPT induction by 100 mM tyramine but also inhibit the phenomenon, although with different efficacy, when it is induced by 100 mM Ca2+ in the absence of tyramine. Taken together, our data suggest that tyramine, clorgyline and pargyline act as modulators of the MPT either through a direct inducing/protective effect or by controlling hydrogen peroxide and aldehyde generation.     

Regulation of Ca2+ transport in brain mitochondria. I. The mechanism of spermine enhancement of Ca2+ uptake and retention

Spermine enhances electrogenic Ca2+ uptake and inhibits Na(+)-independent Ca2+ efflux in rat brain mitochondria. As a result, Ca2+ retention by brain mitochondria increases greatly and the external free Ca2+ level at steady-state can be lowered to physiologically relevant concentrations. The stimulation of Ca2+ uptake by spermine is more pronounced at low concentrations of Ca2+, effectively lowering the apparent Km for Ca2+ uptake from 3 microM to 1.5 microM. However, the apparent Vmax is also increased. At low Ca2+ concentrations, Ca2+ uptake is diffusion-limited. Spermine strongly inhibits Ca2+ binding to anionic phospholipids and it is suggested that this increases the rate of surface diffusion which reduces the apparent Km for uptake. The same effect could inhibit the Na(+)-independent efflux if the rate of efflux is limited by Ca2+ dissociation from the efflux carrier. In brain mitochondria (but not in liver) the spermine effect depends on the presence of ADP. In a medium that contains physiological concentrations of Pi, Mg+, K+, ADP and spermine, brain mitochondria sequester Ca2+ down to 0.1 microM and below, depending on the matrix Ca2+ load. Moreover, brain mitochondria under the same conditions buffer the external medium at 0.4 microM, a concentration at which the set point becomes independent of the matrix Ca2+ content. Thus, mitochondria appear to be capable of modulating calcium oscillations in brain cells.     

Zinc as an inducer of the membrane permeability transition in rat liver mitochondria

It is shown that 2-10 microM Zn2+ induces swelling of rat liver mitochondria incubated in a buffered sucrose medium either with valinomycin or with FCCP, Ca2+, ionophore A23187, oligomycin, and nigericin. This swelling was associated with the release of GSH from mitochondria. Both processes were sensitive to known inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A, and Mg2+. Mitochondrial swelling induced by Zn2+ was also inhibited by rotenone, antymycin A, N-ethylmaleimide, butylhydroxytoluene, and spermine, whereas it was stimulated by tert-butyl hydroperoxide, diamide, and monobromobimane. It did not require the addition of phosphate. The same sensitivity to pH of the mitochondrial swelling induced by Zn2+ and by phenylarsine oxide suggests the same site of the interaction, namely, thiol groups. The ability of Zn2+ to induce mitochondrial swelling gradually decreased along with its increasing concentration above 10 microM. It is concluded that micromolar Zn2+ induces the MPT presumably by the interaction with cysteinyl residues. This process is independent of the mitochondrial membrane potential.     

Effect of Ca2+ on induction of the mitochondrial pore opening in the rat myocardium

The mitochondrial role opening (MPT) induced by Ca2+ has been studied in isolated rat heart mitochondria. MPT was characterized as cyclosporine A-inhibited swelling accompanied by the loss of membrane potential (deltapsim) and Ca2+ efflux after the Ca2+ -loading which was followed spectrophotometrically after the Ca2+ -arsenaso-III complex formation. It has been shown that in suspension of isolated mitochondria MPT was activated by low (with maximum at about 20 microM Ca2+) and high concentrations of Ca2+ (the concentration curve shows a saturation at about 1.0-1.5 mM). In all the cases an access of Ca2+ ions to the matrix space of the mitochondria was necessary for MPT induction. MPT activated by low concentrations of Ca2+ was accompanied by slow decrease of deltapsim and slow release of Ca2+, enhanced by ruthenium red (RR), and was independent of the substrate used (glutamate or succinate). It had not been observed if the respiratory chain was inhibited, even if the Ca2+ access to the inner mitochondrial membrane was provided by Ca2+ -ionophore A23187. At high Ca2+ concentrations rapid Ca2+ -uptake and release via Ca2+ -uniporter (inhibited by ruthenium red) followed by extensive swelling (pore formation) have been observed. It had been supposed that rapid MPT at high concentrations of Ca2+ was the result of Ca2+ entrance to the mitochondrial matrix and depolarisation of the mitochondrial membrane. The data obtained show two different mechanisms of Ca2+ -induced MPT. The one is sensitive to the redox-state of the electron transport chain and is abolished if the respiration is inhibited. The other is independent of mitochondrial respiration and needs only Ca2+ access to the inner mitochondrial membrane and Ca2+ binding to some specific sites leading to MPT opening.     

Arachidonic acid induces specific membrane permeability increase in heart mitochondria

Micromolar concentrations of arachidonic acid cause in Ca2+ loaded heart mitochondria matrix swelling and Ca2+ release. These effects appear to be unrelated to the classical membrane permeability transition (MPT), as they are CsA insensitive, membrane potential independent and can also be activated by Sr2+. Atractyloside potentiated and ATP inhibited the arachidonic acid induced swelling. These observations suggest that the ATP/ADP translocator (ANT) may be involved in the AA induced, CsA insensitive membrane permeability increase. Under the same experimental conditions used for heart mitochondria, arachidonic acid induced the classical CsA sensitive, ADP inhibitable MPT in liver mitochondria.     

Calcium induced release of mitochondrial cytochrome c by different mechanisms selective for brain versus liver.

Increased mitochondrial Ca2+ accumulation is a trigger for the release of cytochrome c from the mitochondrial intermembrane space into the cytosol where it can activate caspases and lead to apoptosis. This study tested the hypothesis that Ca2+-induced release of cytochrome c in vitro can occur by membrane permeability transition (MPT)-dependent and independent mechanisms, depending on the tissue from which mitochondria are isolated. Mitochondria were isolated from rat liver and brain and suspended at 37 degrees C in a K+-based medium containing oxidizable substrates, ATP, and Mg2+. Measurements of changes in mitochondrial volume (via light scattering and electron microscopy), membrane potential and the medium free [Ca2+] indicated that the addition of 0.3 - 3.2 micromol Ca2+ mg-1 protein induced the MPT in liver but not brain mitochondria. Under these conditions, a Ca2+ dose-dependent release of cytochrome c was observed with both types of mitochondria; however, the MPT inhibitor cyclosporin A was only capable of inhibiting this release from liver mitochondria. Therefore, the MPT is responsible for cytochrome c release from liver mitochondria, whereas an MPT-independent mechanism is responsible for release from brain mitochondria.     

Tributyltin causes cytochrome C release from isolated mitochondria by two discrete mechanisms

Using isolated liver mitochondria we show that low concentrations of TBT (0.5 microM) cause the release of mitochondrial cytochrome c, in the presence of Ca(2+). This is reflected in a rapid loss of membrane potential (DeltaPsi(m)), and a large-amplitude swelling characteristic of mitochondrial permeability transition (MPT). Despite this, the inclusion of cyclosporin A could not prevent the release of cytochrome c. Further, in the absence of Ca(2+), low concentrations of TBT (0.5 microM) resulted in a slow sub-maximal shift of DeltaPsi(m), not characteristic of MPT, which was still paralleled by a release of cytochrome c. Further experiments showed that the loss of DeltaPsi(m) in the absence of Ca(2+) was due to a combination of inhibition of respiration and a direct uncoupling effect on the respiratory chain. Under these conditions, rapid swelling of mitochondria could be demonstrated, due to chloride exchange over the inner mitochondrial membrane. Taken together these data suggest that TBT can induce the release of cytochrome c in intact cells by at least two mechanisms. The first and critical mechanism is initiated immediately the mitochondria sense the presence of TBT and involves a slow loss of DeltaPsi(m) and induction of swelling, which allows release of cytochrome c in a relatively non-specific manner and independently from a rise in [Ca(2+)](i). The second mechanism involves the induction of formal MPT as intracellular [Ca(2+)](i) increases. These data help to explain previous observations in intact lymphocytes demonstrating TBT-induced release of mitochondrial cytochrome c in the absence of a rise in [Ca(2+)](i) (Stridh, H., Gigliotti, D., Orrenius, S., and Cotgreave, I. A. (1999) Biochem. Biophys. Res. Commun. 266, 460-465).     

The effect of spermine on transport of Ca2+ ions in brain mitochondria

The effect of spermine (50-400 microM) on the Ca-transporting system of brain mitochondria was studied. In a medium containing Mg2+ and ATP, spermine facilitates the accumulation of Ca2+ by decreasing Km of the uniporter. Spermine inhibits Na-stimulated Ca2+ efflux; this effect is dependent on the ionic strength of the medium--it is decreased when KCl concentration is increased from 20 to 120 mM. Spermine (200 microM) decreases (by 50%) the steady state concentration of Ca2+ maintained by mitochondria. The importance of spermine as a regulator of Ca2+-transport in brain mitochondria is discussed.     

Characteristics of the calcium-triggered mitochondrial permeability transition in nonsynaptic brain mitochondria: effect of cyclosporin A and ubiquinone O

The objective of the present study was to assess the capacity of nonsynaptic brain mitochondria to accumulate Ca2+ when subjected to repeated Ca2+ loads, and to explore under what conditions a mitochondrial permeability transition (MPT) pore is assembled. The effects of cyclosporin A (CsA) on Ca2+ accumulation and MPT pore assembly were compared with those obtained with ubiquinone 0 (Ubo), a quinone that is a stronger MPT blocker than CsA, when tested on muscle and liver mitochondria. When suspended in a solution containing phosphate (2 mM) and Mg2+ (1 mM), but no ATP or ADP, the brain mitochondria had a limited capacity to accumulate Ca2+ (210 nmol/mg of mitochondrial protein). Furthermore, when repeated Ca2+ pulses (40 nmol/mg of protein each) saturated the uptake system, the mitochondria failed to release the Ca2+ accumulated. However, in each instance, the first Ca2+ pulse was accompanied by a moderate release of Ca2+, a release that was not observed during the subsequent pulses. The initial release was accompanied by a relatively marked depolarization, and by swelling, as assessed by light-scattering measurements. However, as the swelling was <50% of that observed following addition of alamethicin, it is concluded that the first Ca2+ pulse gives rise to an MPT in a subfraction of the mitochondrial population. CsA, an avid blocker of the MPT pore, only marginally increased the Ca(2+)-sequestrating capacity of the mitochondria. However, CsA eliminated the Ca2+ release accompanying the first Ca2+ pulse. The effects of CsA were shared by Ubo, but when the concentration of Ubo exceeded 20 microM, it proved toxic. The results thus suggest that brain mitochondria are different from those derived from a variety of other sources. The major difference is that a fraction of the brain mitochondria, studied presently, depolarized and showed signs of an MPT. This fraction, but not the remaining ones, contributed to the chemically and electron microscopically verified mitochondrial swelling.     

Thapsigargin directly induces the mitochondrial permeability transition.

High concentrations of thapsigargin (TG) have been used to study the process of necrotic cell death, which involves mitochondria in the cell rapidly undergoing the mitochondrial permeability transition (MPT). We therefore investigated the effects of TG on MPT in isolated liver and heart mitochondria. Using a matrix swelling assay in combination with a novel enzymatic method based on inner membrane permeability to citrate synthase substrates, TG induced MPT in a concentration-dependent manner, independent of extramitochondrial [Ca2+] and inhibitable by cyclosporin A. Evidence from alamethicin-permeabilized mitochondria suggests that TG induces MPT by causing Ca2+ release from mitochondrial matrix Ca2+-binding sites. These findings suggest that the MPT-inducing effect of TG may contribute to its pro-necrotic and pro-apoptotic effects in various cell types.     

Interactions between spermine and Mg2+ on mitochondrial Ca2+ transport

The effects of the polyamine spermine on the regulation of Ca2+ transport by subcellular organelles from rat liver, heart, and brain were investigated using ion-sensitive minielectrodes and a 45Ca2+ tracer method. Spermine stimulated Ca2+ uptake by mitochondria but not by microsomes. In the presence of spermine, isolated mitochondria could maintain a free extramitochondrial Ca2+ concentration of 0.3-0.2 microM. Stimulation of the initial rates of Ca2+ uptake and 45Ca2+ cycling of mitochondria by spermine shows that this was accomplished through a decrease of the apparent Km for Ca2+ uptake by the Ca2+ uniporter. The half maximally effective concentration of spermine (50 microM) was in the range of physiological concentrations of this polyamine in the cell. Spermidine was five times less effective. Putrescine was ineffective. The stimulation of mitochondrial Ca2+ uptake by spermine was inhibited by Mg2+ in a concentration-dependent manner. However, the diminished contribution of the mitochondria to the regulation of the free extraorganellar Ca2+ concentration could mostly be compensated for by microsomal Ca2+ uptake. Spermine also reversed ruthenium red-induced Ca2+ efflux from mitochondria. It is concluded that spermine is an activator of the mitochondrial Ca2+ uniporter and Mg2+ an antagonist. By this mechanism, the polyamines can confer to the mitochondria an important role in the regulation of the free cytoplasmic Ca2+ concentration in the cell and of the free Ca2+ concentration in the mitochondrial matrix.     

Fluorescamine-induced membrane permeability in mitochondria.

1. Addition of fluorescamine (75 microM) to mitochondria induced an increase in membrane permeability. 2. The leakiness of the inner mitochondrial membrane is characterized by extensive release of accumulated Ca2+, collapse of the transmembrane potential, mitochondrial swelling and efflux of matrix proteins, among them, malate dehydrogenase. 3. These effects were diminished by supplementing the media with 1 mM phosphate, and partially prevented by Mg2+. 4. These results indicate that the primary amino groups of membrane components contribute, partially, to the maintenance of the permeability barrier in mitochondria.     

Free radical scavenging action of the natural polyamine spermine in rat liver mitochondria

The isoflavonoid genistein, the cyclic triterpene glycyrrhetinic acid, and salicylate induce mitochondrial swelling and loss of membrane potential (Delta Psi) in rat liver mitochondria (RLM). These effects are Ca(2+)-dependent and are prevented by cyclosporin A and bongkrekik acid, classic inhibitors of mitochondrial permeability transition (MPT). This membrane permeabilization is also inhibited by N-ethylmaleimide, butylhydroxytoluene, and mannitol. The above-mentioned pro-oxidants also induce an increase in O(2) consumption and H(2)O(2) generation and the oxidation of sulfhydryl groups, glutathione, and pyridine nucleotides. All these observations are indicative of the induction of MPT mediated by oxidative stress. At concentrations similar to those present in the cell, spermine can prevent swelling and Delta Psi collapse, that is, MPT induction. Spermine, by acting as a free radical scavenger, in the absence of Ca(2+) inhibits H(2)O(2) production and maintains glutathione and sulfhydryl groups at normal reduced level, so that the critical thiols responsible for pore opening are also consequently prevented from being oxidized. Spermine also protects RLM under conditions of accentuated thiol and glutathione oxidation, lipid peroxidation, and protein oxidation, suggesting that its action takes place by scavenging the hydroxyl radical.     

Ca2+-release pathways from mitochondria of the yeast Endomyces magnusii

Ca2+-release pathways from Ca2+-preloaded mitochondria of the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca2+ was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Intensive aeration of the mitochondrial suspension rapidly inhibited the efflux of Ca2+ and induced its reuptake. The Ca2+ release was not affected by cyclosporin A, an inhibitor of the nonselective permeability transition of mammalian mitochondria. With acetate as the permeant anion, a spontaneous net Ca2+ efflux began after uptake of about 75% of the added cation. The rate of this efflux was insensitive to cyclosporin A, aeration, and Na+ and was proportional to the Ca2+ load. The Ca2+ release was inhibited by La3+, Mn2+, Mg2+, TPP+, and nigericin (in the presence of KCl) and activated by spermine and hypotonicity. We conclude that Ca2+ efflux from preloaded E. magnusii mitochondria is very similar to the Na+-independent specific pathway for Ca2+ release operative in mitochondria from nonexcitable mammalian tissues.     

Increased permeability of mitochondria during Ca2+ release induced by t-butyl hydroperoxide or oxalacetate. the effect of ruthenium red

Ca2+ release from mitochondria induced by oxalacetate or t-butyl hydroperoxide is accompanied by loss of endogenous Mg2+ and K+, swelling, loss of membrane potential, and other alterations which indicate that Ca2+ release is a result of increased inner membrane permeability. When ruthenium red is added after Ca2+ uptake, but before the releasing agent, the extent of Ca2+ release is diminished as is the extent of Mg2+ and K+ depletion and the extent of swelling. Under these conditions, the membrane potential appears to remain at a high value. When Ca2+ release is induced by oxalacetate or t-butyl hydroperoxide and ruthenium red is added subsequently, an apparent regeneration of membrane potential is observed providing that the associated swelling and Mg2+ loss had not been completed at the time ruthenium red was added. Under these conditions subsequent swelling and Mg2+ loss are inhibited.l Ultrastructural observations show the mitochondria become permeable in response to Ca2+ plus oxalacetate or Ca2+ plus t-butyl hydroperoxide in a heterogeneous manner. Conditions which appear to separate Ca2+ release from a decline in membrane potential or to produce an apparent recovery of membrane potential following partial collapse are shown to prevent a subpopulation of the mitochondria from becoming permeable. It is shown that membrane potential probes will not indicate a decline in potential or the presence of a permeable fraction under these conditions. It is concluded that the presence of Ca2+ accumulation inhibitors does not separate Ca2+ release from the development of increased inner membrane permeability.