Permeability of inner mitochondrial membrane and oxidative stress

The mechanism of increase in the inner membrane permeability induced by Ca2+ plus Pi, diamide and hydroperoxides has been analyzed. (1) The permeability increase is antagonized by oligomycin and favoured by atractyloside. The promoting effect of atractyloside is strongly reduced if the mitochondria are simultaneously treated with oligomycin. (2) Addition of the free-radical scavenger, butylhydroxytoluene, results in a complete protection of the membrane with respect to the permeability increase. (3) Although membrane damage and depression of the GSH concentration are often associated, there is no direct correlation between extent of membrane damage and concentration of reduced glutathione. Abolition of the permeability increase by butylhydroxytoluene or by oligomycin is not accompanied by maintenance of a high GSH concentration in the presence of diamide or hydroperoxides. The membrane damage induced by Ca2+ plus Pi is not accompanied by a depression of the GSH concentration. (4) It is proposed that a variety of processes causing an increased permeability of the inner mitochondrial membrane merge into some ultimate common steps involving the action of oxygen radicals.     

Effect of Ca2+, peroxides, SH reagents, phosphate and aging on the permeability of mitochondrial membranes

The mechanism by which a number of agents such as hydroperoxides, inorganic phosphate, azodicarboxylic acid bis(dimethylamide) (diamide), 2-methyl-1,4-naphthoquinone (menadione) and aging, induce Ca2+ release from rat liver mitochondria has been analyzed by following Ca2+ fluxes in parallel with K+ fluxes, matrix swelling and triphenylmethylphosphonium fluxes (as an index of transmembrane potential). Addition of hydroperoxides causes a cycle of Ca2+ efflux and reuptake and an almost parallel cycle of delta psi depression. The hydroperoxide-induced delta psi depression is biphasic. The first phase is rapid and insensitive to ATP and is presumably due to activation of the transhydrogenase reaction during the metabolization of the hydroperoxides. The second phase is slow and markedly inhibited by ATP and presumably linked to the activation of a Ca2+-dependent reaction. The slow phase of delta psi depression is paralleled by matrix K+ release and mitochondrial swelling. Nupercaine and ATP reduce or abolish also K+ release and swelling. Inorganic phosphate, diamide, menadione or aging also cause a process of Ca2+ efflux which is paralleled by a slow delta psi depression, K+ release and swelling. All these processes are reduced or abolished by Nupercaine and ATP. The slow delta psi depression following addition of hydroperoxide and diamide is largely reversible at low Ca2+ concentration but tends to become irreversible at high Ca2+ concentration. The delta psi depression increases with the increase of hydroperoxide, diamide and menadione concentration, but is irreversible only in the latter case. Addition of ruthenium red before the hydroperoxides reduces the extent of the slow but not of the rapid phase of delta psi depression. Addition of ruthenium red after the hydroperoxides results in a slow increase of delta psi. Such an effect differs from the rapid increase of delta psi due to ruthenium-red-induced inhibition of Ca2+ cycling in A23187-supplemented mitochondria. Metabolization of hydroperoxides and diamide is accompanied by a cycle of reversible pyridine nucleotide oxidation. Above certain hydroperoxide and diamide concentrations the pyridine nucleotide oxidation becomes irreversible. Addition of menadione results always in an irreversible nucleotide oxidation. The kinetic correlation between Ca2+ efflux and delta psi decline suggests that hydroperoxides, diamide, menadione, inorganic phosphate and aging cause, in the presence of Ca2+, an increase of the permeability for protons of the inner mitochondrial membrane. This is followed by Ca2+ efflux through a pathway which is not the H+/Ca2+ exchange.     

Membrane protein thiol cross-linking associated with the permeabilization of the inner mitochondrial membrane by Ca2+ plus prooxidants

In a previous report (Macedo, D.V., Ferraz, V. L., Pereira-da-Silva, L., and Vercesi, A. E. (1988) in Integration of Mitochondrial Functions (Lemasters, J. J., et al., eds) pp. 535-542, Plenum Publishing Corp., New York), we proposed that the alterations in the inner mitochondrial membrane permeability caused by Ca2+ plus prooxidants could be the consequence of membrane protein sulfhydryl-disulfide transitions. In this study, we show that Ca2+ plus diamide, a thiol oxidant, significantly decrease the ability of beef heart submitochondrial particles to build up and sustain a membrane potential generated by succinate oxidation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins indicates that these effects on the membrane potential are associated with the production of protein aggregates due to thiol cross-linking. Evidence is also presented that these protein aggregates can be produced in mitoplasts previously loaded with Ca2+ and that this is potentiated by the presence of either diamide or t-butylhydroperoxide. Furthermore, dithiothreitol, a disulfide reductant, was found to be much more effective than NAD(P)+ reductants in reversing Ca2+ efflux induced by prooxidants. It is concluded that the perturbation of the inner mitochondrial membrane caused by Ca2+ plus prooxidants is associated with protein polymerization due to thiol cross-linking, resulting in the production of high molecular mass protein aggregates.     

Interaction of free fatty acids with mitochondria: coupling, uncoupling and permeability transition

Long chain free fatty acids (FFA) exert, according to their actual concentration, different effects on the energy conserving system of mitochondria. Sub-micromolar concentrations of arachidonic acid (AA) rescue DeltapH-dependent depression of the proton pumping activity of the bc1 complex. This effect appears to be due to a direct interaction of AA with the proton-input mouth of the pump. At micromolar concentrations FFA increase the proton conductance of the inner membrane acting as protonophores. FFA can act as natural uncouplers, causing a mild uncoupling, which prevents reactive oxygen species production in the respiratory resting state. When Ca(2+)-loaded mitochondria are exposed to micromolar concentrations of FFA, the permeability of the inner membrane increases, resulting in matrix swelling, rupture of the outer membrane and release of intermembrane pro-apoptotic proteins. The characteristics of AA-induced swelling appear markedly different in mitochondria isolated from heart or liver. While in the latter it presents the canonical features of the classical permeability transition (PT), in heart mitochondria substantial differences are observed concerning CsA sensitivity, DeltaPsi dependence, reversibility by BSA and specificity for the activating divalent cation. In heart mitochondria, the AA-dependent increase of the inner membrane permeability is affected by ANT ligands such as adenine nucleotides and atractyloside. AA apparently causes a Ca2+-mediated conversion of ANT from a translocator to a channel system. Upon diamide treatment of heart mitochondria, the Ca2+/AA-induced CsA insensitive channel is converted into the classical PT pore. The relevance of these observations in terms of tissue-specific components of the putative PTP and heart ischemic and post-ischemic process is discussed.     

Induction of the non-selective mitochondrial pore in lymphoid cells. 2. Intact rat thymocytes.

Increase of Ca2+ concentration in the cytosol of thymocytes to 400-600 nM causes slow accumulation of Ca2+ in mitochondria. Release of Ca2+ from mitochondria into the cytosol is induced by an uncoupler (FCCP) or by a dithiol cross-linking agent (phenylarsine oxide) and is inhibited by cyclosporin A--a specific inhibitor of the permeability transition pore in the inner mitochondrial membrane. In the presence of oxidizing agents (tert-butyl hydroperoxide and diamide), sub-optimal concentrations of uncoupler induce rapid cyclosporin-sensitive release of Ca2+. 6-Ketocholestanol, a recoupler under these conditions, causes redistribution of Ca2+ from the cytosol into mitochondria. These data indicate that partial uncoupling under conditions of oxidative stress causes opening of the permeability transition pore in a fraction of the mitochondria in intact lymphocytes. This mechanism mediates rapid release of Ca2+ from mitochondria into the cytosol.     

Preparation of an inner membrane-matrix fraction from yeast yeast mitochondria.

Treatment of yeast mitochondria with digitonin was used in order to prepare an inner membrane-matrix fraction preserving its permeability properties. The incubation time of mitochondria with digitonin was an essential parameter for the selective solubilization of the outer membrane. The incubation of mitochondria for l min at different concentrations of digitonin led to a three-step release of mitochondrial enzymes: (a) at low concentrations of digitonin, adenylate kinase was released; (b) higher concentrations were required to solubilize kynurenine hydroxylase, an outer membrane marker; (c) inner membrane markers (succinate dehydrogenase and oligomycin-sensitive adenosine triphosphatase) and matrix markers (fumarase and isocitrate dehydrogenase) were significantly released at concentrations of digitonin higher than 0.4 mg/mg of protein. The electron microscopic aspects of yeast mitoplasts (inner membrane-matrix fraction obtained by treatment with 0.4 mg of digitonin) showed an orthodox and a twisted configuration. These new organelles retained respiratory control when assayed with ethanol as the substrate. Their selective permeability properties were preserved as shown by isoosmotic swelling in potassium or ammonium salt solutions.     

Ca2+ Induces a Cyclosporin A-Insensitive Permeability Transition Pore in Isolated Potato Tuber Mitochondria Mediated by Reactive Oxygen Species

Oxidative damage of mammalian mitochondria induced by Ca²⁺ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca²⁺ uniport, undergo inner membrane permeabilization when treated with Ca²⁺ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg²⁺ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis–trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca²⁺ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.     

A possible mechanism for respiration-dependent efflux of Mg ions from liver mitochondria.

Addition of Pi or diamide to a suspension of rat liver mitochondria induced a net efflux of Mg⁺⁺ which is dependent on coupled respiration. This Mg⁺⁺ efflux is prevented by EGTA and by Ruthenium red, both of which also prevent the increased rate of state 4 respiration induced by Pi or by diamide. It is assumed that an accelerated recycling of endogenous Ca⁺⁺ induced by Pi or by diamide through an altered permeability of inner membrane to Ca⁺⁺ is responsible for Mg⁺⁺ efflux, and accounts for its apparent dependence on coupled respiration.     

The role of glutathione in the retention of Ca2+ by liver mitochondria

Concentrations of rhein and nitrofurantoin in the micromolar range induce Ca2+ release and the development of increased inner membrane permeability in liver mitochondria. Both compounds inhibit the mitochondrial glutathione reductase causing a depletion of GSH and an accumulation of GSSG in energized mitochondria. Under these conditions, the compounds also alter the oxidation state of pyridine nucleotides, NADH becoming oxidized while NADPH remains reduced. Using rhein or nitrofurantoin, together with t-butyl-hydroperoxide and beta-hydroxybutyrate, it is possible to selectively alter the NAD/NADH, the NADP/NADPH, and the GSSG/GSH ratios and to determine the effect of these different states on the ability of Ca2+ to produce a permeable inner membrane. No correlation between pyridine nucleotide ratios and sensitivity to Ca2+ was observed. Mitochondria are stable to Ca2+ when the GSH content is high, but become permeable when Ca2+ is present and GSH is converted to GSSG. It is proposed that the GSSG/GSH ratio, by controlling the reduction state of critical sulfhydryl groups, regulates lysophospholipid acyltransferase activity and, therefore, the ability of mitochondria to remain impermeable upon activation of the intramitochondrial Ca2+ requiring phospholipase A2.     

Effects of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) on mitochondrial bioenergetics and oxidative stress: a comparative study

The potential protective action of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) against oxidative stress was assessed on mitochondrial bioenergetics, inner membrane anion channel (IMAC), Ca2+-induced opening of the permeability transition pore (PTP), and oxidative damage induced by the oxidant pair adenosine diphosphate (ADP)/Fe2+ (lipid peroxidation) of mitochondria isolated from rat liver. By using succinate as the respiratory substrate, respiratory control ratio (RCR), ADP to oxygen ratio (ADP/O), state 3, state 4, and uncoupled respiration rates were not significantly affected by gammapyrone, glutapyrone, and diethone concentrations up to 100 microM. Cerebrocrast at concentrations higher than 25 microM depressed RCR, ADP/O, state 3, and uncoupled respiration rates, but increased three times state 4 respiration rate. The transmembrane potential (deltapsi) and the phosphate carrier rate were also decreased. At concentrations lower than 25 microM, cerebrocrast inhibited the mitochondrial IMAC and partially prevented Ca2+-induced opening of the mitochondrial PTP, whereas gammapyrone, glutapyrone, and diethone were without effect. Cerebrocrast, gammapyrone, and glutapyrone concentrations up to 100 microM did not affect ADP/Fe2+-induced lipid peroxidation of rat liver mitochondria, while very low diethone concentrations (up to 5 microM) inhibited it in a dose-dependent manner, as measured by oxygen consumption and thiobarbituric acid reactive substances formation. Diethone also prevented deltapsi dissipation due to lipid peroxidation initiated by ADP/Fe2+. It can be concluded that: none of the compounds interfere with mitochondrial bioenergetics at concentrations lower than 25 microM; cerebrocrast was the only compound that affected mitochondrial bioenergetics, but only for concentrations higher than 25 microM; at concentrations that did not affect mitochondrial bioenergetics (< or = 25 microM), only cerebrocrast inhibited the IMAC and partially prevented Ca2+-induced opening of the PTP; diethone was the only compound that expressed antioxidant activity at very low concentrations (< or = 5 microM). Cerebrocrast acting as an inhibitor of the IMAC and diethone acting as an antioxidant could provide effective protective roles in preventing mitochondria from oxidative damage, favoring their therapeutic interest in the treatment of several pathological situations known to be associated with cellular oxidative stress.     

Efflux of magnesium and potassium ions from liver mitochondria induced by inorganic phosphate and by diamide

Addition to rat liver mitochondria of 2 mM inorganic phosphate or 0.15 mM diamide, a thiol-oxidizing agent, induced an efflux of endogenous Mg²⁺ linear with time and dependent on coupled respiration. No net Ca²⁺ release occurred under these conditions, while a concomitant release of K⁺ was observed. Mg²⁺ efflux mediated either by P_i or low concentrations of diamide was completely prevented by EGTA, Ruthenium red, and NEM. These reagents also inhibited the increased rate of state 4 respiration induced both by P_i and diamide. At higher concentrations (0.4 mM), diamide induced an efflux of Mg²⁺ which was associated also with a release of endogenous Ca²⁺. Under these conditions EGTA completely prevented Mg²⁺ and K⁺ effluxes, while they were only partially inhibited by Ruthenium red and NEM. It is assumed that Mg²⁺ efflux, occurring at low diamide concentrations or in the presence of phosphate, is dependent on a cyclic in-and-out movement of Ca²⁺ across the inner mitochondrial membrane, in which the passive efflux is compensated by a continuous energy linked reuptake. This explains the dependence of Mg²⁺ efflux on coupled respiration, as well as the increased rate of state 4 respiration. The dependence of Mg²⁺ efflux on phosphate transport is explained by the phosphate requirement for Ca²⁺ movement.Abbreviations Diamide diazenedicarboxylic acidbis-dimethylamide - FCCP p-trifluoromethoxyphenylhydrazone - EGTA ethylene glycol-bis-(2-amino ethyl ether)-N,N-tetracetic acid - P_i inorganic phosphate - Ruthenium red Ru₂(OH)₂Cl₄ · 7NH₃ · 3H₂O - state 4 controlled state of respiration in the presence of substrate - RCI respiratory control index - NEM N-ethyl maleimide A partial and preliminary report of these results has been published inBiochem. Biophys. Res. Comm.,78 (1977) 23.     

Action of La3+ on the systems providing contractility of vertebrate myocardium

The inotropic action of La³⁺ on frog myocardium was studied with taking into account its effect on mitochondria of cardiomyocytes (CM). It has been established that in the range of studied concentrations (0.2–6.0 mM), La³⁺ decreases dose-dependently the force of cardiac contractions (by 3.3–92.2%). In parallel experiments on isolated rat heart mitochondria (RHM), La³⁺ at a concentration of 25 μM has been shown to cause swelling of non-energized and energized mitochondria incubated in isotonic medium with 125 mM NH₄NO₃ and in hypotonic medium with 25 mM CH₃COOK. The study of oxidative processes in mitochondria with aid of polarographic method of measurement of oxygen concentration has shown that La³⁺ at concentrations of 50 and 100 μM increases the oxygen consumption rate by mitochondria in the state 2. However, La³⁺ does not decrease the respiration rate of isolated mitochondria in the state 3, as this takes place in the case of use of Cd²⁺ or at the Ca²⁺-overloading of mitochondria. The rate of endogenous respiration of isolated mitochondria in the medium with La³⁺ was higher than in control, which suggests its effect on ion permeability of the inner membrane. The data obtained in this work indicate that the La³⁺-produced decrease of contractility of cardiac muscle is not only due to the direct blocking effect on the potential-controlled Ca²⁺-channels, but is also mediated by its unspecific action on the CM mitochondria. This action is manifested as an acceleration of the energy-dependent K⁺ transport in matrix and as an increase of ion permeability of the inner mitochondrial membrane (IMM).     

Effect of freezing-thawing rates on the functional state and ionic permeability of rat liver mitochondria

The effects of various rats of freezing-thawing reactions on the functional state and ionic permeability of rat liver mitochondria were studied. The degree of mitochondrial damage during the freezing -- thawing process depended on the rate of thawing rather than on that of freezing. The mitochondria which were slowly or rapidly frozen down to --196 degrees and subsequently slowly thawed revealed a higher membrane permeability for K+ Na+ and H+ and a more than 2-fold increase of the ATPase activity and the maximal rate of NADH oxidation via the antimycin-insensitive pathway in the presence of cytochrome c. This was concomitant with a complete inhibition of the ATP-synthetase activity and a marked inhibition of the respiratory chain function due to the efflux of cytochrome c from the inner mitochondrial membrane. After freezing and rapid thawing the functional activity of mitochondria changed insignificantly. A comparison of different cryoeffects demonstrated that the minimal damaging effects were exerted by rapid freezing -- rapid thawing, when the mitochondria partly restored their ability for oxidative phosphorylation.     

Interaction of tetraphenylphosphonium and dodecyltriphenylphosphonium with lipid membranes and mitochondria

The permeability of a planar lipid membrane (composed of diphytanoylphosphatidylcholine) for tetraphenylphosphonium (TPP) was investigated. The observed level of the diffusion potential generated as a function of the TPP concentration gradient differed from the theoretically expected value, possibly due to proton leakage of the membrane mediated by the traces of fatty acids in the phospholipid forming the membrane. Using the molecular dynamics approach to study movement of TPP and dodecyltriphenylphosphonium (C₁₂TPP) with different affinity to the lipid bilayer through a bilayer lipid membrane, it was found that C₁₂TPP has a greater affinity to the membrane surface than TPP. However, the two cations have the same activation energy for transmembrane transfer. Interaction of TPP and C₁₂TPP with tightly-coupled mitochondria from the yeast Yarrowia lipolytica was also investigated. At low, micromolar concentrations, both cations are “relatively weak, mild uncouplers”, do not shunt electron transfer along the respiratory chain, do not disturb (damage) the inner mitochondrial membrane, and profoundly promote the uncoupling effect of fatty acids. At higher concentrations they inhibit respiration in state 3, and at much higher concentrations they induce swelling of mitochondria, possibly due to their detergent action.     

Effect of protein S-glutathionylation on Ca2+ homeostasis in cultured aortic endothelial cells

Diamide is a membrane-permeable, thiol-oxidizing agent that rapidly and reversibly oxidizes glutathione to GSSG and promotes formation of protein-glutathione mixed disulfides. In the present study, the acute effect of diamide on free cytosolic Ca2+ concentration ([Ca2+]i) was examined in fura-2-loaded bovine aortic endothelial cells. At low concentrations (50, 100 μM), diamide reversibly increased spontaneous, asynchronous Ca2+ oscillations, whereas, at higher concentrations (250, 500 μM), diamide caused an immediate synchronized Ca2+ oscillation in essentially all cells of the monolayer, followed by a time-dependent rise in basal [Ca2+]i. The effects of diamide on [Ca2+]i dynamics were independent of extracellular Ca2+. Inhibition of phospholipase C by U-73122 prevented the observed changes in [Ca2+]i. Additionally, the diamide-induced oscillations, but not the rise in basal [Ca2+]i, were blocked by inhibition of the inositol-1,4,5-trisphosphate (IP3) receptor (IP3R) by 2-aminoethyl diphenyl borate. However, diamide failed to alter the plasmalemmal distribution of a green fluorescent protein-tagged phosphatidylinositol-4,5-bisphosphate binding protein, demonstrating that diamide does not activate phospholipase C. Inhibition of glutathione reductase by N,N'-bis(2-chloroethyl)-N-nitrosourea or depletion of glutathione by l-buthionine-sulfoximine enhanced the effects of diamide, which, under these conditions, could only be reversed by addition of dithiothreitol to the wash buffer. Biochemical assays showed that both the IP3R and the plasmalemmal Ca2+-ATPase pump could be reversibly glutathionylated in response to diamide. These results demonstrate that diamide promotes Ca2+ release from IP3-sensitive internal Ca2+ stores and elevates basal [Ca2+]i in the absence of extracellular Ca2+, effects that may be related to a diamide-induced glutathionylation of the IP3R and the plasmalemmal Ca2+-ATPase Ca2+ pump, respectively.     

Modulation of Ca2+-mediated K+-gating of erythrocyte ghosts by external Ca-EGTA

Using 86Rb+ as a marker for K+ permeability, we find that extracellular Ca-EGTA influences the rate of 86Rb+ efflux from erythrocyte ghosts preloaded with 86Rb+ and "buffered" Ca2+. At an internal free Ca2+, where the rate of 86Rb+ efflux is minimal and uninfluenced by either external EGTA or external Ca2+, external Ca-EGTA at 0.2-0.5 mM can raise the flux rate to as high as can be attained by raising internal Ca2+, in the presence of an excess externally either of Ca2+ or of EGTA. Higher concentrations of Ca-EGTA (up to 1-2 mM) diminish the flux rate. External Ca-EDTA or Mg-EDTA can substitute for Ca-EGTA in enhancing and suppressing flux rate. The peak rate is insensitive to external free Ca2+ but depends on internal Ca2+; internal Mg-EDTA does not substitute for internal Ca-EGTA. Thus, the erythrocyte membrane is asymmetric with respect to its interaction with Ca2+ and Ca-EGTA. Also, 22Na+ does not substitute for 86Rb+. The peak rate of 86Rb+ flux produced by external Ca-EGTA is diminished by chlorpromazine (0.1 mM) and augmented by 1-propranolol (25 microM), in the same way as the rate produced by increasing internal Ca2+. The results suggest that external Ca-EGTA enhances the affinity of internal Ca2+ for its receptor(s) which operate the K+-gate at the inner surface of the membrane. At external concentrations of Ca-EGTA above 1-2 mM, 86Rb+ flux rate again rises with increase of Ca-EGTA. This phenomenon does not depend upon internal Ca2+, is not affected by chlorpromazine or by 1-propranolol, and is associated with an enhanced permeability to 22Na+, inulin, and haemoglobin.     

Lipid peroxidation in mitochondrial inner membranes. I. An integrative kinetic model

An integrative mathematical model was developed to obtain an overall picture of lipid hydroperoxide metabolism in the mitochondrial inner membrane and surrounding matrix environment. The model explicitly considers an aqueous and a membrane phase, integrates a wide set of experimental data, and unsupported assumptions were minimized. The following biochemical processes were considered: the classic reactional scheme of lipid peroxidation; antioxidant and pro-oxidant effects of vitamin E; pro-oxidant effects of iron; action of phospholipase A₂, glutathione-dependent peroxidases, glutathione reductase and superoxide dismutase; production of superoxide radicals by the mitochondrial respiratory chain; oxidative damage to proteins and DNA. Steady-state fluxes and concentrations as well as half-lives and mean displacements for the main metabolites were calculated. A picture of lipid hydroperoxide physiological metabolism in mitochondria in vivo showing the main pathways is presented. The main results are:(a) perhydroxyl radical is the main initiation agent of lipid peroxidation (with a flux of 10⁻⁷Ms⁻¹); (b) vitamin E efficiently inhibits lipid peroxidation keeping the amplification (kinetic chain length) of lipid peroxidation at low values (10); (c) only a very minor fraction of lipid hydroperoxides escapes reduction via glutathione-dependent peroxidases; (d) oxidized glutathione is produced mainly from the reduction of hydrogen peroxide and not from the reduction of lipid hydroperoxides.     

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca²⁺-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.     

Influence of oxidative processes in mitochondria on contractility of the frog Rana temporaria heart muscle. Effects of cadmium

The inotropic Cd2+ action on frog heart is studied with taking into account its toxic effects upon mitochondria. Cd2+ at concentrations of 1, 10, and 20 microM is established to decrease dosedependently (21.3, 50.3, and 72.0%, respectively) the muscle contraction amplitude; this is explained by its competitive action on the potential-controlled Ca2(+)-channels of the L-type (Ca 1.2). In parallel experiments on isolated rat heart mitochondria (RHM) it was shown that Cd2+ at concentrations of 15 and 25 microM produces swelling of non-energized and energized mitochondria in isotonic (with KNO2 and NH4NO3) and hypoosmotic (with 25 mM CH3COOK) media. Study of oxidative processes in RHM by polarographic method has shown 20 microM Cd2+ to disturb activity of respiratory mitochondrial chain. The rate of endogenous respiration of isolated mitochondria in the medium with Cd2+ in the presence of malate and succinate was approximately 5 times lower than in control. In experimental preparations, addition into the medium of DNP-uncoupler of oxidation and phosphorylation did not cause an increase of the oxygen consumption rate. Thus, the obtained data indicate that a decrease in the cardiac muscle contractility caused by Cd2+ is due not only to its direct blocking action on Ca2(+)-channels, but also is mediated by toxic effect on rat heart mitochondria, which was manifested as an increase in ion permeability of the inner mitochondrial membrane (IMM), acceleration of the energy-dependent K+ transport into the matrix of mitochondria, and inhibition of their respiratory chain.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.