The intracellular tyrosine residues of the ATP-gated P2X(1) ion channel are essential for its function

In order to gain a first insight into the effects of reactive oxygen species (ROS) on plant mitochondria, we studied the effect of the ROS producing system consisting of xanthine plus xanthine oxidase on the rate of membrane potential (DeltaPsi) generation due to either succinate or NADH addition to durum wheat mitochondria as monitored by safranin fluorescence. We show that the early ROS production inhibits the succinate-dependent, but not the NADH-dependent, DeltaPsi generation and oxygen uptake. This inhibition appears to depend on the impairment of mitochondrial permeability to succinate. It does not involve mitochondrial thiol groups sensitive to either mersalyl or N-ethylmaleimide and might involve both protein residues and/or membrane lipids, as suggested by the mixed nature. We propose that, during oxidative stress, early generation of ROS can affect plant mitochondria by impairing metabolite transport, thus preventing further substrate oxidation, DeltaPsi generation and consequent large-scale ROS production.     

Role of alpha-tocopherol in the regulation of mitochondrial permeability transition

We previously showed that Ca2+-induced cyclosporin A-sensitive membrane permeability transition (MPT) of mitochondria occurred with concomitant generation of reactive oxygen species (ROS) and release of cytochrome c (Free Rad. Res.38, 29-35, 2004). To elucidate the role of alpha-tocopherol in MPT, we investigated the effect of alpha-tocopherol on mitochondrial ROS generation, swelling and cytochrome c release induced by Ca2+ or hydroxyl radicals. Biochemical analysis revealed that alpha-tocopherol suppressed Ca2+-induced ROS generation and oxidation of critical thiol groups of mitochondrial adenine nucleotide translocase (ANT) but not swelling and cytochrome c release. Hydroxyl radicals also induced cyclosporin A-sensitive MPT of mitochondria. alpha-Tocopherol suppressed the hydroxyl radical-induced lipid peroxidation, swelling and cytochrome c release from mitochondria. These results indicate that alpha-tocopherol inhibits ROS generation, ANT oxidation, lipid peroxidation and the opening of MPT, thereby playing important roles in the prevention of oxidative cell death.     

Involvement of a single thiol group in the conversion of the NAD+-dependent activity of rat liver xanthine oxidoreductase to the O2-dependent activity.

The effects of 2-iodosobenzoic acid, 4-chloromercuribenzoate, 5,5'-dithiobis-(2-nitrobenzoic acid) and tetraethylthioperoxydicarbonic diamide (disulphiram) on the NAD+-dependent activity of xanthine oxidoreductase from rat liver were investigated. Only disulphiram converted the NAD+-dependent activity into the O2-dependent activity quantitatively, without changing the xanthine hydroxylation rate. The modification process was a first-order reaction with respect to time (min) and disulphiram concentration (microM). The kinetic data showed that modification of single thiol group is sufficient for loss of the enzymic activity towards NAD+ as electron acceptor. The complete protection afforded by NAD+ against the action of disulphiram suggests that the essential thiol group may be involved in binding of NAD+ to the xanthine oxidoreductase molecule.     

Proline transport in rat liver mitochondria.

Proline transport across the inner membrane of rat liver mitochondria shows the following properties: (a) It is stereospecific; the penetration of l-proline is two times faster than the penetration of dl-proline. (b) Proline is accumulated against a concentration gradient, (c) The transport of proline is enhanced in the presence of respiratory substrates such as succinate or tetramethylphenylenediamine + ascorbate; it is inhibited by uncouplers of oxidative phosphorylation. (d) Proline transport is inhibited by mersalyl and p-chloromercuribenzoate, but not by hydrophobic thiol blocking reagents; thus, proline transport involves thiol groups located in a very hydrophilic environment. The penetration of several other neutral amino acids (alanine, glycine, serine) is almost insensitive to mersalyl. These results suggest that proline does not travel across the mitochondrial membrane by free diffusion, but that its transport is mediated by a specific carrier. The rate of proline transport has been compared with the rates of the first two steps of proline oxidation: All of these rates are very similar, indicating that proline transport is not a limiting factor of proline metabolism in rat liver mitochondria.     

Oxidative stress underlies the mechanism for Ca(2+)-induced permeability transition of mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca₂₊ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca₂₊ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca₂₊ increased the generation of H₂O₂ by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca₂₊ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca₂₊-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca₂₊ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

Parallel efflux of Ca2+ and Pi in energized rat liver mitochondria.

Addition of Ruthenium Red to energized rat liver mitochondria that have previously accumulated Ca2+ and phosphate from the external medium induces a parallel efflux of both these ions. Mersalyl or dithioerythritol, which decrease Ruthenium Red-insensitive Ca2+ efflux, also decrease phosphate efflux to the same extent. Conversely diazenedicarboxylic acid bis(NN-dimethylamide) (DDBA), which increases the Ruthenium Red-induced Ca2+ efflux concurrently increases phosphate release. Dithioerythritol and DDBA, reducing and oxidizing agents of thiol groups respectively, modify Ca2+ and Pi efflux without penetrating the mitochondrial inner membrane. Under all the adopted conditions the membrane potential is preserved. The release of resting respiration and the parallel efflux of Mg2+ and adenine nucleotides, events closely correlated to Ca2+ cycling, are equally prevented either by mersalyl, which inhibits phosphate transport, or dithioerythritol; DDBA has the opposite effect. These findings and the observation that suggest that Ca2+ and phosphate transport in energized liver mitochondria are closely related and dependent on the redox state of membrane-bound thiol groups.     

Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.     

Oxidative Stress Underlies the Mechanism for Ca2+-induced Permeability Transition of Mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca²⁺ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca²⁺ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca²⁺ increased the generation of H²O² by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca²⁺ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca²⁺-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca²⁺ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

Interaction of genistein with the mitochondrial electron transport chain results in opening of the membrane transition pore

Genistein, a natural isoflavone present in soybeans, is a potent agent in the prophylaxis and treatment of cancer. Addition of genistein to isolated rat liver mitochondria (RLM) induces swelling, loss of membrane potential and release of accumulated Ca2+. These changes are Ca2+-dependent and are prevented by cyclosporin A (CsA) and bongkrekic acid (BKA), two classical inhibitors of the mitochondrial permeability transition (MPT). Induction of the MPT by genistein is accompanied by oxidation of thiol groups and pyridine nucleotides. The reducing agent dithioerythritol and the alkylating agent N-ethylmaleimide (NEM) completely prevent the opening of the transition pore, thereby emphasizing that the effect of the isoflavone correlates with the mitochondrial redox state. Further analyses showed that genistein induces the MPT by the generation of reactive oxygen species (ROS) due to its interaction with the respiratory chain at the level of mitochondrial complex III.     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.     

Steroid hormone receptor fraction stimulation of RNA synthesis: a caution.

Permeant and impermeant labelled thiol reagents were incubated with rat liver mitochondria, and incorporation of reagent into mitochondria estimated. With permeant thiol reagents, incorporation depends on the energetic state of mitochondria; when coupled electron-transfer takes place, incorporation is fairly increased; the stimulation is abolished in the presence of an uncoupler, an electron-transfer inhibitor or inorganic phosphate. With an impermeant thiol reagent, the incorporation is unaffected by the energetic state of the mitochondria. These results favour the view of a participation of thiol groups in the energy-conserving mechanism but it cannot be ruled out that part of the unmasked thiol groups are implicated in the phosphate transport system. The observed stimulation may reflect either an increase in accessibility or in reactivity of some mitochondrial thiol groups.     

Protection of tamoxifen against oxidation of mitochondrial thiols and NAD(P)H underlying the permeability transition induced by prooxidants

The effects of tamoxifen (TAM) were studied on the mitochondrial permeability transition (MPT) induced by the prooxidant tert-butyl hydroperoxide (t-BuOOH) or the thiol cross-linker phenylarsine oxide (PhAsO), in the presence of Ca2+, in order to clarify the mechanisms involved in the MPT inhibition by this drug. The combination of Ca2+ with t-BuOOH or PhAsO induces mitochondrial swelling and depolarization of membrane potential (deltapsi). These events are inhibited by cyclosporine A (CyA), suggesting the inhibition of the MPT. The pre-incubation of mitochondria with TAM also prevents those events and induces a time-dependent reversal of deltapsi depolarization following MPT induction, similarly to CyA. Moreover, TAM inhibits the Ca2+ release and the oxidation of NAD(P)H and protein thiol (-SH) groups promoted by t-BuOOH plus Ca2+. On the other hand, the MPT induced by PhAsO plus Ca2+ does not induce -SH groups oxidation, supporting the notion that MPT induction by this compound is not mediated by the oxidation of specific membrane proteins groups. However, TAM also inhibits the PhAsO induced MPT, suggesting that this drug may inhibit this phenomenon by inhibiting PhAsO binding to -SH vicinal groups, implicated in the MPT induction. These data indicate that the MPT inhibition by TAM may be related to its antioxidant capacity in preventing the oxidation of NAD(P)H and -SH groups or by blocking these groups, since the oxidation of these groups increases the sensitivity of mitochondria to the MPT induction. Additionally, they suggest an MPT-independent pathway for TAM-induced apoptosis and a potential ER-independent mechanism for the effectiveness of this drug in the cancer therapy and prevention.     

The action of tellurite, a reagent for thiol groups, on mitochondria oxidative processes

Tellurite, a reagent for thiol groups, added to rat kidney, or liver mitochondria at the concentration of 1 mM, selectively inhibits the oxidation of NAD dependent substrates (pyruvate, α-ketoglutarate, glutamate, etc.) without affecting the oxidation of succinate, α-glycerophosphate and ascorbate. Moreover NADH oxidation by rat heart or by aged liver mitochondria is not affected by tellurite.     

Peroxynitrite formation from the simultaneous reduction of nitrite and oxygen by xanthine oxidase

One electron reductions of oxygen and nitrite by xanthine oxidase form peroxynitrite. The nitrite and oxygen reducing activities of xanthine oxidase are regulated by oxygen with K(oxygen) 26 and 100 microM and K(nitrite) 1.0 and 1.1 mM with xanthine and NADH as donor substrates. Optimal peroxynitrite formation occurs at 70 microM oxygen with purine substrates. Kinetic parameters: V(max) approximately 50 nmol/min/mg and K(m) of 22, 36 and 70 microM for hypoxanthine, pterin and nitrite respectively. Peroxynitrite generation is inhibited by allopurinol, superoxide dismutase and diphenylene iodonium. A role for this enzyme activity can be found in the antibacterial activity of milk and circulating xanthine oxidase activity.     

A possible cooperation of SOD1 and cytochrome c in mitochondria-dependent apoptosis

A small amount of reactive oxygen species (ROS) is generated through aerobic respiration even under physiological conditions. Because ROS are known to have various deteriorating actions, the way cells could evade the effects of ROS in and around mitochondria would determine the fate of cells. We previously reported that Cu,Zn-superoxide dismutase (SOD1), a cytosolic enzyme, is also localized in mitochondria in various types of cells. Therefore, we undertook this study to elucidate the physiological significance of SOD1 localization in and around mitochondria. We analyzed the effects of various reagents that could modulate mitochondrial respiration, ROS metabolism, and subcellular localization of SOD1 and cytochrome c. Using rat liver mitochondria, we have shown that Ca2+, Fe2+, or long-chain fatty acids increased the mitochondrial generation of ROS and that the resulting ROS oxidized the critical thiol groups in adenine nucleotide translocase (ANT). The oxidation of ANT induced mitochondrial swelling followed by the release of SOD1 and cytochrome c. Although inhibitors of electron transport, such as rotenone, antimycin A, and KCN, also increased ROS generation, they failed to (i) oxidize the critical thiol groups in ANT, (ii) induce swelling, and (iii) release SOD1 and cytochrome c. These results suggest that the oxidation of ANT thiols and the opening of the membrane permeability transition pores induce the release of both SOD1 and cytochrome c. We demonstrated that the loss of SOD1 increases the susceptibility of mitochondria to oxidative stresses and that the simultaneous release of SOD1 enhances the vicious cycle of apoptotic reactions triggered by the released cytochrome c. Therefore, SOD1 must have important roles in protecting mitochondria from ROS-induced injury. Our data also suggest that SOD1 release parallels cytochrome c release under all conditions. We propose that intramembranously localized SOD1 is a third reagent (along with AIF) that will regulate apoptosis.     

Production of thiol groups and retention of calcium ions by cardiac mitochondria.

Efflux of Ca2+ from previously Ca2+-loaded heart mitochondria was measured after inhibiting respiratory activity. The efflux was increased by p-chloromercuribenzoate, methylmercuric chloride, Cu2+, Fe2+, 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (uncoupler). 1,1,1-trifluoro-3-(2-thienylacetone and indomethacin; after such increase it could be diminished by dithiothreitol. The induced loss of the Ca2+ was accompanied by a loss of endogenous adenine nucleotide. Methylmercuric chloride was particularly effective, since it was active at ratios of about 1 nmol/mg of mitochondrial protein. The non-respiring mitochondria were found to regenerate bound thiol groups after their original complement had reacted with thiol-blocking reagent. This regeneration was diminished by the Ca2+-efflux stimulatig agents that were not themselves thiol-blocking reagents, such as thyroxine, uncoupler, trifluorothienylacetone and indomethacin. The external exposure of thiol groups was also diminished by thyroxine, uncoupler and trifluorothienylacetone. The results support the proposal made previously that the membrane is maintained in a state of low permeability by adenine nucleotide and Mg2+ being bound to thiol-dependent sites.     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.     

Comparison of low and high calcium requiring forms of the calcium-activated neutral protease (CANP) from rabbit skeletal muscle

Two distinct calcium-dependent neutral proteases (CANPs) with different sensitivities to calcium ions were purified concurrently by almost the same procedures from rabbit skeletal muscle and their enzymatic properties were compared (sensitivity to various divalent metal ions, the pH dependency and heat-stability of the activity, and the hydrolytic activity towards various substrates). They were further compared chemically in terms of the state of thiol groups, the amino acid compositions of subunits and the peptide fragments by digestion with S. aureus V8 protease. The low calcium requiring form of CANP (microCANP) was more sensitive to other divalent metal ions such as Sr2+ and Ba2+ than the high calcium requiring form of CANP (mCANP). The comparison of the pH dependency of these CANP activities showed that microCANP was active in a broader pH range than mCANP and the former was more heat-stable than the latter. Both CANPs had similar affinity to various substrates, but the hydrolytic velocity was several times higher with microCANP than with mCANP. Although they were inhibited by thiol protease inhibitors to the same extent, the states of thiol groups in them were quite different. The thiol group involved in the catalytic activity of the enzyme was exposed without adding Ca2+ in microCANP, whereas the group in mCANP became exposed only when sufficient Ca2+ was added. The large subunits of these two CANPs were different in their amino acid compositions and in the peptide fragment patterns produced by S. aureus V8 protease but the small subunits were indistinguishable from each other. These results led us to conclude that these two CANPs are quite different in nature and are not in a simple relationship, i.e., one of them is not derived from the other by autolysis or modification.     

Mitochondrial sulfhydryl group modification by adriamycin aglycones

Induction of Ca2+ release from isolated, preloaded rat heart mitochondria by low concentrations (less than 5 micrM) of adriamycin aglycones, has recently been reported [(1988) Biochem. Pharmacol. 37, 803]. Ca2+ release occurs via a generalized, Ca2+-dependent increase in the permeability of the inner mitochondrial membrane to small molecules. The process is antagonized by dithiothreitol, suggesting thiol involvement. This communication demonstrates modification of mitochondrial sulfhydryl groups, detected as decreased 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) reactivity, by adriamycin aglycones. Ca2+ release and sulfhydryl modification are shown to depend similarly on aglycone concentration and on the C-7 substituent of the anthracycline ring. In addition, DTNB elicits Ca2+ release. It can therefore be proposed that adriamycin aglycones alter mitochondrial membrane permeability by altering mitochondrial thiol status.     

Reactions of bis(thiosemicarbazonato) copper(II) complexes with tumor cells and mitochondria.

The respiration of Ehrlich ascites tumor cells is inhibited by 3-ethoxy-2-oxobutyraldehyde bis (thiosemicarbazanato) copper (II). State 3 oxidative phosphorylation in mitochondria from tumor cells is also inhibited, with the effect more pronounced using glutamate or pyruvate-malate as substrates than with succinate. The disruption of oxidative phosphorylation in bovine heart mitochondria is qualitatively similar. The principal site of inhibition is in coupling site one, energetically between the electron transport site chain and the locus of uncoupling by 2,4-dinitrophenol. This appears to contain thiol groups which are oxidized by the complex. For a series of bis (thiosemicarbazonato) copper complexes, the extent of inhibition of heart mitochondrial oxidative phosphorylation is correlated with the reduction potentials of the complexes and with their in vitro cytotoxic effects against Walker 256 carcinoma tumor cells.