Mitochondrial dysfunction induced by different organochalchogens is mediated by thiol oxidation and is not dependent of the classical mitochondrial permeability transition pore opening.
ABSTRACT: Ebselen (Ebs) and diphenyl diselenide [(PhSe)(2)] readily oxidize thiol groups. Here we studied mitochondrial swelling changes in mitochondrial potential (Deltapsim), NAD(P)H oxidation, reactive oxygen species production, protein aggregate formation, and oxygen consumption as ending points of their in vitro toxicity. Specifically, we tested the hypothesis that organochalchogens toxicity could be associated with mitochondrial dysfunction via oxidation of vicinal thiol groups that are known to be involved in the regulation of mitochondrial permeability (Petronilli et al. J. Biol. Chem., 269; 16638; 1994). Furthermore, we investigated the possible mechanism(s) by which these organochalchogens could disrupt liver mitochondrial function. Ebs and (PhSe)(2) caused mitochondrial depolarization and swelling in a concentration-dependent manner. Furthermore, both organochalchogens caused rapid oxidation of the mitochondrial pyridine nucleotides (NAD(P)H) pool, likely reflecting the consequence and not the cause of increased mitochondrial permeability (Costantini, P., Chernyak, B. V., Petronilli, V., and Bernardi, P. (1996). Modulation of the mitochondrial permeability transition pore (PTP) by pyridine nucleotides and dithiol oxidation at two separate sites. J. Biol. Chem. 271, 6746-6751). The organochalchogens-induced mitochondrial dysfunction was prevented by the reducing agent dithiothreitol (DTT). Ebs- and (PhSe)(2)-induced mitochondrial depolarization and swelling were unchanged by ruthenium red (4microM), butylated hydroxytoluene (2.5microM), or cyclosporine A (1microM). N-ethylmaleimide enhanced the organochalchogens-induced mitochondrial depolarization, without affecting the magnitude of the swelling response. In contrast, iodoacetic acid did not modify the effects of Ebs or (PhSe)(2) on the mitochondria. Additionally, Ebs and (PhSe)(2) decreased the basal 2' 7' dichlorofluorescin diacetate (H(2)-DCFDA) oxidation and oxygen consumption rate in state 3 and increased it during the state 4 of oxidative phosphorylation and induced the formation of protein aggregates, which were prevented by DTT. However, DTT failed to reverse the formation of protein aggregates, when it was added after a preincubation of liver mitochondria with Ebs or (PhSe)(2). Similarly, DTT did not reverse the Ebs- or (PhSe)(2)-induced Deltapsim collapse or swelling, when it was added after a preincubation period of mitochondria with chalcogenides. These results show that Ebs and (PhSe)(2) can effectively induce mitochondrial dysfunction and suggest that effects of these compounds are associated with mitochondrial thiol groups oxidation. The inability of cyclosporine A to reverse the Ebs- and (PhSe)(2)-induced mitochondrial effects suggests that the redox-regulated mitochondrial permeability transition (MPT) pore was mechanistically regulated in a manner that is distinct from the classical MPT pore.
Project description:The molecular mechanisms underlying the initiation and control of the release of cytochrome c during mitochondrion-dependent apoptosis are thought to involve the phosphorylation of mitochondrial Bcl-2 and Bcl-x(L). Although the c-Jun N-terminal kinase (JNK) has been proposed to mediate the phosphorylation of Bcl-2/Bcl-x(L) the mechanisms linking the modification of these proteins and the release of cytochrome c remain to be elucidated. This study was aimed at establishing interdependency between JNK signalling and mitochondrial apoptosis. Using an experimental model consisting of isolated, bioenergetically competent rat brain mitochondria, these studies show that (i) JNK catalysed the phosphorylation of Bcl-2 and Bcl-x(L) as well as other mitochondrial proteins, as shown by two-dimensional isoelectric focusing/SDS/PAGE; (ii) JNK-induced cytochrome c release, in a process independent of the permeability transition of the inner mitochondrial membrane (imPT) and insensitive to cyclosporin A; (iii) JNK mediated a partial collapse of the mitochondrial inner-membrane potential (Deltapsim) in an imPT- and cyclosporin A-independent manner; and (iv) JNK was unable to induce imPT/swelling and did not act as a co-inducer, but as an inhibitor of Ca-induced imPT. The results are discussed with regard to the functional link between the Deltapsim and factors influencing the permeability transition of the inner and outer mitochondrial membranes. Taken together, JNK-dependent phosphorylation of mitochondrial proteins including, but not limited to, Bcl-2/Bcl-x(L) may represent a potential of the modulation of mitochondrial function during apoptosis.
Project description:There is a known connection between selenium supplementation and chemo-protective anti-cancer activity. This biological phenomenon may be due to the ability of selenium to instigate cellular apoptosis. However, the mechanism by which selenium promotes cellular apoptosis is still obscure. The present study shows that sodium selenite, a common dietary form of selenium, promotes the mitochondrial permeability transition (MPT) in isolated rat liver mitochondria both in vitro and following in vivo supplementation. A low selenium concentration (0.1-10 microM) strongly induced cyclosporin A-sensitive mitochondrial swelling. Selenium also promoted both calcium release from the matrix of isolated mitochondria and uncoupled respiration. The MPT-inducing effect of selenium provoked the release of cytochrome c, a pro-apoptotic factor, into the incubation medium. Selenium did not increase intra-mitochondrial peroxide production, but did consume endogenous mitochondrial glutathione. Moreover, the effect of MPT induction was greatly potentiated in the presence of thiol-bearing antioxidants, e.g. N -acetylcysteine and lipoamide. During MPT progression, selenium induced NADH oxidation via electron acceptance from complex I. Supplementation for 20 days with 16 p.p.m. selenium in the drinking water of rats increased the propensity of mitochondria to undergo the MPT. More marked mitochondrial swelling in response to calcium and lower calcium-uptake capacity were observed, in the absence of liver damage or the intensive oxidation of reduced glutathione. Therefore selenite facilitates MPT pore opening via its thiol- and NADH/complex I-dependent reduction, and thereby may provide chemo-protection by potentiation of the capacity of the mitochondria to regulate programmed cell death. Data from the present study suggest that selenium can regulate important mitochondrial functions both in vivo and in vitro.
Project description:The iron chelator Deferasirox (DFX) causes severe toxicity in patients for reasons that were previously unexplained. Here, using the kidney as a clinically relevant in vivo model for toxicity together with a broad range of experimental techniques, including live cell imaging and in vitro biophysical models, we show that DFX causes partial uncoupling and dramatic swelling of mitochondria, but without depolarization or opening of the mitochondrial permeability transition pore. This effect is explained by an increase in inner mitochondrial membrane (IMM) permeability to protons, but not small molecules. The movement of water into mitochondria is prevented by altering intracellular osmotic gradients. Other clinically used iron chelators do not produce mitochondrial swelling. Thus, DFX causes organ toxicity due to an off-target effect on the IMM, which has major adverse consequences for mitochondrial volume regulation.
Project description:1 The mitochondrial respiratory chain produces reactive oxygen species (ROS) during normal electron transport. Despite producing ROS, mitochondria are vulnerable to oxidative stress. Mitochondrial dysfunction has been associated with many degenerative diseases, making it important to identify compounds that protect mitochondria from ROS-mediated toxicity. Here we report that ciclopirox (CPX) blocks H2O2-induced mitochondrial injury by maintaining mitochondrial transmembrane potential (Deltapsim). 2 CPX completely blocked H2O2-stimulated release of lactate dehydrogenase (a marker of cell death) and decrease in MTT reduction (a marker of mitochondrial function) in adenocarcinoma SK-HEP-1 cells. 3 H2O2 rapidly depolarized the Deltapsim, and CPX blocked this H2O2-stimulated Deltapsim decrease. Similar data were obtained in experiments using mitochondria isolated from rat liver. 4 Furthermore, CPX effectively inhibited H2O2-induced mitochondrial permeability transition pore (MPTP) opening. In de-energized mitochondria, however, CPX did not inhibit Ca2+-evoked MPTP opening, indicating that CPX is not a direct inhibitor of the MPTP. 5 Oxygen consumption studies showed that in the presence of pyruvate and malate CPX restored the rate of state 3 to state 4 respiration decreased by H2O2. Consistent with this, CPX replenished ATP levels lowered by H2O2. 6 The present results indicate that CPX protects SK-HEP-1 cells from H2O2 cytotoxicity by inhibiting Deltapsim decrease and indirectly preventing MPTP opening.
Project description:1. Rapid choline oxidation and the onset of P(i)-induced swelling by liver mitochondria, incubated in a sucrose medium at or above pH7.0, required the addition of both P(i) and an uncoupling agent. Below pH7.0, P(i) alone was required for rapid choline oxidation and swelling. 2. Choline oxidation was inhibited by each of several reagents that also inhibited P(i)-induced swelling under similar conditions of incubation, including EGTA, mersalyl, Mg(2+), the Ca(2+)-ionophore A23187, rotenone and nupercaine. None of these reagents had any significant effect on the rate of choline oxidation by sonicated mitochondria. There was therefore a close correlation between the conditions required for rapid choline oxidation and for P(i)-induced swelling to occur, suggesting that in the absence of mitochondrial swelling the rate of choline oxidation is regulated by the rate of choline transport across the mitochondrial membrane. 3. Respiratory-chain inhibitors, uncoupling agents (at pH6.5) and ionophore A23187 caused a loss of endogenous Ca(2+) from mitochondria, whereas nupercaine and Mg(2+) had no significant effect on the Ca(2+) content. Inhibition of choline oxidation and mitochondrial swelling by ionophore A23187 was reversed by adding Ca(2+), but not by Mg(2+). It is concluded that added P(i) promotes the Ca(2+)-dependent activation of mitochondrial membrane phospholipase activity in respiring mitochondria, causing an increase in the permeability of the mitochondrial inner membrane to choline and therefore enabling rapid choline oxidation to occur. Nupercaine and Mg(2+) appear to block choline oxidation and swelling by inhibiting phospholipase activity. 4. Choline was oxidized slowly by tightly coupled mitochondria largely depleted of their endogenous adenine nucleotides, suggesting that these compounds are not directly concerned in the regulation of choline oxidation. 5. The results are discussed in relation to the possible mechanism of choline transport across the mitochondrial membrane in vivo and the influence of this process on the pathways of choline metabolism in the liver.
Project description:OBJECTIVE: Recently, several drugs have been shown to exert beneficial effects for metabolic syndrome through mild regulation of mitochondrial function. Hence, we explored a strategy of targeting mitochondrial function to improve glucose and lipid metabolism. RESEARCH DESIGN AND METHODS: Mitochondrial membrane potential (Deltapsim) is a marker of mitochondrial function; therefore, we set up a high-throughput screening assay of Deltapsim in L6 myotubes. The effects of a selected lead compound were investigated in vitro and in vivo in relation to metabolic syndrome. RESULTS: A novel small-molecule compound, C1, was identified through this high-throughput screening. C1 depolarized Deltapsim in L6 myotubes without cytotoxicity and led to increased cellular AMP-to-ATP ratio, activation of AMP-activated protein kinase (AMPK), and enhanced glucose uptake. It also stimulated the AMPK pathway in HepG2 cells, leading to decreased lipid content. Intriguingly, C1 inhibited respiration in L6 myotubes but did not affect respiration in isolated muscle mitochondria, suggesting that it may depolarize Deltapsim indirectly by affecting the supply of electron donors. Acute administration of C1 in C57BL/6J mice markedly increased fat oxidation and the phosphorylation of AMPK and acetyl-CoA carboxylase in the liver. In diabetic db/db mice, chronic administration of C1 significantly reduced hyperglycemia, plasma fatty acids, glucose intolerance, and the mRNA levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in the liver. CONCLUSIONS: Our results demonstrate a novel small molecule that mildly depolarizes Deltapsim and is able to improve glucose and lipid metabolism to exert beneficial effects for metabolic syndrome. These findings suggest that compounds regulating mitochondrial function may have therapeutic potential for type 2 diabetes.
Project description:Mitochondria were simultaneously isolated from striatum and cortex of adult rats and compared in functional assays for their sensitivity to calcium activation of the permeability transition. Striatal mitochondria showed an increased dose-dependent sensitivity to Ca2+ compared with cortical mitochondria, as measured by mitochondrial depolarization, swelling, Ca2+ uptake, reactive oxygen species production, and respiration. Ratios of ATP to ADP were lower in striatal mitochondria exposed to calcium despite equal amounts of ADP and ATP under respiring and nonrespiring conditions. The Ca2+-induced changes were inhibited by cyclosporin A or ADP. These responses are consistent with Ca2+ activation of both low and high permeability pathways constituting the mitochondrial permeability transition. In addition to the striatal supersensitivity to induction of the permeability transition, cyclosporin A inhibition was less potent in striatal mitochondria. Immunoblots indicated that striatal mitochondria contained more cyclophilin D than cortical mitochondria. Thus striatal mitochondria may be selectively vulnerable to the permeability transition. Subsequent mitochondrial dysfunction could contribute to the initial toxicity of striatal neurons in Huntington's disease.
Project description:According to current views, oxidation of aldehyde dehydrogenase-2 (ALDH2) during glyceryltrinitrate (GTN) biotransformation is essentially involved in vascular nitrate tolerance and explains the dependence of this reaction on added thiols. Using a novel fluorescent intracellular nitric oxide (NO) probe expressed in vascular smooth muscle cells (VSMCs), we observed ALDH2-catalyzed formation of NO from GTN in the presence of exogenously added dithiothreitol (DTT), whereas only a short burst of NO, corresponding to a single turnover of ALDH2, occurred in the absence of DTT. This short burst of NO associated with oxidation of the reactive C302 residue in the active site was followed by formation of low-nanomolar NO, even without added DTT, indicating slow recovery of ALDH2 activity by an endogenous reductant. In addition to the thiol-reversible oxidation of ALDH2, thiol-refractive inactivation was observed, particularly under high-turnover conditions. Organ bath experiments with rat aortas showed that relaxation by GTN lasted longer than that caused by the NO donor diethylamine/NONOate, in line with the long-lasting nanomolar NO generation from GTN observed in VSMCs. Our results suggest that an endogenous reductant with low efficiency allows sustained generation of GTN-derived NO in the low-nanomolar range that is sufficient for vascular relaxation. On a longer time scale, mechanism-based, thiol-refractive irreversible inactivation of ALDH2, and possibly depletion of the endogenous reductant, will render blood vessels tolerant to GTN. Accordingly, full reactivation of oxidized ALDH2 may not occur in vivo and may not be necessary to explain GTN-induced vasodilation.
Project description:We evaluated the effects of the phenothiazine derivative thioridazine on mechanisms of mitochondria potentially implicated in apoptosis, such as those involving reactive oxygen species (ROS) and cytochrome c release, as well as the involvement of drug interaction with mitochondrial membrane in these effects. Within the 0 - 100 microM range thioridazine did not reduce the free radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH) nor did it chelate iron. However, at 10 microM thioridazine showed important antioxidant activity on mitochondria, characterized by inhibition of accumulation of mitochondria-generated O2*-, assayed as lucigenin-derived chemiluminescence, inhibition of Fe2+/citrate-mediated lipid peroxidation of the mitochondrial membrane (LPO), assayed as malondialdehyde generation, and inhibition of Ca2+/t-butyl hydroperoxide (t-BOOH)-induced mitochondrial permeability transition (MPT)/protein-thiol oxidation, assayed as mitochondrial swelling. Thioridazine respectively increased and decreased the fluorescence responses of mitochondria labelled with 1-aniline-8-naphthalene sulfonate (ANS) and 1-(4-trimethylammonium phenyl)-6 phenyl 1,3,5-hexatriene (TMA-DPH). The inhibition of LPO and MPT onset correlated well with the inhibition of cytochrome c release from mitochondria. We conclude that thioridazine interacts with the inner membrane of mitochondria, more likely close to its surface, acquiring antioxidant activity toward processes with potential implications in apoptosis such as O2*- accumulation, as well as LPO, MPT and associated release of cytochrome c.
Project description:Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, postmitotic cells, in response to sublethal noxious stress, engage mechanisms that afford protection from subsequent insults. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species (ROS) threshold for the mitochondrial permeability transition (MPT) in cardiomyocytes and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria. Cell protection that exhibits a memory (preconditioning) results from triggered mitochondrial swelling that causes enhanced substrate oxidation and ROS production, leading to redox activation of PKC, which inhibits glycogen synthase kinase-3beta (GSK-3beta). Alternatively, receptor tyrosine kinase or certain G protein-coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3beta, via protein kinase B/Akt and mTOR/p70(s6k) pathways, PKC pathways, or protein kinase A pathways. The convergence of these pathways via inhibition of GSK-3beta on the end effector, the permeability transition pore complex, to limit MPT induction is the general mechanism of cardiomyocyte protection.