S-15176 inhibits mitochondrial permeability transition via a mechanism independent of its antioxidant properties

Mitochondrial Ca(2+) accumulation can induce a sudden increase in the permeability of the inner membrane. This phenomenon is due to the generation of a large nonselective ion channel, termed the permeability transition pore (PTP), which contributes to cellular injury during ischemia and reperfusion. Inhibition of PTP generation constitutes a relevant pharmacological target to protect a cell from death. In this study, we examined the effect of S-15176 ((N-[(3,5-di-tertiobutyl-4-hydroxy-1-thiophenyl)]-3-propyl-N'-(2,3,4-trimethoxybenzyl)piperazine), a novel anti-ischemic agent, on PTP in rat liver mitochondria. S-15176 prevented PTP opening generated by various triggering agents, as attested by the concentration-dependent inhibition of mitochondrial swelling, of mitochondrial membrane potential dissipation and of NADPH oxidation. These effects were associated with an increase in the Ca(2+) loading capacity of mitochondria. S-15176 was a strong inhibitor of lipid peroxidation, but experiments with another trimetazidine derivative devoid of antioxidant activity indicated that this activity was not essential to the inhibitory effect. Binding studies demonstrated that [3H]S-15176 bound to mitochondrial binding sites, especially those localized in the inner membrane. These sites were shared by several well-known inhibitors of PTP opening. These results demonstrate that the mechanism by which S-15176 protects mitochondria against the deleterious effects of ischemia-reperfusion involves inhibition of PTP opening and provide evidence that the drug operates through low structural specificity binding sites located in the inner mitochondrial membrane.     

Attenuation of liver normothermic ischemia--reperfusion injury by preservation of mitochondrial functions with S-15176, a potent trimetazidine derivative.

We investigated the antiischemic properties of a new compound, S-15176, in an experimental model of rat liver subjected to 120-min normothermic ischemia followed by 30-min reperfusion. Rats were divided into groups, pretreated with different doses of S-15176 (1.25, 2.5, 5 and 10 mg/kg/day by intramuscular injection) or solvent alone, and subjected to the ischemia--reperfusion process. Another group served as the sham-operated controls. Ischemia--reperfusion induced huge alterations of hepatocyte functions, namely, a decrease in ATP content and bile flow, and membrane leakage of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT). These effects were associated with alterations in mitochondrial functions characterized by (1) a decrease in ATP synthesis, (2) a decrease in NAD(P)H levels and mitochondrial membrane potential, and (3) an increase in mitochondrial swelling reflecting the generation of permeability transition. Pretreatment of rats with S-15176 alleviated these deleterious ischemia--reperfusion effects at both the cellular and mitochondrial levels in a dose-dependent manner. The protection of mitochondrial functions was almost complete at a dosage of 10 mg/kg/day. In addition, in vitro, S-15176 totally abolished the swelling of isolated mitochondria induced by a calcium overload with an IC(50) value of 10 microM. These data demonstrate that S-15176 protects mitochondria against the deleterious effects of ischemia-reperfusion and suggest that this protective effect could be related to the inhibition of the mitochondrial permeability transition.     

Inhibition of the mitochondrial ATP synthesis by polygodial, a naturally occurring dialdehyde unsaturated sesquiterpene

Polygodial is a naturally occurring sesquiterpene dialdehyde that exhibits several pharmacologically interesting activities. Among them, its antifungal properties have been more thoroughly studied. The mitochondrial ATPase has been suggested as one of the possible targets for polygodial action. However, its mechanism of action is not well defined yet. The effect of polygodial on the mitochondrial energy metabolism is described in this paper. Polygodial inhibited ATP synthesis coupled to succinate oxidation in beef-heart submitochondrial particles at concentrations (IC(50)=2.4+/-0.1 microM) which marginally affected electron transport and ATPase activity (IC(50)=97+/-4 microM). A transitory stimulation of the electron transport in intact rat liver mitochondria in state 4 was also obtained at low polygodial concentrations (EC(50)=20+/-4 microM). These results suggest that polygodial uncouples ATP synthesis from electron transport at low concentrations. Similar concentrations of polygodial partially abolished the ANS fluorescence enhancement (IC(50)=2.2+/-0.4 microM) induced by succinate oxidation in submitochondrial particles but did not collapse the DeltapH. We postulate that polygodial uncouples mitochondrial ATP synthesis by affecting the electrical properties of the membrane surface and consequently collapsing the membrane potential (Deltapsi) and/or the localized transmembrane pH difference (DeltapH(S)) without affecting the DeltapH between the two bulk aqueous phases (DeltapH(B)). The relevance of these findings for the understanding of the biochemical basis of the antifungal activity of polygodial and the evaluation of its potentiality as a therapeutic agent are discussed.     

Inhibition of mitochondrial carnitine palmitoyltransferase-1 by a trimetazidine derivative, S-15176.

The purpose of this study was to investigate the possible effect of the trimetazidine derivative S-15176 on carnitine palmitoyltransferase1 (CPT-1) activity in rat heart and liver mitochondria. S-15176 was compared with the other antianginal agents amiodarone, perhexiline and trimetazidine, which do not show any hemodynamic effects and which are believed to exert their effects by switching the cellular metabolism towards glucose utilization at the expense of lipid metabolism, increasing the yield of oxygen utilization. S-15176 inhibited CPT-1 in vitro and was more effective in heart (IC(50)= 16.8 micro M) than in liver ( 50.8 +/- 3.0 micro M). In the heart, its was less effective than the physiological inhibitor malonyl-CoA (IC(50)= 2.1 micro M), but it was more potent than amiodarone (IC(50)= 140 micro M). Kinetic experiments demonstrated a non-competitive inhibition of CPT-1 by S-15176 indicating that the two compounds did not share the same site of action. CPT-1 inhibition was also obvious ex vivo, in heart and liver tissues, after a 2 week treatment with S-15176. This inhibitory effect may shift heart and liver metabolism from fatty acid to glucose oxidation and contribute to the anti-ischemic effects of the drug.     

Effects of nitrosopropofol on mitochondrial energy-converting system

Nitrosopropofol (NOPR) is a relatively stable compound obtained from the reaction between the general anesthetic 2,6 diisopropylphenol (propofol) and nitrosoglutathione (GSNO) and bearing a more acidic phenol group than propofol. It interfered with mitochondrial energetic metabolism in a concentration-dependent manner. Concentrations as high as 100 or 200 microM disrupted both oxidative phosphorylation and electron transport. Low concentrations of NOPR (50 microM) markedly slowed down the electron transport rate which was insensitive both to ADP and uncoupler stimulation and spontaneously gradually stopped. Consequently, both the transmembrane potential production and the ATP synthesis system were affected. In the presence of 10 or 20 microM NOPR, mitochondria respired but showed a worsening of the respiratory control and produced a transmembrane potential useful to respond to a phosphorylation pulse, but were not able to restore it. These results were consistent with ATP synthesis and swelling experiments. NOPR was effective at concentrations lower than those required by the combination of propofol and GSNO, suggesting that mitochondria might be able to catalyze the reaction between GSNO and propofol and that the resulting metabolite was more active on mitochondrial membrane structure than the parent compounds. Although the details of the process are yet unknown, the mechanism presented may be of potential relevance to rationalize the pathophysiological effects of propofol.     

Calcineurin-independent inhibition of mitochondrial Ca2+ uptake by cyclosporin A

1. Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, derived mainly from the inhibition of calcineurin, and also because of its ability to block the mitochondrial permeability transition pore (PTP). This second effect has been involved in the protection against apoptosis mediated by release of mitochondrial factors. We show here that CsA (1-10 microm) has an additional effect on Ca(2+) homeostasis in mitochondria that cannot be attributed to inhibition of PTP. 2. By measuring specifically mitochondrial [Ca(2+)] with targeted aequorin, we show that CsA inhibited Ca(2+) entry into mitochondria both in intact and in permeabilized cells, and this effect was stronger when Ca(2+) entry was triggered by low cytosolic [Ca(2+)], below 5 microm. 3. Inhibition of mitochondrial Ca(2+) uptake required micromolar concentrations of CsA and was not mimicked by other inhibitors of calcineurin such as FK-506 or cypermethrin, nor by a different inhibitor of the PTP, bongkrekic acid. 4. CsA blocked the increase in mitochondrial Ca(2+) uptake rate induced by the mitochondrial Ca(2+) uniporter activator SB202190. 5. Our results suggest that CsA inhibits Ca(2+) entry through the Ca(2+) uniporter by a mechanism independent of the inhibition of PTP or calcineurin. This effect may contribute to reduce depolarization and Ca(2+) overloading in mitochondria after cell stimulation, and thus cooperate with the direct inhibition of PTP to prevent apoptosis.     

Human mitochondrial and cytosolic branched-chain aminotransferases are cysteine S-conjugate beta-lyases,but turnover leads to inactivation

The mitochondrial and cytosolic branched-chain aminotransferases (BCAT(m) and BCAT(c)) are homodimers in the fold type IV class of pyridoxal 5'-phosphate-containing enzymes that also contains D-amino acid aminotransferase and 4-amino-4-deoxychorismate lyase (a beta-lyase). Recombinant human BCAT(m) and BCAT(c) were shown to have beta-lyase activity toward three toxic cysteine S-conjugates [S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, S-(1,2-dichlorovinyl)-L-cysteine, and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine] and toward beta-chloro-L-alanine. Human BCAT(m) is a much more effective beta-chloro-L-alanine beta-lyase than two aminotransferases (cytosolic and mitochondrial isozymes of aspartate aminotransferase) previously shown to possess this activity. BCAT(m), but not BCAT(c), also exhibits measurable beta-lyase activity toward a relatively bulky cysteine S-conjugate [benzothiazolyl-L-cysteine]. Benzothiazolyl-L-cysteine, however, inhibits the L-leucine-alpha-ketoglutarate transamination reaction catalyzed by both enzymes. Inhibition was more pronounced with BCAT(m). In the presence of beta-lyase substrates and alpha-ketoisocaproate (the alpha-keto acid analogue of leucine), no transamination could be detected. Therefore, with an amino acid containing a good leaving group in the beta position, beta-elimination is greatly preferred over transamination. Both BCAT isozymes are rapidly inactivated by the beta-lyase substrates. The ratio of turnover to inactivation per monomer in the presence of toxic halogenated cysteine S-conjugates is approximately 170-280 for BCAT(m) and approximately 40-50 for BCAT(c). Mitochondrial enzymes of energy metabolism are especially vulnerable to thioacylation and inactivation by the reactive fragment released from toxic, halogenated cysteine S-conjugates such as S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. The present results suggest that BCAT isozymes may contribute to the mitochondrial toxicity of these compounds by providing thioacylating fragments, but inactivation of the BCAT isozymes might also block essential metabolic pathways.     

Dual effect of ebselen on mitochondrial permeability transition

This study reports an investigation on the effect of the seleno-organic compound ebselen on rat liver mitochondria. We show that low concentrations of ebselen induced an increase in rat liver mitochondrial membrane permeability, resulting in swelling and loss of membrane potential. These effects were mediated by the opening of the permeability transition pore. They required Ca(2+), were independent of pyridine nucleotide oxidation, and involved the oxidation of thiol groups. Ebselen pore induction is apparently promoted by the glutathione peroxidase mimicking activity of the drug. Opposite effects, that is, inhibition of both pore opening and thiol oxidation, were observed when concentrations higher than 20 micro M were used. These data demonstrate that ebselen is able to modulate the opening of the permeability transition pore and that it might be a critical event for both the proapoptotic and cytoprotective activities of the drug.     

The Fusarium mycotoxins enniatins and beauvericin cause mitochondrial dysfunction by affecting the mitochondrial volume regulation,oxidative phosphorylation and ion homeostasis

The mechanisms of cell toxicity of mycotoxins of the enniatin family produced by Fusarium sp. enniatin B, a mixture of enniatin homologues (3% A, 20% A₁, 19% B, 54% B1) and beauvericin, were investigated. In isolated rat liver mitochondria, exposure to submicromolar concentrations of the enniatin mycotoxins depleted the mitochondrial transmembrane potential, uncoupled oxidative phosphorylation, induced mitochondrial swelling and decreased calcium retention capacity of the mitochondria. The mitochondrial effects were strongly connected with the potassium (K⁺) ionophoric activity of the enniatins. The observed enniatins induced K⁺ uptake by mitochondria. This shows that the enniatins acted as ionophores highly selective for potassium ions. The effects were observed in potassium containing media whereas less or no effect remained to be observed when K⁺ was partially or totally replaced by isomolar concentrations of Na⁺. The rank order of enniatin induced mitochondrial impairment was beauvericin > enniatin mixture > enniatin B. Exposure to the enniatins depleted the mitochondrial membrane potential also in intact human neural (Paju), murine insulinoma (Min-6) cells as well as boar spermatozoa. Exposure to enniatin B in media with physiological (4 mM) or low (<1 mM) but not in high (60 mM) external concentration of K⁺ induced hyperpolarization of the spermatozoal plasma membrane indicating enniatin that catalysed efflux of the cytosolic K⁺ ions. These results indicate that the cellular toxicity targets of the enniatin mycotoxins are the mitochondrion and the homeostasis of potassium ions.     

Toxic,halogenated cysteine S-conjugates and targeting of mitochondrial enzymes of energy metabolism

Several haloalkenes are metabolized in part to nephrotoxic cysteine S-conjugates; for example, trichloroethylene and tetrafluoroethylene are converted to S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), respectively. Although DCVC-induced toxicity has been investigated since the 1950s, the toxicity of TFEC and other haloalkene-derived cysteine S-conjugates has been studied more recently. Some segments of the US population are exposed to haloalkenes either through drinking water or in the workplace. Therefore, it is important to define the toxicological consequences of such exposures. Most halogenated cysteine S-conjugates are metabolized by cysteine S-conjugate beta-lyases to pyruvate, ammonia, and an alpha-chloroenethiolate (with DCVC) or an alpha-difluoroalkylthiolate (with TFEC) that may eliminate halide to give a thioacyl halide, which reacts with epsilon-amino groups of lysine residues in proteins. Nine mammalian pyridoxal 5'-phosphate (PLP)-containing enzymes catalyze cysteine S-conjugate beta-lyase reactions, including mitochondrial aspartate aminotransferase (mitAspAT), and mitochondrial branched-chain amino acid aminotransferase (BCAT(m)). Most of the cysteine S-conjugate beta-lyases are syncatalytically inactivated. TFEC-induced toxicity is associated with covalent modification of several mitochondrial enzymes of energy metabolism. Interestingly, the alpha-ketoglutarate- and branched-chain alpha-keto acid dehydrogenase complexes (KGDHC and BCDHC), but not the pyruvate dehydrogenase complex (PDHC), are susceptible to inactivation. mitAspAT and BCAT(m) may form metabolons with KGDHC and BCDHC, respectively, but no PLP enzyme is known to associate with PDHC. Consequently, we hypothesize that not only do these metabolons facilitate substrate channeling, but they also facilitate toxicant channeling, thereby promoting the inactivation of proximate mitochondrial enzymes and the induction of mitochondrial dysfunction.     

Reactive oxygen species are involved in the stimulation of the mitochondrial permeability transition by dihydrolipoate

Dihydrolipoic acid (DHLA) has been found to stimulate the Ca(2+)-induced mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) [Biochem. Mol. Biol. Int. 44 (1998) 127] which could be due to its prooxidant properties. We therefore investigated whether DHLA stimulated superoxide anion (O(2)(.-)) generation in RLM and in bovine heart submitochondrial particles (SMP). In RLM DHLA caused a concentration-dependent O(2)(.-) generation assayed by lucigenin chemiluminiscence. The stimulation was seen with the lowest concentrations of DHLA (5 microM) with pyruvate as the respiratory substrate, with 2-oxoglutarate or especially succinate the stimulation was less pronounced. Stimulation of O(2)(.-) production by DHLA was also observed in bovine heart SMP using an electron spin-trapping technique. Radical scavengers (butylhydroxytoluene and TEMPO) decreased O(2)(.-) generation induced by DHLA and inhibited MPT. Slight reduction of the mitochondrial membrane potential by a small amount of a protonophorous uncoupling agent also delayed the DHLA-induced MPT. These data indicate that the stimulation of MPT by DHLA is due to DHLA-derived prooxidants, i.e. stimulated production of O(2)(.-) and possibly other free radicals.     

Role of the permeability transition pore in cytochrome C release from mitochondria during ischemia-reperfusion in rat liver

Ischemia and reperfusion cause mitochondrial dysfunctions that initiate the mitochondrial apoptosis pathway. They involve the release of cytochrome C and the activation of the caspase cascade but the mechanism(s) leading to cytochrome C release is(are) poorly understood. The aim of this study was to analyse the relation between cytochrome C release and the opening of the permeability transition pore (PTP) during in situ liver ischemia and reperfusion. Liver ischemia was induced for 30, 60 and 120 min and blood re-flow was subsequently restored for 30 and 180 min. Ischemia hugely altered mitochondrial functions, i.e., oxidative phosphorylation and membrane potential, and was accompanied by a time-dependent mitochondrial release of cytochrome C into the cytosol and by activations of caspases-3 and -9. PTP opening was not observed during ischemia, as demonstrated by the absence of effect of an in vivo pre-treatment of rats with cyclosporin A (CsA), a potent PTP inhibitor. Cytochrome C release was due neither to a direct effect of caspases onto mitochondria nor to an interaction of Bax or Bid with the mitochondrial membrane but could be related to a direct effect of oxygen deprivation. In contrast, during reperfusion, CsA pre-treatment inhibits cytochrome C release, PTP opening and caspase activation. At this step, cytochrome C release is likely to occur as a consequence of PTP opening. In conclusion, our study reveals that cytochrome C release, and thus the induction of the mitochondrial cell death pathway, occur successively independently and dependent on PTP opening during liver ischemia and reperfusion, respectively.     

Concentration dependent mitochondrial effect of amiodarone

Although, the antiarrhythmic effect of amiodarone is well characterized, its effect on post-ischemic heart and cardiomyocytes, as well as the mechanism of its toxicity on extracardiac tissues is still poorly understood. In this study, we analyzed energy metabolism in situ during ischemia-reperfusion in Langendorff-perfused heart model by measuring the high-energy phosphate metabolites using 31P NMR spectroscopy. The toxicity of amiodarone on cardiomyocytes and cell lines of extracardiac origin, as well as direct effect of the drug on mitochondrial functions in isolated mitochondria was also analyzed. Amiodarone, when was present at low concentrations and predominantly in membrane bound form, protected heart and mitochondrial energy metabolism from ischemia-reperfusion-induced damages in Langendorff-perfused heart model. Toxicity of the drug was significantly higher on hepatocytes and pancreatic cells than on cardiomyocytes. In isolated mitochondria, amiodarone did not induce reactive oxygen species formation, while it affected mitochondrial permeability transition in a concentration dependent way. Up to the concentration of 10 microM, the drug considerably inhibited Ca(2+)-induced permeability transition, while at higher concentrations it induced a cyclosporin A independent permeability transition of its own. At concentrations where it inhibited the Ca(2+)-induced permeability transition (IC(50)=3.9+/-0.8 microM), it did not affect, between 6 and 30 microM it uncoupled, while, at higher concentrations it inhibited the respiratory chain. Thus, the concentration dependent nature of amiodarone's effect on permeability transition together with the different sensitivities of the tissues toward amiodarone can be involved in the beneficial cardiac and the simultaneous toxic extracardiac effects of the drug.     

On the mechanisms of phenothiazine-induced mitochondrial permeability transition: Thiol oxidation, strict Ca dependence, and cyt c release

Phenothiazines (PTZ) are drugs widely used in the treatment of schizophrenia. Trifluoperazine, a piperazinic PTZ derivative, has been described as inhibitor of the mitochondrial permeability transition (MPT). We reported previously the antioxidant activity of thioridazine at relatively low concentrations associated to the inhibition of the MPT (Brit. J. Pharmacol., 2002;136:136-142). In this study, it was investigated the induction of MPT by PTZ derivatives at concentrations higher than 10 μM focusing on the molecular mechanism involved. PTZ promoted a dose-response mitochondrial swelling accompanied by mitochondrial transmembrane potential dissipation and calcium release, being thioridazine the most potent derivative. PTZ-induced MPT was partially inhibited by CsA or Mg²⁺ and completely abolished by the abstraction of calcium. The oxidation of reduced thiol group of mitochondrial membrane proteins by PTZ was upstream the PTP opening and it was not sufficient to promote the opening of PTP that only occurred when calcium was present in the mitochondrial matrix. EPR experiments using DMPO as spin trapping excluded the participation of reactive oxygen species on the PTZ-induced MPT. Since PTZ give rise to cation radicals chemically by the action of peroxidases and cyanide inhibited the PTZ-induced swelling, we propose that PTZ bury in the inner mitochondrial membrane and the chemically generated PTZ cation radicals modify specific thiol groups that in the presence of Ca²⁺ result in MPT associated to cytochrome c release. These findings contribute for the understanding of mechanisms of MPT induction and may have implications for the cell death induced by PTZ.     

Comparative effects of lantadene A and its reduced metabolite on mitochondrial bioenergetics

Lantana (Lantana camara Linn.) is a noxious weed to which certain medicinal properties have been attributed, but its ingestion has been reported to be highly toxic to animals and humans, especially in the liver. The main hepatotoxin in lantana leaves is believed to be the pentacyclic triterpenoid lantadene A (LA), but the precise mechanism by which it induces hepatotoxicity has not yet been established. This work addressed the action of LA and its reduced derivative (RLA) on mitochondrial bioenergetics. At the concentration range tested (5-25 μM), RLA stimulated state-4 respiration, inhibited state-3 respiration, circumvented oligomycin-inhibited state-3 respiration, dissipated membrane potential and depleted ATP in a concentration-dependent manner. However, LA did not stimulate state-4 respiration, nor did it affect the other mitochondrial parameters to the extent of its reduced derivative. The lantadenes didn't inhibit the CCCP-uncoupled respiration but increased the ATPase activity of intact coupled mitochondria. The ATPase activity of intact uncoupled or disrupted mitochondria was not affected by the compounds. We propose, therefore, that RLA acts as a mitochondrial uncoupler of oxidative phosphorylation, a property that arises from the biotransformation (reduction) of LA, and LA acts in other mitochondrial membrane components rather than the ATP synthase affecting the mitochondrial bioenergetics. Such effects may account for the well-documented hepatoxicity of lantana.     

Thenoyltrifluoroacetone, a potent inhibitor of carboxylesterase activity

Thenoyltrifluoroacetone (TTFA), a conventional mitochondrial complex II inhibitor, was found to inhibit purified porcine liver carboxylesterase non-competitively with a K(i) of 0.61x10(-6)M and an IC(50) of 0.54x10(-6)M. Both rat plasma and liver mitochondrial esterases were inhibited in a concentration-dependent fashion. Results indicate that TTFA is a potent inhibitor of carboxylesterase activity, in addition to its ability to inhibit mitochondrial complex II activity. Therefore, caution is warranted in using TTFA as a mitochondrial complex inhibitor in combination with esterase substrates, such as fluorescence probes or vitamin E esters.     

Sanguinarine cytotoxicity on mouse melanoma K1735-M2 cells--nuclear vs. mitochondrial effects

Sanguinarine (SANG) is an alkaloid recognized to have anti-proliferative activity against various human tumour cell lines. No data is available on the susceptibility of advanced malignant melanoma to SANG, although this disease has a very poor prognosis if not detected in time due to the resistance to conventional chemotherapy. The present work was designed to study the nuclear and mitochondrial involvement in the pro-apoptotic effect of SANG in an invasive mouse melanoma cell line. The results obtained show that SANG is primarily accumulated by the cell nuclei, causing inhibition of cell proliferation and inducing cell death, as confirmed by an increase in sub-G1 peaks. At low concentrations, SANG induces mitochondrial depolarization in a sub-population of melanoma cells, which also generally displayed strong nuclear labelling of phosphorylated histone H2AX. Western blotting revealed an increase in p53, but not Bax protein, in both whole-cell extracts and in mitochondrial fractions. Isolated hepatic mitochondrial fractions revealed that SANG affects the mitochondrial respiratory chain, and has dual effects on mitochondrial calcium loading capacity. We suggest that SANG is able to induce apoptosis in metastatic melanoma cells. The knowledge of mitochondrial vs. nuclear effects of SANG is important in the development of this promising compound for clinical use against aggressive melanoma.     

Modulation of mitochondrial metabolic function by phorbol 12-myristate 13-acetate through increased mitochondrial translocation of protein kinase Calpha in C2C12 myocytes

Protein kinase C (PKC) agonists including phorbol 12-myristate 13-acetate (PMA) not only induce the redistribution of cytosolic PKC to various subcellular compartments but also activate the kinase domain of the protein. In the present study we have investigated the nature of mitochondrial PKC pool and its effects on mitochondrial function in cells treated with PMA. Treatment of C2C12 myoblasts, C6 glioma and COS7 cells with PMA resulted in a dramatic redistribution of intracellular PKCalpha pool, with large fraction of the protein pool sequestered in the mitochondrial compartment. We also observed mitochondrial PKCdelta accumulation in a cell restricted manner. The intramitochondrial localization was ascertained by using a combination of protection against protease treatment of isolated mitochondria and immunofluorescence microscopy. PMA-induced mitochondrial localization of PKCalpha was accompanied by increased mitochondrial PKC activity, altered cell morphology, disruption of mitochondrial membrane potential, decreased complex I and pyruvate dehydrogenase activities, and increased mitochondrial ROS production. All of these changes could be retarded by treatment with PKC inhibitors. These results show a direct role for PMA-mediated PKCalpha translocation to mitochondria in inducing mitochondrial toxicity.     

Naringin alleviates cognitive impairment,mitochondrial dysfunction and oxidative stress induced by d-galactose in mice

Role of mitochondrial dysfunction and oxidative stress has been well documented in aging and related disorders such as Alzheimer’s disease. Bioflavonoids have been reported to have a therapeutic potential against several age related processes. Bioflavonoids are being used as a neuroprotectants in the treatment of various neurological disorders including aging. Therefore, present study has been conducted in order to explore the possible role of naringin against d-galactose induced cognitive dysfunction, oxidative damage and mitochondrial dysfunction in mice. Chronic administration of d-galactose (100 mg/kg) for 6 weeks significantly impaired cognitive performance (both in Morris water maze and elevated plus maze), locomotor activity, oxidative defense and mitochondrial complex (I, II and III) enzymes activities as compared to sham group. Six weeks naringin (40 and 80 mg/kg) treatment significantly improved cognitive performance, oxidative defense and restored mitochondria complex enzyme activities as compared to control (d-galactose). Naringin treatment significantly attenuated acetylcholine esterase activity in d-galactose treated mice. In conclusion, present study highlights the potential role of naringin against d-galactose induced cognitive impairment, biochemical and mitochondrial dysfunction in mice.     

S-15176 reduces the hepatic injury in rats subjected to experimental ischemia and reperfusion

The protective effect of N-[(3, 5-di-tertiobutyl-4-hydroxy-1-thiophenyl)]-3-propyl-N'-(2,3, 4-trimethoxybenzyl)piperazine (S-15176) on liver injury induced by warm ischemia-reperfusion was investigated using a rat model. Animals were subjected to 2 h of ischemia followed by different reperfusion times. Hepatocyte integrity was assessed by measuring plasma alanine and aspartate aminotransferase activities, and by determining reduced and oxidized glutathione in plasma and bile. Hepatocyte function was quantitated by determining bile flow and liver ATP content. Ischemia-reperfusion resulted in severe hepatic injury involving a huge increase in alanine and aspartate aminotransferase activities, a drop in ATP content, and a decrease in bile flow. Plasma and bile reduced (GSH) and oxidized (GSSG) glutathione concentrations were inversely related: plasma levels increased when biliary levels decreased. This was associated with a decrease in animal survival (-34%). S-15176 pretreatment (1.25, 2.5, 5 or 10 mg kg(-1) day(-1)) improved the survival rate and limited tissue damages in a dose-dependent manner. The pretreatment also reduced the aminotransferase leakage from hepatocytes and the increase in plasma glutathione levels. In addition, normalization of the plasma GSSG/GSH ratio, a good index of an oxidative stress, was observed in groups treated with the higher dosage, suggesting that the antioxidant properties demonstrated for the compound in vitro (IC(50)=0.3 microM towards lipid peroxidation) could play a role in its protective effect. S-15176 pretreatment also protected the organ from the drop in ATP levels. At the higher dose, ATP content was maintained at a level almost 86% of the sham-operated group after 60 min of reperfusion. This was associated with a restoration of the biliary flow. These data suggest that S-15176 may be a useful drug in liver surgery to prevent ischemia-reperfusion injury.