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PubMed2000

Role of the mitochondrial membrane permeability transition (MPT) in rotenone-induced apoptosis in liver cells.

Rotenone inhibits spontaneously and chemically induced hepatic tumorigenesis in rodents through the induction of apoptosis. However, the mechanism for the induction of apoptosis by rotenone has not been defined. Mitochondrial dysfunction, in particular the induction of the mitochondrial membrane permeability transition (MPT), has been implicated in the cascade of events involved in the induction of apoptosis. Inhibition of the mitochondrial electron-transport chain reduces the mitochondrial transmembrane potential (delta(psi)m), which may induce the formation of the mitochondrial permeability transition pore and the subsequent MPT. Fluorescent microscopy of Hoechst 33258-stained WB-F344 cells, a rat-liver cell line, was utilized to examine the effect of the mitochondrial respiratory chain inhibitor, rotenone (0.5-5 microM), atractyloside (5-10 microM), and cyclosporin A (2.5-10 microM) on apoptosis. A time- and concentration-dependent increase in liver cell apoptosis was observed following treatment with rotenone and atractyloside (11.7- and 7.7-fold, respectively, over solvent control). Cotreatment with 7.5- and 10 microM-cyclosporin A for 12 h inhibited the apoptogenicity of 5-microM rotenone treatment. A similar effect was observed following cyclosporin A cotreatment with atractyloside. Rotenone induced a rapid increase in apoptosis (within 20 min of treatment). By 2 h of treatment, the morphological appearance of apoptosis was similar to that observed in cultures treated continuously with rotenone for 12 h. Inhibition studies demonstrated that cyclosporin A prevented apoptosis if the exposure to it occurred prior to the 20-min threshold necessary to induce apoptosis by rotenone. Mitochondrial function was examined by staining with the mitochondrial membrane potential (delta(psi)m)-sensitive fluorochrome, MitoTracker Red (CMXRos) and confirmed utilizing cytofluorometric analysis of DiOC6(3)-stained cells. Rotenone (5.0-microM) and atractyloside (5.0-microM) reduced the percent of CMXRos or DiOC6(3)-positive (delta(psi)m-positive) liver cells within 15 min and throughout the duration of the study (6 h) to approximately 65-80% and 50-80% of control. However, cotreatment with concentrations of cyclosporin A that inhibited the apoptogenicity of rotenone and atractyloside prevented the rotenone- and atractyloside-induced reduction of the delta(psi)m. Therefore, the apoptogenic effect of rotenone and atractyloside appears to occur rapidly (within 20 min) and is irreversible once mitochondrial damage occurs. The inhibition of the rotenone- and atractyloside-induced apoptosis and mitochondrial dysfunction by cyclosporin A suggests the MPT may be involved in the induction of apoptosis by rotenone.

Animals↗
PubMed1978

Is the adenine nucleotide translocator rate-limiting for oxidative phosphorylation?

1. The effects of atractyloside and carboxyatractyloside (between 5 and 40mum) on O(2) uptake, glucose synthesis, urea synthesis, the adenine nucleotide content and the intracellular K(+) concentration were measured in isolated hepatocytes. 2. Urea synthesis was much less inhibited than glucose synthesis by both atractylosides. Measurements of intermediary metabolites of carbohydrate metabolism in freeze-clamped liver after injection of atractyloside into rats indicate that inhibition of gluconeogenesis is due to interference at the cytosolic reactions requiring ATP (phosphoenolpyruvate carboxykinase and 3-phosphoglycerate kinase). 3. The decrease in [ATP]/[ADP]x[P(i)] after addition of atractyloside or carboxyatractyloside was restricted to the cytosol. 4. Dihydroxyacetone can be converted either into glucose with the consumption of 2mol of ATP (per mol of glucose) or into lactate with the production of 2mol of ATP. In the presence of high concentrations of atractyloside and carboxyatractyloside more ATP was produced than was used for the synthesis of glucose from dihydroxyacetone, probably for the maintenance of intracellular [K(+)]. 5. When the rates of respiration were altered by changing substrates, the degrees of inhibition of respiration and translocation by a given concentration of the atractylosides were the same, whereas at a given concentration of HCN the degree of inhibition was high at higher initial rates, and low at lower initial rates. 6. Inhibition of a complex series of reactions by atractyloside does not necessarily indicate that the translocator is a rate-limiting step in that sequence as Th. P. M. Akerboom, H. Bookelman & J. M. Tager [(1977) FEBS. Lett.74, 50-54] assume. This point is discussed.

Adenine Nucleotides↗
PubMed1980

Kinetic, binding and ultrastructural properties of the beef heart adenine nucleotide carrier protein after incorporation into phospholipid vesicles.

1. ADP/ATP transport has been reconstituted by incorporation of the purified carrier protein in liposomes filled with ATP. The transport was assayed by uptake of [14C]ADP into the liposomes, and by release of ATP as determined by a luminescence technique. [14C]ADP uptake was strictly dependent on internal ATP. 2. The simplest phospholipid system capable of yielding high rates of ADP/ATP transport was a mixture of phosphatidylethanolamine and cariolipin (92: 8, w/w). 3. ADP/ATP transport in the reconstituted system proceeded by exchange-diffusion with a 1/1 stoichiometry. The specificity for aDP and ATP was absolute. The capacity and the rate of exchange depended on the concentration of ATP present in liposomes. The rate of transport at 20 degrees C, at 20 mM internal ATP, routinely ranged between 300 and 1000 nmol of nucleotide exchanged per min/mg of added carrier protein. The apparent Km value for external ADP was around 10 microM. 4. The ADP/ATP exchange in the reconstituted system was rather stable to ageing. It dropped by only 20% after 1 day of ageing at 20 degrees C. Divalent cations (Mg2+, Mn2+, Ca2+) at concentrations higher than 1 to 2 mM had a deleterious effect on ADP/ATP transport, concomitant with the release of internal ATP and accumulation of multilamellar vesicles. 5. Atractyloside behaved as a competitive inhibitor and carboxyatractyloside as a non-competitive inhibitor. Bongkrekic acid required a slightly acidic pH to be inhibitory. The data concerning atractyloside, carboxyatractyloside and bongkrekic acid were similar to those obtained with whole mitochondria, suggesting that the carrier protein in liposomes has the same asymmetrical arrangement as in the mitochondria. 6. The percentage of competent carrier protein in liposomes was calculated from dose-response data concerning the inhibition of ADP/ATP transport by atractyloside or carboxyatractyloside, and from the amount of bound [3H]-atractyloside removable by ADP. By both methods, 3 to 6% of the added carrier protein was found to be competent in ADP/ATP transport, based on the assumption that the binding of one atractyloside or carboxyatractyloside molecule per 30000 molecular weight carrier unit results in complete inhibition of transport. 7. Freeze-fracture electron microscopy showed that the ADP/ATP carrier protein-lipid preparations are formed by small vesicles, most of which give rise to smooth fracture faces (probably pure lipid vesicles). Only a small percentage of the vesicles (2 to 4% depending on the amount of carrier protein added) were clearly particulated. About 90% of the particulated vesicles showed no more than 2 particles per vesicle and only 5% more than 5 particles per vesicle. The distribution of the particles between convex and concave fracture faces was asymmetric; about 2/3 of the protein molecules were anchored at the external surface of the vesicles and only 1/3 at the internal one...

Animals↗
PubMed1983

Characterization of phosphate efflux pathways in rat liver mitochondria.

ATP hydrolysis catalysed by the H+-ATPase of intact mitochondria can be induced by addition of ATP in the presence of valinomycin and KCl. This leads to an increase in intramitochondrial Pi and therefore allows investigation of potential Pi efflux pathways in intact mitochondria. Combining this approach with the direct measurement of both internal and external Pi, we have attempted to determine whether Pi efflux occurs via an atractyloside-sensitive transporter, by the classical operation of the Pi/H+ and Pi/dicarboxylate carriers, and/or by other mechanisms. Initial experiments re-examined the evidence that led to the current view that one efflux pathway for Pi is an atractyloside-sensitive ATP/ADP,0.5Pi transporter. No evidence was found in support of this efflux pathway. Rather, atractyloside-sensitivity of the low rate of Pi efflux observed in previous studies (oligomycin present) was accounted for by ATP entry on the well known ATP/ADP transport system followed by hydrolysis of ATP and subsequent Pi efflux. Thus, under these conditions, where ATP hydrolysis is not completely inhibited, Pi efflux becomes atractyloside sensitive most likely because this inhibitor blocks ATP entry, not because it directly inhibits Pi efflux. Substantial efflux of Pi from rat liver mitochondria is observed on generation of high levels of matrix Pi by ATP hydrolysis induced by valinomycin and K+ (oligomycin absent). A portion of this efflux can be inhibited by thiol-specific reagents at concentrations that normally inhibit the Pi/H+ and Pi/dicarboxylate carriers. However, a significant fraction of efflux continues even in the presence of p-chloromercuribenzoate, N-ethylmaleimide plus n-butylmalonate or mersalyl. The mersalyl-insensitive Pi efflux, which is also insensitive to carboxyatractyloside, is a saturable process, thus suggesting carrier mediation. During this efflux the mitochondrial inner membrane retains considerable impermeability to other low-molecular-weight anions (i.e., malate, 2-oxoglutarate). In conclusion, results presented here rule out an atractyloside-sensitive ATP/ADP,0.5Pi transport system as a mechanism for Pi efflux in rat liver mitochondria. Rather Pi efflux appears to occur on the classical Pi/H+ transport system as well as via a mersalyl-insensitive saturable process. The inhibitor-insensitive Pi efflux may occur on a portion of the Pi/H+ carrier molecules that exist in a state different from that normally catalysing Pi influx. Alternatively, a separate Pi efflux carrier may exist.

Adenosine Triphosphate↗
PubMed2003

Cholesterol impairs the adenine nucleotide translocator-mediated mitochondrial permeability transition through altered membrane fluidity.

Mitochondrial permeability transition (MPT) has been proposed to play a key role in cell death. Downstream MPT events include the release of apoptogenic factors that sets in motion the mitochondrial apoptosome leading to caspase activation. The current work examined the regulation of MPT by membrane fluidity modulated upon cholesterol enrichment. Mitochondria enriched in cholesterol displayed increased microviscosity resulting in impaired MPT induced by atractyloside, a c-conformation stabilizing ligand of the adenine nucleotide translocator (ANT). This effect was dependent on the dose of cholesterol loaded and reversed upon the fluidization of mitochondria by the fatty acid derivative A2C. Mitoplasts derived from cholesterol-enriched mitochondria responded to atractyloside in a similar fashion as intact mitochondria, indicating that a significant amount of cholesterol is still found in the inner membrane. The effects of cholesterol on MPT induced by atractyloside were mirrored by the release of intermembrane proteins, cytochrome c, Smac/Diablo, and apoptosis inducing factor. However, cholesterol loading did not affect the uptake rate of adenine nucleotide hence dissociating the function of ANT as a MPT-mediated protein from its adenine nucleotide exchange function. Thus, these findings indicate that the ability of atractyloside to induce MPT via ANT requires an appropriate membrane fluidity range.

Adenine↗
PubMed1999

Poisoning by plant material: review of human cases and analytical determination of main toxins by high-performance liquid chromatography-(tandem) mass spectrometry.

The authors have reviewed the main toxic plants responsible for human deaths throughout the world. Forty plants (genera or species) were listed in order to establish an inventory of the active molecules that could be identified, the already published analytical methods and the reported human fatal cases. In a second step, the authors have developed a general method for the detection of various toxins in whole blood by high-performance liquid chromatography coupled to mass spectrometry or tandem mass spectrometry. Sample preparation was realized by liquid-liquid extraction at pH 9.5 for oleandrine, taxol and the alkaloids. These latter compounds were divided into two groups following their chemical properties and could be subsequently purified by acid/base clean up. Cyanogenic compounds and atractyloside were isolated by precipitation of the protein content with acetone and purified for atractyloside by washing with chloroform. Separation of the drugs occurred under reversed-phase conditions on a C18 analytical column 150x2 mm I.D. (5 microm particle size) using two different mobile phases. The first one, formiate buffer 2 mM acidified at pH 3.0, was used for the separation of atractyloside, oleandrine, taxol, the cyanogenic molecules and some alkaloids. The second mobile phase, formiate buffer 10 mM made basic at pH 8.2 was used for the majority of other alkaloids. A gradient elution mode was chosen using acetonitrile or acetonitrile-methanol (50:50, v/v) as the eluting solvent. Detection under positive ionization mode was the mode of choice for all compounds except for atractyloside (negative ions) and for taxol (mixed mode available). Application to real forensic cases has been demonstrated.

Child↗
PubMed1997

Oxidative phosphorylation in myocardial mitochondria 'in situ': a calorimetric study on permeabilized cardiac muscle preparations.

A novel flow calorimetric technique was developed to study the energy turnover of myocardial mitochondria. Cylindrical strands of cardiac muscle (trabeculae) weighing 100-500 micrograms were isolated from guinea-pig heart and mounted in a tubular recording chamber which was continuously perfused with physiological salt solution at 37 degrees C. The temperature difference between the upstream and the downstream side of the chamber, which is proportional to the rate of heat production of the trabecula, was measured at high resolution. In this way the rate of energy expenditure of isolated cardiac muscle could be recorded continuously for several hours. When the preparations were superfused with an 'intracellular' solution containing 5 mM pyruvate and 2 mM malate as substrates, permeabilization of the sarcolemma with 25 microM digitonin induced a marked increase in the measured heat rate in the presence of 2 mM ADP. The major fraction of the ADP sensitive heat production (83%) could be blocked with 400 microM atractyloside, an inhibitor of the adeninenucleotide translocase, and by 600 microM alpha-cyano-4-hydroxycinnamate, an inhibitor of monocarboxylate/H+ co-transport. The atractyloside sensitive heat production was abolished in anoxic solution. These results suggest that the atractyloside-sensitive heat production (21.8 +/- 3.5 mW cm-3 of tissue) was attributable to oxidative phosphorylation. The mitochondria apparently remained intact after treatment with digitonin, since application of the uncoupler 2,4-dinitrophenol (DNP) produced a very large increase in heat rate. A minor fraction of the heat rate induced by ADP in permeabilized cardiac muscle preparations (17%) was not sensitive to atractyloside. This component was also seen before application of digitonin and was probably related to ectonucleotidases. In conclusion, our calorimetric technique allows investigation of the energy metabolism of myocardial mitochondria 'in situ', i.e. without destroying the microarchitecture of cardiac muscle cells.

Animals↗
PubMed1981

Phosphoenolpyruvate efflux from kidney cortex mitochondria of rabbit.

(1) The relationship between phosphoenolpyruvate formation and its accumulation in kidney cortex mitochondria of rabbit was studied in the presence of glutamate as substrate. (2) In mitochondria incubated in either State 4 or under uncoupled conditions, both 1,2,3-benzenetricarboxylate and atractyloside resulted in a marked elevation of the intramitochondrial phosphoenolpyruvate accompanied by a 2-4-fold decline in production of this compound. The same effect was induced by n-butylmalonate in uncoupled mitochondria, while both phosphoenolpyruvate efflux and its production were inhibited to a smaller extent in mitochondria incubated with 1,2,3-benzenetricarboxylate in State 3. (3) Citrate, malate or 2-phosphoglycerate caused a fast displacement of phosphoenolpyruvate from atractyloside-inhibited mitochondria to the reaction medium. In contrast, on the addition of ATP to mitochondria incubated with 1,2,3-benzenetricarboxylate, the rate of phosphoenolpyruvate efflux was lower than that induced by either malate or citrate. (4) Despite the presence of both 1,2,3-benzenetricarboxylate and atractyloside, arsenite and rotenone plus antimycin resulted in a leakage of phosphoenolpyruvate from the mitochondria, probably via a carrier-independent mechanism. (5) Based on the present results it seems that depending on the metabolic condition, the tricarboxylate carrier and the adenine nucleotide translocase are functioning to different extents in the efflux of phosphoenolpyruvate from rabbit renal mitochondria to the surrounding medium.

Animals↗
PubMed1990

Plasmodium falciparum: ATP/ADP transport across the parasitophorous vacuolar and plasma membranes.

Previous studies have shown that ATP is required for the growth of the intracellular parasite, Plasmodium, outside its host cell, the erythrocyte, and that bongkrekic acid, an inhibitor of mitochondrial ATP/ADP transporter, inhibits intraerythrocytic Plasmodium maturation. We have characterized ATP/ADP transport of Plasmodium falciparum, isolated by either immune lysis or N2-cavitation. [3H]ATP uptake was due to ATP/ADP exchange since ADP efflux was dependent on exogenous ATP in an approximate 1:1 stoichiometry and both ATP influx and ADP efflux were equally inhibited by atractyloside (Ki = 100 nM). ATP uptake was not inhibited by the nucleoside transport inhibitor, nitrobenzylthioinosine. Conversely, adenosine and hypoxanthine transport were insensitive to atractyloside. ATP influx was characterized by a Km = 0.14 mM and Vmax = 1.2 nmol ATP/min/10(6) cells. Substrate specificity studies for nucleotide-induced ADP efflux indicated a preference for an adenosine ring and triphosphate, but transport did not require a hydrolyzable phosphate bond. Protein synthesis was measured with free parasites starved of glucose. Addition of 1.0 mM ATP resulted in a 40% recovery of total protein synthetic capacity in a process inhibited by 500 nM atractyloside, suggesting that uptake of erythrocyte-derived ATP by P. falciparum may be essential for maintaining maximal rates of protein synthesis during specific stages of intra-erythrocytic parasite maturation.

Adenosine Diphosphate↗
PubMed1990

Cytochemical and morphometric analysis of autophagy in energy depleted rat hepatocytes.

The energy dependence of lysosomal enzyme acquisition by autophagosomes was studied in isolated rat hepatocytes by ultrastructural analysis for acid phosphatase activity. Reduction of the intracellular ATP content by addition of atractyloside or fructose decreased the flux through the autophagic proteolytic pathway to a similar extent (40-50%). Unexpectedly, in the presence of atractyloside the volume density of autophagosomes was reduced by 65%, whereas in the presence of fructose this reduction was only 20%. The volume density of lysosomes was not significantly affected by either of the two compounds. It is concluded that partial ATP depletion by fructose not only inhibits sequestration of cytoplasmic material in autophagosomes, but also affects the fusion between autophagosomes and lysosomes. Since fructose, in contrast to atractyloside, does not affect the cytosolic phosphate potential, it is proposed that autophagic sequestration is more sensitive to changes in the cytosolic phosphate potential whereas the fusion between autophagosomes and lysosomes is more responsive to changes in the ATP concentration.

Acid Phosphatase↗
PubMed2005

Atractylis gummifera L. poisoning: an ethnopharmacological review.

Atractylis gummifera L. (Asteraceae) is a thistle located in the Mediterranean regions. Despite the plant's well-known toxicity, its ingestion continues to be a common cause of poisoning. The toxicity of Atractylis gummifera resides in atractyloside and carboxyatractyloside, two diterpenoid glucosides capable of inhibiting mitochondrial oxidative phosphorylation. Both constituents interact with a mitochondrial protein, the adenine nucleotide translocator, responsible for the ATP/ADP antiport and involved in mitochondrial membrane permeabilization. Poisoned patients manifest characteristic symptoms such as nausea, vomiting, epigastric and abdominal pain, diarrhoea, anxiety, headache and convulsions, often followed by coma. No specific pharmacological treatment for Atractylis gummifera intoxication is yet available and all the current therapeutic approaches are only symptomatic. In vitro experiments showed that some compounds such as verapamil, or dithiothreitol could protect against the toxic effects of atractyloside, but only if administered before atractyloside exposure. New therapeutic approaches could come from immunotherapy research: some studies have already tried to produce polyclonal Fab fragments against the toxic components of Atractylis gummifera.

Atractylis↗
PubMed2006

The neuroprotection conferred by activating the mitochondrial ATP-sensitive K+ channel is mediated by inhibiting the mitochondrial permeability transition pore.

In order to further explore the mechanisms by which activation of mitochondrial ATP-sensitive potassium channels (mitoKATP) confers neuroprotection, we investigated the role of the mitochondrial permeability transition pore (MPTP) in in vivo and in vitro models. Adult male Sprague-Dawley rats were exposed to 90 min of middle cerebral artery occlusion (MCAO) followed by reperfusion for 22 h, when neurological scores and infarct volumes were evaluated. Activating mitoKATP by infusion of 2 mmol/L diazoxide into the ventricles 20 min before MCAO or inhibiting the MPTP by infusion of 1 micromol/L cyclosporin A 15 min before reperfusion significantly increased functional score and reduced infarction volume. Subsequent intracerebroventricular infusion of 2 mmol/L atractyloside, the MPTP opener, 10 min before reperfusion significantly attenuated the neuroprotective effects of diazoxide and cyclosporin A. The swelling of mitochondria isolated from brain was evaluated by spectrophotometry and served as a measure of MPTP opening. In isolated mitochondria, 100 micromol/L atractyloside attenuated the decrease of mitochondrial swelling induced by 30 micromol/L diazoxide or cyclosporin A (0.5 or 1 micromol/L). Furthermore, 100 micromol/L diazoxide or 1 micromol/L cyclosporin A both attenuated the fluorescence intensity in isolated mitochondria loaded with rhod-2 acetoxymethylester, and 100 micromol/L atractyloside abolished the effects of diazoxide and cyclosporin A. These results suggest that activation of mitoKATP protects the brain against injury, and this is probably mediated by attenuating mitochondrial Ca2+ overload and thus inhibiting MPTP opening during brain ischemia and reperfusion.

Animals↗