Expression of specific white adipose tissue genes in denervation-induced skeletal muscle fatty degeneration

To determine whether cationic uncouplers of oxidative phosphorylation induce permeability transition in mitochondria, the effects of the divalent cationic sulfhydryl cross-linker copper-o-phenanthroline (Cu(OP)2) and the cyanine dye tri-S-C4(5) on rat liver mitochondria were examined. Like Ca2+, they accelerated mitochondrial respiration with succinate and induced mitochondrial swelling when inorganic phosphate (Pi) was present in the incubation medium. The acceleration of respiration and swelling were inhibited by the SH-reagent N-ethylmaleimide, and by the specific permeability transition inhibitor cyclosporin A (CsA). In addition, these cations, like Ca2+, induced release of ADP entrapped in the mitochondrial matrix space, and the morphological change of mitochondria induced by these cations was essentially the same as that induced by Ca2+. It is concluded that the uncoupling actions of Cu(OP)2 and tri-S-C4(5) are due to induction of permeability transition in the inner mitochondrial membrane.     

Evaluation of birth cohort patterns in population disease rates

1. The effects of piroxicam, a nonsteroidal anti-inflammatory drug, on rat liver mitochondria were investigated in order to obtain direct evidence about a possible uncoupling effect, as suggested by a previous work with the perfused rat liver. 2. Piroxicam increased respiration in the absence of exogenous ADP and decreased respiration in the presence of exogenous ADP, the ADP/O ratios and the respiratory control ratios. 3. The ATPase activity of intact mitochondria was increased by piroxicam. With 2,4-dinitrophenol uncoupled mitochondria, inhibition was observed. The ATPase activity of freeze-thawing disrupted mitochondria was insensitive to piroxicam. 4. Swelling driven by the oxidation of several substrates and safranine uptake induced by succinate oxidation were inhibited. 5. The results of this work represent a direct evidence that piroxicam acts as an uncoupler, thus, decreasing mitochondrial ATP generation.     

The lipophilic weak base (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421) is a potent protonophore type cationic uncoupler of oxidative phosphorylation in mitochondria

The lipophilic weak base AU-1421 acts as a simple protonophoric uncoupler of oxidative phosphorylation in rat liver mitochondria judging from the following observations. In the absence of any carrier lipophilic anions or P(i), AU-1421 stimulated the rate of state 4 respiration maximally about 7-fold at a concentration of 30 nmol/mg mitochondrial protein. At the same maximum effective concentration, it also inhibited ATP synthesis, released oligomycin-inhibited state 3 respiration, dissipated the proton motive force in the energized state, and activated latent H(+)-ATPase. AU-1421 also allowed proton conduction in both mitochondrial membranes and liposomes. These actions of AU-1421 resemble those of the typical anionic uncoupler SF6847. A marked difference between the two was, however, that ATPase activation by AU-1421 was not suppressed at higher concentrations of AU-1421, whereas ATPase activated by SF6847 was suppressed on increase of the SF6847 concentration. The finding that this simple protonophoric cation acts as an uncoupler at a micromolar concentration is significant, because all true (i.e., protonophore type) uncouplers known so far are anionic not cationic. Thus, AU-1421 is a unique uncoupler of the protonophore type.     

The thiol-specific antioxidant enzyme prevents mitochondrial permeability transition. Evidence for the participation of reactive oxygen species in this mechanism

Mitochondrial swelling and membrane protein thiol oxidation associated with mitochondrial permeability transition induced by Ca2+ and inorganic phosphate are inhibited in a dose-dependent manner either by catalase, the thiol-specific antioxidant enzyme (TSA), a protein recently demonstrated to present thiol peroxidase activity, or ebselen, a selenium-containing heterocycle which also possesses thiol peroxidase activity. This inhibition of mitochondrial permeability transition is due to the removal of mitochondrial-generated H2O2 which can easily diffuse to the extramitochondrial space. Whereas ebselen required the presence of reduced glutathione as a reductant to grant its protective effect, TSA was fully reduced by mitochondrial components. Decrease in the oxygen concentration of the reaction medium also inhibits mitochondrial permeabilization and membrane protein thiol oxidation, in a concentration-dependent manner. The results presented in this report confirm that mitochondrial permeability transition induced by Ca2+ and inorganic phosphate is reactive oxygen species-dependent. The possible importance of TSA as an intracellular antioxidant, avoiding the onset of mitochondrial permeability transition, is discussed in the text.     

Oxidative damage of mitochondria induced by 5-aminolevulinic acid: role of Ca2+ and membrane protein thiols

Reactive oxygen species (ROS) generated by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation have been shown to damage the inner membrane of isolated rat liver mitochondria by a Ca(2+)-dependent mechanism. The present work describes experiments indicating that this damage can be prevented, but not completely reversed by the additions of catalase, ADP, cyclosporin A and dithiothreitol, as judged by the extent of delta psi regeneration by the injured mitochondria. In contrast, the addition of EGTA, which removes free Ca2+ and, possibly, Fe2+ present both in the intra- and extramitochondrial compartments, causes a prompt and complete regeneration of delta psi, even after long periods of mitochondrial incubations in the presence of ALA. This reversibility suggests that protein alterations such as protein thiol cross-linkings, evidenced by SDS-polyacrylamide gel electrophoresis, are the main cause of increased membrane permeability promoted by ALA oxidation. The inhibition of protein aggregation and fast regeneration of delta psi promoted by EGTA suggest that the binding of Ca2+ to some membrane proteins plays a crucial role in the mechanism of both protein polymerization (pore assembly) and pore opening. The implication of these results with the molecular pathology of acute intermittent porphyria is also discussed.     

Respiration and ion permeability of the inner membrane in rat "sodium" liver mitochondria

Ion permeability of internal membrane and a respiration in isolated rat liver mitochondria, further related to as "sodium ones", were studied following replacement of K+ ions for Na+ ones in the mitochondrial matrix. As compared with the control ("potassium mitochondria"), state 4 respiration in the sodium mitochondria, energized by succinate, was shown to be enhanced in KCl or sucrose media. Oxygen consumption rates in the sodium mitochondria, being in state 3 or stimulated by 2,4-dinitrophenol, were lower than rates for the control mitochondria. This effect was much pronounced in the sucrose medium. The coefficients, characterizing the distribution of 137Cs between mitochondria and the medium, were lower for the sodium mitochondria than for the control in the presence of 2.5 mM succinate and 10(-8) M valinomycin. In comparison with the control, a more extensive swelling for the sodium mitochondria was found, first, in the medium containing 25 mM K-acetate and 100 mM sucrose for succinate-energized mitochondria, and second, in the medium containing 125 mM NH4NO3 without mitochondrial energization. Changes disclosed in respiration, swelling and coefficients of 137Cs distribution for the sodium mitochondria are supposed to be caused by non-uniform effects of Na+ and K+ ions on the water structure of mitochondrial matrix, ion permeability of internal membrane, and the activity in oxidative phosphorylation enzymes.     

Mitochondria uncoupling by a long chain fatty acyl analogue

Mitochondria uncoupling by fatty acids in vivo is still questionable, being confounded by their dual role as substrates for oxidation and as putative genuine uncouplers of oxidative phosphorylation. To dissociate between substrate and the uncoupling activity of fatty acids in oxidative phosphorylation, the uncoupling effect was studied here using a nonmetabolizable long chain fatty acyl analogue. beta,beta'-Methyl-substituted hexadecane alpha,omega-dioic acid (MEDICA 16) is reported here to induce in freshly isolated liver cells a saturable oligomycin-insensitive decrease in mitochondrial proton motive force with a concomitant increase in cellular respiration. Similarly, MEDICA 16 induced a saturable decrease in membrane potential, proton gradient, and proton motive force in isolated liver and heart mitochondria accompanied by an increase in mitochondrial respiration. Uncoupling by MEDICA 16 in isolated mitochondria was partially suppressed by added atractyloside. Hence, fatty acids may act as genuine uncouplers of cellular oxidative phosphorylation by interacting with specific mitochondrial proteins, including the adenine nucleotide translocase.     

Membrane potential generation coupled to oxidation of external NADH in liver mitochondria

Oxidation of added NADH by rat liver mitochondria has been studied. It is found that exogenous NADH, when oxidized by rat liver mitochondria in sucrose hypotonic medium supplemented with Mg2+ and EGTA, generates a membrane potential (delta psi) even in the absence of added cytochrome c. ADP and phosphate decrease delta psi, the effect being reversed by oligomycin. Rotenone and myxothiazol do not inhibit delta psi generated by oxidation of exogenous NADH. Added cytochrome c increases the rate of the exogenous NADH oxidation and coupled delta psi formation. In sucrose isotonic medium, or in hypotonic medium without Mg2+, exogenous NADH fails to stimulate respiration and to form a membrane potential. In the presence of Mg2+, exogenous NADH appears to be effective in delta psi generation in isotonic sucrose medium if mitochondria were treated with digitonin. In isotonic KCl without Mg2+, oxidation of exogenous NADH is coupled to the delta psi formation and MgCl2 addition before mitochondria prevents this effect. In hypotonic (but not in isotonic) sucrose medium, Mg2+ makes a portion of the cytochrome c pool reducible by exogenous NADH or ascorbate. It is assumed that (i) hypotonic treatment or digitonin causes disruption of the outer mitochondrial membrane, and, as a consequence, desorption of the membrane-bound cytochrome c in a Mg2+-dependent fashion; (ii) incubation in isotonic KCI without Mg2+ results in swelling of mitochondrial matrix, disruption of the outer membrane and cytochrome c desorption whereas Mg2+ lowers the K+ permeability of the inner membrane and, hence, prevents swelling; (iii) desorbed cytochrome c is reduced by added NADH via NADH-cytochrome b5 reductase and cytochrome b5 or by ascorbate and is oxidized by cytochrome oxidase. The role of desorbed cytochrome c in oxidation of superoxide and cytoplasmic NADH as well as possible relations of these events to apoptosis are discussed.     

Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore. I. Evidence for two separate Me2+ binding sites with opposing effects on the pore open probability

This paper reports an investigation on the regulation of the mitochondrial cyclosporin A-sensitive permeability transition pore (MTP). Energized, coupled rat liver mitochondria incubated in sucrose medium in the presence of phosphate maintain a high proton electrochemical gradient (delta microH) and a low permeability to solutes. Addition of a small (10-20 microM) Ca2+ pulse leads to a transient membrane depolarization. After Ca2+ accumulation, a high delta microH is recovered, and mitochondria remain coupled indefinitely. Yet, addition of fully uncoupling concentrations of carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) brings about MTP opening within seconds. This finding confirms that MTP opening is the consequence rather than the cause of membrane depolarization, and allowed us to study the operation of the MTP in a synchronized population of mitochondria, since pore opening can be triggered by the addition of uncoupler under a series of experimental conditions. We find that three regulatory sites can be defined: (i) an internal Me2+ binding site: when this site is occupied by Ca2+, the pore "open" probability increases, while other Me2+ ions (Sr2+, Mn2+) have an inhibitory effect; (ii) an external Me2+ binding site: when this site is occupied by Me2+ ions, including Ca2+, the pore open probability decreases; (iii) an independent cyclosporin A binding site: when this site is occupied by cyclosporin A the pore open probability decreases. We show that at variance from the case of cyclosporin A, MTP inhibition by the phospholipase A2 inhibitors nupercaine and trifluoperazine is Ca(2+)-competitive and is presumably related to interference by these drugs with Ca2+ binding to the internal regulatory site.     

Mitochondrial permeability transition is altered in early stages of carcinogenesis of 2-acetylaminofluorene

The tumour promoting properties of carcinogenic 2-acetylaminofluorene (AAF) in rat liver are essentially unknown. We proposed that mitochondria are a target for the cytotoxic effects of 2-nitrosofluorene (NOF), a metabolite of AAF, since NOF induces a redox-cycle at complex I and complex III of the respiratory chain, and impairs respiration and oxidative phosphorylation. We now demonstrate that NOF is a potent inducer of the mitochondrial permeability transition pore (PTP) in isolated mitochondria. In the presence of Ca2+, NOF induced rapid swelling of mitochondria in a dose-dependent manner and depolarized the mitochondrial membrane. Permeability transition as well as depolarization were abolished completely by pre-incubation with the PTP inhibitor cyclosporin A. To study whether the PTP is involved in in vivo toxicity, rats were fed a diet containing AAF (0.04%) for 2 weeks. After isolation of mitochondria, permeability transition was induced by high Ca2+ concentrations (150-400 microM) or phosphate plus Ca2+. Swelling was determined as maximal rate of absorption decrease at 540 nm (delta A/delta t). Surprisingly, delta A/delta t-values of mitochondria from AAF-fed rats were significantly lower (16.3 +/- 4.8 x 10(3)/min) than of mitochondria from control animals (32.7 +/- 4.1 x 10(3)/min; P < 0.02). In the presence of phosphate (15 mM), delta A/delta t-values of mitochondria from AAF-fed rats were even lower (10% of control). Moreover, the membrane potential which was dissipated rapidly by the PTP-inducer NOF (30 microM) at a Ca2+ concentration of 80 microM in mitochondria from control animals, remained constant in mitochondria of AAF-treated rats. We therefore propose that the regulation of the PTP is altered on chronic AAF-feeding. The increased resistance of mitochondria against permeability transition may alter the threshold for apoptosis and thus suppress apoptosis. We also discuss the role of epigenetic modifications in early stages of carcinogenesis.     

Modulation of the mitochondrial permeability transition by nitric oxide

The influence of nitric oxide on mitochondrial permeability transition (MPT) phenomenon was studied. NO was generated by photolysis of S-nitroso-N-acetylcysteine, AcCys(NO), with green light (lambda = 550 nm). Two distinct effects of nitric oxide on rat liver mitochondria were identified. First, NO accelerated an onset of swelling in Ca2(+)-loaded mitochondria in a cyclosporin-A-sensitive manner acting as an inducer of permeability transition. This was, apparently, a result of irreversible alteration of mitochondrial function accompanying the inhibition of respiratory chain in the presence of calcium. Formation of ESR-visible iron-sulfur dinitrosyl complexes (g = 2.041) could also contribute to the irreversible changes resulting in MPT induction. Second, NO changed significantly the response of mitochondria to Ca2+/phosphate-induced MPT, acting as a regulator of permeability transition. In this case the action of nitric oxide led to division of the mitochondria into two subpopulations: one which underwent the rapid permeability transition and another in which the MPT was inhibited. The effect of NO on Ca2+/Pi-induced MPT was transient and resulted from reversible inhibition of cytochrome oxidase followed by the changes in transmembrane potential and Ca2+ distribution. The characteristic time of duration of these NO modulated effects depended on nitric oxide as well as on oxygen concentrations. With increasing NO at fixed oxygen concentrations, this time levelled off to reach a maximum value which was inversely related to the oxygen concentration. It is concluded that under physiological condition the duration of reversible NO effects on mitochondrial function could be determined by oxygen concentration.     

Entacapone, a novel catechol-O-methyltransferase inhibitor for Parkinson's disease, does not impair mitochondrial energy production

Entacapone, a novel mainly peripherally acting catechol-O-methyltransferase inhibitor used in the treatment of Parkinson's disease, was evaluated for its possible uncoupling activity in cell culture, in rat liver mitochondria, and in isolated guinea-pig heart. Entacapone did not stimulate respiration in the L1210 murine T cell lymphoma cell line at the concentrations studied (5-40 microM). Furthermore, entacapone neither increased mitochondrial respiration nor impaired cardiac function at pharmacologically relevant concentrations (< 10 microM). In fact, the threshold concentration for increased mitochondrial oxygen consumption was 20 microM and half-maximal stimulation of respiration was not detected until 58 microM. Surprisingly, tolcapone, another catechol-O-methyltransferase inhibitor, which acts both peripherally and centrally, stimulated respiration in L1210 cells at the lowest concentration studied (5 microM). In addition, 1 microM tolcapone increased mitochondrial respiration, indicating that it caused uncoupling at a much lower concentration than that of 2,4-dinitrophenol, a well-known uncoupler of oxidative phosphorylation. Tolcapone also impaired the mechanical function and oxygen consumption of the isolated guinea-pig heart at 1 microM. These results show that peripherally acting entacapone, unlike the brain-penetrating tolcapone, is a safe catechol-O-methyltransferase inhibitor for the treatment of Parkinson's disease, since it does not interfere with mitochondrial energy metabolism at pharmacologically effective concentrations.     

Bax directly induces release of cytochrome c from isolated mitochondria

Bax is a pro-apoptotic member of the Bcl-2 protein family that resides in the outer mitochondrial membrane. It is controversial whether Bax promotes cell death directly through its putative function as a channel protein versus indirectly by inhibiting cellular regulators of the cell death proteases (caspases). We show here that addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria can induce cytochrome c (Cyt c) release, whereas a peptide representing the Bax BH3 domain was inactive. When placed into purified cytosol, neither mitochondria nor Bax individually induced proteolytic processing and activation of caspases. In contrast, the combination of Bax and mitochondria triggered release of Cyt c from mitochondria and induced caspase activation in cytosols. Supernatants from Bax-treated mitochondria also induced caspase processing and activation. Recombinant Bcl-XL protein abrogated Bax-induced release of Cyt c from isolated mitochondria and prevented caspase activation. In contrast, the broad-specificity caspase inhibitor benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(0-methyl)- fluoromethylketone (zVAD-fmk) and the caspase-inhibiting protein X-IAP had no effect on Bax-induced release of Cyt c from mitochondria in vitro but prevented the subsequent activation of caspases in cytosolic extracts. Unlike Ca2+, a classical inducer of mitochondrial permeability transition, Bax did not induce swelling of mitochondria in vitro. Because the organellar swelling caused by permeability transition causes outer membrane rupture, the findings, therefore, dissociate these two events, implying that Bax uses an alternative mechanism for triggering release of Cyt c from mitochondria.     

Cyclosporin A-sensitive release of Ca2+ from mitochondria in intact thymocytes

Release of Ca2+ from mitochondria into cytosol in intact thymocytes was studied using the fluorescent dye Fluo-3. It was shown that the release of Ca2+ induced by the dithiol cross-linking agent phenylarsine oxide or by uncoupler was strongly inhibited by cyclosporin A, a specific inhibitor of the permeability transition pore (PTP) in mitochondria. Oxidative stress sensitized the pore so even partial uncoupling caused rapid cyclosporin A-sensitive release of Ca2+. The experiments on digitonin-permeabilized cells confirmed that uncoupling induced opening of the PTP, which forms the major pathway for rapid release of Ca2+ from thymocyte mitochondria.     

t-Butylhydroperoxide and gliotoxin stimulate Ca2+ release from rat skeletal muscle mitochondria

Rat liver mitochondria have a specific Ca2+ release pathway which operates when NAD+ is hydrolysed to nicotinamide and ADPribose. NAD+ hydrolysis is Ca(2+)-dependent and inhibited by cyclosporine A (CSA). Mitochondrial Ca2+ release can be activated by the prooxidant t-butylhydroperoxide (tbh) or by gliotoxin (GT), a fungal metabolite of the epipolythiodioxopiperazine group. Tbh oxidizes NADH to NAD+ through an enzyme cascade consisting of glutathione peroxidase, glutathione reductase, and the energy linked transhydrogenase, whereas GT oxidizes some vicinal thiols to the disulfide form, a prerequisite for NAD+ hydrolysis. We report now that rat skeletal muscle mitochondria also contain a specific Ca2+ release pathway activated by both tbh and GT. Ca2+ release increases with the mitochondrial Ca2+ load, is completely inhibited in the presence of CSA, and is paralleled by pyridine nucleotide oxidation. In the presence of tbh and GT, mitochondria do not lose their membrane potential and do not swell, provided continuous release and re-uptake of Ca2+ ('Ca2+ cycling') is prevented. These data support the notion that both tbh- and GT-induced Ca2+ release are not the consequence of an unspecific increase of the inner membrane permeability ('pore' formation). Tbh induces Ca2+ release from rat skeletal muscle less efficiently than from liver mitochondria indicating that the coupling between tbh and NADH oxidation is much weaker in skeletal muscle mitochondria. This conclusion is corroborated by a much lower glutathione peroxidase activity in skeletal muscle than in liver mitochondria. The prooxidant-dependent pathway promotes, under drastic conditions (high mitochondrial Ca2+ loads and high tbh concentrations), Ca2+ release to about the same extent and rate as the Na+/Ca2+ exchanger. This renders the prooxidant-dependent pathway relevant in the pathophysiology of mitochondrial myopathies where its activation by an increased generation of reactive oxygen species probably results in excessive Ca2+ cycling and damage to mitochondria.     

Hemostatic risk factors of coronary artery disease in the Chinese

Induction of the mitochondrial permeability transition in vitro is well-characterized and widely implicated in the mechanism of oxidant-induced cell death. Despite an abundance of in vitro evidence, implication of mitochondrial dysfunction in the mechanism of chemical toxicity in vivo awaits demonstration of the induction of the mitochondrial permeability transition in tissues from intoxicated animals. Menadione (2-methyl-1,4-naphthoquinone), an agent known to induce the permeability transition in isolated liver mitochondrial in vitro, was administered as a single bolus to adult male rats, and hepatic mitochondria were isolated 24 h later. Mitochondria from menadione-treated rats exhibited an increased sensitivity to calcium-induced inhibition of state 3 respiration and loss of respiratory control, as well as a greater sensitivity to calcium-induced calcium release that was inhibited by cyclosporine A. Associated with this was the depolarization of membrane potential and swelling of mitochondria from menadione-treated animals, but not control animals. Both the calcium-dependent depolarization and swelling of mitochondria from menadione-treated rats were inhibited by adding either cyclosporine A or ruthenium red. The results are consistent with the induction of the mitochondrial permeability transition and provide the first evidence for the manifestation of an increased sensitivity to this response as a result of chemical exposure in vivo.     

Oxidative metabolism in rat hepatocytes and mitochondria during sepsis

We hypothesized that cellular oxygen consumption is abnormal during sepsis as a result of increased oxidative stress and selective mitochondrial damage. In a rat model of sepsis (cecal ligation and puncture), we studied the respiratory characteristics of isolated hepatocytes and liver mitochondria 16 h after onset of septic injury. Endogenous respiration by isolated cells was decreased during sepsis, while cyanide-resistant (nonmitochondrial) respiration was unaffected. Maximal oxygen consumption in ADP-supplemented, permeabilized hepatocytes was decreased with succinate as the substrate, but not with malate + glutamate or TMPD + ascorbate. In contrast, maximum oxygen consumption (State 3) by isolated liver mitochondria increased up to 35% during sepsis using either succinate or malate + glutamate as substrate. The electrophoretic features and mobility of nondenatured mitochondrial respiratory complexes were similar in control and septic hepatocytes, with the exception of decreased Complex V protein in sepsis. Structural evaluation of mitochondria in fixed liver slices by electron microscopy showed mitochondrial swelling in most of the septic animals. Measurements of oxidative stress during sepsis suggested an increase in hydroxylation of salicylate by isolated hepatocytes, and mitochondrial protein carbonyl content was increased significantly. Induction of iNOS in hepatocytes after 16 h of sepsis was variable, and little release of the oxidation products of NO. was detected. These findings are interpreted to mean that hepatocytes contain a mixed population of injured and hyperfunctional mitochondria during sepsis.     

Increased production of reactive oxygen species by rat liver mitochondria after chronic ethanol treatment

Rat liver microsomes and, to a lesser extent, nuclei were previously shown to produce reactive oxygen species at elevated rates after chronic ethanol treatment. The ability of intact rat liver mitochondria to interact with iron and either NADH or NADPH, and the effects of ethanol treatment, on production of reactive oxygen intermediates was determined. In the presence of ferric-ATP, NADH or NADPH catalyzed mitochondrial lipid peroxidation. Rates were elevated two- to threefold with mitochondria from ethanol-fed rats with both reductants. Mitochondrial lipid peroxidation was insensitive to superoxide dismutase, catalase, or hydroxyl radical scavengers but was sensitive to GSH and anti-oxidants such as trolox. Mitochondrial generation of hydroxyl radical-like species (assayed by oxidation of chemical scavengers) was increased after chronic ethanol treatment, as was H2O2 production. Modifiers of mitochondrial metabolism such as rotenone, cyanide, or an uncoupling agent, had no effect on mitochondrial production of reactive oxygen intermediates. The membrane-impermeable thiol reagent, p-chloromercuribenzoate, was complete inhibitory with both mitochondrial preparations. The activity of the rotenone-insensitive NADH-cytochrome c reductase, an enzyme of the outer mitochondrial membrane, was increased 40 to 60% by the ethanol treatment. These results suggest that NADH acting via the outer membrane NADH reductase can catalyze an iron-dependent production of oxygen radicals by rat liver mitochondria. The outer mitochondrial membrane fraction, prepared by digitonin fractionation, displayed increased rotenone-insensitive NADH-cytochrome c reductase activity after ethanol treatment and was more reactive in catalyzing scission of pBR322 DNA from the supercoiled form to the open circular forms. Rates of oxygen radical production by mitochondria and the extent of increase produced by chronic ethanol treatment are similar to those previously found with microsomes when NADH is the cofactor. Oxidation of ethanol by alcohol dehydrogenase generates NADH, and NADH-dependent production of reactive oxygen species by various organelles is increased after chronic ethanol treatment. These acute metabolic interactions coupled to induction by chronic ethanol treatment may play an important role in the development of a state of oxidative stress in the liver by ethanol.     

Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria

1. The direct effects of diazoxide on mitochondrial membrane potential, Ca2+ transport, oxygen consumption and ATP generation were investigated in mouse pancreatic B-cells and rat liver mitochondria. 2. Diazoxide, at concentrations commonly used to open adenosine 5'-triphosphate (ATP)-dependent K+-channels (K(ATP) channels) in pancreatic B-cells (100 to 1000 microM), decreased mitochondrial membrane potential in mouse intact perifused B-cells, as evidenced by an increase of rhodamine 123 fluorescence. This reversible decrease of membrane potential occurred at non-stimulating (5 mM) and stimulating (20 mM) glucose concentrations. 3. A decrease of mitochondrial membrane potential in perifused B-cells was also caused by pinacidil, but no effect could be seen with levcromakalim (500 microM each). 4. Measurements by a tetraphenylphosphonium-sensitive electrode of the membrane potential of rat isolated liver mitochondria confirmed that diazoxide decreased mitochondrial membrane potential by a direct action. Pretreatment with glibenclamide (2 microM) did not antagonize the effects of diazoxide. 5. In Fura 2-loaded B-cells perifused with the Ca2+ channel blocker, D 600, a moderate, reversible increase of intracellular Ca2+ concentration could be seen in response to 500 microM diazoxide. This intracellular Ca2+ mobilization may be due to mitochondrial Ca2+ release, since the reduction of membrane potential of isolated liver mitochondria by diazoxide was accompanied by an accelerated release of Ca2+ stored in the mitochondria. 6. In the presence of 500 microM diazoxide, ATP content of pancreatic islets incubated in 20 mM glucose for 30 min was significantly decreased by 29%. However, insulin secretion from mouse perifused islets induced by 40 mM K+ in the presence of 10 mM glucose was not inhibited by 500 microM diazoxide, suggesting that the energy-dependent processes of insulin secretion distal to Ca2+ influx were not affected by diazoxide at this concentration. 7. The effects of diazoxide on oxygen consumption and ATP production of liver mitochondria varied depending on the respiratory substrates (5 mM succinate, 10 mM alpha-ketoisocaproic acid, 2 mM tetramethyl phenylenediamine plus 5 mM ascorbic acid), indicating an inhibition of respiratory chain complex II. Pinacidil, but not levcromakalim, inhibited alpha-ketoisocaproic acid-fuelled ATP production. 8. In conclusion, diazoxide directly affects mitochondrial energy metabolism, which may be of relevance for stimulus-secretion coupling in pancreatic B-cells.     

Mechanism of uptake of technetium-tetrofosmin. II: Uptake into isolated adult rat heart mitochondria

BACKGROUND: 99mTc-labeled tetrofosmin is a new myocardial imaging agent that gives stable heart uptake. However, little is known about the mechanism of uptake in heart tissue. The aim of this study was to assess the factors responsible for the uptake and retention of 99mTc-labeled tetrofosmin in isolated heart mitochondria. METHODS AND RESULTS: Mitochondria were isolated from adult rat heart tissue with competent metabolic function (i.e., respiratory control ratio of 3 and adenosine diphosphate/oxygen ratio of 2) for succinate oxidation. Intramitochondrial volume measured by the distribution of 3H-water and 14C-sucrose was 1.16 +/- 0.23 microliters/mg protein (mean +/- SD). In the isolated mitochondria, uptake could be demonstrated within 30 seconds of addition of oxidative substrate, but adenosine triphosphate alone did not stimulate marked uptake. Uptake was proportional to the amount of mitochondrial protein over a range of 0.2 to 3 mg protein but independent of Tc-labeled tetrofosmin concentration over a range of 0.4 to 200 pmol/L (0.1 to 50 microCi/ml). The presence of Tc-labeled tetrofosmin had no effect on the oxidative capability of the mitochondria. Use of the mitochondrial uncoupler 2,4-dinitrophenol caused release of 92% of radioactivity. Addition of Ca2+ to the mitochondria to partially depolarize the membrane resulted in partial release of activity. Application of the Nernst equation to the uptake data gave rise to a value of -163 mV for the mitochondrial membrane potential. CONCLUSION: It was concluded that the accumulation of 99mTc-labeled tetrofosmin by the mitochondria is related to their ability to transduce metabolic energy into electronegative membrane potential.