The interaction of antioxidants and structurally related compounds with mitochondrial oxidative phosphorylation.

The antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), interact with mitochondrial oxidative phosphorylation in two ways. They uncouple phosphorylation from oxidation by making the mitochondrial inner membrane more permeable to protons. They also inhibit respiration by a direct interaction with the electron transport chain. Here we separated out these two properties of BHA and BHT by determining their effects on respiration in coupled and uncoupled mitochondria. Similar experiments were carried out with compounds structurally related to BHA and BHT. Most of these compounds had uncoupling and inhibitory properties essentially similar to BHA and BHT. In contrast, the dimer of BHA had no inhibitory effects on uncoupled respiration and little uncoupling activity. The implications of these results for the interactions of BHA and BHT with mitochondrial oxidative phosphorylation and the design of antioxidants are discussed.     

Interactions of acetaldehyde, ethyl alcohol and oxybarbiturates affecting mitochondrial functions

The effects of combinations of acetaldehyde, ethyl alcohol and an oxybarbiturate on the rate of oxygen consumption, the ADP/O ratio and energy-dependent swelling by rat liver mitochondria have been investigated. The effects of similar combinations on the rate of oxygen uptake by submitochondrial preparations, oxidizing several different substrates, have also been studied. Acetaldehyde at millimolar levels increased the rate of oxygen consumption by rat liver mitochondria which had been preincubated with ADP, inhibited NAD⁺-linked oxidations by mitochondrial and submitochondrial preparations, and increased the rate of the energy-dependent swelling of rat liver mitochondria with suceinate as substrate. Combinations of acetaldehyde, ethyl alcohol and an oxybarbiturate interacted to reduce the rate of oxygen consumption by rat liver mitochondria and the energy-dependent swelling of rat liver mitochondria when dl-3-hydroxybutyrate was the substrate. Ethyl alcohol was not involved in these interactions. The effects of all combinations tested could be ascribed to interactions) between acetaldehyde and an oxybarbiturate or to activities of acetaldehyde or the oxybarbiturate only. In the case of state 3 respiration and uncoupled mitochondrial respiration, acetaldehyde and the tested oxybarbiturates behaved as additive inhibitors. All of the results are consistent with the view that the NADH-ubiquinone segment (Complex 1) of the mitochondrial electron transport chain is the site of an acetaldehyde-oxybarbiturate interaction to inhibit electron transport.     

Halothane impairs the bioenergetic functions of isolated rat liver mitochondria

The effect of halothane, a potent and popular volatile anesthetic, on isolated rat liver mitochondria was examined. Halothane inhibited state 3 and dinitrophenol-induced uncoupled respiration with NAD(+)-linked substrates, but not with FAD-linked substrates, and did not affect the oxidation-reduction state of mitochondrial cytochromes. Moreover, halothane increased state 4 respiration and ATPase activity and decreased the extra-mitochrondrial pH change coupled to ATP synthesis. These results indicate that halothane impairs mitochondrial ATP production by interfering with both the electron transport from NAD+ to FAD and the coupling of oxidative phosphorylation. Halothane only slightly affected the membrane potential, which is commonly dissipated by typical classical uncouplers. Moreover, halothane inhibited both ATP-driven and respiration-driven Ca2+ accumulation in mitochondria and stimulated Ca2+ release from mitochondrial stores at concentrations higher than those at which it inhibited ATP production. These findings indicate that the uncoupling action of halothane is not classical. During halothane anesthesia, these mitochondrial abnormalities may contribute to hepatocyte dysfunctions.     

Cytochrome aa3 depletion is the cause of the deficient mitochondrial respiration induced by chronic valproate administration.

Liver mitochondria from rats fed 1% (w/w) valproic acid for 75 days displayed an approximate 30% decrease in coupled respiration rates with substrates entering the respiratory chain at complexes I and II. Uncoupling the respiration from proton-pumping, or measuring the respiration without complex IV removed this inhibition. The treatment induced a loss of activity of cytochrome oxidase consistent with a decrease in the mitochondrial content of cytochrome aa3. The inhibition induced by long lasting administration of valproate is apparently located at the site of the proton-pumping activity of complex IV. Furthermore, the capacity of electron transport through complex IV, being far in excess of that required for normal functioning in coupled mitochondria, seems to be controlled by the coupling to proton-pumping in which cytochrome aa3 appears to play a major role.     

Mitochondrial complex I dysfunction induced by cocaine and cocaine plus morphine in brain and liver mitochondria

Mitochondrial function and energy metabolism are affected in brains of human cocaine abusers. Cocaine is known to induce mitochondrial dysfunction in cardiac and hepatic tissues, but its effects on brain bioenergetics are less documented. Furthermore, the combination of cocaine and opioids (speedball) was also shown to induce mitochondrial dysfunction. In this work, we compared the effects of cocaine and/or morphine on the bioenergetics of isolated brain and liver mitochondria, to understand their specific effects in each tissue. Upon energization with complex I substrates, cocaine decreased state-3 respiration in brain (but not in liver) mitochondria and decreased uncoupled respiration and mitochondrial potential in both tissues, through a direct effect on complex I. Morphine presented only slight effects on brain and liver mitochondria, and the combination cocaine+morphine had similar effects to cocaine alone, except for a greater decrease in state-3 respiration. Brain and liver mitochondrial respirations were differentially affected, and liver mitochondria were more prone to proton leak caused by the drugs or their combination. This was possibly related with a different dependence on complex I in mitochondrial populations from these tissues. In summary, cocaine and cocaine+morphine induce mitochondrial complex I dysfunction in isolated brain and liver mitochondria, with specific effects in each tissue.     

Structural determinants of fluorochemical-induced mitochondrial dysfunction.

Perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) are thought to induce peroxisome proliferation and interfere with mitochondrial metabolic pathways. Direct measurements revealed that PFOA and the unsubstituted sulfonamide of perfluorooctane (FOSA) uncouple mitochondrial respiration by increasing proton conductance. The purpose of this investigation was to characterize structural determinants responsible for the mitochondrial uncoupling effect of several structurally related fluorochemicals. Included in the study were PFOA, PFOS, FOSA, the N-acetate of FOSA (perfluorooctanesulfonamidoacetate, FOSAA), N-ethylperfluorooctanesulfonamide (N-EtFOSA), and the N-ethyl alcohol [2-(N-ethylperfluorooctanesulfonamido)ethyl alcohol, N-EtFOSE] and N-acetic acid (N-ethylperfluorooctanesulfonamidoacetate, N-EtFOSAA) of N-EtFOSA. Each test compound was dissolved in ethanol and added directly to an incubation medium containing substrate-energized rat liver mitochondria. Mitochondrial respiration and membrane potential were measured concurrently using an oxygen electrode and a TPP+ -selective electrode, respectively. All of the compounds tested, at sufficiently high concentrations, had the capacity to interfere with mitochondrial respiration, albeit via different mechanisms and with varying potencies. At sufficiently high concentrations, the free acids PFOA and PFOS caused a slight increase in the intrinsic proton leak of the mitochondrial inner membrane, which resembled a surfactant-like change in membrane fluidity. Similar effects were observed with the sulfonamide N-EtFOSE. Another fully substituted sulfonamide, N-EtFOSAA, at high concentrations caused inhibition of respiration, the release of cytochrome c, and high-amplitude swelling of mitochondria. The swelling was prevented by cyclosporin A or by EGTA, indicating that this compound induced the mitochondrial permeability transition. The unsubstituted and mono-substituted amides FOSA, N-EtFOSA, and FOSAA all exerted a strong uncoupling effect on mitochondria resembling that of protonophoric uncouplers. Among these compounds, FOSA was a very potent uncoupler of oxidative phosphorylation, with an IC50 of approximately 1 microM. These data suggest that the protonated nitrogen atom with a favorable pKa is essential for the uncoupling action of perfluorooctane sulfonamides in mitochondria, which may be critical to the mechanism by which these compounds interfere with mitochondrial metabolism to induce peroxisome proliferation in vivo.     

Influence of epinephrine on alcohol dehydrogenase activity in rat hepatocyte culture

The effects of epinephrine on alcohol dehydrogenase activity and on rates of ethanol elimination were determined in rat hepatocyte culture. Continuous exposure of the hepatocytes to epinephrine (10 microM) in combination with dexamethasone (0.1 microM) enhanced alcohol dehydrogenase activity on days 4-7 of culture, whereas neither hormone alone had an effect. The increased alcohol dehydrogenase activity was associated with an increased rate of ethanol elimination. Acute addition of 10 microM epinephrine to hepatocytes maintained in culture with 0.1 microM dexamethasone did not change alcohol dehydrogenase activity, but resulted in an immediate marked, but transitory, increase in ethanol elimination within the first 5 min after the addition of the hormone. Prazosin, an alpha 1-adrenergic blocker, and antimycin, an inhibitor of mitochondrial respiration, were powerful inhibitors of the transient increase in ethanol elimination, whereas 4-methylpyrazole was only partially inhibitory. These observations indicate that epinephrine has a chronic effect in increasing alcohol dehydrogenase activity and ethanol elimination and, also, an acute transient effect of increasing ethanol elimination which is not limited by alcohol dehydrogenase activity.     

Effects of guanethidine on electron transport and proton movements in rat heart, brain and liver mitochondria

Guanethidine at 5-25 mM concentrations was found to induce up to 79% inhibition of ADP-stimulated (state III) oxygen consumption in isolated rat heart, brain or liver mitochondria, when the added substrate was glutamate or succinate, but the inhibition was considerably lower (24% or less) when respiration was supported by ascorbate plus tetramethylphenylenediamine (TMPD). Comparable results were seen regarding ADP-stimulated proton uptake, where even greater inhibition (up to 94% with glutamate or succinate, but not ascorbate plus TMPD) was found. Similar but somewhat less marked effects were also seen in resting (state IV) respiration and on the acceptor control ratio (state III/state IV respiration). 2,4-Dinitrophenol was unable to relieve guanethidine-induced inhibition of electron transport. These results indicate that guanethidine inhibits primarily mitochondrial electron transport itself, and that the site where such inhibition is more marked is located in the span between ubiquinone and cytochrome c of the respiratory chain. It is, therefore, suggested that active guanethidine uptake by noradrenergic neurons can lead to a high drug concentration in their cytoplasm and hence to mitochondrial alterations that can contribute to the pharmacological effect of this drug. Our results demonstrate the interaction between guanethidine and the electron transport chain of mitochondria derived from different tissues and, therefore, support this hypothesis.     

Effect of cocaine on mitochondrial electron transport chain evaluated in primary cultures of neonatal rat myocardial cells and in isolated mitochondrial preparations

Cardiotoxicity is commonly associated with cocaine abuse. Previous studies have indicated that cocaine alters myocardial mitochondrial function. This study was undertaken to investigate the effect of cocaine on activity of the mitochondrial electron transport chain in cultures of neonatal rat cardiomyocytes and in isolated myocardial mitochondria. Cocaine concentrations (10(-5) to 10(-3) M) were used, and these concentrations have been reported in human cocaine users and are within a similar range of cocaine concentrations used in studies in vivo. After 24 hr treatment of cocaine, there was a slight increase in lactate dehydrogenase leakage in the cells treated with cocaine (10(-3) M). Reduction of tetrazolium compounds, neotetrazolium chloride (NTC) and triphenyltetrazolium chloride (TTC) was analyzed in intact cells to assess activity of the mitochondrial electron transport chain. Cocaine (10(-3) M) did not significantly change TTC and NTC reduction. In isolated mitochondria, cocaine (10(-3) M) significantly inhibited glutamate/malate-mediated respiration. These data suggest that cocaine at high concentrations may inhibit complex I of the mitochondrial electron transport chain of myocardial cells.     

糖尿病小鼠肝线粒体氧化损伤及其机制

目的:糖尿病是威胁人类健康的主要疾病之一,为了证明糖尿病的发生发展与氧化应激密切相关,从线粒体呼吸链角度探讨其机制。方法:用四氧嘧啶(Alloxan)建立实验性糖尿病小鼠模型,利用比色法分别从整体、组织和线粒体水平测定抗氧化酶活性,用铁氰化钾脉冲法测定线粒体呼吸活链II+III电子传递与质子泵出偶联(H+/2e)。结果:糖尿病小鼠GSH、GSH-Px分别比对照组显著降低(P<0.001),血清和肝组织、线粒体GSH-Px/MDA比值分别降低59.89%、40.69%和59.65%;线粒体呼吸链II+III总H+/2e显著降低24.76%(P<0.05),净H+/2e显著降低32.31%(P<0.05)。结论:实验性糖尿病小鼠肝功能损伤是氧化性损伤,氧化应激又是该损伤的结果,其原因与质子漏增加,电子传递与质子泵出脱偶联有关,导致自由基增加,无效耗氧增加,线粒体受损。     

Effect of ochratoxin A on rat liver mitochondrial respiration and oxidative phosphorylation

The in vitro effects of ochratoxin A on the membrane structure and bioenergetic functions of rat liver mitochondria were studied. It was found that when the toxin was added to the assay medium the respiratory control of the isolated mitochondria was decreased as the concentration of the toxin increased. The mitochondrial respiration was gradually uncoupled by the toxin when its concentration was raised above 1.2 X 10(-6) M, and became fully uncoupled at 6.2 X 10(-4) M. The oxidative phosphorylation was not damaged until the toxin concentration was higher than 9.3 X 10(-5) M. On the other hand, ochratoxin A inhibited the electron transfer functions of the mitochondria. At the concentration above 1.0 X 10(-4) M, ochratoxin A inhibited the succinate dehydrogenase, succinate-cytochrome c reductase, and succinate oxidase activities of the respiratory chain. Fifty percent of succinate-cytochrome c reductase and succinate oxidase activity was lost in the presence of 8.0 X 10(-4) and 6.2 X 10(-4) M ochratoxin A, respectively. The inhibition kinetic studies revealed that ochratoxin A is an uncompetitive inhibitor of both succinate-cytochrome c reductase and succinate dehydrogenase, and the inhibition constants for the 2 enzyme activities were estimated to be 4.4 X 10(-4) and 2.2 X 10(-4) M, respectively. However, the toxin did not inhibit either cytochrome oxidase or NADH dehydrogenase activity of the mitochondrial respiratory chain. It is thus concluded that ochratoxin A exerts its effect on the mitochondrial respiration and oxidative phosphorylation through the impairment of the mitochondrial membrane and inhibition of the succinate-supported electron transfer activities of the respiratory chain.     

Effect of local anesthetic ropivacaine on isolated rat liver mitochondria.

Ropivacaine is a new long-acting aminoamide local anesthetic with a reduced systemic and cardiac toxicity. Since the latter seems to be related, at least partially, to an interference with mitochondrial energy transduction, the effect of ropivacaine on the metabolism of rat liver mitochondria was studied. Ropivacaine alone exhibited little effect on mitochondrial metabolism, whereas effects were strongly enhanced by tetraphenylboron (TPB-) anion. At low drug concentrations, state 4 respiration was stimulated and mitochondrial membrane potential collapsed. At higher concentrations, state 4 and uncoupled respiration were inhibited by impairment of electron transfer from NAD- and flavine adenine dinucleotide-linked substrates to the respiratory chain. The fact that TPB- increased drug effects indicated that stimulation of respiration was due to dissipation of the electrochemical proton gradient caused by its electrophoretic uptake, although a classical uncoupling mechanism cannot be excluded. The mechanism for the lower toxicity of ropivacaine in vivo was ascribed to low liposolubility leading to reduced access to the mitochondrial membrane, resulting in a minimal perturbation of mitochondrial metabolism.     

EFFECT OF COCAINE ON MITOCHONDRIAL ELECTRON TRANSPORT CHAIN EVALUATED IN PRIMARY CULTURES OF NEONATAL RAT MYOCARDIAL CELLS AND IN ISOLATED MITOCHONDRIAL PREPARATIONS

Cardiotoxicity is commonly associated with cocaine abuse. Previous studies have indicated that cocaine alters myocardial mitochondrial function. This study was undertaken to investigate the effect of cocaine on activity of the mitochondrial electron transport chain in cultures of neonatal rat cardiomyocytes and in isolated myocardial mitochondria. Cocaine concentrations (10^?5 to 10^?3 M) were used, and these concentrations have been reported in human cocaine users and are within a similar range of cocaine concentrations used in studies in vivo. After 24 hr treatment of cocaine, there was a slight increase in lactate dehydrogenase leakage in the cells treated with cocaine (10^?3 M). Reduction of tetra zolium compounds, neotetrazolium chloride (NTC) and triphenyltetrazolium chloride (TTC) was analyzed in intact cells to assess activity of the mitochondrial electron transport chain. Cocaine (10^?3 M) did not significantly change TTC and NTC reduction. In isolated mitochondria, cocaine (10^?3 M) significantly inhibited glutamate/malate-mediated respiration. These data suggest that cocaine at high concentrations may inhibit complex I of the mitochondrial electron transport chain of myocardial cells.     

Cadmium exposure affects mitochondrial bioenergetics and gene expression of key mitochondrial proteins in the eastern oyster Crassostrea virginica Gmelin (Bivalvia: Ostreidae)

Cadmium is a ubiquitous and extremely toxic metal, which strongly affects mitochondrial function of aquatic organisms in vitro; however, nothing is known about the in vivo effects of sublethal concentrations of this metal on mitochondrial bioenergetics. We have studied the effects of exposure to 0 (control) or 25 microg L-1 (Cd-exposed) Cd2+ on mitochondrial function and gene expression of key mitochondrial proteins in the eastern oyster Crassostrea virginica. Cadmium exposure in vivo resulted in considerable accumulation of cadmium in oyster mitochondria and in a significant decrease of ADP-stimulated respiration (state 3) by 30% indicating impaired capacity for ATP production. The decrease in state 3 respiration was similar to the level of inhibition expected from the direct effects of cadmium accumulated in oyster mitochondria. On the other hand, while no effect on proton leak was expected based on the mitochondrial accumulation of cadmium, Cd-exposed oysters in fact showed a significant decline of the proton leak rate (state 4+respiration) by 40%. This suggested a downregulation of proton leak, which correlated with a decrease in mRNA expression of a mitochondrial uncoupling protein UCP6 and two other potential uncouplers, mitochondrial substrate carriers MSC-1 and MSC-2. Expression of other key mitochondrial proteins including cytochrome c oxidase, adenine nucleotide transporter and voltage dependent anion channel was not affected by cadmium exposure. Adenylate energy charge (AEC) was significantly lower in Cd-exposed oysters; however, this was due to higher steady state ADP levels and not to the decrease in tissue ATP levels. Our data show that adjustment of the proton leak in cadmium-exposed oysters may be a compensatory mechanism, which allows them to maintain normal mitochondrial coupling and ATP levels despite the cadmium-induced inhibition of capacity for ATP production.     

The effects of some nutritive antibiotics on the respiration of rat liver mitochondria.

(1) Seven antibiotics used as feed additives in animal breeding were investigated for their effects on isolated rat liver mitochondria. Three were found to interfere with mitochondrial energy metabolism. (2) Zinc-bacitracin completely inhibits mitochondrial respiration in the micromolar range. as do other inhibitors known to be highly effective against electron transport system. From studies of this antibiotic on the redox state of cytochromes, as measured by split beam spectra, it is concluded that the site of inhibition is located between cytochrome b and c₁ (antimycin A site). The effect is completely reversed by chelating agents, suggesting that Zn²⁺ ions are required for full activity of the cyclic peptide antibiotic. (3) Flavomycin, a polar glycolipid, linearly stimulates oxygen consumption of mitochondria under state 4 conditions in concentrations greater than 100 μmole/gram of protein. Lower concentrations of the antibiotic inhibits respiration of coupled as well as DNP- or FCCP-uncoupled mitochondria by about 70 per cent. While the uncouplinglike effect at high concentrations of the compound can be attributed to nonspecific surface activity which might facilitate proton conductance, the inhibitory activity seen at lower concentrations is assumed to be located near the second phosphorylation site of the respiratory chain. (4) The influence of chlortetracyclin (aureomycin) on mitochondrial activity was found to be dependent on the identity of the substrate. Succinate respiration was more sensitive to chlortetracyclin (CTC) addition by comparison with NAD-linked substrate oxidation. 45 μmole of CTC/gram of protein decreased succinate respiration to half maximal values, whereas glutamate plus malate or β-hydroxybutyrate respiration were inhibited by only 25 per cent. CTC partially inhibits the dehydrogenation of succinate by the succinate dehydrogenase. Uncoupling of oxidative phosphorylation completely abolished CTC-inhibited respiration of NAD-linked substrates, while succinate respiration remained inhibited by 25 per cent. The results of these experiments are discussed in terms of two sites of action for CTC, one located close to the phosphate carrier, while the second interferes with succinate dehydrogenase.     

Disodium tetrachloropalladate (Na2PdCl4), an inhibitor of rat liver mitochondrial electron transport

The effects of Na2PdCl4 were studied on isolated rat liver mitochondrial electron transport and oxidative phosphorylation in vitro. Significant reductions in ADP-stimulated respiration were observed with increasing Na2PdCl4 concentrations with both succinate and NADH-linked substrate oxidations. Concentration necessary for half-maximal inhibition of oxygen uptake (EC50) for an NADH-linked substrate system was 18 muM while with succinate as substrate the EC50 was 15 muM. At 64 muM both systems were inhibited maximally at 60 and 80%, respectively. At concentrations of Na2PdCl4 sufficient to inhibit acceptor-stimulated oxygen uptake, there was a concomitant decrease in the rate of ADP phosphorylation as measured by proton absorption. Uncoupling agents had no effect on Na2PdCl4 inhibited mitochondria. Mg-ATPase activity and phosphate acceptor limited (State 4) respiratory activity were not stimulated by any Na2PdCl4 concentration used in these investigations. Data from these experiments indicate that Na2PdCl4 inhibits the mitochondrial respiratory chain in vitro.     

Effects of pyrazole, 4-bromopyrazole and 4-methylpyrazole on mitochondrial function

Pyrazole, an inhibitor of alcohol dehydrogenase, has been widely used in studies of ethanol metabolism. Since its specificity has recently been questioned, we studied the effects of pyrazole, methylpyrazole and bromopyrazole on mitochondrial function. These compounds inhibited oxidative phosphorylation, the ATP^?32P exchange reaction, and energy dependent and independent calcium uptake. With α-ketoglutarate as substrate, state 3 (coupled) respiration was inhibited, whereas state 4 (resting) respiration was not affected. By contrast, state 4 respiration was stimulated when succinate or ascorbate served as the substrate, while state 3 respiration was slightly inhibited. Regardless of the substrate, the respiratory control ratio was depressed. The activities of succinic dehydrogenase and cytochrome oxidase were stimulated by pyrazole and its derivatives, which may explain the stimulation of succinate and ascorbate oxidation. The inhibitory effects of these compounds were reversed by washing the mitochondria, indicating that no permanent damage to mitochondria had occurred. This is supported by the lack of stimulation of latent ATPase activity and the unchanged barrier to the penetration of NADH. Pyrazole and its derivatives decreased the uptake of citrate and glutamate, but stimulated that of phosphate and malate. Methylpyrazole and bromopyrazole inhibited the transport of reducing equivalents into the mitochondria, as catalyzed by the malate-aspartate, fatty acid and α-glycerophosphate shuttles. The data mandate caution in advocating the therapeutic use of pyrazole or its derivatives in man, and suggest that the use of pyrazole to assess ethanol metabolism and its sequelae in vivo may have limitations.     

Further studies concerning the effects of clofibrate on respiration and oxidative phosphorylation of rat liver mitochondria

Clofibrate, administered in vitro, inhibited rat liver mitochondrial respiration at two sites within the respiratory chain. One site was between the interaction of NADH with NADH dehydrogenase and the point at which electrons from succinate oxidation enter the electron transport chain; another, less sensitive site, was between the interaction of succinate with succinate dehydrogenase and cytochrome c. In addition to these specific sites, clofibrate inhibited respiration by causing a depletion of pyridine nucleotides that was accompanied or followed by large-amplitude, non-energy-linked swelling. Clofibrate uncoupled oxidative phosphorylation at coupling sites II and III but not at site I. The concentrations required to cause loss of pyridine nucleotides were lower than those required to inhibit at the specific sites. p-Chlorophenoxyisobutyrate (CPIB) also inhibited succinate and β-hydroxybutyrate-linked respiration, and uncoupled oxidative phosphorylation, but at much higher concentrations (50 per cent inhibition of β-hydroxybutyrate oxidation at about 3·7 μmoles/mg of protein) than were required of clofibrate (50 per cent inhibition of β-hydroxybutyrate oxidation at about 0·17 μmole/mg of protein). Clofibrate administration to rats (100 and 300 mg/kg p.o. daily for 1 week) lowered serum lipid levels and increased the liver size, the amount of mitochondrial protein/g of liver, and the oxygen consumption of liver slices. However, mitochondria, isolated from livers of the treated rats, respired normally. A single administration of clofibrate (100 or 300 mg/kg, p.o.) did not affect liver slice respiration.     

Acetaldehyde metabolism by isolated hepatic mitochondria from rats consuming different amounts of ethanol

We have previously reported that in rats, ethanol intake as low as 4 g/kg per day induced mitochondrial alterations, detected by a decrease in mitochondrial O2 uptake supported by substrates entering at the three sites of coupled phosphorylation. Since it has been reported that acetaldehyde oxidation occurs mainly inside the mitochondria, linked to the respiration chain, the effect of daily previous administration of low doses of ethanol during a month, on the acetaldehyde oxidation rate by intact rat liver mitochondria was studied. Determination of acetaldehyde oxidation rate and O2 uptake accompanying acetaldehyde removal by liver mitochondria indicates that the mean value of these parameters studied in rats consuming low amount of ethanol (0.5-3.0 g/kg per day) was significantly higher than that of controls drinking only water (P less than 0.001, in state 3, and P less than 0.05, in state 4). This enhancing effect of ethanol cannot be ascribed to an uncoupling of oxidative phosphorylation and also not to a change in aldehyde dehydrogenase activity which was measured in disrupted mitochondria. On the other hand, high ethanol consumption (4-7 g/kg per day), which alters mitochondrial function, did not decrease mitochondrial acetaldehyde oxidation, as compared to rats drinking water only. This result differs from the decrease induced by higher levels of previous ethanol intake (12-14 g/kg per day) as reported by several authors, showing that possibly this effect is dose dependent.     

The effects of the peripheral-type benzodiazepine acceptor ligands, Ro 5-4864 and PK 11195, on mitochondrial respiration.

The role of the peripheral-type benzodiazepine acceptor is unclear. It has been suggested that the acceptor ligands, Ro 5-4864 and PK 11195, stimulate mitochondrial respiration by binding to the peripheral-type benzodiazepine acceptor. We determined the effect of the benzodiazepine Ro 5-4864 and of the isoquinoline carboxamide PK 11195 on the respiration rates of liver, kidney and adrenal mitochondria during coupled, uncoupled and phosphorylating respiration. These ligands inhibited uncoupled and phosphorylating respiration, but only at concentrations substantially greater than their KD values for binding to the acceptor. There was a slight stimulation of coupled respiration by these ligands at concentrations similar to their KD values for the acceptor, but this stimulation was markedly greater at higher concentrations. These results suggest that the ligands Ro 5-4864 and PK 11195 affect respiration in a non-specific way, independently of binding to the acceptor. There was no correlation between the effect of these ligands on respiration and the density of the acceptor in mitochondria from liver, kidney and adrenals. We suggest that the earlier reported alteration of respiration by these ligands was due to non-specific effects and was not mediated by the peripheral-type benzodiazepine acceptor.