Rat Liver Mitochondrial Function Research Articles

Mechanism of primary Cd 2+-induced rat liver mitochondria dysfunction: discrete modes of Cd 2+ action on calcium and thiol-dependent domains

We attempted to discern discrete sites of Cd 2+ deleterious action on rat liver mitochondrial function. In particular, EGTA, ADP, and cyclosporin A (potent mitochondrial permeability transition antagonists) affected mainly Cd 2+ -induced changes in resting state respiration, eliminating its stimulation in KCl medium, while dithiothreitol (DTT, a dithiol reductant) produced its effect both on Cd 2+ activation of the basal respiration and Cd 2+ depression of uncoupler-stimulated respiration, evoking its restoration. Substantial differences in DTT influence on mitochondrial respiration at low and high [Cd 2+] were revealed, namely, an enhanced mitochondrial permeabilization in the presence of saturated [DTT] at high [Cd 2+] took place. Besides, DTT only partially reversed Cd 2+ -induced swelling in NH 4NO 3 medium when glutamate plus malate or succinate without rotenone was used. Contrarily, DTT produced complete reversal of the swelling of succinate-energized mitochondria when rotenone was present in the medium. In addition, in the presence of rotenone both Cd 2+ -produced activation of the resting state respiration in KCl medium and Cd 2+-induced swelling in sucrose medium of succinate-energized mitochondria were more sensitive to cyclosporin A than the same Cd 2+ effects obtained on mitochondria oxidizing succinate (without rotenone) or glutamate plus malate. We have concluded that Cd 2+, producing primary mitochondrial dysfunction, acts both as a thiol and Me 2+ binding site reagent. Suppositions about possible localization of separate sites of direct Cd 2+ effects on mitochondrial function were made.

Acrolein-induced toxicity--defective mitochondrial function as a possible mechanism.

Administration of acrolein (2.5 mg/kg body weight/day) to rats for 45 days depleted the glutathione level in liver, which triggered an imbalance in the antioxidant defense, resulting in lipid peroxidation. Enhanced lipid peroxidation damaged the membranous structure of mitochondria, which was indicated by the loss of lamellae, and increased the oxidation of exogenously added NADH. Loss in membrane integrity altered the activities of the tricarboxylic acid cycle enzymes and levels of cytochromes. Decreased rate of ADP-stimulated oxygen uptake, respiratory coupling ratio, and ATP synthesis-were also observed. We report that the acrolein-induced toxicity is mediated through the depletion of GSH leading to impairment of rat liver mitochondrial function.

Mitochondrial permeability transition during hypothermic to normothermic reperfusion in rat liver demonstrated by the protective effect of cyclosporin A.

The purpose of this study was to test the hypothesis that mitochondrial permeability transition might be implicated in mitochondrial and intact organ dysfunctions associated with damage induced by reperfusion after cold ischaemia. Energetic metabolism was assessed continuously by 31P-NMR on a model system of isolated perfused rat liver; mitochondria were extracted from the livers and studied by using top-down control analysis. During the temperature transition from hypothermic to normothermic perfusion (from 4 to 37 degrees C) the ATP content of the perfused organ fell rapidly, and top-down metabolic control analysis of damaged mitochondria revealed a specific control pattern characterized by a dysfunction of the phosphorylation subsystem leading to a decreased response to cellular ATP demand. Both dysfunctions were fully prevented by cyclosporin A, a specific inhibitor of the mitochondrial transition pore (MTP). These results strongly suggest the involvement of the opening of MTP in vivo during the transition to normothermia on rat liver mitochondrial function and organ energetics.

Effect of MI-D, a new mesoionic compound, on energy-linked functions of rat liver mitochondria

MI-D (4-phenyl-5-(4-nitro-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride), a new mesoionic compound, depressed the phosphorylation efficiency of liver mitochondria as deduced from an accentuated decrease of the respiratory control coefficient and ADP/O ratio. Analysis of segments of the respiratory chain suggested that the MI-D inhibition site is further on than complex I and between complexes II and III. The transmembrane electrical potential (Δψ) was collapsed dependent on MI-D concentration. ATPase activity was dramatically increased by MI-D in intact mitochondria, but inhibited in carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled mitochondria. These results suggest that MI-D acts as an uncoupler agent, a property closely related to its structural characteristics.

Protective effect of vitamin E against ethionine toxicity.

The effect of administration of ethionine on rat liver mitochondrial functions and the protective effect of vitamin E on ethionine induced damage was studied. Ethionine treatment decreased the rate of respiration, respiratory control ratio and P/O ratio. There was a significant decrease in the activities of NADH dehydrogenase, succinate cytochrome C reductase and cytochrome oxidase. A significant decrease was seen on membrane potential and on the levels of ATP. Among the mitochondrial phospholipids only cardiolipin decreased significantly. The lipid peroxide level increased significantly in ethionine treated rats. Administration of vitamin E prior to ethionine treatment relieved the effects (induced by ethionine) on all the parameters studied. This study shows that vitamin E protects against ethionine toxicity.

Protective Effect of Phyllanthus fraternus against Thioacetamide-Induced Mitochondrial Dysfunction.

The effect of administration of thioacetamide on rat liver mitochondrial functions and the protective effect of an aqueous extract of Phyllanthus fraternus against thioacetamide-induced damage were studied. When rats were treated with thioacetamide, the rate of mitochondrial respiration was decreased significantly with both NAD+ linked and FAD linked substrates, and the respiratory control ratio, an index of membrane integrity and the P/O ratio, a measure of phosphorylation efficiency, decreased significantly. Also, there was a significant decrease in the activities of NADH dehydrogenase, succinate cytochrome c reductase, and cytochrome oxidase; whereas succinate dehydrogenase was not affected. A significant decrease was seen in membrane potential and in the level of mitochondrial ATP. There was a significant decrease in the levels of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin by thioacetamide treatment. The lipid peroxide level increased significantly in thioacetamide-treated rats. Administration of P. fraternus prior to thioacetamide treatment relieved the inhibition of all the parameters studied, and brought down the lipid peroxide levels significantly in liver homogenates and mitochondria. This study shows that P. fraternus protects against thioacetamide-induced toxicity by its ability to suppress the elevated lipid peroxide levels in the mitochondrial membrane.

Inhibitory effect of Mg 2+ on the protonophoric activity of palmitic acid

To discriminate whether fatty acids are uncouplers that cause acceleration of State-4 respiration, associated with a decrease in the protonmotive force, or decouplers that increase respiration without associated decrease in the protonmotive force, we examined the effect of palmitate on functions of rat-liver mitochondria under various conditions. We found that palmitate itself increases State-4 respiration, releases oligomycin-inhibited State-3 respiration, inhibits ATP synthesis and ATP ⇌ P i exchange reaction, and increases H + permeability in mitochondrial and model bilayer phospholipid membranes. Thus, palmitate is a classical uncoupler of oxidative phosphorylation. However, these effects were inhibited by Mg 2+, due to rapid formation of a stable complex between palmitate and Mg 2+.

Improvement of Rat Survival and Liver Mitochondrial Function in Biliary Obstruction After Treatment With Sodium Thiosulfate

The exact cause of liver failure occurring after long standing biliary obstruction is not known. Impairment of hepatic mitochondrial respiration was postulated in some studies. Sodium thiosulphate (STS) is known to have a protective effect on liver function during administration of hepatotoxic chemotherapy. In the present experimental study the effect of treatment with STS in the presence of obstructive jaundice was studied by determination of the survival rate of rats subjected to biliary obstruction and by polarographic determination of the hepatic mitochondrial function. Treatment with STS was found to result in a significant improvement in rats' survival rate (p < 0.05). Polarography demonstrated significant preservation of mitochondrial respiratory capacity after treatment with STS. The results of the present study show that the deterioration in liver function in the presence of biliary obstruction is probably caused by impairment of mitochondrial respiration. This may be preserved by treatment with STS. The exact explanation of its effect is not yet clear.

Catabolism of 5-Aminolevulinic Acid to CO2 by Rat Liver Mitochondria

5-Aminolevulinic acid (ALA), the heme precursor accumulated in plasma and several organs of carriers of acute intermittent porphyria, hereditary tyrosinemia, and saturnism, was previously shown to yield reactive oxygen species upon metal-catalyzed aerobic oxidation and to cause the in vivo and in vitro impairment of rat liver mitochondrial functions. We have studied the uptake and catabolism of [5-14C]ALA to CO2 by isolated rat liver mitochondria (RLM) with the aim of determining whether possible ALA-driven oxidative injury to mitochondria can also occur into the matrix. Using silicone oil centrifugation of [5-14C]ALA-treated RLM, ALA was found to partition evenly into the intra- and extramatrix space of the mitochondrial preparations. The yield of evolved 14CO2 is very low (0.2%), responds to the concentration of added ADP, and is inhibited by malonate (75% at 2 mM), iproniazid (45% at 2 mM), β-chloroalanine (36% at 1 mM), and aminooxyacetate (55% at 0.1 mM). With both iproniazid and aminooxyacetate, the percentage of inhibition is the same as that observed with the latter inhibitor alone. These data indicate that ALA decarboxylation by the Krebs cycle is a minor process and that it is initiated enzymically (transaminase) and not by metal-catalyzed ALA autoxidation.

MOLECULAR DAMAGE TO RAT LIVER MOTOCHONDRIAL H+-ATPASE DURING COLD PRESERVATION WITH UW SOLUTION

Deterioration of rat liver mitochondrial function during cold preservation with UW solution was studied. Mitochondria were isolated from control liver (0-hr preservation), 24-hr preserved liver, and 48-hr preserved liver with UW solution at 4 degrees C. Respiration assay revealed that phosphorylation was damaged more rapidly than oxidation. Inside-out submitochondrial particles prepared from each sample by sonication in the presence of EDTA were subjected to ATPase assay. ATP hydrolyzing activity of H(+)-ATPase (ATP synthase), which plays a key role in phosphorylation in mitochondria, decreased markedly to 58% after 24-hr preservation. After 48-hr preservation, reduction to 40% of control was noted. When an intrinsic H(+)-ATPase inhibitor protein was removed from ESMP by Sephadex gel filtration, decrease of the ATPase activity was enhanced down to 49% and 29% of the control for 24-hr and 48-hr preserved liver, respectively. Molecular damage of the enzyme was confirmed by SDS-PAGE. Alpha subunit of the enzyme decreased time-dependently, and H(+)-ATPase molecules that lost alpha subunit seemed to lose their catalytic activity. Although the cause of the molecular damage of H(+)-ATPase is not clear yet, some mitochondrial protease(s) may be involved.

Effect of thyroid state and cold exposure on rat liver mitochondrial protein mass and function

The effects of thyroid state on liver mitochondrial protein mass was investigated in rats at 24 and 4 degrees C, as was oxidative phosphorylation using substrates which represent the final catabolic products of the metabolic fuels. In rats at 24 degrees C, a significant increase in mitochondrial protein mass (about +40%) was observed only in hyperthyroid animals, while a significant increase due to cold exposure was found in hypothyroid (+45%) and euthyroid (+35%) rats. In rats at 24 degrees C, hypothyroidism significantly decreased the oxidation of glutamate and palmitoyl carnitine but not of pyruvate, while hyperthyroidism only increased the oxidation of palmitoyl carnitine. On the other hand, exposure to cold significantly increased the oxidation of glutamate and pyruvate only in the presence of tri-iodothyronine. Our results underline not only the fact that a simple and single hypothesis for thyroid effects cannot be adopted, but also that any study concerning oxidative metabolism should be carried out using different substrates and involving different pathways of oxidation.

Postnatal mitochondrial differentiation in rat liver. Regulation by thyroid hormones of the beta-subunit of the mitochondrial F1-ATPase complex.

Postnatal development of rat liver mitochondrial functions, i.e. differentiation of pre-existing mitochondria, is a key regulatory process that is accomplished during the first postnatal hour (Valcarce, C., Navarrete, R. M., Encabo, P., Loeches, E., Satrústegui, J., and Cuezva, J. M. (1988) J. Biol. Chem. 263, 7767-7775). The general mechanism that appears to control this process is the rapid (2-fold) increase in inner mitochondrial membrane proteins, as a result of a rapid increase in the rates of protein synthesis for mitochondrial proteins. Fetal hypothyroidism induced by administration of methimazole to pregnant rats resulted in half the ATPase activity and amount of beta-F1-ATPase protein in liver homogenates compared with normal fetuses. Furthermore, hypothyroid neonates showed no postnatal increase in the amount of mitochondrial beta subunit of the F1-ATPase complex. Administration of thyroid hormones to hypothyroid neonates at birth promoted a 2-fold increase in liver ATPase activity and in the amount of beta-F1-ATPase protein both in liver homogenates and in isolated mitochondria. The rapid increase in beta-F1-ATPase protein observed in thyroid hormone-treated hypothyroid neonates was paralleled by a 2-fold increase in the relative rates of beta-F1-ATPase synthesis and by a 2-fold increase in the relative amount of hepatic beta-F1-ATPase mRNA. The results show that the basal expression of the beta-F1-ATPase gene is regulated by thyroid hormones at either a transcriptional and/or post-transcriptional level. Thus, these results show the requirement of thyroid hormones for proliferation and differentiation of mitochondria in the fetal and neonatal liver, respectively. On the other hand, the fact that liver mRNA level of beta-F1-ATPase protein does not change between 0 and 1 postnatal h in the normal neonatal liver indicates that postnatal mitochondrial differentiation is regulated at a translational level.

Dual inhibitory effects of the peptide antibiotics leucinostatins on oxidative phosphorylation in mitochondria.

The effects of the hydrophobic peptide antibiotics leucinostatins A and B, originally isolated by Arai, T., Y. Mikami, K. Fukushima, T. Utsumi, and K. Yazawa. (J. Antibiotics (1973) 26: 157-161), on the functions of rat liver mitochondria were examined. At a concentration of 240 nM, these compounds completely inhibited state 3 respiration and ATPase activity that was stimulated by weakly acidic uncouplers. However, at higher concentrations, they induced uncoupling, probably by their protonophoric action. The uncoupling action was potentiated by known phosphoryl transfer inhibitors such as venturicidin, DCCD and oligomycin. The binding site of leucinostatins at lower concentrations was suggested to be located at, or very close to that of venturicidin. The potencies of the two analogues of leucinostatin were almost the same for all their actions. Their effects were very similar to those of the peptide antibiotics A20668's, which have been used as leucinostatins without any chemical and biological confirmation that they are in fact leucinostatins. Thus, the chemical structures of leucinostatins are thought to be analogues to those of the A20668's.

The effect of pancreatitis associated ascitic fluid on some functions of rat liver mitochondria

The damage to the liver during acute pancreatitis (AP) could be partly dependent on depressive action of pancreatitis associated ascitic fluid (PAAF) on the energy metabolism of hepatocytes. The aim of the study was to assess the effect of PAAF from dogs with acute experimental pancreatitis (AEP) and from humans with AP on the respiratory function of isolated rat liver mitochondria (RLM). The mitochondrial oxygen consumption rate in state 3 respiration (with ADP) and in state 4 (without ADP) using sodium succinate as substrate and oxygen Clark's electrode was estimated. Respiratory control ratio (RCR) and P/O ratio were calculated. PAAF was collected after 6 h of AEP induced by Elliott's method in 8 dogs, and from 4 patients with AP, intraoperatively. Both animal and human PAAFs increase the oxygen consumption rate by RLM in state 4 dose dependently (by 65% with 50 microL to 150% with 200 microL of canine PAAF). This uncoupling effect of human PAAF was twice more potent than the canine. Dialysis of PAAF reduced this effect almost completely. The mitochondrial ATPase activity in RLM treated with PAAF was stimulated and this effect was also reduced by dialysis. The conclusion was that the damage to the liver in AEP could be partly dependent on the toxicity of dializable component(s) of PAAF on the energy metabolism of mitochondria. These findings may partly explain the beneficial effects of peritoneal lavage in acute pancreatitis.

The mechanism of acute cytotoxicity of triethylphosphine gold(I) complexes: III. Chlorotriethylphosphine gold(I)-induced alterations in isolated rat liver mitochondrial function

Chlorotriethylphosphine gold(I) (TEPAu) is an organo-gold compound that has therapeutic activity in animal models of rheumatoid arthritis. Initial studies have suggested that TEPAu is a potent cytotoxic compound in vitro against a variety of cultured cell types and isolated hepatocytes. Mitochondrial dysfunction induced by this compound has been suggested as a primary biochemical alteration which may result in lethal cell injury in isolated hepatocytes. The purpose of this study was, therefore, to determine the mechanism of TEPAu-induced dysfunction of isolated rat liver mitochondria. TEPAu induced a rapid, concentration-related collapse of the mitochondrial inner membrane potential (EC50 = 24.7 ± 2.5 μ m) which was potentiated in Ca 2+ loaded mitochondria (EC50 = 11.3 ± 3.8 μ m). TEPAu-induced collapse of the membrane potential was partially inhibited in the presence of ruthenium red or EGTA. TEPAu caused the rapid release of mitochondrially sequestered Ca 2+ which was not inhibited by ruthenium red and, thus, was not via a reversal of the Ca 2+ uniporter. TEPAu caused mitochondrial swelling, increased permeability of the inner membrane, and the oxidation/hydrolysis of endogenous mitochondrial pyridine nucleotides. Addition of exogenous ATP slightly reversed the effects of TEPAu on pyridine nucleotides. TEPAu-induced mitochondrial alterations were reversed or inhibited by exposure to the sulfhydryl reducing agent, dithiothreitol. Also, the TEPAu-induced collapse of the mitochondrial membrane potential was partially inhibited by dibucaine, a non-specific inhibitor of phospholipases. These data suggest that TEPAu-induced mitochondrial dysfunction is sulfhydryl dependent. TEPAu-induced mitochondrial dysfunction results in dissipation of the potential difference across the inner mitochondrial membrane which inhibits mitochondrial oxidative phosphorylation. The mechanism by which TEPAu induces the collapse of the membrane potential may be mediated by a sulfhydryl-dependent increase in permeability of the inner membrane to protons.

Effects of moderate chronic ethanol consumption on rat liver mitochondrial functions

The biochemical consequences of moderate chronic ethanol ingestion has been scarcely investigated in spite of the fact that most of the human population drinks ethanol on a moderate basis. This paper describes some metabolic effects produced by moderate ethanol consumption. The substitution of drinking water in rats for a 10% ethanol solution during 4 weeks resulted in: a) a decrease of blood urea and citrulline synthesis in liver mitochondria; b) a slight inhibition in state 3 and state 4 respiration either with glutamate-malate as substrates or succinate as substrate; c) no change in ADP/O ratio with succinate but slight increase with glutamate-malate; d) a reduction of the cytochrome oxidase activity and cytochromes a+a 3 content; e) a 42% increase in the succinate dehydrogenase activity and a small but constant increase in the V max (no change in the K m) of the adenin nucleotide translocase activity in liver mitochrondria. These results show that even moderate, but continuous ethanol ingestion, produces metabolic responses that must be carefully evaluated to define health risk in larger human groups.

A comparative study of the effect of ten non-steroidal anti-inflammatory drugs (NSAIDS) upon some mitochondrial and platelet functions

1. 1. Ten different NSAIDs were used in a comparative study of their effects upon rat liver mitochondrial and human platelet functions. 2. 2. The drugs behaved like uncouplers of mitochondrial respiration, decreased membrane potential, stimulated ATPase activity and had an ionophore-like effect upon calcium transport. 3. 3. In vitro experiments showed the same effect with 45Ca as well as 22Na. 4. 4. In platelets, aggregation was considerably reduced by most NSAIDs. 5. 5. Addition of ADP (mitochondrial calcium uptake) or magnesium (platelet aggregation and in vitro cation transference) contributed to protect from those effects.

Ｉｓｏｆｌｏｘｙｔｈｅｐｉｎのラット肝ミトコンドリア膜に対する作用

The effect of isofloxythepin (IFT) on isolated rat liver mitochondria was examined.In the presence of β-hydroxybutyric acid, succinic acid or ascorbic acid as respiratory substrate, IFT accelerated the state 4 respiration with a dose-dependent manner at low concentrations (≤100 μM), but did not show any noticeable influence on the state 3 respiration.IFT stimulated the latent ATPase activity at low concentrations. But at higher concentrations, the DNP-stimulated ATPase activity was decreased remarkably. The activities of the latent and DNP-stimulated ATPases were at the same level when IFT at a concentration of 400uM was added. These results suggest that IFT influences isolated rat liver mitochondrial functions as an uncoupler.

Comparative toxicity of 4-chlorobiphenyl and its metabolite 4-chloro-4′-biphenylol in isolated rat liver mitochondria

4-Chlorobiphenyl (4-CB) is converted by the microsomal cytochrome P-450 system to its hydroxylated metabolite 4-chloro-4'-biphenylol (4'-OH-4-CB). A study of the effects of 4-CB and 4'-OH-4-CB on the energy-linked functions of rat liver mitochondria was carried out. 4'-OH-4-CB was more effective than 4-CB in causing the inhibition of state 3 respiration of mitochondria with both succinate and glutamate/malate. As a substrate specificity, with glutamate/malate the inhibition by each compound (ID 50, 30 μM for 4'-OH-4-CB, 76 μM for 4.CB) was more significant than that with succinate (ID 50, 200 μM for 4'-OH-4-CB. never reached 50% for 4-CB). From the effects on DNP-stimulated respiration, it was indicated that the electron transport from both glutamate/malate and succinate to oxygen was more sensitively inhibited by 4'-OH-4-CB than by 4-CB, with the same substrate specificity as for state 3 respiration (i.e. the inhibition by both compounds was greater with glutamate/malate than with succinate). Since there existed a good coincidence in the inhibition between state 3 and DNP-stimulated respiration with both substrates, the inhibition of state 3 respiration by both compounds was due to the inhibition of the electron transport. With succinate, the uncoupling of oxidative phosphorylation by both compounds was observed, the extent of which was greater with 4'-OH-4-CB than with 4-CB, although the uncoupling by higher concentrations of 4'-OH-4-CB was masked because of the increased inhibition in respiration. With glutamate/malate, the uncoupling action of 4-CB was largely, while that of 4'-OH-4-CB was completely, masked by progressive respiratory inhibition. 4'-OH-4-CB was more effective than 4-CB in causing stimulation of latent ATPase in mitochondria. These results indicate that both 4-CB and 4'-OH-4-CB impair mitochondrial energy-transducing functions, but 4'-OH-4-CB is more effective than 4-CB in damaging these functions. Thus, the product of the metabolism is more biologically active than the parent compound. The impairment of energy-linked mitochondrial reactions by the metabolite as well as of the parent compound may be an important factor in the toxicity of 4-CB.

Mechanism of alterations in isolated rat liver mitochondrial function induced by gold complexes of bidentate phosphines.

Au(DPPE)+2 (bis[1,2-bis(diphenylphosphino)ethane] gold(I] is an organo-gold antineoplastic agent that has anti-tumor activity in a variety of in vitro cell lines and in vivo rodent tumor models. Preliminary studies suggested that this compound represented a novel class of inhibitors of mitochondrial function. The purpose of this study was, therefore, to determine the mechanism of mitochondrial dysfunction induced by Au(DPPE)+2. Au(DPPE)+2 induced a rapid, dose-related collapse of the inner mitochondrial membrane potential (EC50 = 28.0 microM) that was not potentiated by Ca2+ preloading. Au(DPPE)+2-induced dissipation of mitochondrial membrane potential was accompanied by an efflux of Ca2+ from mitochondria upon exposure to Au(DPPE)+2. Ca2+ efflux in these experiments was via a reversal of the Ca2+ uniporter as efflux could be inhibited with ruthenium red. Au(DPPE)+2 did not increase the permeability of mitochondria to oxalacetate, indicating that the collapse of membrane potential may not be a result of gross increased inner membrane permeability. However, Au(DPPE)+2 may mediate an increased permeability of the inner membrane to cations and protons. Au(DPPE)+2 caused passive swelling in potassium acetate buffer in the absence of valinomycin, suggesting Au(DPPE)+2 facilitated the exchange of H+ and K+. Ca2+ cycling was not extensive and did not contribute to the decrease in membrane potential. These data suggest that one possible mechanism of Au(DPPE+2-induced uncoupling of mitochondrial oxidative phosphorylation is via increased permeability of the inner mitochondrial membrane to cations. The disruption of mitochondrial function may be a key process leading to hepatocyte cell injury by this drug.