Effects of tamoxifen on the electron transport chain of isolated rat liver mitochondria

Tamoxifen (and 4-hydroxytamoxifen), a nonsteroidal triphenylethylene antiestrogenic drug widely used in the treatment of breast cancer, interacts strongly with the respiratory chain of isolated rat liver mitochondria. The drug acts as both an uncoupling agent and a powerful inhibitor of electron transport. Tamoxifen brings about a collapse of the membrane potential. Enzymatic assays and spectroscopic studies indicate that tamoxifen inhibits electron transfer in the respiratory chain at the levels of complex III (ubiquinol–cytochrome-c reductase) and, to a lesser extent, of complex IV (cytochrome-c oxidase). The activities can be restored by the addition of diphosphatidylglycerol, a phospholipid implicated in the functioning of the respiratory chain complexes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Anandamide inhibits oxidative phosphorylation in isolated liver mitochondria

A study on the effect of anandamide (AEA) in energy coupling of rat liver mitochondria is presented. Micromolar concentrations of AEA, while almost ineffective on substrate supported oxygen consumption rate and on uncoupler stimulated respiration, strongly inhibited the respiratory state III. AEA did not change the rate and the extent of substrate generated membrane potential, but markedly delayed rebuilding by respiration of the potential collapsed by ADP addition. Overall, these data suggest that anandamide inhibits the oxidative phosphorylation process. Direct measurement of the F_oF₁ ATP synthase activity showed that the oligomycin sensitive ATP synthesis was inhibited by AEA, (IC₅₀, 2.5 μM), while the ATP hydrolase activity was unaffected. Consistently, AEA did not change the membrane potential generated by ATP hydrolysis.     

Oxidative activity in mitochondria isolated from rat liver at different stages of development

The purpose of this study was to evaluate the oxidative capacities in hepatic mitochondria isolated from prepubertal, young adult and adult rats (40, 90 and 180 days of age, respectively). In these rats, mitochondrial respiratory rates using FAD- and NAD-linked substrates as well as mitochondrial protein mass were measured. The results show that only the oxidative capacity of FAD-linked pathways significantly declined in mitochondria from 180-day-old rats compared with those from younger animals. When we consider FAD-linked respiration expressed per g liver, no significant difference was found among rats of different ages because of an increased mitochondrial protein mass found in 180-day-old rats. However, when FAD-linked and lipid-dependent respiratory rates were expressed per 100 g body weight, significant decreases occurred in 180-day-old rats. Therefore, the decrease in liver weight expressed per 100 g body weight rather than an impaired hepatic cellular activity may be the cause of body energy deficit in 180-day-old rats. Copyright © 1998 John Wiley & Sons, Ltd.     

Allatostatin-C reversibly blocks the transport of citrate out of the mitochondria and inhibits juvenile hormone synthesis in mosquitoes

Aedes aegypti allatostatin-C (AeaAST-C or PISCF-AST) is a strong and fast reversible inhibitor of juvenile hormone III (JH III) synthesis by the corpora allata (CA) of mosquitoes; however, its mechanism of action remains poorly understood. AeaAST-C showed no inhibitory activity in the presence of any of the intermediate precursors of JH III indicating that the AeaAST-C target is located before the entry of acetyl-CoA in the pathway. Stimulation experiments using different sources of carbon (glucose, pyruvate, acetate and citrate) suggest that AST-C acts after pyruvate is transformed to citrate in the mitochondria. In vitro inhibition of the citrate mitochondrial carrier (CIC) mimicked the effect of AeaAST-C, and was overridden by addition of citrate or acetate. Our results provide compelling evidence that AeaAST-C inhibits JH III synthesis by blocking the CIC carrier that transports citrate from the mitochondria to the cytosol, obstructing the production of cytoplasmic acetyl-CoA that sustains JH III synthesis in the CA of mosquitoes.     

Glyceollin: a site-specific inhibitor of electron transport in isolated soybean mitochondria

The glyceollin inhibition of electron transport by isolated soybean and corn mitochondria was similar to that of rotenone, acting at site I between the internal NADH dehydrogenase and coenzyme Q. Coupled state 3 malate oxidation was inhibited by glyceollin and rotenone with apparent K_i values of about 15 and 5 micromolar, respectively. Carbonylcyanide m-chlorophenyl hydrazone uncoupled state 4 malate oxidation was also inhibited by glyceollin and rotenone, but uncoupled succinate and exogenous NADH state 4 oxidation was only slightly inhibited by both compounds. Glyceollin also inhibited ferricyanide reduction with malate as the electron donor, with an apparent K_i of 5.4 micromolar, but failed to inhibit such reduction with succinate or externally added NADH as electron donors. Glyceollin did not inhibit state 4 oxidation of malate, succinate, or exogenous NADH. Glyceollin did not act as a classical uncoupler or as an inhibitor of oxidative phosphorylation.     

Thyroid hormone effects on the State IV proton motive force in rat liver mitochondria

In order to further investigate the mechanisms regulating the control of mitochondrial respiration by thyroid hormone, the proton motive force was measured during State IV respiration in liver mitochondria isolated from euthyroid, hyperthyroid, hypothyroid and T₃-treated hypothyroid rats. The proton motive force was significantly higher in the hyperthyroid group due to an increased pH. The proton motive force of hypothyroid mitochondria was lower than controls due to a decreased membrane potential. The proton motive force for the T3-treated hypothyroid group did not differ from the euthyroid group due to negating changes in the pH gradient and the membrane potential. The intramitochondrial volume was decreased in the hyperthyroid group and unchanged in the other groups. The results indicate that the thyroid status alters the proton motive force in State IV through individual changes in the pH and membrane potential components of the force. The component that changes in hyperthyroid mitochondria is different from that changing in hypothyroid mitochondria.     

Vitamin A supplementation inhibits chemiluminescence and lipid peroxidation in isolated rat liver microsomes and mitochondria

In the present study we investigated if administration of vitamin A could protect rat liver microsomes and mitochondria from in vitro peroxidation. Appreciable decrease of chemiluminescence and lipid peroxidation was measured in microsomal membranes from rats receiving vitamin A, with respect to control animals. In membranes derived from control animals, the fatty acid composition was profoundly modified when subjected to in vitro peroxidation mediated by ascorbate-Fe⁺⁺, with a considerable decrease of 20:4 n6 and 22:6 n3 in mitochondria and 18:2 n6 and 20:4 n6 in microsomes. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in supplemented animals than in control group when both kind of membranes were analyzed. These changes were less pronounced in membranes derived from rats receiving vitamin A. These results are in agreement with previous results that indicated that vitamin A may act as an antioxidant protecting membranes from deleterious effects.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - CL chemiluminescence - PI peroxidizability index Member of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Cientificas y Técnicas de la Republica Argentina     

The transport of L-cysteinesulfinate in rat liver mitochondria.

1. The mechanism of L-cysteinesulfinate permeation into rat liver mitochondria has been investigated. 2. Mitochondria do not swell in ammonium or potassium salts of L-cysteinesulfinate in all the conditions tested, including the presence of valinomycin and/or carbonylcyanide p-trifluoromethoxyphenylhydrazone. 3. The activation of malate oxidation by L-cysteinesulfinate is abolished by aminooxyacetate, an inhibitor of the intramitochondrial aspartate aminotransferase, it is not inhibited by high concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone (in contrast to the oxidation of malate plus glutamate) and it is decreased on lowering the pH of the medium. 4. All the aspartate formed during the oxidation of malate plus L-cysteinesulfinate is exported into the extramitochondrial space. 5. Homocysteinesulfinate, cysteate and homocysteate, which are all good substrates of the mitochondrial aspartate aminotransferase, are unable to activate the oxidation of malate. Homocysteinesulfinate and homocysteate have no inhibitory effect on the L-cysteinesulfinate-induced respiration, whereas cysteate inhibits it competitively with respect to L-cysteinesulfinate. 6. In contrast to D-aspartate, D-cysteinesulfinate and D-glutamate, L-aspartate inhibits the oxidation of malate plus L-cysteinesulfinate in a competitive way with respect to L-cysteinesulfinate. Vice versa, L-cysteinesulfinate inhibits the influx of L-aspartate. 7. Externally added L-cysteinesulfinate elicits efflux of intramitochondrial L-aspartate or L-glutamate. The cysteinesulfinate analogues homocysteinesulfinate, cysteate and homocysteate and the D-stereoisomers of cysteinesulfinate, aspartate and glutamate do not cause a significant release of internal glutamate or aspartate, indicating a high degree of specificity of the exchange reactions. External L-cysteinesulfinate does not cause efflux of intramitochondrial Pi, malate, malonate, citrate, oxoglutarate, pyruvate or ADP. The L-cysteinesulfinate-aspartate and L-cysteinesulfinate-glutamate exchanges are inhibited by glisoxepide and by known substrates of the glutamate-aspartate carrier. 8. The exchange between external L-cysteinesulfinate and intramitochondrial glutamate is accompanied by translocation of protons across the mitochondrial membrane in the same direction as glutamate. The L-cysteinesulfinate-aspartate exchange, on the other hand, is not accompanied by H+ translocation. 9. The ratios delta H+/delta glutamate, delta L-cysteinesulfinate/delta glutamate and delta L-cysteinesulfinate/delta aspartate are close to unity. 10. It is concluded that L-cysteinesulfinate is transported by the glutamate-aspartate carrier of rat liver mitochondria. The present data suggest that the dissociated form of L-cysteinesulfinate exchanges with H+-compensated glutamate or with negatively charged aspartate.     

Mechanism of citrinin-induced dysfunction of mitochondria. II. Effect on respiration, enzyme activities, and membrane potential of liver mitochondria.

The mycotoxin citrinin, depressed the phosphorylation efficiency of liver mitochondria as deduced from a decrease of respiratory coefficient and of the ADP/O ratio. Citrinin (1.0 mM) inhibited some enzymes linked to the respiratory chain, namely NADH oxidase and NADH cytochrome c reductase involved with complex I. The activities of enzymes related with other enzymatic complexes of the respiratory chain were either unaffected or enhanced. ATPase activity was inhibited by the mycotoxin. Malate, glutamate, and 2-oxoglutarate dehydrogenases were also inhibited. The transmembrane potential (delta psi), developed by energized mitochondria and depolarization on the addition of ADP, was decreased. The results suggest that citrinin promotes a partial dissipation of the transmembrane potential, different from that resulting from a classical uncoupler such as 2,4-dinitrophenol.     

The effect of ferric iron complex on Ca2+ transport in isolated rat liver mitochondria

The in vitro effects of iron (III)-gluconate complex on the production of malondialdehyde and on the Ca2+ transport in isolated rat liver mitochondria were studied. A correlation between the concentration of iron added and the formation of malondialdehyde was found. The enhancement by iron of lipid peroxidative process in the mitochondrial membrane brought about the induction of Ca2+ release from mitochondria. Experimental evidence based on the membrane potential pattern of mitochondria pre-loaded with a low pulse of Ca2+ suggested that Ca2+ efflux was not due to a nonspecific increase in the inner membrane permeability, i.e. to a collapse of membrane potential, but rather to the activation of an apparently selective pathway for Ca2+ release.     

The effects of cryopreservation on protein synthesis and membrane transport in isolated rat liver mitochondria.

Protein synthesizing activity and membrane transport were examined in fresh and cryopreserved isolated rat liver mitochondria. In the presence of 0.6, 1.2, and 1.8 M final concentrations of dimethyl sulfoxide (Me2SO), both metabolic parameters were considerably inhibited in the fresh samples and even more inhibited in the cryopreserved specimens. However, simple exposure to this penetrating cryoprotectant, followed by its subsequent removal by washing, did not seem to affect significantly the examined functions. When different freeze-thaw regimes were investigated, it was observed that optimal recovery of protein synthesis and membrane transport functions were obtained when fast freezing took place in the absence of Me2SO.     

Effect of theophylline and theobromine on cell respiration and calcium transport in isolated rat liver mitochondria

The influence of theophylline and theobromine on cellular respiration and on membrane transport of calcium has been studied in isolated rat liver mitochondria, using oxygen and Ca2+ selective electrodes. A linear decrease in respiratory coefficients, in the total amount and rate of "extra" oxygen consumption induced by ADP is observed with drug concentration. Theobromine does not show any appreciable effect on these respiratory parameters, but this result is similar to that observed with theophylline for the same concentration range. Calcium uptake coupled to respiration is inhibited by both drugs depending on their concentrations. Theobromine is more effective than theophylline. Calcium saturation of the mitochondria takes place in all cases after 36 +/- 2 s but only a 20% of the maximum calcium uptake observed in the absence of the drugs is determined in the presence of 15 mM theophylline or only 1.8 mM theobromine. Comparative studies show direct correlation between the pharmacological activities as stimulants of caffeine, theophylline and theobromine and their behaviour as inhibitors of calcium uptake coupled to respiration by mitochondria.     

A novel nuclease from mitochondria of rat liver

Alkaline RNase partially purified from rat liver mitochondria hydrolyzes both RNA and denatured DNA. The behaviors of RNase activity of the nuclease are closely similar to those of the DNase activity. The nuclease has a pH optimum between 9.0 and 9.5, and the activity is absolutely dependent on Mg²⁺ and reversibly inhibited by $\text{p}$-hydroxymercuribenzoate.     

Hepatic mitochondrial transport of glutathione: studies in isolated rat liver mitochondria and H4IIE rat hepatoma cells

Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxoglutarate carriers (OGC; Slc25a11). To determine whether these carriers function similarly in liver mitochondria, we assessed the effect of competition with specific substrates or inhibitors on GSH uptake in isolated rat liver mitochondria. GSH uptake was uniphasic, independent of ATP hydrolysis, and exhibited K_m and V_max values of 4.08 mM and 3.06 nmol/min per mg protein, respectively. Incubation with butylmalonate and phenylsuccinate inhibited GSH uptake by 45-50%, although the individual inhibitors had no effect, suggesting in rat liver mitochondria, the DIC and OGC are only partially responsible for GSH uptake. H4IIE cells, a rat hepatoma cell line, were stably transfected with the cDNA for the OGC, and exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by H₂O₂, methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver.     

Phosphate transport,membrane potential,and movements of calcium in rat liver mitochondria

The membrane potential and calcium accumulation of mitochondria were followed by ion-specific electrodes in the presence of the proton-donor anions phosphate, acetate, glutamate, and beta-hydroxybutyrate. Phosphate was the only anion which allowed rapid and complete restoration of both the membrane potential and the steady-state extramitochondrial calcium concentration after the uptake of 100–200 nmol calcium per mg protein. If there was no influx of any proton-donor anion, the extent of calcium uptake depended on the intramitochondrial phosphate content. Both the fall of the membrane potential and the increase of the external calcium concentration brought about by a given amount of uncoupler were counteracted by phosphate transported into the mitochondria.