The action of defoliants on the energetics of rat liver mitochondria in vitro]

The effect of defoliants butyphos (I), dropp (II), butylcaptacs (III), hinazopin (IV), syhat (V), tetra-n-butylammonium bromide (VI), etrel (VII), gemetrel (VIII), allyl-4-methylpyridinium bromide (IX), 1-amino-cyclopropan-1-carbonate (ACPC) (X) at various concentrations (1 x 10(-5)-2 x 10(-4) M) on respiration, oxidative phosphorylation (OP) and permeability of the inner mitochondrial membrane from rat liver has been studied. It has been established that some of the compounds uncouple OP by increasing the inner mitochondrial membrane permeability for H+ (II) inhibit the respiration in V3 condition and induce less selective permeability for a number of ions (I, III). The other defoliants either induce respiration generally in metabolic states 3 and 4 (IV, VI, IX) or have no effect on the respiration and OP (V, VII, VIII, X). On the whole a good correlation between the common toxicity of the studied preparation (LD50) and their mitochondrial effect has been revealed, therefore the latter can be considered as intracellular "targets" involved in the realization of pesticide action.     

The effect of bile salts and calcium on isolated rat liver mitochondria

Intact mitochondria were incubated with and without calcium in solutions of chenodeoxycholate, ursodeoxycholate, or their conjugates. Glutamate dehydrogenase, protein and phospholipid release were measured. Alterations in membrane and organelle structure were investigated by electron paramagnetic resonance spectroscopy. Chenodeoxycholate enhanced enzyme liberation, solubilized protein and phospholipid, and increased protein spin label mobility and the polarity of the hydrophobic membrane interior, whereas ursodeoxycholate and its conjugates did not damage mitochondria. Preincubation with ursodeoxycholate or its conjugate tauroursodeoxycholate for 20 min partially prevented damage by chenodeoxycholate. Extended preincubation even with 1 mM ursodeoxycholate could no longer prevent structural damage. Calcium (from 0.01 mM upward) augmented the damaging effect of chenodeoxycholate (0.15-0.5 mM). The combined action of 0.01 mM calcium and 0.15 mM chenodeoxycholate was reversed by ursodeoxycholate only, not by its conjugates tauroursodeoxycholate and glycoursodeoxycholate. In conclusion, ursodeoxycholate partially prevents chenodeoxycholate-induced glutamate dehydrogenase release from liver cell mitochondria by membrane stabilization. This holds for shorter times and at concentrations below 0.5 mM only, indicating that the different constitution of protein-rich mitochondrial membranes does not allow optimal stabilization such as has been seen in phospholipid- and cholesterol-rich hepatocyte cell membranes, investigated previously.     

Effects of gamma-irradiation on isolated rat liver mitochondria

Gamma-irradiation of isolated rat liver mitochondria with doses of up to 475 Gy leading to hydrated electrons (G = 1.9, corrected for reaction with solutes), 30 Gy leading to carbohydrate radicals, (G = 5.6), 100 Gy leading to superoxide radicals (G = 6.2), and 130 Gy leading to formate radicals (G = 6.2) showed, within the error of the measurements, no effects on the rate of oxygen uptake in the various respiratory states, the respiratory control ratio, or the adenosine diphosphate to atomic oxygen ratio. Typical values obtained were 0.020-0.100 nmol O2 s-1 mg protein-1 for State 1 respiration, 0.25-0.33 nmol O2 s-1 mg protein-1 for State 4 respiration and 0.65-1.10 nmol O2 s-1 mg protein-1 for State 3 respiration. Typical respiratory control ratios ranged from 2.0-3.5 for succinate and 4.0-6.5 for a 1:1 glutamate: malate substrate mixture. Adenosine diphosphate to atomic oxygen ratios with succinate as substrate varied from 1.6 to 1.9. Because these results are unexpected, in situ and in vitro irradiated mitochondria were examined in an electron microscope and compared to mitochondria in situ, non-irradiated mitochondria and mitochondria isolated after whole liver irradiation. Irradiation of isolated mitochondria with 375 Gy results in the partial destruction of the mitochondrial outer membrane with no significant changes in respiratory rates.     

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.     

The oxidation of fatty acids combined with albumin by isolated rat liver mitochondria

Long chain fatty acids at concentrations inhibiting mitochondrial respiration were, in the presence of serum albumin, found to produce almost as high a rate of oxygen uptake as alpha-ketoglutarate, succinate, or acetate. This oxidation was characterized in terms of its coupling to phosphorylation, need for cofactors, and production of different metabolites during the reactions. Fatty acids were oxidized to carbon dioxide, acetoacetate, beta-hydroxybutyrate, and other water-soluble metabolites, tentatively identified as intermediates of the citric acid cycle. An agent to spark the citric acid cycle and adenosine tri- or monophosphate were necessary for optimal oxidation rate, as described for other fatty acid oxidation systems. Balance experiments with different amounts of malate were performed with incubations lasting as long as oxygen uptake took place. In the presence of 1 mumole of malate, practically all added palmitic acid was used up and found to be converted primarily to carbon dioxide, acetoacetate, and other water-soluble metabolites of which the major part was tentatively identified as succinate. A significant portion was found in mitochondrial phospholipids. With 10 mumoles of malate some palmitic acid remained in the system, while a comparatively small amount was converted to carbon dioxide, and a major part was found as succinate. Here also incorporation into phospholipids occurred. With no malate added, fatty acid oxidation was much smaller than with malate, although significant conversion to carbon dioxide took place. Only a little succinate and phospholipid were found. Oxygen uptake was greater than a theoretical value calculated from radioactive balance experiments. It was concluded that albumin contains oxidizable material even after extraction and dialysis. Albumin at high concentrations inhibited both fatty acid and alpha-ketoglutarate oxidation. The oxidation of long chain fatty acids in high concentrations in the form of albumin-fatty acid complex was coupled to phosphorylation. Thus P:O ratios above 2 were found as well as evidence for respiratory control. It was concluded that oxidation of long chain fatty acids by isolated mitochondria occurs from their albumin complex. This process can also be studied at high concentrations of fatty acids, where high rates of oxygen uptake are obtained from oxidation which is coupled to phosphorylation.     

Ammonia formation in isolated rat liver mitochondria

Studies of isolated rat liver mitochondria were undertaken in order to evaluate the importance of glutamate transport, oxidation reduction state, and product inhibition on the rates of formation of ammonia from glutamate. Uptake and efflux of glutamate across the mitochondrial membrane were measured isotopically in the presence of rotenone. Efflux was stimulated by H+ in the mitochondrial matrix and was found to be first order with respect to matrix glutamate except when the matrix pH was unphysiologically low. The data suggest that the Km of matrix glutamate for efflux is decreased by H+. Matrix H+ also appeared to stimulate glutamate uptake, but the effect was to increase both the Km of medium glutamates and Vmax. Mitochondria were incubated at 15 and 28 degrees C with glutamate and malonate. Under these conditions, glutamate was metabolized only by the deamination pathway. Flux was evaluated by assay of ammonia formation. Oxidation reduction state was varied with ADP and uncoupling agents. Matrix alpha-ketoglutarate was varied either by the omission of malonate from the incubation media or by adding alpha-ketoglutarate to the external media. Influx and efflux of glutamate could be calculated from previously determined transport parameters. The difference between calculated influx and efflux was found to be equal to ammonia formation under all conditions. It was, therefore, possible to evaluate the relative contributions of oxidation reduction state, transport, and product inhibition as effectors of ammonia formation. The contribution of transport was relatively small while oxidation reduction state exerted a large influence. alpha-Ketoglutarate was found to be a potent competitive inhibitor of ammonia production and glutamate dehydrogenase. Inhibition of glutamate dehydrogenase by alpha-ketoglutarate was judged to be a potentially important modulator of metabolic fluxes.     

Metformin promotes isolated rat liver mitochondria impairment

Metformin, a drug widely used in the treatment of type 2 diabetes, has recently received attention due to the new and contrasting findings regarding its effects on mitochondrial function. In the present study, we evaluated the effect of metformin in isolated rat liver mitochondria status. We observed that metformin concentrations ≥8 mM induce an impairment of the respiratory chain characterized by a decrease in RCR and state 3 respiration. However, only metformin concentrations ≥10 mM affect the oxidative phosphorylation system by decreasing the mitochondrial transmembrane potential and increasing the repolarization lag phase. Moreover, our results show that metformin does not prevent H₂O₂ production, neither protects against lipid peroxidation induced by the pro-oxidant pair ADP/Fe²⁺. In addition, we observed that metformin exacerbates Ca²⁺-induced permeability transition pore opening by decreasing the capacity of mitochondria to accumulate Ca²⁺ and increasing the oxidation of thiol groups. Taken together, our results show that metformin can promote liver mitochondria injury predisposing to cell death. Cristina Carvalho and Sónia Correia contributed equally to this work.     

Arginine uptake by isolated rat liver mitochondria

The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-¹⁴C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [¹⁴C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin-¹⁴C]carboxylic acid or [¹⁴C]sucrose There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH₄Cl, glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0°C. Mitochondria did not concentrate citrulline.     

Effects of Cd2+ and two cadmium organic complexes on isolated rat liver mitochondria

Effects of Cd2+ and two complexes of bivalent cadmium with 1,3-bis(4-chlorbenzylidenamino)-guanidine and anabasine on ion permeability of the inner membrane and respiration of isolated rat liver mitochondria were studied. Starting from 5 microM, Cd2+ decreased state 3 and DNP-stimulated respiration of mitochondria and increased their state 4 respiration. At 30 microM, Cd2+ decreased state 4 respiration. The complexes, particularly complex of Cd2+ with 1,3-bis(4-chlorbenzylidenamino)-guanidine, inhibited the mitochondrial respiration at lower concentration of Cd2+. Nonenergized mitochondria incubated in media containing 125 mM of NH4NO3 or KNO3 showed more pronounced swelling in experiments with 10 microM of the complexes than with Cd2+. The complexes produced swelling of the mitochondria energized by 5 mM of succinate and incubated in medium containing 25 mM K-acetate and 100 mM sucrose. Uptake of 137-Cs by succinate-energized mitochondria in the presence of 10(-8) M of valinomycin was substantially decreased in experiments with 10 microM of the complexes than with Cd2+. Ruthenium red (7.5 microM) prevented this effect with 10 microM of complex of Cd2+ with 1,3-bis(4-chlorbenzylidenamino)-guanidine and especially complex of Cd2+ with anabasine and Cd2+. These results indicate that the cadmium organic complexes affect respiration and perturb ion permeability significantly stronger than Cd2+.     

Porphyrin-sensitized photodynamic damage of isolated rat liver mitochondria

The respiration rates and the respiratory control ratios of isolated rat liver mitochondria have been measured following exposure to 0–160 kJ/m² of near-ultraviolet radiation (blacklight) in the presence of low concentrations of porphyrins (0.1–0.2 μmol/l).

Depending on the light dose, the concentration and the type of porphyrin, the following sequence of reactions occurred: uncoupling and inhibition of oxidative phosphorylation, energy dissipation, inhibition of respiration and swelling and disruption of the mitochondria.

The detrimental effects could not be elicited in the absence of oxygen, neither could they be elicited by porphyrins or light alone.

At equimolar concentrations, the effectiveness of the porphyrins as photosensitizers were: deuteroporphyrin > protoporphyrin coproporphyrin > murophorphyrin.

The results may be of importance to explain the skin lesions seen when porphyrins of different hydrophobicity accumulate in the skin.     

Effects of piperine on enzyme activities and bioenergetic functions in isolated rat liver mitochondria and hepatocytes

The influence of piperine on the enzymes and bioenergetic functions in isolated rat liver mitochondria and hepatocytes was studied. Piperine at lower concentrations (<50 μM) did not affect the RCR and ADP:O ratios, state 4 and 3 respirations supported by site-specific substrates, viz. glutamate + malate, succinate, and ascorbate + TMPD. The site-specific effects became significantly apparent only at higher concentrations. Only the state 3 respiration supported by NAD-linked substrates was impaired equipotently in mitochondria and permeabilized hepatocytes; the effect appeared to be localized at energy-coupling site 1. In hypotonic treated mitochondria, respiration supported by three kinds of substrates was not affected. Among the respiratory chain-linked enzymes, the activity of NADH-dehydrogenase registered a significant decrease of about 25, 42, and 53% at 100, 150, and 180 μM piperine, respectively. The activity of Mg⁺⁺-ATPase, however, was stimulated at concentrations above 150 μM. Among the matrix enzymes, only malate and succinate dehydrogen-ases were studied. Malate dehydrogenase only showed a strong concentration-related inhibition in both the forward and backward directions. Enzyme kinetics indicated noncompetitive inhibition with a very low Ki of 10 μM. The presence of unsaturated double bonds in the side chain of piperine appeared essential for producing this strong inhibition. The studies suggested that piperine produces concentration related site-specific effects on mitochondrial bioenergetics and enzymes of energy metabolism.