Reversible depression of oxygen consumption in isolated liver mitochondria during hibernation

The biochemical mechanisms by which hibernators cool as they enter torpor are not fully understood. In order to examine whether rates of substrate oxidation vary as a function of hibernation, liver mitochondria were isolated from telemetered ground squirrels (Spermophilus lateralis) in five phases of their annual hibernation cycle: summer active, and torpid, interbout aroused, entrance, and arousing hibernators. Rates of state 3 and state 4 respiration were measured in vitro at 25 degrees C. Relative to mitochondria from summer-active animals, rates of state 3 respiration were significantly depressed in mitochondria from torpid animals yet fully restored during interbout arousals. These findings indicate that a depression of ADP-dependent respiration in liver mitochondria occurs during torpor and is reversed during the interbout arousals to euthermia. Because this inhibition was determined to be temporally independent of entrance and arousal, it is unlikely that active suppression of state 3 respiration causes entrance into torpor by facilitating metabolic depression. In contrast to the observed depression of state 3 respiration in torpid animals, state 4 respiration did not differ significantly among any of the five groups, suggesting that alterations in proton leak are not contributing appreciably to downregulation of respiration in hibernation.     

Retention of oxidized glutathione by isolated rat liver mitochondria during hydroperoxide treatment

The addition of tert-butyl hydroperoxide (t-BuOOH) to isolated mitochondria resulted in oxidation of approximately 80% of the mitochondrial reduced glutathione (GSH) independently of the dose of t-BuOOH (1-5 mM). Concomitant with the oxidation of GSH inside the mitochondria was the formation of GSH-protein mixed disulfides (protein-SSG), with approximately 1% of the mitochondrial protein thiols involved. A dose-dependent rate of GSH recovery was observed, via the reduction of oxidized GSH (GSSG) and a slower reduction of protein-SSG. Although t-BuOOH administration affected the respiratory control ratio, the mitochondria remained coupled and loss of the matrix enzyme, citrate synthase, was not increased over the control and was less than 3% over 60 min. A slow loss of GSH out of the coupled non-treated mitochondria was not increased by t-BuOOH treatment, in fact, a dose-dependent drop of GSH levels occurred in the medium. However, no GSSG was found outside the mitochondria, indicating the necessary involvement of enzymes in the t-BuOOH-induced conversion of GSH to GSSG. The absence of GSSG in the medium also suggests that, unlike the plasma membrane, the mitochondrial membranes do not have the ability to export GSSG as a response to oxidative stress. Our results demonstrate the inability of mitochondria to export GSSG during oxidative stress and may explain the protective role of mitochondrial GSH in cytotoxicity.     

The pathway of inorganic-phosphate efflux from isolated liver mitochondria during adenosine triphosphate hydrolysis

1. The distribution of P_i between mitochondria and suspending medium during uncoupler-stimulated hydrolysis of ATP by rat liver mitochondria [Tyler (1969) Biochem. J. 111, 665–678] has been reinvestigated, by using either mersalyl or N-ethylmaleimide as inhibitors of P_i transport and either buffered sucrose/EDTA or LiCl/EGTA solutions as suspending medium. More than 75% of the total P_i liberated was retained in mitochondria treated with either inhibitor at all ATP concentrations tested (0.2–2.5mm). With low ATP concentrations and mersalyl-treated mitochondria incubated in sucrose/EDTA, virtually all the P_i liberated was retained in the mitochondria. 2. Larger amounts of P_i appeared in the suspending medium during ATPase activity, despite the presence of N-ethylmaleimide, when LiCl/EGTA was used as suspending medium compared with sucrose/EDTA. Two sources of this P_i were identified: (a) a slow efflux of P_i from mitochondria to suspending medium despite the presence of N-ethylmaleimide; (b) a slow ATPase activity insensitive to carboxyatractyloside, which was stimulated by added Mg²⁺, partially inhibited by oligomycin or efrapeptin and strongly inhibited by EDTA. 3. It is concluded that liver mitochondria preparations contain two distinct forms of ATPase activity. The major activity is associated with coupled mitochondria of controlled permeability to adenine nucleotides and P_i and is stimulated strongly by uncoupling agents. The minor activity is associated with mitochondria freely permeable to adenine nucleotides and P_i, is unaffected by uncoupling agents and is activated by endogenous or added Mg²⁺. 4. When mitochondria treated with mersalyl were incubated in buffered sucrose solution, almost all the P_i liberated was recovered in the suspending medium, unless inhibitors of P_i-induced large-amplitude swelling such as EDTA, EGTA, antimycin, rotenone, nupercaine or Mg²⁺ were added. Thus the loss of the specific permeability properties of the mitochondrial inner membrane associated with large-amplitude swelling also influences the extent of P_i retention during ATPase activity. 5. The results confirm the previous conclusion (Tyler, 1969) that the P_i transporter provides the sole pathway for P_i efflux during uncoupler-stimulated ATP hydrolysis by mitochondria. It is concluded that more recent hypotheses concerning the influence of Mg²⁺ on mersalyl inhibition of the P_i transporter [Siliprandi, Toninello, Zoccaroto & Bindoli (1975) FEBS Lett. 51, 15–17] and a postulated role of the adenine nucleotide exchange carrier in P_i efflux [Reynafarje & Lehninger (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4788–4792] are erroneous and should be discarded.     

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.     

Uptake of thiamin by isolated rat liver mitochondria

Investigation is made here of 14C-thiamin uptake by rat liver mitochondria in vitro. Following incubation with mitochondria, 14C-thiamin remained in the mitochondrial pellet in spite of several washings of the organelles. Accordingly, externally added thiamin produced intra/extra-mitochondrial concentration ratios up to 5.4 and 14C-thiamin space/3H2O space ratios higher than one. These ratios decreased with increasing vitamin concentrations, thus suggesting the occurrence of saturation characteristics for vitamin uptake into mitochondria. Thiamin was proven to enter both intermembrane and matrix spaces, where neither binding to intramitochondrial protein nor phosphorylation were found to occur. Moreover thiamin uptake inhibition by both metal ions and certain thiamin analogues was also found.     

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 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.     

Valinomycin-induced chloride permeability in isolated rat liver mitochondria

1. Ionophore-induced osmotic swelling was used to study Cl- transport in isolated rat liver mitochondria. 2. Energy-dependent, neutral ionophore-induced swelling in Cl- salts at pH 7.2 required K+ and was preceded by a brief lag phase that was absent in chlorotributyltin-induced swelling. 3. Treatments that stimulated or inhibited mitochondrial K+/H+ exchange had qualitatively similar effects on both valinomycin-induced swelling and the associated lag phase. 4. The results suggest that valinomycin-induced Cl- permeability results from an interaction between the K+/H+ antiporter and neutral ionophore K+ complexes.     

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.     

Gramicidin and ion transport in isolated liver mitochondria

1. Eight distinct acid-hydrolase activities present in cytoplasmic extracts from bone tissue occur in latent form to the extent of 50-70% of their total activity, depending on the enzyme. 2. This latency can be decreased or suppressed by exposure to Triton X-100 or to media of low osmotic pressure, by treatment in the Waring Blendor, and by freezing and thawing, but not by increasing the substrate concentration in the assay medium up to 10-fold the Michaelis constant of the enzymes. 3. Latency is the property of the particle-bound enzymes, and treatments that suppress latency simultaneously cause solubilization of the enzymes. Most enzymes show an excess of free over soluble activity; the magnitude of this excess seems to depend largely on the nature of the enzyme, and sometimes also on the kind of treatment suffered by the preparations; it is attributed mainly to adsorption artifacts. 4. In preparations subjected to graded activating treatments, seven of the eight acid hydrolases studied are released in closely parallel fashion, suggesting that they are associated with particles possessing similar properties. Acid phenylphosphatase is released less readily than the other enzymes by Triton X-100 and by exposure to media of low osmotic pressure. 5. It is concluded from these and previous published fractionation experiments that, with the possible exception of part of the acid-phenylphosphatase activity, the eight acid hydrolases studied belong to lysosome-like particles. Bone lysosomes exhibit a relatively high degree of biochemical and physical heterogeneity. Their possible functions are discussed. Part of the acid-phenylphosphatase activity could be linked to another group of particles. 6. Catalase is also partly (30%) latent in cytoplasmic extracts of bone. Latent catalase can be released by some of the treatments that suppress the latency of the lysosomal enzymes, but differs from the latter by a greater resistance to Triton X-100, and, especially, by a complete insensitivity to exposure to media of low osmotic pressure. It is concluded from these results that the catalase-containing particles are probably different from lysosomes, as they are in liver. 7. Cytochrome oxidase, which is presumably associated with the mitochondria, and alkaline phenylphosphatase, an enzyme occurring predominantly in the microsomal fraction, exhibited no latency under the conditions of the present experiments.