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.     

The interaction of MgADP with H-ATPase in rat liver mitochondria

The activating anions are found to induce an unexpectedly high (up to 8-fold for sulphite) increase of ATPase activity in intact rat liver mitochondria. This effect is not determined by the observed changes in K_m and K_i (ADP) values. The stimulation seems to be caused by dissociation of the inactive complex of ATPase with Mg·ADP. The quantity of this complex formed in the course of ATP hydrolysis is approx. 90% of the total ATPase content in intact mitochondria. The data on toluene-permeabilized mitochondria suggest that the high content of the complex is a result of the stabilizing effect of some matrix macromolecules.     

Control of oxidative phosphorylation by Complex I in rat liver mitochondria: implications for aging

We compared NAD-dependent state 4 and state 3 respiration, NADH oxidation and Complex I specific activity in liver mitochondria from 4- and 30-month-old rats. All the activities examined were significantly decreased with aging. In both groups of animals, the flux control coefficients measured by rotenone titration indicated that Complex I is largely rate controlling upon NADH aerobic oxidation while, in state 3 respiration, it shares the control with other steps in the pathway. Moreover, we observed a trend wherein flux control coefficients of Complex I became higher with age. This indication was strengthened by examining the rotenone inhibition thresholds showing that Complex I becomes more rate controlling, over all the examined activities, during aging. Our results point out that age-related alterations of the mitochondrial functions are also present in tissues considered less prone to accumulate mitochondrial DNA mutations.     

Chenodeoxycholate is a potent inducer of the permeability transition pore in rat liver mitochondria

Several reports support the concept that bile acids may be cytotoxic during cholestatic disease process by causing mitochondrial dysfunction. Here we report additional data and findings aimed at a better understanding of the involvement of the permeability transition pore (PTP) opening in bile acids toxicity. The mitochondrial PTP is implicated as a mediator of cell injury and death in many situations. In the presence of calcium and phosphate, chenodeoxycholic acid (CDCA) induced a permeability transition in freshly isolated rat liver mitochondria, characterized by membrane depolarization, release of matrix calcium, and osmotic swelling. All these events were blocked by cyclosporine A (CyA) and the calcium uniporter inhibitor ruthenium red (RR). The results suggest that CDCA increases the sensitivity of isolated mitochondria in vitro to the calcium-dependent induction of the PTP.     

Inhibition of sulfate-forming activity in rat liver mitochondria by (aminooxy)acetate

Summary The effect of (aminooxy)acetate, an inhibitor of aminotransferases, on the sulfate formation froml-cysteine andl-cysteinesulfinate in rat liver mitochondria was studied. Incubation of 10 mMl-cysteine with rat liver mitochondria at 37°C in the presence of 10 mM 2-oxoglutarate and 10 mM glutathione resulted in the formation of 4.60 and 1.52µmol of sulfate and thiosulfate, respectively, per 60 min per mitochondria obtained from 1 g of liver. Under the same conditions sulfate formation froml-cysteinesulfinate was 24.96µmol, but thiosulfate was not formed. The addition of (aminooxy)acetate at 2 mM or more completely inhibited the sulfate and thiosulfate formation froml-cysteine and the sulfate formation froml-cysteinesulfinate. These findings support our previous conclusion that cysteine transamination and 3-mercaptopyruvate pathway (MP pathway) are involved in the sulfate formation froml-cysteine in rat liver mitochondria (Ubuka et al., 1992).     

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO₃ and KNO₃ resulted in the Tl⁺-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca²⁺ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl⁺-induced MPTP opening in the inner membrane of Ca²⁺-loaded rat liver mitochondria.     

Studies on the mobilization of iron from ferritin by isolated rat liver mitochondria

Rat liver mitochondria and rat liver mitoplasts mobilize iron from ferritin by a mechanism which depends on a respiratory substrate (preferentially succinate), a small molecular weight electron mediator (FMN, phenazine methosulphate or methylene blue) and (near) anaerobic conditions.The release process under optimized conditions (approx. 50 μmol/l FMN, 1 mmol/l succinate, 0.35 mmol/l Fe(III) (as ferritin iron), 37°C and pH 7.40) amounts to 0.9–1.2 nmol iron/mg protein per min.The results suggest that ferritin might function as an intermediate in the cytosolic transport of iron to the mitochondria.     

On the mechanism of action of polyether XXVIII at site I of the electron-transfer chain in rat liver mitochondria

In rat liver mitochondria, the macrocyclic polyether, dibenzo-18-crown-6 (polyether XXVIII) inhibits the oxidation of NAD-dependent substrates, as stimulated by ADP, uncouplers and valinomycin plus K⁺. It does not inhibit the oxidation of succinate. It is concluded that polyether XXVIII inhibits electron transfer in the NADH-CoQ span of the respiratory chain. This is a process that is reversed by menadione. Inhibition of oxidation of NAD-dependent substrates in K⁺-depleted mitochondria induced by the polyether is reversed by concentrations of K⁺ higher than 60 mM, and also by Li⁺, a cation that does not complex with polyether XXVIII. As assayed by swelling mitochondria, reversal of the inhibition of electron transfer is accompanied by influx of monovalent cations. Polyether XXVIII also inhibits in submitochondrial particles the aerobic oxidation of NADH, but not that of succinate; this inhibition is also reversed by K⁺ at high concentrations, and Li⁺. The data are consistent with the hypothesis that a monovalent cation is required for maximal rates of electron transport in the NADH-CoQ span of the respiratory chain.     

The targets of 2,2',5,5'-tetrachlorobiphenyl in the respiratory chain of rat liver mitochondria revealed by modular kinetic analysis

The response of the respiratory subsystem of oxidative phosphorylation to the environmental pollutant, 2,2',5,5'-tetrachlorobiphenyl (2,2',5,5'-TCB) was investigated by modular kinetic approach. The effects of 20 M 2,2',5,5'-TCB on the activity of the respiratory chain modules in rat liver mitochondria oxidizing succinate (+ rotenone) in state 3 were assessed. The toxin inhibited the rate of respiration by 23%. Analysis around cytochrome c revealed that 2,2',5,5'-TCB inhibited both cytochrome c-oxidizing and - reducing modules. The toxin inhibited also CoQ-oxidizing module, however it did not affect the kinetics of CoQ-reducing module. Taken together, these data indicated that 2,2',5,5'-TCB inhibited cytochrome bc₁ but had no effect on succinate dehydrogenase.     

Electroneutral H+-K+ exchange in liver mitochondria Regulation by membrane potential

The paper analyzes the factors affecting the H⁺-K⁺ exchange catalyzed by rat liver mitochondria depleted of endogenous Mg²⁺ by treatment with the ionophore A23187. The exchange has been monitored as the rate of K⁺ efflux following addition of A23187 in low-K⁺ media. (1) The H⁺-K⁺ exchange is abolished by uncouplers and respiratory inhibitors. The inhibition is not related to the depression of ΔpH, whereas a dependence is found on the magnitude of the transmembrane electrical potential, Δψ. Maximal rate of K⁺ efflux is observed at 180–190 mV, whereas K⁺ efflux is inhibited below 140–150 mV. (2) Activation of H⁺-K⁺ exchange leads to depression of ΔpH but not of Δψ. Respiration is only slightly stimulated by the onset of H⁺-K⁺ exchange in the absence of valinomycin. These findings indicate that the exchange is electroneutral, and that the Δψ control presumably involves conformational changes of the carrier. (3) Incubation in hypotonic media at pH 7.4 or in isotonic media at alkaline pH results in a marked activation of the rate of H⁺-K⁺ exchange, while leaving unaffected the level of Mg²⁺ depletion. This type of activation results in partial ‘uncoupling’ from the Δψ control, suggesting that membrane stretching and alkaline pH induce conformational changes on the exchange carrier equivalent to those induced by high Δψ. (4) The available evidence suggests that the activity of the H⁺-K⁺ exchanger is modulated by the electrical field across the inner mitochondrial membrane.     

Heavy metal intracellular balance and relationship with metallothionein induction in the liver of carp after contamination by silver,cadmium and mercury following or not pretreatment by zinc

Determination of metal levels (copper, zinc, cadmium, silver and mercury) in soluble and insoluble fractions of liver homogenates has been performed after 7 days exposure of carps (Cyprinus carpio) to moderate concentrations of cadmium, silver and mercury in water. Metallothionein (MT) levels have been quantified by a polarographic method before and after the contamination and a subsequent decontamination phase (7 days). The influence of pretreatment by zinc (7 days) has also been evaluated. MT level variations have been interpreted as having regard to inter-related flows of metal between subcellular fractions. Special interest has been focused on heat-stable compound (HSC)-bound heavy metal flows within the cytosol, taking in account that MT is the major component of these ligands. Our data showed differences between the ability of metals to bind cytosolic ligands and HSCs, and their respective potency for MT induction in liver. Regardless of pretreatment, mercury gave the highest increase of liver MT, but the MT level decreased during the decontamination step, especially after pretreatment by zinc. Cadmium and silver gave similar increases, but a significant difference with the control appeared only after the decontamination step with cadmium, while 1 week of contamintion was enough for silver. However, silver binding with MT was achieved only by the end of the decontamination step, while cadmium depicted the highest ratio for HSC-bound toxic metals after the contamination. Our experimental conditions gave the following order of potency for MT induction in liver: mercury silver > cadmium > zinc. Results are discussed comparatively with data obtained with carp gills.     

Biological effects of anionic meso-tetrakis (para-sulfonatophenyl) porphyrins modulated by the metal center. Studies in rat liver mitochondria

In this paper, we present a study about the influence of the porphyrin metal center and meso ligands on the biological effects of meso-tetrakis porphyrins. Different from the cationic meso-tetrakis 4-N-methyl pyridinium (Mn(III)TMPyP), the anionic Mn(III) meso-tetrakis (para-sulfonatophenyl) porphyrin (Mn(III)TPPS4) exhibited no protector effect against Fe(citrate)-induced lipid oxidation. Mn(III)TPPS4 did not protect mitochondria against endogenous hydrogen peroxide and only delayed the swelling caused by tert-BuOOH and Ca²⁺. Fe(III)TPPS4 exacerbated the effect of the tert-BuOOH, and both porphyrins did not significantly affect Fe(II)citrate-induced swelling. Consistently, Fe(III)TPPS4 predominantly promotes the homolytic cleavage of peroxides and exhibits catalytic efficiency ten-fold higher than Mn(III)TPPS4. For Mn(III)TPPS4, the microenvironment of rat liver mitochondria favors the heterolytic cleavage of peroxides and increases the catalytic efficiency of the manganese porphyrin due to the availability of axial ligands for the metal center and reducing agents such as glutathione (GSH) and proteins necessary for Compound II (oxomanganese IV) recycling to the initial Mn(III) form. The use of thiol reducing agents for the recycling of Mn(III)TPPS4 leads to GSH depletion and protein oxidation and consequent damages in the organelle.