Ferruccio Guerrieri

Effect of acetaminophen administration on hepatic glutathione compartmentation and mitochondrial energy metabolism in the rat

Changes in cell energy metabolism and mitochondrial dysfunction have been observed after acetaminophen administration. Because consumption of hepatic glutathione is closely related to acetaminophen toxicity, we investigated the kinetics of: 1. glutathione depletion in liver mitochondria and cytosol; 2. State 3 and 4 respiratory rates of succinate-supplemented mitochondria; 3. rate of ATP synthesis; 4. oligomycin-sensitive ATP hydrolase activity and passive proton conductivity of inside-out vesicles of the inner mitochondrial membrane; and 5. changes in hepatic and mitochondrial malondialdehyde in the rat after in vivo acetaminophen administration. Two hours after acetaminophen injection, hepatic glutathione decreased and malondialdehyde increased. In the same interval, an increase in both State 3 and 4 respiratory rates of succinate-supplemented mitochondria was observed. This was accompanied by a decrease in the rate of ATP synthesis and the P/O ratio and by an increase in the passive proton permeability of the inner mitochondrial membrane, which was insensitive to oligomycin. No significant change in oligomycin-sensitive ATP hydrolase activity was observed. Four hours after APAP injection, the respiratory rates, as well as the proton conductivity, decreased, the rate of ATP synthesis was restored, and the mitochondrial glutathione started to increase; the cytosolic levels of glutathione were still low and the cytosolic and mitochondrial levels of malondialdehyde remained high for 2 more hr. The concentrations of these indices were completely restored 24 hr postdosing. Our findings suggest that acetaminophen administration selectively depletes (within 2 hr) mitochondrial glutathione, and produces local toxicity by altering membrane permeability and decreasing the efficiency of oxidative phosphorylation. This renders mitochondria more susceptible to oxidative damage, especially during increased free radical production, as in the case of enhanced mitochondrial respiration in State 4. The concomitant restoration of mitochondrial respiration, oxidative phosphorylation, membrane permeability, and glutathione levels is consistent with the importance of the mitochondrial glutathione pool for the protection of the mitochondrial membrane against oxidative damage.

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Kinetics of proton translocation and role of cations

Abstract The kinetics and mechanism of passive and active proton translocation in submitochondrial vesicles, obtained by sonication of beef heart mitochondria, have been studied. Analysis of the anaerobic release of the protons taken up by submitochondrial particles in the respiring steady state shows that proton diffusion consists of two parallel, apparent first-order processes: a fast reaction which, on the basis of its kinetic properties and response to cations and various effectors, is considered to consist of a proton/monovalent cation exchange; and a slow process which, on analogous grounds, is considered as a single electrogenic flux. The study of the various parameters of the respiration-linked active proton translocation and of the accompanying migration of permeant anions and K+ led to the following conclusions: (i) The oxidoreduction-linked proton translocation is electrogenic. (ii) Cation counterflow is not a necessary factor in the respiration-driven proton translocation. (iii) The membrane potential developed by active proton translocation exerts a coupling with respect to permeant cations and anions. (iv) The respiration-driven proton translocation is secondarily coupled, through the ΔμH component of the electrochemical proton gradient and at the level of a proton-cation exchange system of the membrane, to the flow of K+ and Na+.

Mitochondrial oxidative alterations following partial hepatectomy

Mitochondria, isolated from rat livers during the early phase of liver regeneration (7-24 h after partial hepatectomy), show: (i) decrease in the rate of ATP synthesis; (ii) increase of malondialdehyde and of oxidized protein production; (iii) decrease of the content of intramitochondrial glutathione and of protein thiols on mitochondrial proteins; (iv) increase of the glutathione bound to mitochondrial proteins by disulfide bonds. These observations suggest an increase of production of oxygen radicals in liver mitochondria, following partial hepatectomy, which can alter the function of the enzymes involved in the oxidative phosphorylation. Blue-native gel electrophoresis of rat liver mitochondria, isolated after partial hepatectomy, shows, during the early phase of liver regeneration (0-24 h after partial hepatectomy), a progressive decrease of the content of F0F1-ATP synthase complex. The amount of glutathione bound to the F0F1-ATP synthase, electroeluted from the blue-native gels, progressively increased during the early phase of liver regeneration. It is concluded that partial hepatectomy causes mitochondrial oxidative stress that, in turn, modifies proteins (such as F0F1-ATP synthase) involved in the mitochondrial oxidative phosphorylation.

Proton translocation and energy transduction in mitochondria

Summary Data are presented showing that respiration-linked proton translocation in the inner mitochondrial membrane consists of a vectorial, single, electrogenic flow, mediated by system(s) different from cation carriers. The proton pump is, however, secondarily coupled through the ΔpH component of the proton gradient and at the level of a proton-cation antiporter, to flow of Na + or K + . The redox proton pump appears to be directly coupled to oxido-reductions of respiratory carriers without the intervention of uncoupler-sensitive chemical intermediates. Two mechanism will be discussed for this direct coupling : (1) the oxido-reductase proton translocator of Mitchell ; (2) a so-called membrane Bohr effect, based on shifts of the pK of protonable groups of the apoprotein of electron carriers of the respiratory chain which accompany the redox changes of the electrony carrying metal centers.

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Analysis of proton translocation associated with oxidation of endogenous ubiquinol.

A study is presented of the kinetics and stoichiometry of fast proton translocation associated to aerobic oxidation of components of the mitochondrial respiratory chain. 1. Aerobic oxidation of ubiquinol and b cytochromes is accompanied in EDTA particles, obtained by sonication of beef-heart mitochondria, by synchronous proton uptake. 2. The rapid proton uptake associated to oxidation and b cytochromes is greatly stimulated by valinomycin plus K+, but is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. 3. 4 gion H+ are taken up per mol ubiquinol oxidized by oxygen. This H+/2e- ratio, measured in the rapid anaerobic-aerobic transition of the particles is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. 4. Intact mitochondria aerobic oxidation of oxygen-terminal electron carriers is accompanied by antimycin-insensitive synchronous proton release, oxidation of ubiquinol and reduction of b cytochromes. The amount of protons released is in excess with respect to the amount of ubiquinol oxidized. 5. It is concluded that electron flow along complex III, from ubiquinol to cytochrome c, is directly coupled to vectorial proton translocation. The present data suggest that there exist(s) between ubiquinol and cytochrome c one (or two) respiratory carrier(s), whose oxido-reduction is directly linked to effective transmembrane proton translocation.

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Analysis of proton translocation associated to oxidoreductions of the oxygen-terminal respiratory carriers

Abstract The kinetics and stoicheiometry of fast proton translocation associated to aerobic oxidation of the oxygen-terminal components of the mitochondrial respiratory chain have been analyzed by means of continuous- and stopped-flow techniques. 1. 1. In intact mitochondria the aerobic oxidation of the respiratory carriers situated on the oxygen side of the antimycin site was accompanied by synchronous release of protons. When the proton conductivity of the membrane was increased by carbonyl cyanide p -trifluoromethoxyphenylhydrazone, oxidation of the terminal respiratory carriers was accompanied by stoicheiometric consumption of protons. This proton disappearance from the medium was, however, much slower than the oxidation of the respiratory carriers. 2. 2. In sonic sub-mitochondrial particles oxidation of the terminal respiratory carriers was accompanied by synchronous and stoicheiometric proton consumption. This proton uptake was practically unaffected by carbonyl cyanide p -trifluoromethoxyphenylhydrazone; its rate was markedly increased by valinomycin plus K + . 3. 3. The results presented provide functional evidence that cytochrome oxidase is a transmembranous molecule with haeme a 3 reacting with oxygen at the matrix side of the inner mitochondrial membrane and haeme a reacting with cytochrome c at the outer side. 4. 4. The fast proton release accompanying the oxidation of the terminal respiratory carriers in intact mitochondria appears to be associated to antimycin-insensitive oxidation of a hydrogen carrier.

A Possible Role of Slips in Cytochrome C Oxidase in the Antioxygen Defense System of the Cell

Evidence is available showing that the coupling efficiency of the proton pump in cytochrome c oxidase of mitochondria can under certain conditions decrease significantly below the maximum attainable value. The view is developed that slips in the proton pump of cytochrome c oxidase represent an intrinsic switch mechanism which regulates the relative contribution of energy transfer and respiratory protection against oxygen toxicity by the oxidase.

Redox Bohr-effects in the cytochrome system of mitochondria.

Redox titrations show that the midpoint potential (E;,) of various electron carriers in coupling membranes decreases as the pH of the reaction medium is raised within certain ranges [ 1 J. As far as the cytochrome system of mitochondria is concerned the b cytochromes [2-41, the heme groups of cytochrome c oxidase (5-71 and the Rieske Fe-sulfur center, g 1.90 (8 J have been shown to exhibit such a property. This feature indicates the occurrence in electron carriers of cooperative linkage between electron transfer by the metal and proton transfer by ionizable groups, possibly in the apoprotein, owing to increase of their pK upon reduction ([ 1,3,9] cf. [lo]). Thus at pH values in the order of the pK, values oxidation of the electron carrier should result in the release of protons and its reduction in the uptake of protons [9,1 I]. These considerations, as well as the inherent ambig~ties of redox titrations of membr~e bound systems [IO], has led us to investigate these linkage phenomena denominated, by analogy to those described for hemoglobin [12,13], redox Bohr effects or membrane Bohr effects [ 141 by an independent approach based on direct measurements of scalar proton release and uptake associated to redox transitions of electron carriers in the respiratory chain of mitochondria. The results presented here show that this approach provides direct demonstration for the occurrence of redox Bohr effects in the cytochrome system and is particularly suitable for their characterization.

ATPase activity, IF1 content, and proton conductivity of ESMP from control and ischemic slow and fast heart-rate hearts.

Earlier studies by Rouslin and coworkers showed that, during myocardial ischemia in slow heart-rate species which include rabbits and all larger mammals examined including humans, there is an IF1-mediated inhibition of the mitochondrial ATPase due to an increase in the amount of IF1, bound to the ATPase (Rouslin, W., and Pullman, M.E.,J. Mol. Cell. Cardiol.19, 661–668, 1987). Earlier work by Guerrieri and colleagues demonstrated that IF1 binding to bovine heart ESMP was accompanied by parallel decreases in ATPase activity and in passive proton conduction (Guerrieri, F.,et al., FEBS Lett.213, 67–72, 1987). In the present study rabbit was used as the slow heart-rate species and rat as the fast heart-rate species. Rat is a fast heart-rate species that contains too little IF1 to down regulate the ATPase activity present. Mitochondria were prepared from control and ischemic hearts and ESMP were made from aliquots by sonication at pH 8.0 with 2 mM EDTA. Oligomycin-sensitive ATPase activity and IF1 content were measured in SMP prepared from the control and ischemic mitochondrial samples. After identical incubation procedures, oligomycin-sensitive ATPase activity, oligomycin-sensitive proton conductivity, and IF1 content were also measured in ESMP samples. The study was undertaken to corroborate further what appear to be fundamental differences in ATPase regulation between slow and fast heart-rate mammalian hearts evident during total myocardial ischemia. Thus, passive proton conductivity was used as an independent measure of these regulatory differences. The results show that, consistent with the low IF1 content of rat heart cardiac muscle mitochondria, control rat heart ESMP exhibit approximately twice as much passive proton conductivity as control rabbit heart ESMP regardless of the pH of the incubation and assay. Moreover, while total ischemia caused an increase in IF1 binding and a commensurate decrease in passive proton conductivity in rabbit heart ESMP regardless of pH, neither IF1 content nor proton conductivity changed significantly in rat heart ESMP as a result of ischemia.

On the proton translocation system of the inner mitochondrial membrane

Oxygen or ATP pulses cause a reversible pH decrease in mitochondriaI suspension 1 I, 21. In submitochon&a\ partides, obttin& by sonjcaljon and consjsling of vescicles of the inner mitochondrial membrane turlnect “inside out” I33 , respiration UC ATP hydrolysis causes proton movements in the reverse direction [4-61 Tnis inversion of poiariry, and of!rservarions on iniramitochondrial pH changes [7-91 indicate that the energy-linked pH changes represent, at least in part, effective translocation of protons across the inner mitochondrial membrane. Potassium salts stimulate the respiration-driven proton uptake in sonic particles [6]. This effect is potentiated by valinomycin, indicating that, at least in the presence of this antibiotic, the stimulation of proton uptake is due to K’ translocation (cf. [ 1 O] ). Since, however, the activity of r-salts varies with the anions used, these must also be involved. In this paper a study of the effect of a series of salts on proton translocation in submitochondrial particles, is presented.