Michele Lorusso

Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function.

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs

Arachidonic acid interaction with the mitochondrial electron transport chain promotes reactive oxygen species generation.

A study has been carried out on the interaction of arachidonic acid and other long chain free fatty acids with bovine heart mitochondria. It is shown that arachidonic acid causes an uncoupling effect under state 4 respiration of intact mitochondria as well as a marked inhibition of uncoupled respiration. While, under our conditions, the uncoupling effect is independent of the fatty acid species considered, the inhibition is stronger for unsaturated acids. Experiments carried out with mitochondrial particles indicated that the arachidonic acid dependent decrease of the respiratory activity is caused by a selective inhibition of Complex I and III. It is also shown that arachidonic acid causes a remarkable increase of hydrogen peroxide production when added to mitochondria respiring with either pyruvate+malate or succinate as substrate. The production of reactive oxygen species (ROS) at the coupling site II was almost double than that at site I. The results obtained are discussed with regard to the impairment of the mitochondrial respiratory activity as occurring during the heart ischemia/reperfusion process.

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+.

Role of mitochondria and reactive oxygen species in dendritic cell differentiation and functions

Dendritic cells (DC) are potent antigen-presenting cells capable of inducing T and B responses and immune tolerance. We have characterized some aspects of energy metabolism accompanying the differentiation process of human monocytes into DC. Compared to precursor monocytes, DC exhibited a much larger number of mitochondria and consistently (i) a higher endogenous respiratory activity and (ii) a more than sixfold increase in ATP content and an even larger increase in the activity of the mitochondrial marker enzyme citrate synthase. The presence in the culture medium of rotenone, an inhibitor of the respiratory chain Complex I, prevented the increase in mitochondrial number and ATP level, without affecting cell viability. Rotenone inhibited DC differentiation, as revealed by the observation that the expression of CD1a, which is a specific surface marker of DC differentiation, was strongly reduced. Cells cultured in the presence of rotenone displayed a lower content of growth factor-induced, mitochondrially generated, hydrogen peroxide. A similar drop in ROS was observed upon addition of catalase, which caused functional effects similar to those produced by rotenone treatment. These results suggest that ROS play a crucial role in DC differentiation and that mitochondria are an important source of ROS in this process.

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.

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.

Arachidonic acid induces specific membrane permeability increase in heart mitochondria

Micromolar concentrations of arachidonic acid cause in Ca2+ loaded heart mitochondria matrix swelling and Ca2+ release. These effects appear to be unrelated to the classical membrane permeability transition (MPT), as they are CsA insensitive, membrane potential independent and can also be activated by Sr2+. Atractyloside potentiated and ATP inhibited the arachidonic acid induced swelling. These observations suggest that the ATP/ADP translocator (ANT) may be involved in the AA induced, CsA insensitive membrane permeability increase. Under the same experimental conditions used for heart mitochondria, arachidonic acid induced the classical CsA sensitive, ADP inhibitable MPT in liver mitochondria.

Action of local anaesthetics on passive and energy-linked ion translocation in the inner mitochondrial membrane

The effect of dibucaine on passive and respiration-driven ion translocation and oxidative phosphorylation in submitochondrial particles from beef-heart has been studied.Dibucaine inhibited the nigericin-mediated H+/K+ exchange diffusion and the electrogenic, valinomycin-mediated K+ translocation in submitochondrial particles.The local anaesthetic exerted a direct stimulatory effect on the respiration-driven proton uptake and on the passive proton-diffusion reactions. The increase of the respiration-linked proton turnover caused by dibucaine was accompanied by uncoupling of oxidative phosphorylation. It is concluded that spontaneous noncoupled as well as ionophoremediated K+ translocation in mitochondria occurs across phospholipid bilayer regions of the membrane whilst other components of the membrane would be specifically involved in active and passive proton translocation across the membrane.The results indicate that polar groups of membrane phospholipids play an important role in energy conservation and transfer in the mitochondrial membrane.