Marcantonio Bragadin

Proton electrochemical gradient and rate of controlled respiration in mitochondria.

The correlation between deltamuH, the proton electrochemical potential difference, and the rate of controlled respiration is analyzed. deltamuH (the proton concentration gradient) is measured on the distribution of [3H]acetate, and deltapsi (the membrane potential) on the distribution of 86Rb+, 45Ca2+ and [3H]triphenylmethylphosphonium used either alone or simultaneously. The effects of the addition of ADP + hexokinase (state-3 ADP) and of carbonylcyanide trifluoromethoxyphenylhydrazone (state-3 uncoupler) on respiration and deltamuH are not equivalent: the uncoupler depresses deltamuH more than ADP at equivalent respiratory rates. The effects of the additions of nigericin-valinomycin and of ionophore A23187 (state-3 cation transport) and of carbonylcyanide trifluoromethoxy-phenylhydrazone (state 3-uncoupler) on respiration and deltamuH are also not equivalent: the uncoupler depresses deltamuH more than A23187 and nigericin + valinomycin at equivalent respiratory rate. A23187 is very efficient in stimulating respiration with negligible deltamuH changes.

Cobalt induces oxidative stress in isolated liver mitochondria responsible for permeability transition and intrinsic apoptosis in hepatocyte primary cultures

It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process. Cobalt, in the ionized species Co(2+), is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co(2+) induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca(2+) transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co(2+) also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent DeltaPsi drop, suggesting that Co(2+) also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co(2+) of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co(2+) also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co(2+) results in apoptosis induction with caspase activation and increased level of expression of HIF-1alpha. All these observations allow us to state that, in the presence of calcium, Co(2+) is an inducer of apoptosis triggered by mitochondrial oxidative stress.

H+/site ratio and steady state distribution of divalent cations in mitochondria.

Mitochondria as well as bacteria are known to operate as proton pumps, i.e. the metabolic force is used to transport primarily protons across the membrane. The primary transport of protons is then coupled through various mechanisms to the movement of other species, cations, anions and water. The mechanism of proton transport has been assumed to occur via either respiratory loops [ 1 ] or membrane Bohr effect [2,3] or H’ carrier or channel [4] . Since the membrane Bohr effect and the proton channel may accomodate a stoicheometry of 4 H’/ site while the respiratory loops are compatible with a ratio only of 2 H/site, much interest has been concentrated over the exact determination of the H/site ratio. While Mitchell and Moyle [5 ] reported a ratio of 2 H/site, recently Lehninger and associates [6,7] reported that when the movement of endogenous phosphate is restricted a ratio of 3 or 4 H/site could be measured. This observation supports the earlier conclusions of Azzone and associates [8-lo], who not only measured a ratio of 4 K’/site but also indicated the opportunity of measuring the stoicheometry on cation rather than on H’ charges [ 1 l] . This is due to the masking of the H’ fluxes because of overlapping anion fluxes. A role of endogenous phosphate in affecting the H/site ratio is confirmed also by the appearance of a spin exchange signal in the ESR spectrum during Mr? uptake [ 121; the signal is attributed to (Mn)@O& precipitate. While the view of 4 H/site is gaining increasing consensus, Moyle and Mitchell [ 131 have brought a new argument in favour of 2 H/site, based on the

A proposal for a new mechanism of interaction of trialkyltin (TAT) compounds with mitochondria

The interactions of trialkyltin (TAT) compounds with the mitochondria were largely studied. The current view of this phenomenon is that these compounds, by exploiting the Cl− and OH− gradient in energised mitochondria, behave as electroneutral OH−/Cl− exchangers. Experiments performed with triethyltin-, tripropyltin-, and tributyltin-chloride, allow us to propose an alternative mechanism. The crucial point of this new mechanism is that TATs enter the mitochondria as lipophilic cations (alkyl)3Sn+ and not as electroneutral compounds. The influx is followed by extrusion of the trialkyltin compounds as electroneutral hydroxi compounds (alkyl)3SnOH. Measurements of membrane potential show that the uptake of the TAT cation (alkyl)3Sn+ is followed by membrane depolarisation measured as the release of the trimethylphenylphoshonium probe. The depolarization occurs in the order: (Et)3SnCl < (Pro)3SnCl < (Bu)3SnCl in agreement with the increasing lipophilicity of the tested compounds from ethyltin toward butylin derivative. The uptake of TAT induces the opening of a selective anionic channel which could explain the swelling of mitochondria observed in a chloride medium.

Effect of Metal Complexes on Thioredoxin Reductase and the Regulation of Mitochondrial Permeability Conditions

Abstract: Gold(I) compounds such as auranofin, chloro(triethylphosphine) gold(I), and aurothiomalate act on mitochondrial functional parameters by determining an extensive permeability transition and a decrease of membrane potential. On the contrary, pyridine nucleotides and glutathione are not modified, whereas a slight but significant decrease of total thiols is apparent. The effect of gold(I) compounds is essentially referable to the inhibition, in the nanomolar range, of thioredoxin reductase activity and to an increase of hydrogen peroxide production. Metal ions and metal complexes (zinc and cadmium acetate, cisplatin, tributyltin) are also good inhibitors of thioredoxin reductase, although in the micromolar range, and in addition, they act as inducers of permeability transition and of membrane potential decrease. At variance with gold(I) compounds, which appear to work almost exclusively on thioredoxin reductase, metal ions and complexes are less specific, since they are active on different mitochondrial targets, including the respiratory chain.

AN IN VITRO STUDY ON THE TOXIC EFFECTS OF NONYLPHENOLS (NP) IN MITOCHONDRIA

This paper is focused on alkylphenols, compounds which are formed by the biodegradation of polyethoxilatedalkylphenols detergents. Our experiments show that alkylphenols act not only as detergents, but also as uncouplers of the oxidative phosphorylation. This effect, can be observed at very low doses, thus suggesting that the preferential target of nonylphenols in living organisms are mitochondria.

Thallium Induces Apoptosis in Jurkat Cells

Abstract: Thallium induces swelling in mitochondria and apoptosis in Jurkat cells. Both the swelling and the apoptosis are inhibited by means of cyclosporine A (CsA), thus suggesting that these phenomena result from the opening of the membrane transition pore (MTP) in mitochondria. Therefore, apoptosis can be explained as due to the opening of the MTP in mitochondria.

Mitochondrial bioenergetics as affected by cationic detergents

This paper examines the accumulation and toxicity mechanism of a cationic detergent, cetyltrimethylammonium (bromide) (CTAB), in energized rat liver mitochondria. The results suggest that: (1) the CTAB ion is accumulated in the mitochondrial matrix by a membrane potential-driven uptake mechanism; (2) accumulation may lead to a toxic effect, since it gives rise to collapse of the mitochondrial membrane potential (ΔΦ) which is correlated with ATP synthesis and which regulates Ca++ uptake; (3) collapse of ΔΦ may be due to enhanced permeability of the mitochondrial membrane to the ions (detergent effect); and (4) ΔΦ collapse and Ca++ and K+ release were also observed in another cationic detergent, NTAB, but not in the presence of anionic detergents.

ESR determination of Mn++ uptake and binding in mitochondria.

Active transport in energy transducing membranes involves the utilization of metabolic energy to move ions against electrochemical gradients. In the case of the divalent cations which are actively transported by mitochondria, namely Ca++, Mn++ and Sr++ [l] , the problem arises as to the determination of the matrix concentration of osmotically active cations. Massari et al. [2] have calculated the ion uptake from absorbancy changes. Gunther and Puskin [3-51 have used the ESR signal of Mn++ to distinguish between free and bound cations and to calculate the matrix concentration of free Mn++. According to Gunther and Puskin [3], during active uptake the Mn++ spectrum can be split into two components E and S belonging to membrane bound and matrix free Mn++, respectively. The binding of divalent cations to the membrane has also been followed qualitatively through the enhancement of the fluorescent chelate of tetracycline with divalent cations [6]. In the present paper the changes of e.s.r. signal of Mn++ during active uptake have been correlated with absorbance and.rhatrix volume changes. On the basis of the combined volume and e.s.r. data a procedure is described for the determination of free Mn++ concentration in the mitochondrial matrix.

Effects of nitrosopropofol on mitochondrial energy-converting system.

Nitrosopropofol (NOPR) is a relatively stable compound obtained from the reaction between the general anesthetic 2,6 diisopropylphenol (propofol) and nitrosoglutathione (GSNO) and bearing a more acidic phenol group than propofol. It interfered with mitochondrial energetic metabolism in a concentration-dependent manner. Concentrations as high as 100 or 200 microM disrupted both oxidative phosphorylation and electron transport. Low concentrations of NOPR (50 microM) markedly slowed down the electron transport rate which was insensitive both to ADP and uncoupler stimulation and spontaneously gradually stopped. Consequently, both the transmembrane potential production and the ATP synthesis system were affected. In the presence of 10 or 20 microM NOPR, mitochondria respired but showed a worsening of the respiratory control and produced a transmembrane potential useful to respond to a phosphorylation pulse, but were not able to restore it. These results were consistent with ATP synthesis and swelling experiments. NOPR was effective at concentrations lower than those required by the combination of propofol and GSNO, suggesting that mitochondria might be able to catalyze the reaction between GSNO and propofol and that the resulting metabolite was more active on mitochondrial membrane structure than the parent compounds. Although the details of the process are yet unknown, the mechanism presented may be of potential relevance to rationalize the pathophysiological effects of propofol.