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G F Azzone

Publications and source records attributed to G F Azzone.

At least 37 records · Page 2Linked to original sources
PubMed1991

Phenylarsine oxide induces the cyclosporin A-sensitive membrane permeability transition in rat liver mitochondria.

This paper reports an investigation on the effects of the hydrophobic, bifunctional SH group reagent phenylarsine oxide (PhAsO) on mitochondrial membrane permeability. We show that PhAsO is a potent inducer of the mitochondrial permeability transition in a process which is sensitive to both the oxygen radical scavanger BHT and to cyclosporin A. The PhAsO-induced permeability transition is stimulated by Ca2+ but takes place also in the presence of EGTA in a process that maintains its sensitivity to BHT and cyclosporin A. Our findings suggest that, at variance from other known inducers of the permeability transition, PhAsO reacts directly with functional SH groups that are inaccessible to hydrophilic reagents in the absence of Ca2+.

Animals↗
PubMed1990

Tracking of proton flow during transition from anaerobiosis to steady state in rat liver mitochondria.

(1) The hydrophobic pH indicator Bromthymol blue and the hydrophilic pH indicator Phenol red have been used to follow the redox-pump-linked proton flows during transition from anaerobiosis to static head. The domains monitored by the pH indicators, whether external or internal, and the localization of the dye, whether free or membrane bound, have been identified by recording the absorbance changes following addition of nigericin or valinomycin to anaerobic or aerobic mitochondria and the effects of permeant and impermeant buffers. (2) After addition of the H+/K+ exchanger, nigericin, to anaerobic mitochondria. Phenol red and Bromthymol blue record an alkalinization and an acidification, respectively, indicating that while the hydrophilic pH indicator faces an external domain, the hydrophobic pH indicator faces, at least partly, an internal domain. The latter effect is sensitive to phosphate and to phosphate carrier inhibitors. On the other hand, addition of nigericin to aerobic mitochondria leads to an increased Bromthymol blue absorbance, which reflects an alkalinization, indicating that the pH indicator faces an external domain. The reorientation of the dye from the internal to the external domain is a function of the uncoupler concentration and thus of the membrane potential (cf. Mitchell et al. (1968) Eur. J. Biochem. 4, 9-19). (3) The amount of oxygen required for the transition from anaerobiosis to static head has been determined by following in parallel the extent of oxidation of cytochrome aa3 and the rise of delta mu H+. With succinate as substrate, 50% levels of cytochrome oxidation are obtained at 0.125 ngatom oxygen/mg and 50% of Safranine response at about 0.2 ngatom oxygen/mg. These amounts of oxygen correspond to an H+ displacement of about 0.8-1.2 ngatom/mg on the basis of the H+/O stoichiometry. It is concluded that mitochondria are in presteady state below, and in static head above, displacement of 2-3 ngatom H+/mg. This figure is very close to the original calculation of Mitchell (Mitchell, P. (1966) Biol. Rev. 41, 445-502). (4) Transition, by oxygen pulses, of EGTA-supplemented mitochondria from anaerobiosis to either presteady state or static head state results in a response of the hydrophilic pH indicator, Phenol red, which is negligible in amount and/or kinetically unrelated to the delta mu H+ rise. The fact that H+ extrusion in the bulk aqueous phase is negligible also in presteady state excludes proton cycling as an explanation. Addition of oxygen pulses to Sr2(+)-supplemented anaerobic mitochondria results in an H+ extrusion whose amount and rate is proportional to the Sr2+ concentration.(ABSTRACT TRUNCATED AT 400 WORDS)

Anaerobiosis↗
PubMed1990

A gated pathway for electrophoretic Na+ fluxes in rat liver mitochondria. Regulation by surface Mg2+.

Addition of EDTA to mitochondria incubated aerobically in a phosphate-supplemented medium containing Na+ ions results in activation of cation uptake which is accompanied by membrane depolarization and stimulation of respiration. The same results are obtained in media containing Li+ but not K+, indicating that this pathway for cation transport is selective. The activation of Na+ transport is not accompanied by changes of matrix Mg2+, indicating that cation transport is controlled by surface-bound rather than intramitochondrial Mg2+. Na+ transport in respiring mitochondria is competitively inhibited by Mg2+ with a Ki in the nanomolar range. A Na+ current can also be induced by a K+ diffusion potential in the absence of respiration. The K(+)-diffusion-driven Na+ current has the same magnitude in the absence or presence of inorganic phosphate, suggesting that Na+ transport is mediated by Na+ uniport rather than by electrogenic nNa+/H+ antiport with n greater than 1. Analysis of the flow/force relationship indicates that the putative Na+ uniporter has a conductance of about 0.2 nmol Na+ x mg protein-1 x min-1 x mV-1, and that it is active only when the membrane potential exceeds about 150 mV.

Animals↗
PubMed1990

On the nature of the uncoupling effect of fatty acids.

The effect of palmitic acid on the electrical potential differences delta psi across the inner mitochondrial membrane appears to depend on the medium in which mitochondria are incubated. In medium A (cf. Luvisetto et al. (1987), Biochemistry, 26, 7332-7338) delta psi decreases much more than in medium B (cf. Rottenberg and Hashimoto (1986), Biochemistry, 25, 1747-1755) at concentrations of fatty acid which equally stimulate the rate of respiration in state 4. Valinomycin and NaCl were both present in medium B and absent in medium A. However, in both media the pattern of the P/O ratio as a function of antimycin in the presence of a constant amount of palmitic acid or of FCCP shows similar behaviour. We conclude that in both media palmitic acid increases the membrane conductance to protons, but for unclear reasons the delta psi assay fails to measure the decline of delta psi in medium B. However, the increase in membrane conductance induced by palmitic acid does not quantitatively account for the stimulation of the rate of respiration.

Adenosine Triphosphate↗
PubMed1989

Activation of latent K+ uniport in mitochondria treated with the ionophore A23187.

Addition of A23187 plus EDTA to energized mitochondria in KCl medium determines a rapid osmotic swelling due to K+ uptake. The swelling is fully reversed by uncoupler, is stimulated by quinine, and is accompanied by membrane depolarization and increased rate of respiration. A23187-treated mitochondria passively swell in K+ thiocyanate at neutral pH, under conditions where the H+-K+ antiporter appears to be silent. These data indicate that A23187 activates electrophoretic K+ flux, supporting the notion that Mg2+ depletion unmasks several ionic conductance pathways whose concerted interplay could provide a sensitive regulation of mitochondrial volume homeostasis.

Calcimycin↗
PubMed1989

Nature of proton cycling during gramicidin uncoupling of oxidative phosphorylation.

Addition of gramicidin D to liver mitochondria, incubated in low- or high-salt media, results in stimulation of respiration in the absence or presence of depression of delta muH, respectively. Gramicidin D concentrations 2 orders of magnitude higher are required in the low-salt media with full uncoupling at 1 nmol of gramicidin.mg-1. The stimulation of respiration is not accompanied by increased passive proton influx in low-salt media. In high-salt media, the extent of respiratory stimulation and the extent of delta muH depression differ according to the nature and concentration of cation. The flow-force relationship is very steep when gramicidin D induced uncoupling occurs in low-salt media and much less steep in high-salt media. A multiplicity of flow-force relationship, respiratory rate vs delta muH, is obtained, the slope of which depends on the nature and concentration of cation, and which can be reproduced by computer simulation by introducing a variable extent of proton cycling either in the membrane or in the pump. The apparent proton conductance, as analyzed in the relationship of Je/delta muH vs delta muH, increases in the so-called ohmic and nonohmic regions according to whether gramicidin D is added in high-salt or low-salt media, respectively. Titration with antimycin of the respiratory control ratio (RCR) in gramicidin D treated mitochondria leads to a depression of the RCR in high-salt but not in low-salt media. The view is discussed that in low-salt media the gramicidin D induced uncoupling is due to a cycling of protons within a proton domain operationally located at or near the proton pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Local protons and uncoupling of aerobic and artificial delta muH-driven ATP synthesis.

Gramicidin D causes inhibition of ATP synthesis either in the absence or in the presence of depression of delta muH, in low-salt and in high-salt media, respectively, at concentrations 2 orders of magnitude higher in the former with respect to the latter case. When the number of active redox pumps is reduced by increasing the antimycin concentration, the P/O ratio of respiring, gramicidin-treated mitochondria either is slightly increased in low-salt media or is first decreased and then constant in high-salt media. Addition of gramicidin D in low-salt media to mitochondria synthesizing ATP by means of artificially imposed delta muH gradients results in (a) no effect on the K+ efflux ratio +/- ADP (equivalent to the aerobic respiratory control ratio) and (b) no effect on the ATP/K+ ratio (equivalent to the P/O ratio) except at the low gramicidin D concentrations where there is also a slight enhancement of the rate of ATP hydrolysis. During respiration-driven ATP synthesis, addition of valinomycin plus K+ causes depression of delta muH with little inhibition of ATP synthesis while addition of gramicidin D causes inhibition of ATP synthesis with little depression of delta muH. The view is discussed that the gramicidin-accessible protons which uncouple aerobic ATP synthesis in a delta muH-independent manner are of a different class from the gramicidin-inaccessible protons which uncouple diffusion potential driven ATP synthesis in a delta muH-dependent manner. The gramicidin-accessible protons are suggested to be pump associated and to reflect primary events in energy transduction.

Adenosine Triphosphate↗
PubMed1988

Mechanism of nitrofurantoin toxicity and oxidative stress in mitochondria.

5-Nitrofuran derivatives change the inner mitochondrial membrane permeability as indicated by the transmembrane potential, the rate of spontaneous K+ efflux and the basal respiratory rate: (a) at low concentrations nitrofurantoin prevents the increase of inner membrane permeability due to hydroperoxides or to diamide; (b) at higher concentrations or after longer times of incubation, nitrofurantoin enhances the membrane damage due to hydroperoxides or to diamide; the damage due Ca2+ plus Pi is enhanced by nitrofurantoin at all concentrations; (c) higher nitrofurantoin concentrations cause membrane damage independently of the presence of hydroperoxides or of diamide. The effect of nitrofurantoin is cancelled by the addition of free-radical scavengers. The above effects of nitrofurantoin are compatible with the observations of Mason and colleagues that nitrofurantoin is reduced by a NADPH nitroreductase to a nitro anion radical which can then undergo subsequent reactions, among which are (a) initiation of a free-radical reaction chain and (b) reduction of hydroperoxides and diamide.

Animals↗
PubMed1988

Permeability of inner mitochondrial membrane and oxidative stress.

The mechanism of increase in the inner membrane permeability induced by Ca2+ plus Pi, diamide and hydroperoxides has been analyzed. (1) The permeability increase is antagonized by oligomycin and favoured by atractyloside. The promoting effect of atractyloside is strongly reduced if the mitochondria are simultaneously treated with oligomycin. (2) Addition of the free-radical scavenger, butylhydroxytoluene, results in a complete protection of the membrane with respect to the permeability increase. (3) Although membrane damage and depression of the GSH concentration are often associated, there is no direct correlation between extent of membrane damage and concentration of reduced glutathione. Abolition of the permeability increase by butylhydroxytoluene or by oligomycin is not accompanied by maintenance of a high GSH concentration in the presence of diamide or hydroperoxides. The membrane damage induced by Ca2+ plus Pi is not accompanied by a depression of the GSH concentration. (4) It is proposed that a variety of processes causing an increased permeability of the inner mitochondrial membrane merge into some ultimate common steps involving the action of oxygen radicals.

Adenosine Triphosphate↗
PubMed1988

Analysis of mechanisms of free-energy coupling and uncoupling by inhibitor titrations: theory, computer modeling and experiments.

The rates of ATP synthesis and of ATP-driven NAD reduction have been measured in bovine heart submitochondrial particles as a function of the fraction of inhibited redox pumps (in titrations with either antimycin or rotenone) and of the fraction of inhibited ATPases (in titrations with DCCD). The flux control coefficients of the redox and ATPase proton pumps on the rates of ATP synthesis and of ATP-driven NAD reduction have been derived and found to be equal to 1 for both pumps; i.e., both pumps appear to be 'completely rate limiting'. A theoretical analysis of the inhibitor titration approach based on kinetic models of chemiosmotic coupling and on the theory of metabolic control is presented. This analysis (i) shows that the results of the single inhibitor titrations are incompatible with a delocalized chemiosmotic mechanism of energy coupling if the proton conductance of the membrane is sufficiently low with respect to the conductances of the pumps; and (ii) suggests an experimental approach based on the determination of the P/O and the respiratory control ratios at different degrees of inhibition of the proton pumps to establish the origin of the 'loose coupling' of submitochondrial particle preparations. Three independent types of observation show that the 'loose coupling' of the particle preparation is not mainly due to an increased membrane proton conductance. The same and other independent observations are consistent with the view that the loose coupling of submitochondrial particle preparation is due mainly to inhomogeneity, i.e. to the presence of a subpopulation of highly leaky non-phosphorylating vesicles respiring at maximal rate. The results as a whole together with the simulations and analysis presented lead to the conclusion that the mechanism of free-energy coupling in submitochondrial particles is not completely delocalized.

Adenosine Triphosphatases↗
PubMed1987

Uncoupling of oxidative phosphorylation. 1. Protonophoric effects account only partially for uncoupling.

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxy)phenylhydrazone (FCCP), a typical weak acid protonophore, oleic acid, a fatty acid, and chloroform, a general anesthetic, has been investigated by measuring in mitochondria their effect on (i) the transmembrane proton electrochemical potential gradient (delta mu H) and the rates of electron transfer and adenosine 5'-triphosphate (ATP) hydrolysis in static head, (ii) delta mu H and the rates of electron transfer and ATP synthesis in state 3, and (iii) the membrane proton conductance. Both FCCP and oleic acid increase the membrane proton conductance, and accordingly, they cause a depression of delta mu H [generated by either the redox proton pumps or the adenosinetriphosphatase (ATPase) proton pumps]. Although their effects on ATP synthesis/hydrolysis, respiration, and delta mu H are qualitatively consistent with a pure protonophoric uncoupling mechanism and an additional inhibitory action of oleic acid on both the ATPases and the electron-transfer enzymes, a quantitative comparison between the dissipative proton influx and the rate of either electron transfer or ATP hydrolysis (multiplied by either the H+/e- or the H+/ATP stoichiometry, respectively) at the same delta mu H shows that the increase in membrane conductance induced by FCCP and oleic acid accounts for the stimulation of the rate of ATP hydrolysis but not for that of the rate of electron transfer. Chloroform (at concentrations that fully inhibit ATP synthesis) only very slightly increases the proton conductance of the mitochondrial membrane and causes only a little depression of delta mu H.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1987

Uncoupling of oxidative phosphorylation. 2. Alternative mechanisms: intrinsic uncoupling or decoupling?

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), oleic acid, and chloroform is further investigated by measuring in the presence of a certain concentration of each type of uncoupler (i) the mitochondrial P/O and respiratory control ratios upon progressive inhibition of the redox pumps and (ii) delta mu H and the rate of either electron transfer or adenosine 5'-triphosphate (ATP) hydrolysis in static head upon progressive inhibition of either the redox or the adenosine triphosphatase (ATPase) proton pumps. Chloroform exhibits in all the experiments a behavior very different from that of FCCP and oleic acid. For example, upon addition of antimycin to chloroform-supplemented mitochondria, the respiratory control ratio remains unchanged and the P/O ratio slightly increases (in a certain range of inhibition) instead of decreasing as expected for an increased membrane conductance (and as indeed measured in the presence of either FCCP or oleic acid). From the kinetic model of chemiosmotic free energy coupling described by Pietrobon and Caplan [Pietrobon, D., & Caplan, S.R. (1986) Biochemistry 25, 7690-7696] all the results can be simulated by making the assumptions that (i) chloroform acts specifically at the level of the proton pumps and intrinsically uncouples electron transfer and ATP hydrolysis/synthesis from proton translocation and (ii) FCCP and oleic acid have a mixed behavior and act both as protonophores and as intrinsic uncouplers of the redox pumps (but not of the ATPases). The consistency of the results with the alternative hypothesis that the three agents interfere either with localized energy coupling sites or with a direct interaction between proton pumps is discussed.

Adenosine Triphosphatases↗
PubMed1987

Pathways for Ca2+ efflux in heart and liver mitochondria.

1. Two processes of Ruthenium Red-insensitive Ca2+ efflux exist in liver and in heart mitochondria: one Na+-independent, and another Na+-dependent. The processes attain maximal rates of 1.4 and 3.0 nmol of Ca2+.min-1.mg-1 for the Na+-dependent and 1.2 and 2.0 nmol of Ca2+.min-1.mg-1 for the Na+-independent, in liver and heart mitochondria, respectively. 2. The Na+-dependent pathway is inhibited, both in heart and in liver mitochondria, by the Ca2+ antagonist diltiazem with a Ki of 4 microM. The Na+-independent pathway is inhibited by diltiazem with a Ki of 250 microM in liver mitochondria, while it behaves as almost insensitive to diltiazem in heart mitochondria. 3. Stretching of the mitochondrial inner membrane in hypo-osmotic media results in activation of the Na+-independent pathway both in liver and in heart mitochondria. 4. Both in heart and liver mitochondria the Na+-independent pathway is insensitive to variations of medium pH around physiological values, while the Na+-dependent pathway is markedly stimulated parallel with acidification of the medium. The pH-activated, Na+-dependent pathway maintains the diltiazem sensitivity. 5. In heart mitochondria, the Na+-dependent pathway is non-competitively inhibited by Mg2+ with a Ki of 0.27 mM, while the Na+-independent pathway is less affected; similarly, in liver mitochondria Mg2+ inhibits the Na+-dependent pathway more than it does the Na+-independent pathway. In the presence of physiological concentrations of Na+, Ca2+ and Mg2+, the Na+-independent and the Na+-dependent pathways operate at rates, respectively, of 0.5 and 1.0 nmol of Ca2+.min-1.mg-1 in heart mitochondria and 0.9 and 0.2 nmol of Ca2+.min-1.mg-1 in liver mitochondria. It is concluded that both heart and liver mitochondria possess two independent pathways for Ca2+ efflux operating at comparable rates.

Animals↗
PubMed1987

Effect of Ca2+, peroxides, SH reagents, phosphate and aging on the permeability of mitochondrial membranes.

The mechanism by which a number of agents such as hydroperoxides, inorganic phosphate, azodicarboxylic acid bis(dimethylamide) (diamide), 2-methyl-1,4-naphthoquinone (menadione) and aging, induce Ca2+ release from rat liver mitochondria has been analyzed by following Ca2+ fluxes in parallel with K+ fluxes, matrix swelling and triphenylmethylphosphonium fluxes (as an index of transmembrane potential). Addition of hydroperoxides causes a cycle of Ca2+ efflux and reuptake and an almost parallel cycle of delta psi depression. The hydroperoxide-induced delta psi depression is biphasic. The first phase is rapid and insensitive to ATP and is presumably due to activation of the transhydrogenase reaction during the metabolization of the hydroperoxides. The second phase is slow and markedly inhibited by ATP and presumably linked to the activation of a Ca2+-dependent reaction. The slow phase of delta psi depression is paralleled by matrix K+ release and mitochondrial swelling. Nupercaine and ATP reduce or abolish also K+ release and swelling. Inorganic phosphate, diamide, menadione or aging also cause a process of Ca2+ efflux which is paralleled by a slow delta psi depression, K+ release and swelling. All these processes are reduced or abolished by Nupercaine and ATP. The slow delta psi depression following addition of hydroperoxide and diamide is largely reversible at low Ca2+ concentration but tends to become irreversible at high Ca2+ concentration. The delta psi depression increases with the increase of hydroperoxide, diamide and menadione concentration, but is irreversible only in the latter case. Addition of ruthenium red before the hydroperoxides reduces the extent of the slow but not of the rapid phase of delta psi depression. Addition of ruthenium red after the hydroperoxides results in a slow increase of delta psi. Such an effect differs from the rapid increase of delta psi due to ruthenium-red-induced inhibition of Ca2+ cycling in A23187-supplemented mitochondria. Metabolization of hydroperoxides and diamide is accompanied by a cycle of reversible pyridine nucleotide oxidation. Above certain hydroperoxide and diamide concentrations the pyridine nucleotide oxidation becomes irreversible. Addition of menadione results always in an irreversible nucleotide oxidation. The kinetic correlation between Ca2+ efflux and delta psi decline suggests that hydroperoxides, diamide, menadione, inorganic phosphate and aging cause, in the presence of Ca2+, an increase of the permeability for protons of the inner mitochondrial membrane. This is followed by Ca2+ efflux through a pathway which is not the H+/Ca2+ exchange.

Animals↗
PubMed1986

ATP synthase-mediated proton fluxes and phosphorylation in rat liver mitochondria: dependence on delta mu H.

The dependence of the proton flux through the ATP synthases of rat liver mitochondria on a driving force composed mainly of a potassium diffusion potential was determined and compared with the relationship between rate of phosphorylation and delta mu H given by titrations with the respiratory inhibitor malonate. The two functions are in good agreement in the lower part of the delta mu H range covered. However, the maximal proton fluxes through the ATP synthases are much lower than needed to account for the rate of State 3 phosphorylation sustained by the same mitochondria oxidizing succinate. Possible reasons for this behavior are discussed.

Animals↗
PubMed1986

Free energy coupling between H+-generating and H+-consuming pumps. Ratio between output and input forces.

The delta Gp/delta mu H ratio has been measured in mitochondria close to state 4 in the presence of various uncoupler or K+/valinomycin concentrations in media containing either 1 mM or 50 mM Pi. Care has been taken to control the factors affecting delta Gp and delta mu H which could lead to an artefactual increase of the delta Gp/delta mu H ratio above the highest accepted value for the H+/ATP stoichiometry (n = 4, synthesis + transport). In particular, to avoid overestimation of delta Gp due to inactivation of the ATPases at low delta mu H or to the presence of adenylate kinase, the static head state was approached from the side of net ATP synthesis and delta Gp was measured in a state close to static head but still maintaining a residual rate of aerobic phosphorylation. For each concentration of uncoupler or K+, the Pi concentration and/or the adenylate energy charge (EC) as a function of time have been measured as indicators of net ATP synthesis. Only the values of delta Gp measured during a decrease in Pi concentration and/or an increase in EC have been considered to be meaningful for calculations of delta Gp/delta mu H ratios. Both uncouplers and K+ transport cause a marked depression of delta mu H and a parallel depression of the rate of ATP synthesis. However the low rate of ATP synthesis taking place under conditions of low delta mu H eventually results, especially at high Pi concentrations, in a relatively large delta Gp. The delta Gp/delta mu H ratios obtained at the lower delta mu H values exceed 4 and approach 6. Although slightly higher delta Gp/delta mu H ratios are obtained with valinomycin-treated than with uncoupler-treated mitochondria, the pattern of the rise of the force ratio as delta mu H decreases is similar in both cases. An increase of the delta Gp/delta mu H ratio above 4, the maximal accepted H+/ATP stoichiometry is thermodynamically incompatible with the delocalized protonic coupling model.

Adenosine Triphosphate↗