Regulation of the mitochondrial Na+/Ca2+ antiport by matrix pH

The effect of matrix pH (pHi) on the activity of the mitochondrial Na+/Ca2+ antiport has been studied using the fluorescence of SNARF-1 to monitor pHi and Na(+)-dependent efflux of accumulated Ca2+ to follow antiport activity. Heart mitochondria respiring in a KCl medium maintain a large delta pH (interior alkaline) and show optimal Na+/Ca2+ antiport only when the pH of the medium (pH0) is acid. Addition of nigericin to these mitochondria decreases delta pH and increases the membrane potential (delta psi). Nigericin strongly activates Na+/Ca2+ antiport at values of pH0 near 7.4 but inhibits antiport activity at acid pH0. When pHi is evaluated in these protocols, a sharp optimum in Na+/Ca2+ antiport activity is seen near pHi 7.6 in the presence or absence of nigericin. Activity falls off rapidly at more alkaline values of pHi. The effects of nigericin on Na+/Ca2+ antiport are duplicated by 20 mM acetate and by 3 mM phosphate. In each case the optimum rate of Na+/Ca2+ antiport is obtained at pHi 7.5 to 7.6 and changes in antiport activity do not correlate with changes in components of the driving force of the reaction (i.e., delta psi, delta pH, or the steady-state Na+ gradient). It is concluded that the Na+/Ca2+ antiport of heart mitochondria is very sensitive to matrix [H+] and that changes in pHi may contribute to the regulation of matrix Ca2+ levels.     

The entrapment of the Ca2+ indicator arsenazo III in the matrix space of rat liver mitochondria by permeabilization and resealing. Na+-dependent and -independent effluxes of Ca2+ in arsenazo III-loaded mitochondria.

The permeabilization-resealing technique [Al-Nasser & Crompton, Biochem. J. (1986) 239, 19-29] has been applied to the entrapment of arsenazo III in the matrix compartment of rat liver mitochondria. The addition of 10 mM-arsenazo III to mitochondria permeabilized with Ca2+ partially restores the inner-membrane potential (delta psi) and leads to the recovery of 3.9 nmol of arsenazo III/mg of protein in the matrix when the mitochondria are washed three times. The recovery of entrapped arsenazo III is increased 2-fold by 4 mM-Mg2+, which also promotes repolarization. ATP with or without Mg2+ decreased arsenazo III recovery. Under all conditions, less arsenazo III than [14C]sucrose is entrapped, in particular in the presence of ATP. The amount of arsenazo III entrapped is proportional to the concentration of arsenazo III used as resealant, and is equally distributed between heavy and light mitochondria. Arsenazo III-loaded permeabilized and resealed (PR) mitochondria develop delta psi values of 141 +/- 3 mV. PR mitochondria retain arsenazo III and [14C]sucrose for more than 2 h at 0 degrees C. At 25 degrees C, and in the presence of Ruthenium Red, PR mitochondria lose arsenazo III and [14C]sucrose at equal rates, but Ca2+ efflux is more rapid; this indicates that Ca2+ is released by an Na+-independent carrier in addition to permeabilization. The Na+/Ca2+ carrier of PR mitochondria is partially (60%) inhibited by extramitochondrial free Ca2+ stabilized with Ca2+ buffers; maximal inhibition is attained with 2 microM free Ca2+. A similar inhibition occurs in normal mitochondria with 3.5 nmol of matrix Ca2+/mg of protein, but the inhibition is decreased by increased matrix Ca2+. The data suggest the presence of Ca2+ regulatory sites on the Na+/Ca2+ carrier that change the affinity for matrix free Ca2+.     

t-Butylhydroperoxide-induced Ca2+ efflux from liver mitochondria in the presence of physiological concentrations of Mg2+ and ATP

Isolated rat liver mitochondria, energized either by succinate oxidation or by ATP hydrolysis, present a transient increase in the rate of Ca2+ efflux concomitant to NAD(P)H oxidation by hydroperoxides when suspended in a medium containing 3 mM ATP, 4 mM Mg2+ and acetate as permeant anion. This is paralleled by an increase in the steady-state concentration of extramitochondrial Ca2+, a small decrease in delta psi and an increase in the rate of respiration and mitochondrial swelling. With the exception of mitochondrial swelling all other events were found to be reversible. If Ca2+ cycling was prevented by ruthenium red, the changes in delta psi, the rate of respiration and the extent of mitochondrial swelling were significantly diminished. In addition, there was no significant decrease in the content of mitochondrial pyridine nucleotides. Mitochondrial coupling was preserved after a cycle of Ca2+ release and re-uptake under these experimental conditions. It is concluded that hydroperoxide-induced Ca2+ efflux from intact mitochondria is related to the redox state of pyridine nucleotides.     

Investigation of Ca2+ -induced changes of membrane potential of smooth muscle mitochondria using flow cytometric analysis

Using flow cytometric analysis and potential-sensitive fluorescent dye TMRM Ca2+ -induced changes of membrane potential of isolated smooth muscle mitochondria were studied. It was shown, that Ca2+ (100 microM) addition to the incubation medium induced mitochondrial membrane depolarization that probably could be explained by Ca2+/H+ -exchanger activation which functioning lead to membrane potential dissipation. In the case of ruthenium red (10 microM) preliminary presence in incubation medium, Ca2+ (100 microM) addition did not lead to membrane potential dissipation. Hence, membrane potential dissipation was caused by an increase of matrix Ca2+ concentration. In the presence of Mg2+ (3 mM) and ATP (3 mM), Ca2+ addition did not cause depolarization. It was supposed that in this case ATP synthase acted in the opposite direction as H+ -pump and prevented from mitochondrial membrane potential dissipation. Thus, the flow cytometry method allows to register membrane potential of isolated smooth muscle mitochondria and also to test the effectors, capable to modulate this parameter.     

Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore by the proton electrochemical gradient. Evidence that the pore can be opened by membrane depolarization.

This paper reports an investigation on the relationship between the proton electrochemical gradient (delta mu H+) and the cyclosporin A-sensitive permeability transition pore (PTP) in rat liver mitochondria. Using the SH group cross-linker phenylarsine oxide as the inducer, we show that both matrix pH and the membrane potential can modulate the process of PTP induction independently of Ca2+. We find that membrane depolarization induces the PTP per se when pHi is above 7.0, while at acidic matrix pH values PTP induction is effectively prevented. Since Ca2+ uptake leads to major modifications of the delta mu H+ (i.e. matrix alkalinization and membrane depolarization), we have explored the possibility that the Ca(2+)-induced changes of the delta mu H+ may contribute to PTP induction by Ca2+. Our data in mitochondria treated with Ca2+ plus N-ethylmaleimide and Ca2+ plus phosphate show that membrane depolarization is a powerful inducer of the PTP. Taken together, our observations indicate that the PTP can be controlled directly by the delta mu H+ both in the absence and presence of Ca2+, and suggest that a collapse of the membrane potential may be the cause rather than the consequence of PTP induction under many experimental conditions. Thus, many inducers may converge on dissipation of the membrane potential component of the delta mu H+ by a variety of mechanisms.     

Alterations in mitochondrial Ca2+ flux by the antibiotic X-537A (lasalocid-A).

A previous communication (Pereira da Silva, L., Bernardes, C.F. and Vercesi, A.E. (1984) Biochem. Biophys. Res. Commun. 124, 80-86) presented evidence that lasalocid-A, at concentrations far below those required to act as a Ca2+ ionophore, significantly inhibits Ca2+ efflux from liver mitochondria. In the present work we have studied the mechanism of this inhibition in liver and heart mitochondria. It was observed that lasalocid-A (25-250 nM), like nigericin, promotes the electroneutral exchange of K+ for H+ across the inner mitochondrial membrane and as a consequence can cause significant alterations in delta pH and delta psi. An indirect effect of these changes that might lead to inhibition of mitochondrial Ca2+ release was ruled out by experiments showing that the three observed patterns of lasalocid-A effect depend on the size of the mitochondrial Ca2+ load. At low Ca2+ loads (5-70 nmol Ca2+/mg protein), under experimental conditions in which Ca2+ release is supposed to be mediated by a Ca2+/2H+ antiporter, the kinetic data indicate that lasalocid-A inhibits the efflux of the cation by a competitive mechanism. The Ca2+/2Na+ antiporter, the dominant pathway for Ca2+ efflux from heart mitochondria, is not affected by lasalocid-A. At intermediate Ca2+ loads (70-110 nmol Ca2+/mg protein), lasalocid-A slightly stimulates Ca2+ release. This effect appears to be due to an increase in membrane permeability caused by the displacement of a pool of membrane bound Mg2+ possibly involved in the maintenance of membrane structure. Finally, at high Ca2+ loads (110-140 nmol Ca2+/mg protein) lasalocid-A enhances Ca2+ retention by liver mitochondria even in the presence of Ca2(+)-releasing agents such as phosphate and oxidants of the mitochondrial pyridine nucleotides. The maintenance of a high membrane potential under these conditions may indicate that lasalocid-A is a potent inhibitor of the Ca2(+)-induced membrane permeabilization. Nigericin, whose chemical structure resembles that of lasalocid-A, caused similar results.     

Calcium-Dependent Nonspecific Permeability of the Inner Mitochondrial Membrane Is Not Induced in Mitochondria of the Yeast Endomyces magnusii

Mitochondria of the yeast Endomyces magnusii were examined for the presence of a Ca2+- and phosphate-induced permeability of the inner mitochondrial membrane (pore). For this purpose, coupled mitochondria were incubated under conditions known to induce the permeability transition pore in animal mitochondria, i.e., in the presence of high concentrations of Ca2+ and P(i), prooxidants (t-butylhydroperoxide), oxaloacetate, atractyloside (an inhibitor of ADP/ATP translocator), SH-reagents, by depletion of adenine nucleotide pools, and deenergization of the mitochondria. Large amplitude swelling, collapse of the membrane potential, and efflux of the accumulated Ca2+ were used as parameters for demonstrating pore induction. E. magnusii mitochondria were highly resistant to the above-mentioned substances. Deenergization of mitochondria or depletion of adenine nucleotide pools have no effect on low-amplitude swelling or the other parameters. Cyclosporin A, a specific inhibitor of the nonspecific permeability transition in animal mitochondria, did not affect the parameters measured. It is thus evident that E. magnusii mitochondria lack a functional Ca2+-dependent pore, or possess a pore differently regulated as compared to that of mammalian mitochondria.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Regulation of calcium transport in bovine spermatozoa

Calcium uptake into bovine epididymal spermatozoa is enhanced by introducing phosphate in the suspending medium (Babcock et al. (1975) J. Biol. Chem. 250, 6488-6495). This effect of phosphate is found even at a low extracellular Ca2+ concentrations (i.e., 5 microM) suggesting that phosphate is involved in calcium transport via the plasma membrane. Bicarbonate (2 mM) cannot substitute for phosphate, and a relatively high bicarbonate concentration (20 mM) causes partial inhibition of calcium uptake in absence of Pi. In the presence of 1-2 mM phosphate, 20 mM bicarbonate enhances Ca2+ uptake. The data indicate that the plasma membrane of bovine spermatozoa contains two carriers for Ca2+ transport: a phosphate-independent Ca2+ carrier that is stimulated by bicarbonate and a phosphate-dependent Ca2+ carrier that is inhibited by bicarbonate. Higher phosphate concentrations (i.e., 10 mM) inhibit Ca2+ uptake into intact cells (compared to 1.0 mM phosphate) and this inhibition can be relieved partially by 20 mM bicarbonate. This effect of bicarbonate is inhibited by mersalyl. Calcium uptake into the cells is enhanced by adding exogenous substrates to the medium. There is no correlation between ATP levels in the cells and Ca2+ transport into the cell. ATP levels are high even without added exogenous substrate and this ATP level is almost completely reduced by oligomycin, suggesting that ATP can be synthesized in the mitochondria in the absence of exogenous substrate. Calcium transport into the sperm mitochondria (washed filipin-treated cells) is absolutely dependent upon the presence of phosphate and mitochondrial substrate. Bicarbonate cannot support Ca2+ transport into sperm mitochondria. There is good correlation between Ca2+ uptake into intact epididymal sperm and into sperm mitochondria with the various substrates used. This indicates that the rate of calcium transport into the cells is determined by the rate of mitochondrial Ca2+ uptake and respiration with the various substrates.     

Control processes in oxidative phosphorylation: kinetic constraints and stoichiometry

Control processes in oxidative phosphorylation have been studied in three experimental models. (1) In isolated yeast mitochondria, external ATP is a regulatory effector of cytochrome-c oxidase activity. In phosphorylating or uncoupling states, the relationships between respiratory rate and delta mu H+, and the respiratory rate and cytochrome-c oxidase reduction level are dependent on this kinetic regulation. (2) In rat liver mitochondria, the response of the respiratory rate to uncoupler addition is age-dependent: liver mitochondria isolated from young rats maintain a greater delta mu H+ than liver mitochondria isolated from adults, with the same respiratory rate obtained with the same concentration of uncoupler. This behaviour is linked to redox proton pump properties, i.e., to the degree of intrinsic uncoupling induced by uncoupler addition. (3) The effect of almitrine, a new kind of ATPase/ATPsynthase inhibitor, was studied in mammalian mitochondria. (i) Almitrine inhibits oligomycin-sensitive ATPase - it decreases the ATPase/O value without any change in delta mu H+; (ii) almitrine increased the mechanistic H+/ATP stoichiometry of ATPase/ATPsynthase; (iii) almitrine-induced changes in H+/ATPase stoichiometry depend on the flux magnitude through ATPase. These results are discussed in terms of the following interdependent parameters; flux value, force, pump efficiency and control coefficient.     

Effect of Ca ions on the transmembrane electric potential, synthesis and hydrolysis of ATP in brain mitochondria

The transmembrane potential (delta psi) of rabbit brain mitochondria was measured with the fluorescent dye dis--C2--5. During oxidative phosphorylation a fall in delta psi in the order of 20% was observed. In the presence of inhibitors of ATP synthesis, there was a good correlation between the fall in delta psi and the ADP-stimulated increase in respiration rate. The influence of endogenous calcium on the energetic metabolism of mitochondria was studied by measuring the changes of delta psi. An amount of 12 nmol Ca2+/mg protein cause half-inhibition of the ATP synthesis rate; 50 nmol/mg completely inhibits oxidative phosphorylation. The effect of the Ca2+ load on the ATPase activity of intact mitochondria was studied. It was found that endogenous calcium inhibits in a similar degree synthesis and hydrolysis of ATP. It was shown that both Ca ATP and Mg ATP can serve as a substrate for the mitochondrial ATPase.     

The relationship between extramitochondrial Ca2+ concentration, respiratory rate, and membrane potential in mitochondria from brown adipose tissue of the rat

The ability of isolated mitochondria from rat brown-adipose tissue to regulate extramitochondrial Ca2+ (measured by arsenazo) was studied in relation to their ability to produce heat (measured polarographically). The energetic state of the mitochondria was expressed as a membrane potential, delta psi (estimated with safranine), and was varied semi-physiologically by the use of different GDP concentrations. In these mitochondria GDP binds to the 32-kDa polypeptide, thermogenin, which regulates coupling. Ca2+ uptake (at 5 microM extramitochondrial Ca2+) was maximal at delta psi greater than 150 mV. Basal Ca2+ release increased from 1 to 2 nmol x min-1 x mg-1 below 150 mV. Na+ -stimulated rate of Ca2+ release was stable within the investigated delta psi span (100-160 mV). Initial Ca2+ levels were maintained below 0.2 microM for 100 mV less than delta psi less than 160 mV. Ca2+ levels maintained after Ca2+ challenge (20 nmol Ca2+ x mg-1) were below 0.4 microM for delta psi greater than 135 mM. Respiration was unstimulated for delta psi greater than 150 mV and was maximal at delta psi less than or equal to 135 mV. In the presence of well-oxidised substrates, the respiration at maximally activated thermogenin was markedly below fully uncoupled respiration and was probably limited by thermogenin activity--i.e. by a limited H+ reentry (OH- exit) and therefore by a membrane potential maintained at about 135 mV. It is concluded that at membrane potentials of 135 mV and above the mitochondria exhibit full Ca2+ control and are able to regulate thermogenic output up to maximum without interfering with this Ca2+ control. Membrane potential probably does not decrease below 135 mV in vivo. Therefore, Ca2+ homeostasis and thermogenesis are non-interfering and can be hormonally independently regulated, e.g. by alpha-adrenergic and beta-adrenergic stimuli, respectively.     

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.     

The proton stoichiometry of electron transport in Ehrlich ascites tumor mitochondria.

Initial rate measurements of the stoichiometric relationships between H+ ejection, K+ and Ca2+ uptake, and electron transport were carried out on mitochondria from Ehrlich ascites tumor cells grown in mice. With succinate as substrate and N-ethylmaleimide to prevent interfering H+ reuptake via the phosphate carrier, close to 8 H+ were ejected per oxygen atom reduced (H+/O ejection ratio = 8.0); with the NAD-linked substrates pyruvate or pyruvate + malate, the H+/O ejection ratio was close to 12. The average H+/site ratio (H+ ejected/2e-/energy-conserving site) was thus close to 4. The simultaneous uptake of charge-compensating cations, either K+ (in the presence of valinomycin) or Ca2+, was also measured, yielding average K+/site uptake ratios of very close to 4 and Ca2+/site ratios close to 2. It was also demonstrated that each calcium ion enters the respiring tumor mitochondria carrying two positive electric charges. These stoichiometric data observed in mitochondria from Ehrlich ascites tumor cells thus are in complete agreement with similar data on normal rat liver and rat heart mitochondria and suggest that the H+/site ratio of mitochondrial electron transport may be 4 generally. It was also observed that the rate of deltaH+ back-decay in anaerobic tumor mitochondria following oxygen pulses is some 6- to 8-fold greater than in rat liver mitochondria tested at equal amounts of mitochondrial protein.     

The reversible Ca2+-induced permeabilization of rat liver mitochondria.

Rat liver mitochondria became permeabilized to sucrose according to an apparent first-order process after accumulating 35 nmol of Ca2+/mg of protein in the presence of 2.5 mM-Pi, but not in its absence. A fraction (24-32%) of the internal space remains sucrose-inaccessible. The rate constant for permeabilization to sucrose decreases slightly when the pH is decreased from 7.5 to 6.5, whereas the rate of inner-membrane potential (delta psi) dissipation is markedly increased, which indicates that H+ permeation precedes sucrose permeation. Permeabilization does not release mitochondrial proteins. [14C]Sucrose appears to enter permeabilized mitochondria instantaneously. Chelation of Ca2+ with EGTA restores delta psi and entraps sucrose in the matrix space. With 20 mM-sucrose at the instant of resealing, about 21 nmol of sucrose/mg of protein becomes entrapped. The amount of sucrose entrapped is proportional to the degree of permeabilization. Entrapped sucrose is not removed by dilution of the mitochondrial suspension. Resealed mitochondria washed three times retain about 74% of the entrapped sucrose. In the presence of Ruthenium Red and Ca2+ buffers permeabilized mitochondria reseal only partially with free [Ca2+] greater than 3 microM. [14C]Sucrose enters partially resealed mitochondria continuously with time, despite maintenance of delta psi, in accordance with continued interconversion of permeable and impermeable forms. Kinetic analyses of [14C]sucrose entry indicate two Ca2+-sensitive reactions in permeabilization. This conclusion is supported by the biphasic time courses of resealing and repolarization of permeabilized mitochondria and the acute dependence of the rapid repolarization on the free [Ca2+]. A hypothetical model of permeabilization and resealing is suggested and the potential of the procedure for matrix entrapment of substances is discussed.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

The proton motive force lowers the level of ATP required for the in vitro translocation of a secretory protein in Escherichia coli

The role of the electrochemical potential difference of proton (delta mu H+) in protein translocation across the membrane of Escherichia coli was examined in detail using an efficient in vitro assay system (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). Delta mu H+ reduced the level of ATP necessary for the efficient translocation of OmpF-Lpp, a chimeric model secretory protein. The apparent Km value of the translocation reaction for ATP was lower by 2 orders of magnitude in the presence of delta mu H+ than in its absence. The membrane potential and delta pH, both of which are components of delta mu H+, independently lowered the apparent Km value of the translocation reaction for ATP. An ATP-generating system also lowered the level of ATP required for translocation in the absence of delta mu H+ but not in its presence. It is proposed that ADP formed during protein translocation lowers the affinity of the putative translocation machinery for ATP and that the removal of ADP from the secretory machinery, a possible critical step in the translocation reaction, is stimulated in the presence of either delta mu H+, an ATP-generating system, or a higher concentration of ATP.     

Evaluation of the H+/site ratio of mitochondrial electron transport from rate measurements.

The mitochondrial H+/site ratio (i.e. the number of protons ejected per pair of electrons traversing each of the energy-conserving sites of the respiratory chain) has been evaluated employing a new experimental approach. In this method the rates of oxygen uptake and H+ ejection were measured simultaneously during the initial period of respiration evoked by addition of succinate to aerobic, rotenone-inhibited, de-energized mitochondria. Either K+, in the presence of valinomycin, or Ca2+, was used as mobile cation to dissipate the membrane potential and allow quantitative H+ ejection into the medium. The H+/site ratio observed with this method in the absence of precautions to inhibit the uptake of phosphate was close to 2.0, in agreement with values obtained using the oxygen pulse technique (Mitchell, P. and Moyle, J. (1967) Biochem. J. 105, 1147-1162). However, when phosphate movements were eliminated either by inhibition of the phosphate-hydroxide antiporter with N-ethylamaleimide or by depleting the mitochondria of their endogenous phosphate content, H+/site ratios close to 4.0 were consistently observed. This ratio was independent of the concentration of succinate, of mitochondrial protein, of pH between 6 and 8, and of ionic composition of the medium, provided that sufficient K+ (plus valinomycin) or Ca2+ were present. Specific inhibitors of the hydrolysis of endogenous ATP or transport of other ions (adenine nucleotides, tricarboxylates, HCO3-, etc.) were shown not to affect the observed H+/site ratio. Furthermore, the replacement of succinate by alpha-glycerol phosphate, a substrate which is oxidized on the outer surface of the inner membrane and thus does not need to enter the matrix, gave the same H+/site ratios as did succinate. It is concluded that the H+/site ratio of mitochondrial electron transport, when phosphate movements are eliminated, may be close to 4.0.     

The relative proton stoichiometries of the mitochondrial proton pumps are independent of the proton motive force

Several different proton pumps were used to generate a proton motive force (delta p, proton motive force across the mitochondrial inner membrane) in isolated rat liver mitochondria, and the relationship between delta p and pump rate was investigated by titrating with various inhibitors of the pumps. It was found that this relationship was the same for mitochondria respiring on succinate irrespective of whether respiration was inhibited with malonate, antimycin or cyanide, indicating that the relationship was independent of the redox state of the respiratory chain. When delta p was generated by either the cytochrome bc1 complex, cytochrome oxidase, both together, or ATP hydrolysis (and transport), the reaction rates (in moles of electrons or ATP) were in the ratio of close to 3:1.5:1:1, respectively, at all accessible values of delta p. This suggests that the proton stoichiometries (H+/e and H+/ATP, where H+/e is the number of protons translocated vectorially across the inner membrane per electron transferred by the respiratory chain and H+/ATP is the number of protons translocated vectorially per ATP molecule hydrolyzed externally) were in the ratio of close to 1:2:3:3, respectively, at all values of delta p. Possible reasons for previous contradictory results are suggested.     

Superstoichiometric Ca2+ uptake supported by hydrolysis of endogenous ATP in rat liver mitochondria.

The nature of the energy store causing rapid superstoichiometric leads to H+/2e minus ejection and leads to Ca2+/2e minus uptake ratios in rat liver mitochondria pulsed with Ca2+ has been investigated. The extent and the rate of the initial fast superstoichiometric phase of H plus ejection were greatly reduced by oligomycin and other ATPase inhibitors; the subsequent shoichiometric phase was unaffected. No such inhibition was seen with atractyloside. Similarly, the initial fast phase of Ca2+ uptake was reduced in extent by oligomycin, whereas the slower stoichiometric phase was unaffected. Moreover, the ATP content of mitochondria previously incubated with succinate decreased by about 80% within 5 s after pulsing with Ca2+. The energy store for superstoichiometric Ca2+ uptake and H plus injection is thus identified as endogenous ATP.