Regulation of oxidative activity and delta psi of liver mitochondria of active and hibernating gophers. The role of phospholipase A2.

1. In the present work the initially lowered oxidase activity of liver mitochondria of hibernating gophers is shown to increase upon Ca(2+)-loading, after freezing-thawing repeated three times and after swelling in a medium containing potassium acetate as well as in a hypotonic sucrose medium. 2. In all cases the inhibition of phospholipase A2 hindered the increase of the oxidase activity of mitochondria. 3. Mitochondria of hibernating gophers have a lowered delta psi in comparison with active animals, which is restored in the hypotonic medium.     

Activation of oxidative phosphorylation and energy-dependent absorption of Ca2+ and K+ ions by liver mitochondria of hibernating ground squirrels in hypotonic media]

Activation of initially suppressed oxidative phosphorylation and energy-dependent uptake of Ca2+ and K+ ions by liver mitochondria of hibernating gophers which is prevented by phospholipase A2 inhibitors, has been shown to occur in hypotonic media. Partial inhibition of the respiratory chain of liver mitochondria of active gophers by antimycin A which causes a decrease in the uncoupled respiration rate and delta psi down to values typical of mitochondria of hibernating gophers, practically exactly reproduced the suppression of oxidative phosphorylation and energy-dependent uptake of cations observed during hibernation. It was concluded that partial deenergization arising as a result of inhibition of the respiratory chain is the main and unique cause of suppression of energy-dependent functions of liver mitochondria of hibernating gophers.     

Analysis of the causes of the suppression of oxidative phosphorylation and energy-dependent cationic transport into liver mitochondria of hibernating gophers, Citellus undulatus.

1. The causes of the suppression of oxidative phosphorylation and energy-dependent cationic transport into liver mitochondria of hibernating gophers have been analysed. 2. The decrease of the ATP synthesis rate and suppression of the energy-dependent K(+)- and Ca(2+)-transport into mitochondria during hibernation has been found to be mainly related to a delta psi decrease in mitochondria of hibernating gophers. 3. The increase delta psi upon incubation of the mitochondria of hibernating animals in a hypotonic medium results in an essential acceleration of ATP synthesis and energy-dependent cationic transport.     

Participation of the ATP/ADP antiporter and fatty acids in oxidative phosphorylation uncoupling in squirrel liver mitochondria during winter hibernation and awakening]

The parameters of energy coupling of mitochondria isolated from the livers of hibernating and awakening gophers were studied. The ATP/ADP-antiporter inhibitor carboxyatractylate slowed down the respiration rate, increased delta psi and decreased the ionic conductivity of the inner mitochondrial membrane as measured by the rate of the delta psi decline after addition of cyanide (in the presence of oligomycin and EGTA). A similar effect was produced by BSA, carboxyatractylate being fairly ineffective in the presence of BSA. In hibernating gophers the maximal rate of the uncoupled respiration and the ionic conductivity of the inner mitochondrial membrane were markedly decreased as compared with awakening gophers. The data obtained suggest that in awakening animals fatty acids induce the uncoupling of oxidative phosphorylation by the ATP/ADP-antiporter, this process being simultaneous with the activation of the respiratory chain.     

The effect of medium tonicity on the rate of respiration and oxidative phosphorylation in liver mitochondria of active and hibernating ground squirrels

The rate of respiration and ATP synthesis in liver mitochondria (M) isolated from hibernating ground squirrels and incubated in the medium with normal tonicity (250 mosm) was shown to be considerably lower than the rate of respiration and ATP synthesis in liver M from active animals. The increase of the medium tonicity to 600 mosm simulated the state of M from hibernating animals, resulting in a decrease of the respiration rate of M from active ground squirrels. On the contrary, the decrease of the tonicity to 60 mosm caused the activation of the respiration and increase of the ATP synthesis in M from hibernating ground squirrels. Bromophenacylbromide (BPhB), an inhibitor of phospholipase A2, prevented the activation of the respiration of M from hibernating animals incubated in the medium with low tonicity. BPhB had practically no effect on the respiration of M from both hibernating and active ground squirrels as well as on the swelling of M in hypotonic medium. It was concluded that the activation of the respiration and increase of the ATP synthesis rate in M from hibernating ground squirrels incubated in the medium with low tonicity is related to the activation of phospholipase A2. It was assumed that decrease of phospholipase A2 activity and change in the lipid composition of mitochondrial membrane may be one of the reasons for inhibition of the respiration rate in M from hibernating ground squirrels.     

Regulation of Ca2+ efflux in rat liver mitochondria. Role of membrane potential

The paper analyzes the relationship between membrane potential (delta psi), steady state pCao (-log [Ca2+] in the outer aqueous phase) and rate of ruthenium-red-induced Ca2+ efflux in liver mitochondria. Energized liver mitochondria maintain a pCao of about 6.0 in the presence of 1.5 mM Mg2+ and 0.5 mM Pi. A slight depression of delta psi results in net Ca2+ uptake leading to an increased steady state pCao. On the other hand, a more marked depression of delta psi results in net Ca2+ efflux, leading to a decreased steady-state pCao. These results reflect a biphasic relationship between delta psi and pCao, in that pCao increases with the increase of delta psi up to a value of about 130 mV, whereas a further increase of delta psi above 130 mV results in a decrease of pCao. The phenomenon of Ca2+ uptake following a depression of delta psi is independent of the tool used to affect delta psi whether by inward K+ current via valinomycin, or by inward H+ current through protonophores or through F1-ATP synthase, or by restriction of e- flow. The pathway for Ca2+ efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCao at steady state and by an increased rate of ruthenium-red-induced Ca2+ efflux. By restricting the rate of e- flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium-red-induced Ca2+ efflux on the value of delta psi is observed, in that the rate of Ca2+ efflux increases with the value of delta psi. The pCao is linearly related to the rate of Ca2+ efflux. Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP-supplemented mitochondria, is followed by a phase of Ca2+ uptake, which is reversed by atractyloside. These findings support the view that Ca2+ efflux in steady state mitochondria occurs through an independent, delta psi-controlled pathway and that changes of delta psi during oxidative phosphorylation can effectively modulate mitochondrial Ca2+ distribution by inhibiting or activating the delta psi-controlled Ca2+ efflux pathway.     

Mechanism of citrinin-induced dysfunction of mitochondria. II. Effect on respiration, enzyme activities, and membrane potential of liver mitochondria.

The mycotoxin citrinin, depressed the phosphorylation efficiency of liver mitochondria as deduced from a decrease of respiratory coefficient and of the ADP/O ratio. Citrinin (1.0 mM) inhibited some enzymes linked to the respiratory chain, namely NADH oxidase and NADH cytochrome c reductase involved with complex I. The activities of enzymes related with other enzymatic complexes of the respiratory chain were either unaffected or enhanced. ATPase activity was inhibited by the mycotoxin. Malate, glutamate, and 2-oxoglutarate dehydrogenases were also inhibited. The transmembrane potential (delta psi), developed by energized mitochondria and depolarization on the addition of ADP, was decreased. The results suggest that citrinin promotes a partial dissipation of the transmembrane potential, different from that resulting from a classical uncoupler such as 2,4-dinitrophenol.     

Phospholipase A activity determines the rate of respiration of the mitochondria in hibernating animals

The mechanisms for regulating the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels were studied. The microviscosity of the mitochondrial membrane in hibernating squirrels was found to be higher than that in active animals. Probably, a high microviscosity of the membrane causes a decreases in the rate of the transport of oxidation substrates into the mitochondrial matrix, which in turn may be one of the main reasons for the inhibition of mitochondrial respiration in hibernating squirrels. The activation of phospholipase A2 in a hypotonic medium results in the acceleration of the respiration and phosphorylation in the mitochondria from hibernating squirrels and is accompanied by the increase of the transport of substrates across the mitochondrial membrane. The inhibition of phospholipase A2 decreases Ca2+--induced acceleration of the transport of substrates and prevents the activation of the respiration and phosphorylation in a hypotonic medium.     

Biochemical bases of the inhibition and activation of liver mitochondrial respiration in hibernating susliks

The possible mechanisms in regulation of the respiration rate of mitochondria from liver of hibernating ground squirrels have been investigated. The inhibition of respiration has been shown to be mainly due to the inhibition of electron transfer to the respiratory chain from flavoproteins to cytochrome c. Calcium ions evoke the gradual increase in the respiration rate of mitochondria from liver of hibernating ground squirrels which is abolished by adding albumin, ruthenium red and sovcaine. The lower content of free fatty acids and decreased rate of the oxidation of exogenic NADH in the external pathway indicate the decrease in the activity of phospholipase A2 in mitochondria from liver of hibernating ground squirrels. The decreased calcium capacity of mitochondria indicates the higher sensitivity to calcium ions. A conclusion concerning the leading role of phospholipase A2 in Ca-induced activation of respiration of mitochondria from liver of hibernating ground squirrels is made.     

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.     

Electroneutral H+-K+ exchange in liver mitochondria. Regulation by membrane potential

The paper analyzes the factors affecting the H+-K+ exchange catalyzed by rat liver mitochondria depleted of endogenous Mg2+ by treatment with the ionophore A23187. The exchange has been monitored as the rate of K+ efflux following addition of A23187 in low-K+ media. (1) The H+-K+ exchange is abolished by uncouplers and respiratory inhibitors. The inhibition is not related to the depression of delta pH, whereas a dependence is found on the magnitude of the transmembrane electrical potential, delta psi. Maximal rate of K+ efflux is observed at 180-190 mV, whereas K+ efflux is inhibited below 140-150 mV. (2) Activation of H+-K+ exchange leads to depression of delta pH but not of delta psi. Respiration is only slightly stimulated by the onset of H+-K+ exchange in the absence of valinomycin. These findings indicate that the exchange is electroneutral, and that the delta psi control presumably involves conformational changes of the carrier. (3) Incubation in hypotonic media at pH 7.4 or in isotonic media at alkaline pH results in a marked activation of the rate of H+-K+ exchange, while leaving unaffected the level of Mg2+ depletion. This type of activation results in partial 'uncoupling' from the delta psi control, suggesting that membrane stretching and alkaline pH induce conformational changes on the exchange carrier equivalent to those induced by high delta psi. (4) The available evidence suggests that the activity of the H+-K+ exchanger is modulated by the electrical field across the inner mitochondrial membrane.     

The interaction of glucagon treatment and uncoupler concentration on ATPase activity of rat liver mitochondria

Mitochondria isolated from livers of rats treated briefly with glucagon show an increased ATPase activity in the presence of appropriate concentrations of protonophoric uncouplers (Yamazaki, R. K., Sax, R.D., and Hauser, M.A. (1977) FEBS Lett. 75, 295-299). With the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) the effect of glucagon treatment was most evident at concentrations of uncoupler higher than required for maximal stimulation of ATPase in control mitochondria. In this range of FCCP concentrations that produced the greatest contrast in ATPase activity of control and hormone-stimulated mitochondria, there were no significant differences in delta pH, delta psi, or delta p between the two groups. The presence of added succinate in the ATPase assay system mimicked the effect of glucagon treatment, permitting greater activity at high concentrations of uncoupler without significantly affecting delta p. No significant effect of glucagon treatment or uncoupler concentrations on mitochondrial volumes was observed. Following treatment with glucagon, the mitochondria retained a greater content of Mg+ and K+ throughout the range of FCCP concentrations tested. In the upper range of FCCP concentrations there was appreciable loss of K+ from the mitochondria which was greater in control mitochondria than in mitochondria from glucagon-treated rats or in mitochondria assayed in the presence of succinate. The activity of the uncoupler-dependent ATPase was greatly stimulated by increased concentrations of potassium chloride in the assay medium without significantly diminishing the hormone effect. It is proposed that the intrinsic peptide inhibitor of ATPase is dissociated from the enzyme to an increased degree following glucagon treatment and that high levels of uncoupler inhibit by causing an increased association of the enzyme and its inhibitor.     

Unique relationships between the rates of oxidation and phosphorylation and the protonmotive force in rat-liver mitochondria

The rate of ATP synthesis (JP) in isolated rat-liver mitochondria was strongly dependent on the magnitude of the protonmotive force (delta mu H+) across the mitochondrial inner membrane. Addition of different concentrations of various uncouplers or malonate to mitochondrial incubations in State 3 led to a depression of delta mu H+ and a concomitant decrease in JP. A unique relationship between JP and delta mu H+ was obtained, which was independent of the way in which delta mu H+ was varied. This unique relationship was observed when K+ (in the presence of valinomycin) was used as a probe for delta psi. Different relationships between JP and delta mu H+ were observed when K+ was used as a probe for delta psi and when K+ was measured after separation of the mitochondria by centrifugation without silicone oil. This led to a serious underestimation of delta psi, specifically when uncouplers were present, and non-unique flow-force relationships were thus obtained. Anomalous relationships between JP and delta mu H+ were also found when TPMP+ was used as a probe for delta psi. However, in uncoupler incubations the presence of TBP- strongly affected the TPMP+ accumulation ratio without any effect on the K+ accumulation or on JP and in the presence of TBP- unique relationships between JP and delta mu H+ were again obtained. This indicates that the accumulation of TPMP+ inside the mitochondria is not a straightforward function of delta psi but also depends on conditions like the presence of TBP- or uncouplers. We conclude that there is a unique relationship between the rate of phosphorylation and the protonmotive force in mitochondria and that under some conditions the behaviour of TPMP+ is anomalous.     

Regulation of the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels, Citellus undulatus

1. The rates of oxidation of various substrates (beta-hydroxybutyrate, succinate, ascorbate + TMPD) and the rate of ATP synthesis in liver mitochondria from active and hibernating ground squirrels were measured. 2. It was shown that the rate of mitochondrial respiration is significantly lower in hibernating animals than in active animals. 3. The degree of inhibition of mitochondrial respiration in hibernating ground squirrels was found to correlate with the length of the respiratory chain fragment involved in the oxidation of a given substrate. 4. The inhibition of mitochondrial respiration in hibernating animals was accompanied by a decrease in the rate of ATP synthesis. 5. The activity of phospholipase A2 in liver mitochondria from hibernating ground squirrels was found to be decreased. The activation of phospholipase A2 by Ca2+ ions eliminated the inhibition of respiration almost completely. 6. It was assumed that the inhibition of mitochondrial respiration during hibernation is (a) related to the suppression of phospholipase A2 activity and (b) caused by the reduced rates of electron transport through the respiratory chain and/or of substrate transport across the mitochondrial membrane.     

Effects of adrenergic agonists and mitochondrial energy state on the Ca2+ transport systems of mitochondria

This study investigates the effects of adrenergic agonists and mitochondrial energy state on the activities of the Ca2+ transport systems of female rat liver mitochondria. Tissue perfusion with the alpha-adrenergic agonist phenylephrine and with adrenaline, but not with the beta-adrenergic agonist isoprenaline, induced significant activation of the uniporter and the respiratory chain. Uniporter activation was evident under two sets of experimental conditions that excluded influences of delta psi, i.e., at high delta psi, where uniporter activity was delta psi independent, and at low delta psi, where uniporter conductance was measured. Preincubation of mitochondria with extracts from phenylephrine-perfused tissue quantitatively reproduced uniporter activation when comparison was made with mitochondria treated similarly with extracts from tissue perfused without agonist. Similar, but more extensive, data were obtained with heart mitochondria pretreated with extracts from hearts perfused with the alpha-adrenergic agonist methoxamine. Phenylephrine did not affect Ca2+ efflux mediated by the Na+-Ca2+ carrier or the Na+-independent system. In contrast, the liver mitochondrial Na+-Ca2+ carrier was activated by tissue perfusion with isoprenaline; the Na+-independent system was unaffected. Na+-Ca2+ carrier activation was not associated with any change in a number of basic bioenergetic parameters. It is concluded that the Ca2+ transport systems of liver mitochondria may be controlled in an opposing manner by alpha-adrenergic agonists (promotion of Ca2+ influx) and beta-adrenergic agonists (promotion of Ca2+ efflux). At delta psi values greater than 110 mV, the Na+-independent system was activated by increase in delta psi; the uniporter and Na+-Ca2+ carrier activities were insensitive to delta psi changes in this range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of cellular redox potential as measured in a steady-state, cell-free system

A cell-free system consisting of rat liver mitochondria, liver cytosol, lactate, and the substrates intrinsic to the malate-aspartate shuttle was reconstituted for studies of steady-state substrate fluxes and, more specifically, to evaluate further the mechanism of control of the intra- and extramitochondrial steady states of the free NAD+/NADH ratios. Soluble (F1) ATPase or 2,4-dinitrophenol (DNP) were added in varying amounts to alter substrate fluxes and the constant energy state of this 'open' metabolizing system. The steady-state redox segregation (1.36 log NAD+/NADH ratio out vs NAD+/NADH in the mitochondrial matrix) was maximally about 3 kcal, and declined together with the membrane potential (delta psi) and log ATP/ADP, which obtain on imposing an increasing energy load on the system. It is concluded that transmembrane movement of reducing equivalents is coupled to electron transfer through delta psi, mediated by the electrogenic exchange of glutamate and aspartate. When delta psi was high (near State 4), delta G redox was approximately the same as that generated without flux of reducing equivalents [E. J. Davis, J. Bremer, and K. E. Akerman (1980) J. Biol. Chem. 255, 2277-2283], suggesting that delta Gredox is in near thermodynamic equilibrium with delta psi. If the steady-state ATP/ADP ratio was altered with an energy load (F1-ATPase), delta Gredox decreased more steeply than delta psi (tetraphenyl phosphonium-sensitive electrode used to measure delta psi). At comparable ranges of ATP/ADP, both delta Gredox and delta psi decreased more steeply with uncoupler than with an external ADP-regenerating system.     

Sites of action of glucagon and other Ca2+ mobilizing hormones on the malate aspartate cycle

Data from a number of laboratories suggest that the exchange of glutamate for aspartate across the mitochondrial inner membrane is stimulated by glucagon and by Ca2+-mobilizing hormones. The purpose of this study was to determine the site of action of these hormones. Two possibilities were considered and tested. The first hypothesis is that the mitochondrial membrane electrical potential gradient (delta psi m) in the cells is increased by the hormones; and that the putative increase in delta psi m stimulates aspartate efflux. The second possibility is that Ca2+ mediates decreases in cellular levels of alpha-ketoglutarate, secondary to stimulation of alpha-ketoglutarate dehydrogenase, and that the decrease in alpha-ketoglutarate stimulates aspartate production by mitochondria. The effect of glucagon on delta psi m was estimated in intact hepatocytes using the lipophilic cation tetraphenyl phosphonium. No increase in delta psi m was observed due to hormone treatment. On the other hand, alpha-ketoglutarate was found to be an effective competitive inhibitor of aspartate formation via glutamate transamination by isolated liver mitochondria (Ki = 0.55 mM).     

Control of proteoliposomal cytochrome c oxidase: the overall reaction

The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (delta psi) and pH gradient (delta pH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse delta psi and increase delta pH; the respiration rate decreases. High levels of valinomycin, however, decrease delta pH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases delta psi and collapses delta pH; the respiration rate increases. On a millivolt equivalent basis delta pH is a more effective inhibitor of activity than is delta psi. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both delta pH and delta psi components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing delta pH but in a nonlinear way to delta psi ("non-ohmically"). High levels of both delta psi and delta pH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both delta pH and delta psi components of the proton-motive force, but is more sensitive to delta pH than to delta psi at an equivalent delta mu H+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella.     

A general characteristic of tumor mitochondria: Leakage of endogenous Mg2+ on incubation with uncoupler and resultant reduction of uncoupler-stimulated ATPase activity

Previous studies showed that stimulation of mouse mitochondrial ATPase activity of tumor cells, fetal liver, and adult brain by the uncoupler 2,4-dinitrophenol was markedly suppressed during incubation of the mitochondria with the uncoupler (J.-I. Hayashi et al., 1980, Biochem. Biophys. Res. Commun.92, 261–267). The present work showed the reason for this suppression. More than half the endogenous Mg²⁺ leaked from mitochondria of all tumor cells tested, and of fetal liver and adult brain during incubation with the uncoupler, while only about 30% of the endogenous Mg²⁺ leaked from mitochondria of other normal tissues. The effect of the uncoupler on Mg²⁺ leakage from liver mitochondria changed from the fetal to the adult type within about 30 min after birth. In hypotonic medium, normal liver mitochondria also lost more than half their total Mg²⁺ and concomitantly stimulation of their ATPase activity by uncoupler was considerably reduced. Exogenously added Mg²⁺ could reverse this reduced effect of the uncoupler on ATPase activity of mitochondria from normal tissues and tumor cells. These results show that the endogenous Mg²⁺ content of mitochondria directly affects the stimulation by uncoupler of ATPase activity of mitochondria from both normal tissues and tumor cells. Thus, mitochondria of all tumor cells tested, and of fetal liver and adult brain are leaky to Mg²⁺ during incubation with uncoupler and as a result of the leakage, the stimulatory effect of the uncoupler on their ATPase activity is greatly reduced.     

Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences.

The transport of precursor proteins into mitochondria requires an energized inner membrane. We report here that the import of various precursor proteins showed a differential sensitivity to treatment of the mitochondria with the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The differential inhibition by carbonyl cyanide m-chlorophenylhydrazone was not influenced by the length of the precursor, the presence of mature protein parts, or the folding state of the precursor but was specific for the presequence. Moreover, only the membrane potential delta psi and not the total proton motive force was required for the transport of precursors, indicating that protein translocation across the inner membrane is not driven by a movement of protons. We conclude that delta psi (negative inside) is needed for the translocation of the positively charged presequences, possibly via an electrophoretic effect.