Adenine nucleotide content of liver mitochondria increases after glucagon treatment of rats or isolated hepatocytes

Glucagon injected into rats via tail vein or incubated with isolated hepatocytes in vitro caused a 22–35% increase in the intramitochondrial ATP+ADP+AMP content. There was no statistically significant change in state 3 or uncoupled respiratory rates in these mitochondria. Similar glucagon effects were noted irrespective of the medium used or time required for isolation of mitochondria. There has been recent controversy over possible artifacts in assessing the effects of glucagon on mitochondrial function. The present results suggest that the glucagon-induced increase in the matrix adenine nucleotide content is a reproducible phenomenon with probable physiological significance.     

Effects of beta-bungarotoxin on mitochondrial respiration are caused by associated phospholipase A activity.

Mitochondria prepared from tissue that had been incubated with β-bungarotoxin exhibited abnormal respiration. The respiratory rate in the presence of substrate only was apparently normal, but it did not increase upon the addition of ADP. This effect could also be obtained by treatment with $\text{V. russelli}$ phospholipase A or oleate. Treatment with lesser amounts of these agents caused the mitochondria to become uncoupled.     

Effect of salicylic acid on the metabolic activity of plant mitochondria

The effects of salicylic acid (SA) on mitochondrial respiration and generation of membrane potential across the inner membrane of mitochondria isolated from stored taproots of sugar beet (Beta vulgaris L.) and etiolated seedling cotyledons of yellow lupine (Lupinus luteus L.) were studied. When malate was oxidized in the presence of glutamate, low SA concentrations (lower than 1.0 mM) exerted predominantly uncoupling action on the respiration of taproot mitochondria: they activated the rate of oxygen uptake in State 4 (in the absence of ADP) and did not affect oxidation in State 3 (in the presence of ADP). In contrast, in lupine cotyledon mitochondria these SA concentrations inhibited oxygen uptake in the presence of ADP and much weaker activated substrate oxidation in State 4. Thus, SA (0.5 mM) reduced the respiratory control ratio according to Chance (RCR) by 25% in the taproots and 35% in cotyledons. When the concentration of phytohormone was increased (above 1.0 mM), malate oxidation in State 3 was inhibited and in State 4 — activated independently of the plant material used. In this case, the values of RCR and ADP/O were reduced by 50–60%. The effect of high SA concentrations (2 mM and higher) on malate oxidation depended on the duration of incubation and had a biphasic pattern: the initial activation of oxygen uptake was later replaced by its inhibition. The parallel studying the SA effect on the generation of membrane potential (ΔΨ) at malate oxidation in the mitochondria of beet taproots and lupine cotyledons showed that ΔΨ dissipation was observed because of SA uncoupling and inhibiting action on respiration. The degree of ΔΨ dissipation depended on the phytohormone concentration and duration on mitochondria treatment, especially at its high concentrations. In general, a correlation was found between the effects of SA on mitochondrial respiration and ΔΨ values in the coupling membranes. Furthermore, these results show that the responses of mitochondria to SA were determined not only by its concentration but also by treatment duration and evidently by the sensitivity to the phytohormone of mitochondria isolated from different plant tissues.     

The phosphorylation of intramitochondrial AMP: A suggestion for the compartmentation of endogenous Pi pool

1. The mitochondrial level of AMP gradually diminishes during incubation of mitochondria with glutamate but does not with succinate. This decline of AMP, associated with stoichiometric increase in ADP and/or ATP, is accelerated by the addition of electron acceptors or 2,4-dinitrophenol, while arsenite, arsenate and rotenone are inhibitory. These results are in agreement with the view that AMP is phosphorylated to ADP in the inner space of rat liver mitochondria via succinyl-CoA synthetase (succinate: CoA ligase (GDP), EC 6.2.1.4) and GTP:AMP phosphotransferase dependent on the oxidation of 2-oxoglutarate, which is promoted by the transfer of electron from NADH to the respiratory chain.2. Studies of the periodical changes of chemical quantities of adenine nucleotides as well as of their labelling with ³²P_i reveals the following characteristics concerning mitochondrial phosphorylation. (i) In contrast to the mass action ratio of ATP to ADP, the ratio of ADP to AMP is not affected by the intramitochondrial concentration of P_i. (ii) ³²P_i, externally added, is incorporated into ADP much more slowly than into γ-phosphate of ATP. (iii) Conversely, ATP loses its radioactivity from γ-phosphate position more rapidly than [³²P]ADP when ³²P-labelled mitochondria are incubated with non-radioactive P_i.3. In order to elucidate the above characteristic properties of phosphorylation, a hypothetical scheme is proposed which postulates the two separate compartments in the intramitochondrial pool of P_i; one readily communicates with external P_i and is utilized for the phosphorylation of ADP in oxidative phosphorylation, while the other less readily communicates with external P_i and serves as the precursor of ADP via succinyl-CoA synthetase and GTP:AMP phosphotransferase.     

Influence of oxidative processes in mitochondria on contractility of the frog Rana temporaria heart muscle. Effects of cadmium

The inotropic Cd2+ action on frog heart is studied with taking into account its toxic effects upon mitochondria. Cd2+ at concentrations of 1, 10, and 20 microM is established to decrease dosedependently (21.3, 50.3, and 72.0%, respectively) the muscle contraction amplitude; this is explained by its competitive action on the potential-controlled Ca2(+)-channels of the L-type (Ca 1.2). In parallel experiments on isolated rat heart mitochondria (RHM) it was shown that Cd2+ at concentrations of 15 and 25 microM produces swelling of non-energized and energized mitochondria in isotonic (with KNO2 and NH4NO3) and hypoosmotic (with 25 mM CH3COOK) media. Study of oxidative processes in RHM by polarographic method has shown 20 microM Cd2+ to disturb activity of respiratory mitochondrial chain. The rate of endogenous respiration of isolated mitochondria in the medium with Cd2+ in the presence of malate and succinate was approximately 5 times lower than in control. In experimental preparations, addition into the medium of DNP-uncoupler of oxidation and phosphorylation did not cause an increase of the oxygen consumption rate. Thus, the obtained data indicate that a decrease in the cardiac muscle contractility caused by Cd2+ is due not only to its direct blocking action on Ca2(+)-channels, but also is mediated by toxic effect on rat heart mitochondria, which was manifested as an increase in ion permeability of the inner mitochondrial membrane (IMM), acceleration of the energy-dependent K+ transport into the matrix of mitochondria, and inhibition of their respiratory chain.     

The possible role of palmitoyl-CoA in the regulation of the adenine nucleotides transport in mitochondria under different metabolic states. I. Comparison of liver mitochondria from starved and fed rats.

It has been shown that KM values for ADP when rat liver mitochondria oxidized succinate were strictly dependent on the values of the respiratory control ratios. The Ki values for palmitoyl-CoA inhibition of the ADP-stimulated succinate oxidation and the inhibition of the uncoupler-stimulated ATPase activity were equal to 0.5 muM. Mitochondria from livers of starved rats showed 30% inhibition of the state 3 respiratory rate (compared to the uncoupled respiratory rate) which was abolished by addition of carnitine. It was supposed that this inhibition was due to the influence of acyl-CoAs bound to the inner mitochondrial membrane on the adeninenucleotide translocase. Mitochondria from livers of fed rats showed a strong inhibition of succinate oxidation both in state 4 and state 3, although the rate of uncoupled respiration was normal. It was assumed that in this case the changes in mitochondrial behaviour was caused by the decrease in the concentration of ADP and ATP in the matrix space of mitochondria.     

Biochemical and ultrastructural properties of osmotically lysed rat-liver mitochondria

Isolated rat-liver mitochondria were osmotically lysed by suspension and washing 3 times in cold, distilled water. Pellets obtained by centrifugation at 105,000 g for 30 min were resuspended, fixed with glutaraldehyde and OsO₄, and embedded in Epon 812. Thin sections show the presence of two distinct membranous populations, each of which is relatively homogeneous in size and appearance. Swollen mitochondria (∼1.5 µ in diameter), which have been stripped of their outer membranes, are largely devoid of matrix and normal matrix granules and are referred to as "ghosts." The smaller (0.2 to 0.4 µ in diameter), empty appearing, vesicular elements, derived primarily from the outer mitochondrial membrane, can be differentiated from the ghosts on the basis of their smaller size and complete absence of internal structures, especially cristae. Each membranous element is enclosed by a single, continuous membrane; the "double membrane" organization typical of intact mitochondria is not observed. These findings indicate that the outer membrane of rat-liver mitochondria is spatially dissociated from the inner mitochondrial membrane by osmotic lysis of the mitochondria in distilled water. Three parameters of structural and functional significance in freshly isolated rat-liver mitochondria have been correlated with the structural alterations observed: (a) chemical composition (total protein, lipid phosphate and total phosphate), (b) specific and total activities of marker enzymes for mitochondrial matrix and membranes (malate dehydrogenase (MDH), D-β-hydroxybutyrate dehydrogenase (BDH) and cytochromes), and (c) integrated multienzyme functions (respiration, phosphorylation, and contraction). The data presented indicate that all mitochondrial membranes are completely conserved in the crude ghost preparation and that, in addition, about ⅓ of the matrix proteins (estimated by assays for MDH activity and protein) are retained. The study of integrated mitochondrial functions shows that a number of physiologically important multienzyme activities also are preserved in the water-washed preparation. The respiratory rate of ghosts per milligram of protein is 1.5 to 2.0 times that of intact mitochondria, which shows that the respiratory chain in the ghosts is functionally intact. The rate of phosphorylation is reduced, however, to about 25% of that measured in freshly isolated mitochondria and accounts for lowered P:O ratios using succinate as substrate (P:O ranges from 0.4 to 0.9). The phosphorylation of ADP to ATP is the only biochemical function, so far investigated, that is greatly affected by osmotic lysis. In addition, two lines of evidence suggest that the ghosts undergo an energy-dependent transformation resulting in contraction: (a) suspensions of the crude ghost preparation in 0.02 M Tris-0.125 M KCl medium show a marked increase in optical density upon the addition of ATP, and (b) ghost preparations incubated in ion-uptake medium in the absence of added calcium but in the presence of added ATP contain a large number of highly condensed ghosts (about 50% of the total profiles) when viewed as thin sections in the electron microscope. The correlated biochemical and morphological study presented here shows that the outer membrane of rat-liver mitochondria can be removed by controlled osmotic lysis without greatly impairing a number of integrated biochemical functions associated with the inner membrane.     

The effect of glucagon on hepatic respiratory capacity

Data from numerous laboratories show that mitochondria isolated from livers treated acutely with glucagon have higher rates of state 3 respiration than control mitochondria. The purpose of the present study was to learn whether this phenomenon is an isolation artifact resulting from a stabilization of the mitochondrial membrane or whether it represents a real increase in the maximal respiratory capacity of liver cells due to glucagon treatment. Electron transport was measured through different spans of the electron transport chain by using ferricyanide as an alternate electron acceptor to O2. With isolated intact liver mitochondria, pretreatment with glucagon was found to cause an increase in electron flow, through both Complex I and Complex III, suggesting that the effect of glucagon was not specific for a single site in the electron transport chain. Using intact isolated hepatocytes, different results are obtained. Respiration was measured in isolated hepatocytes after quantitation of the hepatocyte mitochondrial content by assay of citrate synthase. Hepatocyte respiration could therefore be reported per mg of mitochondrial protein. By providing durohydroquinone to the cells, it was possible to measure electron flow from coenzyme Q to O2 in the absence of the physiological regulation of substrate supply. Likewise, the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone released the in situ mitochondria from control by the cytosolic ATP/ADP ratio and it was possible to measure maximal electron flow rates through Complex III. In the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, electron flow was higher in mitochondria in the cell than in isolated mitochondria. Glucagon caused no increase in mitochondrial respiration in situ either in the presence of the physiological substrates or in the presence of durohydroquinone. The data obtained do not support a role for the electron transport chain as a target of glucagon action in hepatocytes.     

Energy-dependent accumulation of iron by isolated rat liver mitochondria. IV. Relationship to the energy state of the mitochondria

1. The energy-dependent accumulation of iron by isolated rat liver mitochondria, respiring on endogenous substrates, is strongly dependent on the efficiency of energy coupling in the respiratory chain as measured by respiratory control with ADP and the endogenous energy dissipation. The accumulation reached a saturation level at respiratory control with ADP values (with succinate as the substrate) of approx. 4.0.2. In the presence of exogenous substrate, the energy-dependent accumulation of iron was markedly reduced, primarily due to binding of iron as carboxylate complexes having less favourable dissociation constants than the iron(III)-sucrose complex(es).3. The effect of added ATP was at least 2-fold, i.e. that of providing energy and that of chelating iron. When the mitochondria respired on endogenous substrate, the energy-dependent accumulation of iron increased at low concentrations of ATP, whereas higher concentrations (> 50 μM) gradually inhibited the uptake.4. Energization of the mitochondria by the generation of an artificial K⁺ gradient across the inner membrane with valinomycin in a K⁺-free medium increased the energy-dependent accumulation of iron.     

Studies on the interactions of Ca2+ and pyruvate in the regulation of rat heart pyruvate dehydrogenase activity. Effects of starvation and diabetes.

1. Previous studies showed that the activation of pyruvate dehydrogenase within intact rat heart mitochondria of pyruvate is much diminished in mitochondria from starved or diabetic animals [see Kerbey, Randle, Cooper, Whitehouse, Pask & Denton (1976) Biochem. J. 154, 327-348]. In the present study, diminished responses to added Ca2+ and ADP were also found in these mitochondria. 2. Starvation or diabetes did not affect the mitochondrial respiratory control ratio of the ATP content. Moreover, starvation and diabetes did not alter the response of the intramitochondrial Ca2+-sensitive enzyme, 2-oxoglutarate dehydrogenase, to changes in the extramitochondrial concentration of Ca2+ and 2-oxoglutarate, thus indicating that there were no appreciable changes in the distribution of Ca2+ and H+ across the mitochondrial inner membrane. 3. Pyruvate, Ca2+ and ADP were found to have synergistic effects on pyruvate dehydrogenase activity, particularly in mitochondria from starved and diabetic rats. 4. The results suggest that the effects of diabetes and starvation on pyruvate dehydrogenase are not brought about by changes in the distribution of these effectors across the mitochondrial inner membrane or by changes in the intrinsic sensitivity of the kinase or phosphatase of the pyruvate dehydrogenase system to pyruvate, Ca2+ or ADP; rather it is probably that there is an increase in the maximum activity of kinase relative to that of the phosphatase. 6. The results also lend further support to the hypothesis that adrenaline may bring about the activation of pyruvate dehydrogenase in the rat heart by an increase in the intramitochondrial concentration of Ca2+.     

Respiratory State and Phosphatidylserine Import in Brain Mitochondria In Vitro

The mechanism of phosphatidylserine (PS) movement from donor membranes into rat brain mitochondria was investigated. Mitochondria were incubated with liposomes and subjected to density gradient centrifugation. The energized state was monitored by flow cytometry measuring the fluorescence of membrane-potential-sensitive rhodamine-123 dye. Mitochondria density decreased upon increase of the respiratory rate, as a consequence of their association with liposomes. After interaction of mitochondria with ¹⁴C-PS containing liposomes, ¹⁴C-PS became a substrate of PS decarboxylase, as monitored by the formation of ¹⁴C-phosphatidylethanolamine (PE), indicating translocation of ¹⁴C-PS to the inner membrane. The kinetics of ¹⁴C-PE formation showed a high rate upon addition of ADP, malate and pyruvate (state 3) compared to control (state 1). In state 3, ¹⁴C-PE formation decreased in the presence of NaN₃. Mitochondria-associated membranes (MAM) are the major site of PS synthesis. However, their role in the translocation of PS to mitochondria has not been completely elucidated. A crude mitochondrial fraction (P₂) containing MAM, synaptosomes and myelin was prelabeled with ¹⁴C-PS and incubated in different respiratory states. At a high respiratory rate, low-density labeled mitochondria, whose band overlaps that of synaptosomes, were obtained by centrifugation. A parallel decrease of both radioactivity and protein in MAM fraction was observed, indicating that the association of MAM and mitochondria had occurred. Synthesis and translocation of ¹⁴C-PS in P₂ membranes were also studied by incubating P₂ with ¹⁴C-serine. In the resting state ¹⁴C-PS accumulated in MAM, indicating that the transfer to mitochondria was a limiting step. In state 3 both the transfer rate of ¹⁴C-PS and its conversion to ¹⁴C-PE increased. Respiratory mitochondrial activity modulated the association of MAM and mitochondria, triggering a mechanism that allowed the transport of PS across the outer mitochondrial membrane. Received: 7 April 1999/Revised: 21 September 1999     

Functional relationship between the ADP/ATP-carrier and the F1-ATPase in mitochondria.

1. The distribution of labeled and unlabeled adenine-nucleotides inside and outside mitochondria was followed after addition of [14C]ADP to rat liver mitochondria. Two types of mitochondria were used: 1, respiring mitochondria which were carrying out oxidative phosphorylation and which had been replenished in ATP by incubation in a medium supplemented with succinate and phosphate; 2, non-respiring mitochondria which had been partially depleted of ATP by incubation in a medium supplemented with rotenone and phosphate. During the first minute following addition of [14C]ADP to the respiring mitochondria, the pre-existing intramitochondrial (internal) [12C]ATP was released into the medium and replaced by newly synthesized [14C]ATP. No [14C]ADP accumulated in the mitochondria. It is suggested that extramitochondrial (external) ADP entering respiring mitochondria in exchange for internal ATP is phosphorylated to ATP before its complete release in the matrix space. In non-respiring mitochondria, the entry of [14C]ADP into the mitochondria was accompanied by the appearance in the external space of [12C]ADP and [12C]ATP, with a marked predominance of [12C]ADP. Thus in non-respiring mitochondria, the residual internal ATP is dephosphorylated to ADP in the inner membrane before being released outside the mitochondria. 2. When mitochondria were incubated with glutamate, ADP and [32P]phosphate, the [32P]ATP which accumulated in the matrix space became rapidly labeled in both the P gamma and P beta groups of the ATP, due to the presence of a transphosphorylation system in the mitochondrial matrix. The [32P]ATP which accumulated outside the mitochondria was also labeled in the P beta group, although less rapidly than the internal ATP. Our data show that a large fraction (75-80%) of the ATP produced by phosphorylation of added ADP within the inner mitochondrial membrane is released into the matrix space before being transported out from the mitochondria; only a small part (20-25%) is released directly outside the mitochondria without penetrating the matrix space. 3. In respiring and phosphorylating mitochondria, the value of the Km of the ADP-carrier for external ADP was 2-4 times lower than its value in non-respiring and non-phosphorylating mitochondria. 4. The above experimental data are discussed with reference to the topological and functional relationships between the ADP-carrier and the oxidative phosphorylation complex in the inner mitochondrial membrane. They strongly suggest that the ADP-carrier comes to the close neighbourhood of the ATP synthetase on the matrix side of the inner membrane.     

Intrinsic and extrinsic uncoupling of oxidative phosphorylation

This article reviews parameters of extrinsic uncoupling of oxidative phosphorylation (OxPhos) in mitochondria, based on induction of a proton leak across the inner membrane. The effects of classical uncouplers, fatty acids, uncoupling proteins (UCP1-UCP5) and thyroid hormones on the efficiency of OxPhos are described. Furthermore, the present knowledge on intrinsic uncoupling of cytochrome c oxidase (decrease of H⁺/e⁻ stoichiometry=slip) is reviewed. Among the three proton pumps of the respiratory chain of mitochondria and bacteria, only cytochrome c oxidase is known to exhibit a slip of proton pumping. Intrinsic uncoupling was shown after chemical modification, by site-directed mutagenesis of the bacterial enzyme, at high membrane potential ΔΨ, and in a tissue-specific manner to increase thermogenesis in heart and skeletal muscle by high ATP/ADP ratios, and in non-skeletal muscle tissues by palmitate. In addition, two mechanisms of respiratory control are described. The first occurs through the membrane potential ΔΨ and maintains high ΔΨ values (150-200 mV). The second occurs only in mitochondria, is suggested to keep ΔΨ at low levels (100-150 mV) through the potential dependence of the ATP synthase and the allosteric ATP inhibition of cytochrome c oxidase at high ATP/ADP ratios, and is reversibly switched on by cAMP-dependent phosphorylation. Finally, the regulation of ΔΨ and the production of reactive oxygen species (ROS) in mitochondria at high ΔΨ values (150-200 mV) are discussed.     

ADENINE NUCLEOTIDE-INDUCED CONTRACTION OF THE INNER MITOCHONDRIAL MEMBRANE : I. General Characterization

The inner membranes of isolated bovine heart mitochondria undergo pronounced contraction upon being exposed to exogenous adenosine diphosphate (ADP), adenosine triphosphate (ATP), and certain other high-energy phosphate compounds. Contraction results in decrease of inner membrane expanse which in turn results in decrease of intracristal space and increase of mitochondrial optical density (OD). The magnitude of the OD change appears to be proportional to the degree of contraction Half-maximal contraction can be achieved with ADP or ATP at concentrations as low as about 0 3 µM. Atractyloside at concentrations as low as about 1.2 nmol/mg mitochondrial protein completely inhibits the contraction. It is concluded from these and other observations that inner membrane contraction occurs as a result of adenine nucleotide binding to the carrier involved in the exchange of adenine nucleotides across the inner mitochondrial membrane.     

Adenine nucleotide translocase as a site of regulation by ADP of the rat liver mitochondria permeability to H+ and K+ lons

In the presence of oligomycin ADP inhibits the osmotic swelling of the nonenergized rat liver mitochondria in the NH₄NO₃ medium. With the energized mitochondria ADP enhances contraction of the mitochondria swollen in the NH₄NO₃ medium. Carboxyatractyloside and atractyloside abolish or prevent the effects of ADP. The direct measurements of the proton conductance of rat liver mitochondria shows that the inhibitory action of ADP + oligomycin on the H⁺ permeability does not depend on the energization of mitochondria. In these experiments the local anesthetic nupercaine and ADP additively inhibit the inner membrane conductance for protons, but carboxyatractyloside abolishes only the effect of ADP. In the presence of oligomycin ADP also inhibits the osmotic swelling of the nonenergized liver mitochondria in the KNO₃ medium, and the energy-dependent swelling of rat liver mitochondria in the medium with K⁺ ions and P_i. The inhibition by ADP of the membrane passive permeability for K⁺ is also sensitive to carboxyatractyloside. It is concluded that rat liver mitochondria possess an ADP-regulated channel for H⁺ and K⁺. The properties of this pathway for protons and potassium ions favor the idea that ADP regulates the mitochondrial permeability via adenine nucleotide translocase. It is assumed that the adenine nucleotides carrier should operate according to the “gated pore” mechanism.     

Glucagon treatment of rats activates the respiratory chain of liver mitochondria at more than one site

The rate of reduction of ferricyanide in the presence and absence of antimycin and ubiquinone-1 was measured using liver mitochondria from control and glucagon treated rats. Glucagon treatment was shown to increase electron flow from both NADH and succinate to ubiquinone, and from ubiquinone to cytochrome c. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was shown to inhibit the oxidation of glutamate + malate to a much greater extent than that of succinate or duroquinol. Spectral and kinetic studies confirmed that electron flow between NADH and ubiquinone was the primary site of action but that the interaction of the ubiquinone pool with complex 3 was also affected. The effects of various respiratory chain inhibitors on the rate of uncoupled oxidation of succinate and glutamate + malate by control and glucagon treated mitochondria were studied. The stimulation of respiration seen in the mitochondria from glucagon treated rats was maintained or increased as respiration was progressively inhibited with DCMU, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO) and colletotrichin, but greatly reduced when inhibition was produced with malonate or antimycin. These data were also shown to support the conclusion that glucagon treatment may cause some stimulation of electron flow through NADH dehydrogenase, succinate dehydrogenase and through the bc1 complex, probably at the point of interaction of the complexes with the ubiquinone pool. The effects of glucagon treatment on duroquinol oxidation and the inhibitor titrations could not be mimicked by increasing the matrix volume, nor totally reversed by aging of mitochondria. These are both processes that have been suggested as the means by which glucagon exerts its effects on the respiratory chain (Armston, A.E., Halestrap, A.P. and Scott, R.D., 1982, Biochim. Biophys. Acta 681, 429-439). It is concluded that an additional mechanism for regulating electron flow must exist and a change in lipid peroxidation of the inner mitochondrial membrane is suggested.     

Acetoacetate as regulator of palmitic acid-induced uncoupling involving liver mitochondrial ADP/ATP antiporter and aspartate/glutamate antiporter

The effect of acetoacetate on palmitate-induced uncoupling with the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter has been studied in liver mitochondria. The incubation of mitochondria with acetoacetate during succinate oxidation in the presence of rotenone, oligomycin, and EGTA suppresses the accumulation of conjugated dienes. This is considered as a display of antioxidant effect of acetoacetate. Under these conditions, acetoacetate does not influence the respiration of mitochondria in the absence or presence of palmitate but eliminates the ability of carboxyatractylate or aspartate separately to suppress the uncoupling effect of this fatty acid. The action of acetoacetate is eliminated by β-hydroxybutyrate or thiourea, but not by the antioxidant Trolox. In the absence of acetoacetate, the palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter); in its presence, it is limited by a stage insensitive to the effect of these agents. In the presence of Trolox, ADP suppresses the uncoupling action of palmitate to the same degree as carboxyatractylate. Under these conditions, acetoacetate eliminates the recoupling effects of ADP and aspartate, including their joint action. This effect of acetoacetate is eliminated by β-hydroxybutyrate or thiourea. It is supposed that the stimulating effect of acetoacetate is caused both by increase in the rate of transfer of fatty acid anion from the inner monolayer of the membrane to the outer one, which involves the ADP/ATP antiporter and aspartate/glutamate antiporter, and by elimination of the ability of ADP to inhibit this transport. Under conditions of excessive production of reactive oxygen species in mitochondria at a high membrane potential and in the presence of small amounts of fatty acids, such effect of acetoacetate can be considered as one of the mechanisms of antioxidant protection.     

Effect of ATP translocation on citrulline and oxaloacetate synthesis by isolated rat liver mitochondria.

The possibility that the availability of ATP may affect the rate of synthesis of carbamoyl phosphate (measured as citrulline) by carbamoyl phosphate synthase (ammonia) was studied using respiring isolated rat liver mitochondria incubated with added ADP, with hexokinase, glucose, and ATP, or with atractylate, in order to enhance or prevent the efflux of mitochondrial ATP. The effects of these agents were compared with those on oxaloacetate synthesis from pyruvate. Addition of hexokinase, glucose, and ATP to isolated mitochondria resulted in an inhibition of citrulline synthesis which was proportional to the amounts of glucose 6-phosphate formed; under these conditions, matrix ATP and ATP/ADP tended to decrease. The addition of increasing amounts of ADP also resulted in proportional inhibition of citrulline synthesis, but in this case the matrix content of ATP and ADP increased, and ATP/ADP decreased very slightly. In the presence of atractylate, citrulline synthesis was maximal despite a 30% decrease in matrix ATP and ATP/ADP. These effects were observed whether pyruvate, succinate, glutamate, or β-OH-butyrate was used as the respiratory substrate. ADP, the hexokinase system, and atractylate had qualitatively similar but much less pronounced effects on oxaloacetate synthesis from pyruvate. Within the limits of variation observed in these experiments, the rate of synthesis of citrulline appears not to be affected by the matrix content of total ATP, total ADP, or by ATP/ADP. It is affected, however, by the velocity of translocation of ATP into the extramitochondrial medium. These findings suggest that carbamoyl phosphate synthase (ammonia) may be loosely associated with the mitochondrial inner membrane, and may compete for ATP with the ATP-ADP translocator to an extent determined by the extramitochondrial demands for ATP.     

Control of oxidative phosphorylation in rat heart mitochondria: The role of the adenine nucleotide carrier

1. Inhibitor titration experiments carried out with carboxyatractyloside, oligomycin and rotenone show that in the case of heart mitochondria the membrane-bound ATPase and the respiratory chain are the major factors controlling the rate of oxidative phosphorylation whereas the adenine nucleotide carrier exhibits no control strength. 2. As shown by carboxyatractyloside titration curves under different conditions, the relative importance of the adenine nucleotide carrier depends on the mode of regeneration (F₁-ATPase or glucose plus hexokinase) of ADP from ATP exported outside mitochondria, on the total concentration of adenine nucleotides present in the medium and on the mode of limitation of the rate of respiration (cyanide, rotenone, oligomycin or mersalyl). 3. Concomitantly with the inhibition of O₂ consumption, carboxyatractyloside brings about a rise in membrane potential. The inverse relationship between the two processes is observed for carboxyatractyloside concentrations ranging between 0.7 and 1.5 nmol per mg protein. Carboxyatractyloside concentrations below and above this range increase the membrane potential without affecting significantly the rate of respiration. 4. Titration experiments aimed at comparing the effects of ADP, carboxyatractyloside and the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, corroborate the conclusion that in heart mitochondria a major limiting factor in oxidative phosphorylation is the capacity of the respiratory chain.     

Liver mitochondrial pyrophosphate concentration is increased by Ca2+ and regulates the intramitochondrial volume and adenine nucleotide content.

1. The matrix pyrophosphate (PPi) content of isolated energized rat liver mitochondria incubated in the presence of ATP, Mg2+, Pi and respiratory substrate was about 100 pmol/mg of protein. 2. After incubation with sub-micromolar [Ca2+], this was increased by as much as 300%. There was a correlation between the effects of Ca2+ on PPi and on the increase in matrix volume reported previously [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. Half-maximal effects were seen at 0.3 microM-Ca2+. 3. Coincident with these effects, the total adenine nucleotide content increased in a carboxyatractyloside-sensitive manner. 4. Incubation with 0.2-0.5 mM-butyrate induced similar but smaller effects on mitochondrial swelling and matrix PPi and total adenine nucleotide content. Addition of butyrate after Ca2+, or vice versa, caused Ca2+-induced mitochondrial swelling to stop or reverse, while matrix PPi increased 30-fold. 5. Addition of atractyloside or the omission of ATP from incubations greatly enhanced swelling induced by Ca2+ without increasing matrix PPi. 6. Swelling of mitochondria incubated under de-energized conditions in iso-osmotic KSCN was progressively enhanced by the addition of increasing concentrations of PPi (1-20 mM) or valinomycin. 7. In iso-osmotic potassium pyrophosphate swelling was slow initially, but accelerated with time. This acceleration was inhibited by ADP, whereas carboxyatractyloside induced rapid swelling. Swelling in other iso-osmotic PPi salts showed that the rate of entry decreased in the order NH4+ greater than K+ greater than Na+ greater than Li+, whereas choline, tetramethylammonium and Tris did not enter. It is suggested that the adenine nucleotide translocase transports small univalent cations when PPi is bound and that PPi can also be transported when the transporter is in the conformation induced by carboxyatractyloside. 8. It is concluded that Ca2+ and butyrate cause swelling of energized mitochondria through this effect of PPi on K+ permeability of the mitochondrial inner membrane. 9. Freeze-clamped livers from rats treated with glucagon or phenylephrine show 30-50% increases in tissue PPi. It is proposed that Ca2+-mediated increases in mitochondrial PPi are responsible for the increase in matrix volume and total adenine nucleotide content observed after hormone treatment.