Citrulline synthesis: Regulation by alterations in the total mitochondrial adenine nucleotide content

The total adenine nucleotide content of rat liver mitochondria was varied in vitro over a wide range in order to investigate a possible relationship between net changes in the total matrix ATP + ADP + AMP content and the overall rate of citrulline synthesis. Isolated mitochondria were specifically depleted of matrix adenine nucleotides by incubating with inorganic pyrophosphate (G. K. Asimakis and J. R. Aprille, 1980, Arch. Biochem. Biophys.203, 307–316); alternatively, matrix adenine nucleotides were increased by incubating mitochondria with 1 mm ATP at 30 °C. No exogenous ATP or ADP was included in the subsequent incubations for the determination of citrulline synthesis. Rates varied from 0.1 to 1.6 μmol citrulline/mg protein/h as a linear function of total adenine nucleotide content in the range 2–15 nmol (ATP + ADP + AMP)/mg protein. Further increases in the matrix ATP + ADP + AMP content caused no further increase in citrulline synthesis rates. Changes in the total adenine nucleotide content were reflected in proportional changes in both the ATP and ADP content of the matrix. The $\text{ATP}\text{ADP}$ ratio did not change significantly. Therefore, the variations in citrulline synthesis were most simply explained as the effect of different concentrations of ATP on the activity of carbamoyl-phosphate synthetase. It was concluded that net changes in the total adenine nucleotide content can contribute to the control of citrulline synthesis. These findings are significant in the context of recent evidence which shows that the matrix adenine nucleotide pool size is under hormonal control.     

The stimulation of the mitochondrial respiration by citrulline synthesis.

1. The influence of ammonia and ornithine on the oxygen uptake and the formation of citrulline was investigated with isolated rat liver mitochondria. The experiments were performed in a cytosol-like saline medium at 38 degrees C. 2. Under these conditions an increase of the respiration rate by ammonia and ornithine was observed, but a small response to external ADP, only. The missing stimulation by ADP was due to a partial inhibition of the respiratory chain by traces of zinc (approximately 1 microM) present in the medium. This inhibition was only detected at low concentrations of mitochondria. 3. For activation of respiration by ammonia plus ornithine two different processes were responsible: (i) chelation of the inhibiting zinc by ornithine, which could be prevented by EDTA; (ii) ADP production in the matrix space during formation of carbamoyl phosphate, which could be prevented by oligomycin but not by carboxyatractyloside. 4. This stimulus of the carbamoyl phosphate formation and of the equivalent citrulline synthesis on the mitochondrial respiration ran to 12% of that increase caused by phosphorylation of external ADP. The maximum rate of citrulline formation was limited by the activity of carbamoyl phosphate synthetase. 5. Added ADP suppresses the production of citrulline probably by the exchange of extramitochondrial ADP versus intramitochondrial ATP. The data suggest a common adenine nucleotide pool delivering ATP to the adenine nucleotide translocase as well as to the carbamoyl phosphate synthetase.     

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Characteristics related to quantitative studies of RNA metabolism

The atractyloside-insensitive accumulation of adenine nucleotides by rat liver mitochondria (as opposed to the exchange-diffusion catalysed by the adenine nucleotide translocase) has been measured by using the luciferin/luciferase assay as well as by measuring [14C]ATP uptake. In foetal rat liver mitochondria ATP is accumulated more rapidly than ADP, whereas AMP is not taken up. The uptake of ATP occurs against a concentration gradient, and the rate of ATP uptake is greater in foetal than in adult rat liver mitochondria. The accumulated [14C]ATP is shown to be present within the mitochondrial matrix space and is freely available to the adenine nucleotide translocase for exchange with ATP present in the external medium. The uptake is specific for ATP and ADP and is not inhibited by adenosine 5'-[beta gamma-imido] triphosphate, GTP, CTP, cyclic AMP or Pi, whereas dATP and AMP do inhibit ATP accumulation. The ATP accumulation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, KCN and mersalyl but is insensitive to atractyloside. The ATP uptake is concentration-dependent and exhibits Michaelis-Menten kinetics. The divalent cations Mg2+ and Ca2+ greatly enhance ATP accumulation, and the presence of hexokinase inhibits the uptake of ATP by foetal rat liver mitochondria. These latter effects provide an explanation for the low adenine nucleotide content of foetal rat liver mitochondria and the rapid increase that occurs in the mitochondrial adenine nucleotide concentration in vivo immediately after birth.     

Competition between extramitochondrial and intramitochondrial ATP-consuming processes.

The relationship between intra- and extramitochondrial ATP utilization was investigated in liver mitochondria isolated from normally fed, starved and high-protein fed rats. ATP export was provoked by adding a hexokinase-glucose-trap and intramitochondrial ATP consumption by adding ammonia, bicarbonate and ornithine in order to stimulate citrulline synthesis. Both processes compete for ATP produced via oxidative phosphorylation; the rate of citrulline formation declines as the extramitochondrial [ATP]/[ADP] ratio decreases. It is concluded that ATP for adenine nucleotide translocation and that for carbamoyl phosphate synthesis are delivered from a common intramitochondrial pool of adenine nucleotides. In mitochondria from rats with a high-protein diet, citrulline synthesis greatly stimulates the rate of oxidative phosphorylation (about two thirds of state 3 respiration). Under these conditions the intramitochondrial [ATP]/[ADP] ratio is significantly reduced. The intramitochondrial [ATP]/[ADP] ratio is not in thermodynamic equilibrium with the extramitochondrial one.     

Effect of adenine nucleotide pool size in mitochondria on intramitochondrial ATP levels.

Net adenine nucleotide transport into and out of the mitochondrial matrix via the ATP-Mg/Pi carrier is activated by micromolar calcium concentrations in rat liver mitochondria. The purpose of this study was to induce net adenine nucleotide transport by varying the substrate supply and/or extramitochondrial ATP consumption in order to evaluate the effect of the mitochondrial adenine nucleotide pool size on intramitochondrial adenine nucleotide patterns under phosphorylating conditions. Above 12 nmol/mg protein, intramitochondrial ATP/ADP increased with an increase in the mitochondrial adenine nucleotide pool. The relationship between the rate of respiration and the mitochondrial ADP concentration did not depend on the mitochondrial adenine nucleotide pool size up to 9 nmol ADP/mg mitochondrial protein. The results are compatible with the notion that net uptake of adenine nucleotides at low energy states supports intramitochondrial ATP consuming processes and energized mitochondria may lose adenine nucleotides. The decrease of the mitochondrial adenine nucleotide content below 9 nmol/mg protein inhibits oxidative phosphorylation. In particular, this could be the case within the postischemic phase which is characterized by low cytosolic adenine nucleotide concentrations and energized mitochondria.     

On the mechanism of the so-called uncoupling effect of medium- and short-chain fatty acids

Octanoate applied to rat liver mitochondria respiring with glutamate plus malate or succinate (plus rotenone) under resting-state (State 4) conditions stimulates oxygen uptake and decreases the membrane potential, both effects being sensitive to oligomycin but not to carboxyatractyloside. Octanoate also decreases the rate of pyruvate carboxylation under the same conditions, this effect being correlated with the decrease of intramitochondrial content of ATP and increase of AMP. The decrease of pyruvate carboxylation and the change of mitochondrial adenine nucleotides are both reversed by 2-oxoglutarate. Fatty acids of shorter chain length have similar effects, though at higher concentrations. Addition of octanoate in the presence of fluoride (inhibitor of pyrophosphatase) produces intramitochondrial accumulation of pyrophosphate, even under conditions when oxidation of octanoate is prevented by rotenone. In isolated hepatocytes incubated with lactate plus pyruvate, octanoate also increases oxygen uptake and produces a shift in the profile of adenine nucleotides similar to that observed in isolated mitochondria. It decreases the ‘efficiency’ of gluconeogenesis, as expressed by the ratio between an increase of glucose production and an increase of oxygen uptake upon addition of gluconeogenic substrates (lactate plus pyruvate), and increases the reduction state of mitochondrial NAD. These effects taken together are not compatible with uncoupling, but point to intramitochondrial hydrolysis of octanoyl-CoA and probably also shorter chain-length acyl-CoAs. This mechanism probably functions as a ‘safety valve’ preventing a drastic decrease of intramitochondrial free CoA under a large supply of medium- and short-chain fatty acids.     

Decrease in mitochondrial levels of adenine nucleotides and concomitant mitochondrial dysfunction in ischemic rat liver

The process of mitochondrial dysfunction in ischemic rat liver was studied. A close correlation was found between decrease in the mitochondrial adenine nucleotide content and deterioration of oxidative phosphorylation capacity. The level of total adenine nucleotides, which was 15--20 nmol/mg protein in mitochondria isolated from normal liver, fell to 1--2 nmol/mg protein with concomitant loss of oxidative phosphorylation capacity after anoxic incubation in vitro or in vivo for 120 min. However, neither the permeability barrier to adenine nucleotides nor matrix enzymes were affected under these conditions. The loss of adenine nucleotides was ascribed to degradation of AMP to adenosine and then leakage of the latter. Conventional procedures for maintenance of oxidative phosphorylation capacity of isolated mitochondria, preservation in the cold and addition of ATP or a respiratory substrate under aerobic conditions, were very effective in maintaining the intramitochondrial levels of adenine nucleotides. Of the three species of adenine nucleotides, only AMP was ineffective in maintaining mitochondrial function; mitochondria containing more than 5 nmol of ATP plus ADP/mg protein exhibited normal activity of oxidative phosphorylation, but with less than 2 nmol they showed no activity.     

The stimulatory effect of alloxan diabetes on citrulline formation in rabbit liver mitochondria

The effect of alloxan diabetes on citrulline formation from NH₄Cl and bicarbonate was studied in rabbit liver mitochondria incubated with glutamate or succinate as respiratory substrate, as well as with exogenous ATP in the presence of uncoupler and oligomycin. In contrast to ornithine transcarbamoylase, the activity of carbamoyl-phosphate synthetase (ammonia) was higher in mitochondria from diabetic animals than in those from normal ones. In diabetic rabbits the rates of citrulline synthesis were stimulated under all conditions studied. In contrast, levels of N-acetyglutamate, an activator of carbamoyl-phosphate synthetase (ammonia), were significantly increased only in the presence of glutamate, while the highest rates of citrulline formation occurred in uncoupled mitochondria incubated with exogenous ATP as energy source. Treatment of animals with alloxan resulted in an increase of both the intramitochondiral ATP level and the rate of adenine nucleotide translocation across the mitochondrial membrane. The results indicate that the stimulation of citrulline formation in liver mitochondria of diabetic rabbits is mainly due to an increase in carbamoyl-phosphate synthetase (ammonia) activity and an elevation of content of intramitochondrial ATP, a substrate of this enzyme.     

Adenine nucleotide pool size,adenine nucleotide translocase activity,and respiratory activity in newborn rabbit liver mitochondria

The adenine nucleotide content (ATP+ADP+AMP) of newborn rabbit liver mitochondria was 6.0±0.5 nmol/mg mitochondrial protein at birth, increased rapidly to 14.5±1.7 nmol/mg protein by 2 h postnatal, peaked at 6 h, then decreased gradually to 7.8±0.6 nmol/mg protein by 4 days postnatal. There was a strong positive correlation (r=0.82) between the total adenine nucleotide pool size and adenine nucleotide translocase activity in these mitochondria. In contrast, glutamate + malate-supported State 3 respiratory rates remained constant from birth through the first week of life. State 4 rates also remained constant, as did the respiratory control index and uncoupled respiratory rates. The following conclusions are suggested: (1) The maximum rate of translocase activity is limited by the intramitochondrial adenine nucleotide pool size. (2) In newborn rabbit liver mitochondria, the State 3 respiratory rate is not limited by either the adenine pool size or the maximum capacity for translocase-mediated adenine exchange. (3) In contrast to rat, rabbit liver mitochondria are fully functional at birth with regard to respiratory rates and oxidative phosphorylation. (4) The rapid postnatal accumulation of adenine nucleotides by liver mitochondria, now documented in two species, may be a general characteristic of normal metabolic adjustment in neonatal mammals.     

Effect of pent-4-enoic acid, propionic acid and other short-chain fatty acids on citrulline synthesis in rat liver mitochondria.

19 The effect of pent-4-enoic acid, propionic acid and several other short-chain fatty acids on citrulline synthesis in rat liver mitochondria was studied. 2.Pent-4-enoate at 1 mM inhibited mitochondrial citulline synthesis by about 80-90%. It is concluded that pent-4-enoate inhibits citrulline synthesis by interfering with some aspect of mitochondrial energy metabolism. This results in impairment of mitochondrial ornithine uptake or depletion of mitochondrial ATP, which, in turn, impairs carbamoyl phosphate synthesis or both. Evidence in support of this conclusion includes: pent-4-enoate has no effect on citrulline synthesis supported by succinate or exogenous ATP; pent-4-enoate lowers the medium plus mitochondrial ATP concentration; finally, when glutamate is the oxidizable substrate, pent-4-enoate decreases the carbamoyl phosphate concentration in mitochondria incubated without ornithine to minimize citrulline synthesis and impairs the mitochondrial uptake of ornithine, but it has neither effect when succinate is the oxidizable substrate. 4. Propionate, butyrate and crotonate also inhibit mitochondrial citrulline synthesis, but much less than pent-4-enoate. 5. Acetate, pentanoate, pent-2-enoate, hexanoate, octanoate, isovalerate, tiglylate and alpha-methylbutyrate have little or no effect on mitochondrial citrulline synthesis.     

Changes in intramitochondrial adenine nucleotides in blowfly flight-muscle mitochondria

1. With freshly isolated blowfly mitochondria 38% of the intramitochondrial adenine nucleotide was present as AMP. 2. On incubation with oxidizable substrates the AMP and ADP concentrations fell and that of ATP rose; with pyruvate together with proline the ATP concentration reached its maximum value at 6min; with glycerol phosphate the phosphorylation of endogenous nucleotide was more rapid. 3. Addition of the uncoupling agent carbonyl cyanide phenylhydrazone caused a rapid fall of ATP and a parallel rise in ADP, then ADP was converted into AMP. 4. This was in contrast with rat liver mitochondria endogenous AMP concentrations, which were always lower than those of blowfly mitochondria and changed little under different metabolic conditions. 5. Evidence is presented that adenylate kinase (EC 2.7.4.3) has a dual distribution in blowfly mitochondria, a part being located in the matrix space and a part in the space between the outer and inner mitochondrial membranes, as in liver and other mitochondria. 6. The possible regulatory role of changing AMP concentrations in the mitochondrial matrix was investigated. Partially purified pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.3.7) were inhibited 30% by 2mm-AMP, whereas pyruvate dehydrogenase (EC 1.2.4.1) was unaffected. 7. AMP activated the NAD(+)-linked isocitrate dehydrogenase (EC 1.1.1.41) activity of blowfly mitochondria in the absence of ADP, but in the presence of ADP, AMP caused inhibition. 8. It is suggested that AMP may exert a controlling effect on the oxidative activity of blowfly mitochondria.     

Relation between glutamate and adenine nucleotide levels of heart mitochondria during hypoxia

The effect of acute respiratory hypoxia in rats on mitochondrial respiration, adenine nucleotides and some amino acids of the heart was studied. The decrease in the total (ATP + ADP + AMP) and exchangeable (ATP + ADP) adenine nucleotide pool of the mitochondria was accompanied by a pronounced loss of state 3 respiration with glutamate plus malate and a slight decrease with succinate plus rothenone. The uncoupled respiration of mitochondria with glutamate and malate was decreased in the same degree as in the absence of 2,4-dinitrophenol. State 4 respiration with substrates of both types was unaffected by hypoxia. These data point to a hypoxia-induced impairment of complex I of the respiratory chain. The decrease of tissue and mitochondrial glutamate was accompanied by the elevation of alanine content in the heart and an increase in intramitochondrial aspartate. The ADP-stimulated respiration of mitochondria was correlated with mitochondrial glutamate and ATP as well as with exchangeable adenine nucleotide pools during hypoxia. The experimental results suggest that mitochondrial dysfunction induced by hypoxia may also be attributed to the low level of mitochondrial glutamate.     

Permeability transition in rat liver mitochondria is modulated by the ATP-Mg/Pi carrier

Mitochondrial permeability transition, due to opening of the permeability transition pore (PTP), is triggered by Ca2+ in conjunction with an inducing agent such as phosphate. However, incubation of rat liver mitochondria in the presence of low micromolar concentrations of Ca2+ and millimolar concentrations of phosphate is known to also cause net efflux of matrix adenine nucleotides via the ATP-Mg/Pi carrier. This raises the possibility that adenine nucleotide depletion through this mechanism contributes to mitochondrial permeability transition. Results of this study show that phosphate-induced opening of the mitochondrial PTP is, at least in part, secondary to depletion of the intramitochondrial adenine nucleotide content via the ATP-Mg/Pi carrier. Delaying net adenine nucleotide efflux from mitochondria also delays the onset of phosphate-induced PTP opening. Moreover, mitochondria that are depleted of matrix adenine nucleotides via the ATP-Mg/Pi carrier show highly increased susceptibility to swelling induced by high Ca2+ concentration, atractyloside, and the prooxidant tert-butylhydroperoxide. Thus the ATPMg/Pi carrier, by regulating the matrix adenine nucleotide content, can modulate the sensitivity of rat liver mitochondria to undergo permeability transition. This has important implications for hepatocytes under cellular conditions in which the intramitochondrial adenine nucleotide pool size is depleted, such as in hypoxia or ischemia, or during reperfusion when the mitochondria are exposed to increased oxidative stress.     

In vitro alteration of the size of the liver mitochondrial adenine nucleotide pool: Correlation with respiratory functions

Mitochondrial respiration was studied as a function of the total adenine nucleotide content of rat liver mitochondria. The adenine nucleotide content was varied by treating isolated mitochondria with pyrophosphate or by incubating pyrophosphate-treated mitochondria with ATP. Mitochondria with at least 4 nmol adenine nucleotides/mg protein maintained at least 80% of the State 3 activity of control mitochondria, which had approximately 10 nmol/mg protein. However, State 3 decreased rapidly once the adenine nucleotide content fell below 4 nmol/mg protein. Between 2 and 4 nmol adenine nucleotides/mg, State 3 was not limited by the maximal capacity of electron flow as measured by the uncoupled respiration. However, at very low adenine nucleotide levels (<2 nmol/mg), the uncoupled rates of respiration were markedly depressed. State 4 was not affected by changes in the mitochondrial adenine nucleotide content. Adenine translocase activity varied in almost direct correlation with changes in the adenine nucleotide content. Therefore, adenine translocase activity was more sensitive than State 3 to changes in total adenine nucleotides over the range of 4 to 10 nmol/mg protein. The results suggest that (i) State 3 is dependent on the level of intramitochondrial adenine nucleotides, particularly in the range below 4 nmol/mg protein, (ii) adenine translocase activity is not rate-limiting for oxidative phosphorylation in mitochondria with the normal complement of adenine nucleotides, however, at low adenine nucleotide levels, depressed State 3 rates may be explained in part by the low rate of ADP translocation, and (iii) a mechanism of net ATP uptake exists in mitochondria with low internal adenine nucleotides.     

Control of the energizing of liver mitochondria at the adenine nucleotide carrier level under hypotonic conditions

The effects of ADP, carboxyatractyloside (CAT) and the local anaesthetic nupercaine on the energy-dependent Ca2+ uptake by rat liver mitochondria oxidizing succinate in the presence of oligomycin were compared, using incubation media of 320 mosM and 120 mosM tonicities. In hypotonic media the mitochondrial Ca2+ capacity was increased by 50%, and the mitochondria were more stable to the damaging effects of Ca + Pi. In the presence of ADP the Ca2+ capacities of mitochondria increased both in normotonic and hypotonic media; however, the absolute amounts of calcium consumed were levelled off. CAT abolished the effect of ADP on the mitochondrial Ca2+ uptake and equalized the Ca2+ capacities of rat liver mitochondria in the both media. The local anaesthetic nupercaine also increased the Ca2+ capacity of mitochondria. The effects of nupercaine and ADP were additive. CAT abolished the effect of ADP but not that of nupercaine. Measurements of the intramitochondrial contents of adenine nucleotides showed that in 120 mosM media there was a significant increase in the intramitochondrial content of ATP and the total pool of adenine nucleotides. It was concluded that in hypotonic media the mitochondrial adenine nucleotide carrier exists predominantly in the m-conformation thus facilitating the energization of mitochondria.     

Investigation of the dependence of the intramitochondrial [ATP]/[ADP] ratio on the respiration rate

The relationship between the respiration rate and the intra- and extramito-chondrial adenine nucleotides was investigated in isolated rat liver mitochondria.

For the determination of adenine nucleotide patterns in both compartments a new procedure was developed, based on the evaluation of these metabolites from incubation of various amounts of mitochondria under identical stationary states of oxidative phosphorylation. These identical states were adjusted by addition of appropriate amounts of hexokinase to a glucose-containing incubation mixture.

Adenine nucleotides were measured in aliquots of the total extract of the incubation mixture without any separation. The concentrations of the adenine nucleotides in both compartments were obtained from a plot of the total concentration of these species versus mitochondrial protein. Disturbances of this method by unspecific efflux of adenine nucleotides could be excluded.

The results obtained for the total adenine nucleotide content (12 nmol · mg⁻¹ protein) and the intramitochondrial [ATP]/[ADP] ratio (about 4 in the resting state) are in good agreement with data obtained by other methods.

Strong evidence is provided for a decrease of the intramitochondrial [ATP]/[ADP] ratio with increasing rate of oxygen consumption. Therefore it is not necessary to assume a microcompartmentation of the intramitochondrial adenine nucleotide pool in respect to the ATPase reaction and the adenine nucleotide translocation.     

The effects of altered sterol composition on the mitochondrial adenine nucleotide transporter of Saccharomyces cerevisiae.

1. The membrane sterol composition of mitochondria of the ole-3 mutant of Saccharomyces cerevisiae was manipulated by growing the organism in the presence of Tween 80 (1%, W/V) plus defined supplements o- delta-aminolaevulinate. 2. Changes in mitochondrial sterol content induced considerable changes in the adenine nucleotide transporter. 3. As the sterol content was decreased, the affinity of the transporter for ATP did not alter significantly, but the rate of ATP uptake was greatly decreased, the total number of atractylate-sensitive binding sites diminished, and the proportion of high-affinity binding sites was decreased. 4. Since sterol depletion also uncouples oxidative phosphorylation [Astin & Haslam (1977) Biochem. J., 166, 287-298] and prevents the intramitochondrial generation of ATP, the decrease in the rate of ATP uptake by sterol-depleted mitochondria will cause a decrease in intramitochondrial ATP concentrations in vivo. This probably explains the inhibition of mitochondrial macromolecular synthesis that has previously been reported in lipid-depleted yeast mitochondria.     

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.     

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.     

Relationship of the intra- and extramitochondrial adenine nucleotide ratios during synthesis of phosphoenolpyruvate using extramitochondrial ATP

Mitochondria prepared from the livers of guinea pig, chicken, and pigeon all actively synthesize phosphoenolpyruvate from oxalacetate and GTP, utilizing phosphoenolpyruvate carboxykinase. It was previously shown (Wilson, D. F., Erecińska, M., and Schramm, V. L. (1983). J. Biol. Chem. 258, 10464-10473) that phosphoenolpyruvate carboxykinase is freely reversible and that, in conjunction with nucleoside diphosphate kinase and malate dehydrogenase, which are also present in the mitochondria, it can be used to measure the intramitochondrial [ATPfree]/[ADPfree]. In this study, synthesis of phosphoenolpyruvate by guinea pig liver mitochondria was studied under conditions for which the only source of GTP was extramitochondrial ATP via adenine nucleotide translocase and nucleoside diphosphate kinase (the mitochondria were treated with rotenone, oligomycin, uncoupler, and fluorocitrate). When the extramitochondrial [ATP]/[ADP] was greater than the intramitochondrial [ATPfree]/[ADPfree] calculated from the phosphoenolpyruvate carboxykinase reaction, there was net synthesis of phosphoenolpyruvate, but when it was less, there was net disappearance of phosphoenolpyruvate. Thus, the intramitochondrial [ATPfree]/[ADPfree] was equal to the extramitochondrial value at the point of reversal of the phosphoenolpyruvate carboxykinase reaction. This equality of the intra- and extramitochondrial adenine nucleotide ratios occurred with a measured mitochondrial membrane potential of approximately -36 mV, whereas in the previous experiments, equality was observed for conditions in which the measured membrane potential was -111 to -125 mV. Thus, adenine nucleotide translocation was not dependent on the transmembrane electrical potential and must, therefore, have occurred by electroneutral exchange.