A 1H NMR study of succinate synthesis from exogenous precursors in oxygen-deprived rat heart mitochondria

Succinate synthesis from exogenous malate, alpha-ketoglutarate, oxaloacetate and L-glutamate in isolated oxygen-deprived rat heart mitochondria was studied using 1H NMR. The highest rate of succinate synthesis was observed during incubation of mitochondria with a mixture of L-glutamate and oxaloacetate. When mitochondria were incubated with [U-13C] glutamate and oxaloacetate the [U-13C] succinate/succinate and aspartate/succinate ratios were equal to 2. This suggests that the succinate produced from [U-13C] alpha-keto-glutarate formed via transamination of [U-13C] glutamate with oxaloacetate by aspartate aminotransferase exceeds twofold that synthesized via oxaloacetate reduction. It may thus be expected that GTP yield in a reaction catalyzed by the succinic thiokinase will be 2 times higher that of ATP production coupled with NADH-dependent fumarate reduction.     

The formation of ornithine from proline in animal tissues

1. Homogenates of liver or kidney from rat, mouse, dog and guinea pig formed ornithine from proline but not from glutamate. Rat kidney was most active in this reaction and was used for further studies. 2. The overall reaction was found to be catalysed by proline oxidase to yield glutamic gamma-semialdehyde, followed by transamination of this product with glutamate as catalysed by ornithine-keto acid aminotransferase. 3. The unfavourable equilibrium of the ornithine-keto acid aminotransferase reaction was overcome chiefly by glutamate dehydrogenase in the tissue, which removed the alpha-oxoglutarate produced, by reduction with endogenous ammonia and NADH. 4. Aspartate aminotransferase in these preparations also aided in the removal of alpha-oxoglutarate. In this case the overall reaction was driven also by the rapid decarboxylation of oxaloacetate. 5. No evidence could be found for a pathway of ornithine synthesis involving acylated intermediates as has been observed in some micro-organisms. 6. The rate of ornithine synthesis in homogenates of several rat tissues paralleled the activity of ornithine-keto acid aminotransferase in these tissues, indicating that this enzyme was rate-determining for the synthesis. 7. The possible influence of these reactions on urea synthesis is discussed.     

Glutamate Dehydrogenase Reaction as a Source of Glutamic Acid in Synaptosomes

The role of the glutamate dehydrogenase reaction as a pathway of glutamate synthesis was studied by incubating synaptosomes with 5 mM 15NH4Cl and then utilizing gas chromatography-mass spectrometry to measure isotopic enrichment in glutamate and aspartate. The rate of formation of [15N]glutamate and [15N]aspartate from 5 mM 15NH4Cl was approximately 0.2 nmol/min/mg of protein, a value much less than flux through glutaminase (4.8 nmol/min/mg of protein) but greater than flux through glutamine synthetase (0.045 nmol/min/mg of protein). Addition of 1 mM 2-oxoglutarate to the medium did not affect the rate of [15N]glutamate formation. O2 consumption and lactate formation were increased in the presence of 5 mM NH3, whereas the intrasynaptosomal concentrations of glutamate and aspartate were unaffected. Treatment of synaptosomes with veratridine stimulated reductive amination of 2-oxoglutarate during the early time points. The production of ([15N]glutamate + [15N]aspartate) was enhanced about twofold in the presence of 5 mM beta-(+/-)-2-aminobicyclo [2.2.1]heptane-2-carboxylic acid, a known effector of glutamate dehydrogenase. Supplementation of the incubation medium with a mixture of unlabelled amino acids at concentrations similar to those present in the extracellular fluid of the brain had little effect on the intrasynaptosomal [glutamate] and [aspartate]. However, the enrichment in these amino acids was consistently greater in the presence of supplementary amino acids, which appeared to stimulate modestly the reductive amination of 2-oxoglutarate. It is concluded: (a) compared with the phosphate-dependent glutaminase reaction, reductive amination is a relatively minor pathway of synaptosomal glutamate synthesis in both the basal state and during depolarization; (b) NH3 toxicity, at least in synaptosomes, is not referable to energy failure caused by a depletion of 2-oxoglutarate in the glutamate dehydrogenase reaction; and (c) transamination is not a major mechanism of glutamate nitrogen production in nerve endings.     

Activation of Glutamate Dehydrogenase by Leucine and Its Nonmetabolizable Analogue in Rat Brain Synaptosomes

Leucine and beta-(+/-)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) stimulated, in a dose-dependent manner, reductive amination of 2-oxoglutarate in rat brain synaptosomes treated with Triton X-100. The concentration dependence curves were sigmoid, with 10-15-fold stimulations at 15 mM leucine (or BCH); oxidative deamination of glutamate also was enhanced, albeit less. In intact synaptosomes, leucine and BCH elevated oxygen uptake and increased ammonia formation, consistent with stimulation of glutamate dehydrogenase (GDH). Enhancement of oxidative deamination was seen with endogenous as well as exogenous glutamate and with glutamate generated inside synaptosomes from added glutamine. With endogenous glutamate, the stimulation of oxidative deamination was accompanied by a decrease in aspartate formation, which suggests a concomitant reduction in flux through aspartate aminotransferase. Activation of reductive amination of 2-oxoglutarate by BCH or leucine could not be demonstrated even in synaptosomes depleted of internal glutamate. It is suggested that GDH in synaptosomes functions in the direction of glutamate oxidation, and that leucine may act as an endogenous activator of GDH in brain in vivo.     

Influence of the Malate-Aspartate Shuttle on Oxidative Metabolism in Synaptosomes

beta-Methyleneaspartate, a specific inhibitor of aspartate aminotransferase (EC 2.6.1.1.), was used to investigate the role of the malate-aspartate shuttle in rat brain synaptosomes. Incubation of rat brain cytosol, "free" mitochondria, synaptosol, and synaptic mitochondria, with 2 mM beta-methyleneaspartate resulted in inhibition of aspartate aminotransferase by 69%, 67%, 49%, and 76%, respectively. The reconstituted malate-aspartate shuttle of "free" brain mitochondria was inhibited by a similar degree (53%). As a consequence of the inhibition of the aspartate aminotransferase, and hence the malate-aspartate shuttle, the following changes were observed in synaptosomes: decreased glucose oxidation via the pyruvate dehydrogenase reaction and the tricarboxylic acid cycle; decreased acetylcholine synthesis; and an increase in the cytosolic redox state, as measured by the lactate/pyruvate ratio. The main reason for these changes can be attributed to decreased carbon flow through the tricarboxylic acid cycle (i.e., decreased formation of oxaloacetate), rather than as a direct consequence of changes in the NAD+/NADH ratio. Malate/glutamate oxidation in "free" mitochondria was also decreased in the presence of 2 mM beta-methyleneaspartate. This is probably a result of decreased glutamate transport into mitochondria as a result of low levels of aspartate, which are needed for the exchange with glutamate by the energy-dependent glutamate-aspartate translocator.     

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.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: pathways of C4 acid decarboxylation in bundle sheath cells of Urochloa panicoides

The mechanism of C4 acid decarboxylation was studied in bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C4 plant. Added malate was decarboxylated to give pyruvate and this activity was often increased by adding ADP. Added oxaloacetate or aspartate plus 2-oxoglutarate (which produce oxaloacetate via aspartate aminotransferase) gave little metabolic decarboxylation alone but with added ATP there was a rapid production of PEP. For this activity ADP could replace ATP but only when added in combination with malate. In addition, the inclusion of aspartate plus 2-oxoglutarate with malate plus ADP often increased the rate of pyruvate production from malate by more than twofold. Experiments with respiratory chain inhibitors showed that the malate-dependent stimulation of oxaloacetate decarboxylation (PEP production) was probably due to ATP generated during the oxidation of malate in mitochondria. We could provide no evidence that photophosphorylation could serve as an alternative source of ATP for the PEP carboxykinase reaction. We concluded that both PEP carboxykinase and mitochondrial NAD-malic enzyme contribute to C4 acid decarboxylation in these cells, with the required ATP being derived from oxidation-linked phosphorylation in mitochondria.     

Glutamate metabolism in relation to glutamate transport in kidney cortex mitochondria of rabbit

1. The metabolism of glutamate was followed by measurements of phosphoenolpyruvate production, aspartate synthesis and ammonia release, whereas the transport of glutamate across the inner membrane of kidney cortex mitochondria was studied using an oxygen electrode and the swelling technique.2. When added separately, avenaciolide and aminooxyacetate only partially inhibited both State 3 and uncoupled respiration of the mitochondria, as studied in the presence of glutamate as substrate. In contrast, the addition of both inhibitors to the reaction medium resulted in an almost complete inhibition of glutamate oxidation.3. Swelling of kidney mitochondria in an isosmotic solution of ammonium glutamate was accelerated by uncoupler and inhibited by avenaciolide, while the swelling of mitochondria in potassium glutamate was stimulated by valinomycin and inhibited by uncoupler.4. When glutamate was used as the sole substrate, inhibition of aspartate formation by aminooxyacetate resulted in a stimulation of both ammonia release and phosphoenolpyruvate production. In contrast, with glutamate plus malate as substrate an elevation of the rate of glutamate deamination on the addition of aminooxyacetate was accompanied by an inhibition of phosphoenolpyruvate synthesis in both State 3 and uncoupled conditions.5. In the presence of valinomycin to induce K⁺-permeability a marked enhancement of glutamate deamination was accompanied by a significant inhibition of glutamate transamination.6. Based on the presented results it was concluded that in rabbit renal mitochondria utilizing glutamate as substrate the rates of ammonia production, phosphoenolpyruvate formation and aspartate synthesis vary in response to different metabolic conditions, in which both the glutamate—H⁺ symport and the glutamate—aspartate exchange systems are functioning to different extents.     

Glucose and Synaptosomal Glutamate Metabolism: Studies with [15N]Glutamate

The metabolism of [15N]glutamate was studied with gas chromatography-mass spectrometry in rat brain synaptosomes incubated with and without glucose. [15N]Glutamate was taken up rapidly by the preparation, reaching a steady-state level in less than 5 min. 15N was incorporated predominantly into aspartate and, to a much lesser extent, into gamma-aminobutyrate. The amount of [15N]ammonia formed was very small, and the enrichment of 15N in alanine and glutamine was below the level of detection. Omission of glucose substantially increased the rate and amount of [15N]aspartate generated. It is proposed that in synaptosomes (a) the predominant route of glutamate nitrogen disposal is through the aspartate aminotransferase reaction; (b) the aspartate aminotransferase pathway generates 2-oxoglutarate, which then serves as the metabolic fuel needed to produce ATP; (c) utilization of glutamate via transamination to aspartate is greatly accelerated when flux through the tricarboxylic acid cycle is diminished by the omission of glucose; (d) the metabolism of glutamate via glutamate dehydrogenase in intact synaptosomes is slow, most likely reflecting restriction of enzyme activity by some unknown factor(s), which suggests that the glutamate dehydrogenase reaction may not be near equilibrium in neurons; and (e) the activities of alanine aminotransferase and glutamine synthetase in synaptosomes are very low.     

Anaerobic metabolism of goldfish,Carassius auratus (L.). Influence of anoxia on mass-action ratios of transaminase reactions and levels of ammonia and succinate

Summary Changes in the concentrations of ammonia, glutamate, alanine, aspartate, -ketoglutarate, oxaloacetate and succinate were measured in freeze-clamped lateralred muscle, dorsal white muscle and liver, and in rapidly cooled blood of goldfish after 12 h of anoxia. Alanine accumulation, succinate accumulation and aspartate depletion are observed in all tissues examined; in the liver the concentrations of glutamate increase and those of ammonia decrease. The mass-action ratio of the glutamate-pyruvate transaminase-catalyzed reaction stays within one order of magnitude from thermodynamic equilibrium in the direction of alanine formation. The mass-action ratio of the glutamate-oxaloacetate transaminase reaction is far from equilibrium when measured oxaloacetate concentrations are used. When levels of free oxaloacetate are calculated from LDH and MDH equilibrium constants, the mass-action ratio of glutamate-oxaloacetate transamination is close to equilibrium in the direction of aspartate formation. Since neither alanine nor glutamate decreases, and since ammonia gradients suggest a continuous ammonia production in all tissues examined, anaerobic proteolysis is assumed. A possible coupling between amino acid catabolism and ethanol production is discussed.Abbreviations ALA alanine - ASP aspartate - EDTA ethylene diamine tetraacetate - FP _ox oxidated flavoprotein - FP _red reduced flavoprotein - FUM fumarate - GLU glutamate - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - IMP inosine monophosphate - KG -ketoglutarate - LDH lactate dehydrogenase - MAL malate - MAR mass action ratio - MDH malate dehydrogenase - OAA oxaloacetate - PYR pyruvate - sAMP adenylosuccinate - SDH succinate dehydrogenase - SUCC succinate     

The urea cycle in the liver of arthritic rats

The urea cycle in the liver of adjuvant-induced arthritic rats was investigated using the isolated perfused liver. Urea production in livers from arthritic rats was decreased during substrate-free perfusion and also in the presence of the following substrates: alanine, alanine + ornithine, ammonia, ammonia + lactate, ammonia + pyruvate and glutamine but increased when arginine and citrulline + aspartate were the substrates. No differences were found with ammonia + aspartate, ammonia + aspartate + glutamate, aspartate, aspartate + glutamate and citrulline. Ammonia consumption was smaller in the arthritic condition when the substance was infused together with lactate or pyruvate but higher when the substance was simultaneously infused with aspartate or aspartate + glutamate. Glucose production tended to correlate with the smaller or higher rates of urea synthesis. Blood urea was higher in arthritic rats (+25.6%), but blood ammonia was lower (–32.2%). Critical for the synthesis of urea from various substrates in arthritic rats seems to be the availability of aspartate, whose production in the liver is probably limited by both the reduced gluconeogenesis and aminotransferase activities. This is indicated by urea synthesis which was never inferior in the arthritic condition when aspartate was exogenously supplied, being even higher when both aspartate and citrulline were simultaneously present. Possibly, the liver of arthritic rats has a different substrate supply of nitrogenous compounds. This could be in the form of different concentrations of aspartate or other aminoacids such as citrulline or arginine (from the kidneys) which allow higher rates of hepatic ureogenesis.     

The regulation and glutamate metabolism by tricarboxylic acid-cycle activity in rat brain mitochondria

1. The interrelationship of metabolism of pyruvate or 3-hydroxybutyrate and glutamate transamination in rat brain mitochondria was studied. 2. If brain mitochondria are incubated in the presence of equimolar concentrations of pyruvate and glutamate and the K(+) concentration is increased from 1 to 20mm, the rate of pyruvate utilization is increased 3-fold, but the rate of production of aspartate and 2-oxoglutarate is decreased by half. 3. Brain mitochondria incubated in the presence of a fixed concentration of glutamate (0.87 or 8.7mm) but different concentrations of pyruvate (0 to 1mm) produce aspartate at rates that decrease as the pyruvate concentration is increased. At 1mm-pyruvate, the rate of aspartate production is decreased to 40% of that when zero pyruvate was present. 4. Brain mitochondria incubated in the presence of glutamate and malate alone produce 2-oxoglutarate at rates stoicheiometric with the rate of aspartate production. Both the 2-oxoglutarate and aspartate accumulate extramitochondrially. 5. Externally added 2-oxoglutarate has little inhibitory effect (K(i) approx. 31mm) on the production of aspartate from glutamate by rat brain mitochondria. 6. It is concluded that the inhibitory effect of increased C(2) flux into the tricarboxylic acid cycle on glutamate transamination is caused by competition for oxaloacetate between the transaminase and citrate synthase. 7. Evidence is provided from a reconstituted malate-aspartate (or Borst) cycle with brain mitochondria that increased C(2) flux into the tricarboxylic acid cycle from pyruvate may inhibit the reoxidation of exogenous NADH. These results are discussed in the light of the relationship between glycolysis and reoxidation of cytosolic NADH by the Borst cycle and the requirement of the brain for a continuous supply of energy.     

Pyruvate decarboxylation in astrocytes and in neurons in primary cultures in the presence and the absence of ammonia

Oxidative decarboxylation of [1-¹⁴C]pyruvate was studied in primary cultures of neurons and of astrocytes. The rate of this process, which is a measure of carbon flow into the tricarboxylic acid (TCA) cycle and which is inhibited by its end product, acetyl CoA, was determined under conditions which would either elevate or reduce the components of the malate-aspartate shuttle (MAS). Addition of aspartate (1 mM) was found to stimulate pyruvate decarboxylation in astrocytes whereas addition of glutamate (or glutamine) had no effect. Since aspartate is a precursor for extramitochondrial malate, and thus intramitochondrial oxaloacetate, whereas glutamate and glutamine are not, this suggests that an increase in oxaloacetate level stimulates TCA cycle activity. Conversely, a reduction of the glutamate content by 3 mM ammonia, which might reduce exchange between glutamate and aspartate across the mitochondrial membrane, suppressed pyruvate decarboxylation. This effect was abolished by addition of glutamate or glutamine or exposure to methionine sulfoximine (MSO). These findings suggest that impairment of MAS activity by removal of MAS constituents decreases TCA cycle activity whereas replenishment of these compounds restores the activity of the TCA cycle. No corresponding effects were observed in neurons.     

Effects of ischaemia on metabolite concentrations in rat liver

1. Changes in the concentrations of ammonia, glutamine, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, alanine, aspartate, malate, lactate, pyruvate, NAD(+), NADH and adenine nucleotides were measured in freeze-clamped rat liver during ischaemia. 2. Although the concentrations of most of the metabolites changed rapidly during ischaemia the ratios [glutamate]/[2-oxoglutarate][NH(4) (+)] and [3-hydroxybutyrate]/[acetoacetate] changed equally and the value of the expression [3-hydroxybutyrate][2-oxoglutarate][NH(4) (+)]/[acetoacetate][glutamate] remained approximately constant, indicating that the 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase systems were at near-equilibrium with the mitochondrial NAD(+) couple. 3. The value of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] was about 0.7 in vivo and remained fairly constant during the ischaemic period of 5min, although the concentrations of alanine and oxoglutarate changed substantially. No explanation can be offered why the value of the ratio differed from that of the equilibrium constant of the alanine aminotransferase reaction, which is 1.48. 4. Injection of l-cycloserine 60min before the rats were killed increased the concentration of alanine in the liver fourfold and decreased the concentration of the other metabolites measured, except that of pyruvate. During ischaemia the concentration of alanine did not change but that of aspartate almost doubled. 5. After treatment with l-cycloserine the value in vivo of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] rose from 0.7 to 2.4. During ischaemia the value returned to 0.8. 6. The effects of l-cycloserine are consistent with the assumption that it specifically inhibits alanine aminotransferase. 7. Most of the alanine formed during ischaemia is probably derived from pyruvate and from ammonia released by the deamination of adenine nucleotides and glutamine. The alanine is presumably formed by the combined action of glutamate dehydrogenase and alanine aminotransferase. 8. The rate of anaerobic glycolysis, calculated from the increase in the lactate concentration, was 1.3mumol/min per g fresh wt. 9. Although the concentrations of the adenine nucleotides changed rapidly during ischaemia, the ratio [ATP][AMP]/[ADP](2) remained constant at 0.54, indicating that adenylate kinase established near-equilibrium under these conditions.     

Synaptosomal bioenergetics. The role of glycolysis, pyruvate oxidation and responses to hypoglycaemia

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.     

The urea cycle and related pathways in the liver of Walker-256 tumor-bearing rats

The urea cycle was evaluated in perfused livers isolated from cachectic tumor-bearing rats (Walker-256 tumor). Urea production in livers of tumor-bearing rats was decreased in the presence of the following substrates: alanine, alanine + ornithine, alanine + aspartate, ammonia, ammonia + lactate, ammonia + pyruvate and glutamine. Urea production from arginine was higher in livers of tumor-bearing rats. No difference was found with aspartate, aspartate + ammonia, citrulline, citrulline + aspartate and glutamine + aspartate. Ammonia consumption was smaller in livers from cachectic rats when the substance was infused together with lactate and pyruvate. Glucose production was smaller in the cachectic condition only when alanine was the gluconeogenic substrate. Blood urea was higher in tumor-bearing rats, suggesting higher rates of urea production. The availability of aspartate seems to be critical for urea synthesis in the liver of tumor-bearing rats, which is possibly unable to produce this amino acid in sufficient amounts from endogenous sources. The liver of tumor-bearing rats may have a different exogenous substrate supply of nitrogenous compounds. Arginine could be one of these compounds in addition to aspartate which seems to be essential for an efficient ureogenesis in tumor-bearing rats.     

Effective Mechanism for Synthesis of Neurotransmitter Glutamate and its Loading into Synaptic Vesicles

Glutamate accumulation into synaptic vesicles is a pivotal step in glutamate transmission. This process is achieved by a vesicular glutamate transporter (VGLUT) coupled to v-type proton ATPase. Normal synaptic transmission, in particular during intensive neuronal firing, would demand rapid transmitter re-filling of emptied synaptic vesicles. We have previously shown that isolated synaptic vesicles are capable of synthesizing glutamate from α-ketoglutarate (not from glutamine) by vesicle-bound aspartate aminotransferase for immediate uptake, in addition to ATP required for uptake by vesicle-bound glycolytic enzymes. This suggests that local synthesis of these substances, essential for glutamate transmission, could occur at the synaptic vesicle. Here we provide evidence that synaptosomes (pinched-off nerve terminals) also accumulate α-ketoglutarate-derived glutamate into synaptic vesicles within, at the expense of ATP generated through glycolysis. Glutamine-derived glutamate is also accumulated into synaptic vesicles in synaptosomes. The underlying mechanism is discussed. It is suggested that local synthesis of both glutamate and ATP at the presynaptic synaptic vesicle would represent an efficient mechanism for swift glutamate loading into synaptic vesicles, supporting maintenance of normal synaptic transmission.     

Effects of probes of membrane potential on metabolism in synaptosomes

Effects of three probes for measuring membrane potential, tetraphenylphosphonium (TPP+), rhodamine 6G and 3,3'-dipropylthiocarbocyanine (diS-C3-(5)) on energy metabolism in synaptosomes were investigated. None of the three probes had any effect on lactate production in synaptosomes. TPP+ and rhodamine 6G did not inhibit the respiration of synaptosomes with pyruvate and succinate as exogenous substrate and were only weakly inhibitory with endogenous substrates. In contrast, diS-C3-(5) markedly inhibited the respiration of synaptosomes with glucose, pyruvate and endogenous substrates. All three probes reduced ATP content in synaptosomes and depolarized the membrane potential in synaptosomes with increasing concentrations of the probes. It is, therefore, preferable to estimate membrane potential with TPP+ or rhodamine 6G at their low concentrations where their effect on metabolism is negligible.     

Ammonia Intoxication: Effects on Cerebral Cortex and Spinal Cord

The effect of an acute systemic ammonia intoxication on the metabolic states of the cerebral cortex and the spinal cord of the same animal was studied in the cat. The intravenous infusion of ammonium acetate (2 and 4 mmol/kg body weight/30 min) increased the gross levels of tissue NH4+, glutamine, glutamine/glutamate ratio, lactate, and the lactate/pyruvate ratio in the cerebral cortex and the spinal cord. Pyruvate increased, but significantly only in the spinal cord; aspartate decreased, but significantly only in the cerebral cortex. The infusion of ammonium acetate did not significantly change the levels of phosphocreatine, ATP, ADP, AMP, total adenine nucleotides, adenylate energy charge, glucose, glutamate, alpha-ketoglutarate, and malate in either tissue. The changes of NH4+, glutamine, and lactate levels as well as glutamine/glutamate and lactate/pyruvate ratios in the spinal cord correlated significantly with the corresponding changes of these metabolites in the cerebral cortex. Thus, cerebral cortex and spinal cord show certain specific and comparable metabolic changes in response to a systemic ammonia intoxication. The effect of ammonia intoxication on the increases of glutamine and lactate levels is discussed.