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Notes: Abstract: We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used 15NH4Cl as a metabolic tracer to measure the production of both [5-15N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-15N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [15N]-glutamate to [2-15N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-15N]glutamine and stimulated the formation of [2-15N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-15N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-15N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-15N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of 14CO2 from [U-14C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the Km of glutamine synthetase for glutamate, which we determined to be ∼7 mM.

Notes: Abstract: The aim was to study the extent to which leu-cine furnishes α-NH2 groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [15N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [15N]glutamate/[15N]leucine and [2-15N]glutamine/[15N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [15N]leucine declined, reflecting dilution of the 16N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [16N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of 15N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ∼50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [15N]glutamate. After 45 min, the most highly labeled amino acid was [15N]alanine, which was closely followed by [15N]leucine and [15N]isoleucine. Relatively little 15N was detected in any other amino acids, except for [15N]serine. The transamination of leucine was ∼17 times greater than the rate of [1-14C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.

Notes: Abstract: Gas chromatography-mass spectrometry was used to evaluate the metabolism of [I5N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate 〉9 nmol/min/mg of protein. The metabolism of [15NJglutamine generated l5N-labelled glutamate, aspartate, and γ-aminobutyric acid (GABA). An efflux of both [15N]gIutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of ,5N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.

Notes: Abstract: Stable isotopes were used to measure both the rate of GABA formation by glutamic acid decarboxylase (GAD) and the rate of utilization by GABA-transaminase (GABA-T). The initial rate of GABA accumulation, determined with either [2-15N]glutamine or [2H5]glutamine as precursor, was 0.3–0.4 nmol/min/mg of protein. Addition of the calcium ionophore A23187 enhanced GAD activity, whereas changes in levels of inorganic phosphate and H+ were without influence. Flux through GABA-T (GABA → glutamate), measured with [15N]GABA as precursor, was 0.82 nmol/min/mg of protein, whereas the reamination of succinic acid semialdehyde (reverse flux through GABA-T) was almost sixfold faster, 4.8 nmol/min/mg of protein. The rate of GABA metabolism via the tricarboxylic acid cycle was very slow, with the upper limit on flux being 0.03 nmol/min/mg of protein. Addition of either acetoacetate or β-hydroxybutyrate raised the internal content of glutamate and reduced that of aspartate; the GABA concentration and the rate of its formation increased. It is concluded that in synaptosomes (a) GABA-T is a primary factor in regulating the turnover of GABA, (b) a major regulator of GAD activity is the concentration of internal calcium, (c) GAD in nerve endings may not be saturated with its substrate, glutamate, and the concentration of the latter is a determinant of flux through this pathway, and (d) levels of ketone bodies increase, and maintain at a higher value, the synaptosomal content of GABA, a phenomenon that may contribute to the beneficial effect of a ketogenic diet in the treatment of epilepsy.

Notes: Abstract: We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a Vmax of 53.6 ± 3.2 nmol/mg of protein/min and a Km of 449.2 ± 94.9 µM. Virtually all leucine transport was found to be Na+ independent. Astrocytic accumulation of leucine was much greater (3×) in the presence of α-aminooxyacetic acid (5 mM), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to α-ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent Km for leucine conversion to extracellular KIC was a medium [leucine] of 58 µM with a Vmax of ∼2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine + α-ketoglutarate ? KIC + glutamate) in astrocytes, but flux from leucine → glutamate is more active than that from glutamate → leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the “buffering” of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.

Notes: Abstract: The pathways of nitrogen transfer from 50 μM [15N]aspartate were studied in rat brain synaptosomes and cultured primary rat astrocytes by using gas chromatography-mass spectrometry technique. Aspartate was taken up rapidly by both preparations, but the rates of transport were faster in astrocytes than in synaptosomes. In synaptosomes, 15N was incorporated predominantly into glutamate, whereas in glial cells, glutamine and other 15N-amino acids were also produced. In both preparations, the initial rate of N transfer from aspartate to glutamate was within a factor of 2-3 of that in the opposite direction. The rates of transamination were greater in synaptosomes than in astrocytes. Omission of glucose increased the formation of [15N]-glutamate in synaptosomes, but not in astrocytes. Rotenone substantially decreased the rate of transamination. There was no detectable incorporation of 15N from labeled aspartate to 6-amino-15N-labeled adenine nucleotides during 60-min incubation of synaptosomes under a variety of conditions; however, such activity could be demonstrated in glial cells. The formation of 15N-labeled adenine nucleotides was marginally increased by the presence of 1 mM aminooxyacetate, but was unaffected by pretreatment with 1 mM 5-amino-4-imidazolecarboxamide ribose. It is concluded that (1) aspartate aminotransferase is near equilibrium in both synaptosomes and astrocytes under cellular conditions, but the rates of transamination are faster in the nerve endings; (2) in the absence of glucose, use of amino acids for the purpose of energy production increases in synaptosomes, but may not do so in glial cells because the latter possess larger glycogen stores; and (3) nerve endings have a very limited capacity for salvage of the adenine nucleotides via the purine nucleotide cycle.

Notes: Abstract: The role of the glutamate dehydrogenase reaction as a pathway of glutamate synthesis was studied by incubating synaptosomes with 5 mM15NH4Cl and then utilizing gas chromatography-mass spectrometry to measure isotopic enrichment in glutamate and aspartate. The rate of formation of I15N]glutamatc and [15N]aspartate from 5 mM15NH4Cl was ∼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 mMNH3, 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 β-(±)- 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.

Notes: Abstract: The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mMKCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na+-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasyn-aptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.

Notes: Abstract: 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 〈5 min. 15N was incorporated predominantly into aspartate and, to a much lesser extent, into γ-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 transami-nation 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 de-hydrogenase in intact synaptosomes is slow, most likely reflecting restriction of enzyme activity by some unknown factors), 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.

Notes: Abstract: The metabolism of branched-chain amino acids (BCAAs) was studied in cortical synaptosomes. With [15N]leucine (1 mM) as precursor, the cumulative appearance of 15N in [15N]glutamate and [15N]aspartate was 0.2 nmol/min/mg of protein without supplemental α-ketoglutarate and 0.3 nmol/min/mg of protein in the presence of α-ketoglutarate (0.5 mM). The BCAA aminotransferase reaction also proceeded in the “reverse” direction [α-ketoisocaproate (KIC) + glutamate → leucine + α-ketoglutarate]. This was documented by incubating synaptosomes with [15N]glutamate and measuring the formation of [15N]leucine. Without KIC in the medium, the rate of [15N]leucine production was 0.13 nmol/min/mg of protein. In the presence of 25 µM KIC the rate was 0.79 nmol/min/mg of protein and even greater (1.0 nmol/min/mg of protein) in the presence of 500 µM KIC. The reamination of KIC was two- to threefold faster with [2-15N]glutamine as precursor compared with [15N]glutamate. The ketoacid of valine, α-ketoisovalerate (KIV), was reaminated to [15N]valine at a rate comparable to that observed with respect to KIC. The BCAA transaminase mediated not only the bidirectional transfer of amino groups between leucine or valine and glutamate, but also the direct transfer of nitrogen between leucine and valine. This was ascertained in studies in which the incubation medium was supplemented with either [15N]leucine and KIV or [15N]valine and KIC (amino acids at 1 mM and ketoacids at 25 or 500 µM). The rate was faster in the direction of leucine formation at both the lower (6.1-fold) and higher (1.7-fold) KIC concentration. It is suggested that in synaptosomes the BCAA transaminase (a) functions predominantly in the direction of leucine formation and (b) maintains a constant ratio of BCAAs and ketoacids to one other.