Inhibition of glutamine synthetase in the mouse kidney: a novel mechanism of adaptation to metabolic acidosis

As part of a study on the regulation of renal ammoniagenesis in the mouse kidney, we investigated the effect of chronic metabolic acidosis on glutamine synthesis by isolated mouse renal proximal tubules. The results obtained reveal that, in tubules from control mice, glutamine synthesis occurred at high rates from glutamate and proline and, to a lesser extent, from ornithine, alanine, and aspartate. A 48 h, metabolic acidosis caused a marked inhibition of glutamine synthesis from near-physiological concentrations of both alanine and proline that were avidly metabolized by the tubules; metabolic acidosis also greatly stimulated glutamine utilization and metabolism. These effects were accompanied by a large increase (i) in alanine, proline, and glutamine gluconeogenesis and (ii) in ammonia accumulation from proline and glutamine. In the renal cortex of acidotic mice, the activity of phosphoenolpyruvate carboxykinase increased 4-fold, but that of glutamate dehydrogenase did not change; in contrast with what is known in the rat renal cortex, metabolic acidosis markedly diminished the glutamine synthetase activity and protein level, but not the glutamine synthetase mRNA level in the mouse renal cortex. These results strongly suggest that, in the mouse kidney, glutamine synthetase is an important regulatory component of the availability of the ammonium ions to be excreted for defending systemic acid-base balance. Furthermore, they show that, in rodents, the regulation of renal glutamine synthetase is species-specific.     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

Stimulation of glutamine metabolism by 3-aminopicolinate in isolated dog kidney-cortex tubules

1. The effects of 3-aminopicolinate, a known hyperglycaemic agent in the rat, on glutamine metabolism were studied in isolated dog kidney tubules. 2. 3-Aminopicolinate greatly stimulated glutamine (but not glutamate) removal and glutamate accumulation from glutamine as well as formation of ammonia, aspartate, lactate, alanine and glucose. 3. The increased accumulation of aspartate from glutamine and glutamate, and the inhibition of glucose synthesis from various non-nitrogenous gluconeogenic substrates, as well as the increased accumulation of malate from succinate, support the proposal that 3-aminopicolinate is an inhibitor rather than a stimulator of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) in dog kidney tubules. 4. With glutamine as substrate, the increase in flux through glutamate dehydrogenase (EC 1.4.1.3) could not explain the large increase in glutamine removal caused by 3-aminopicolinate. 5. Inhibition by amino-oxyacetate of accumulation of aspartate and alanine from glutamine caused by 3-aminopicolinate did not prevent the acceleration of glutamine utilization. 6. These data are consistent with a direct stimulation of glutaminase (EC 3.5.1.2) by 3-aminopicolinate in dog kidney tubules.     

Effect of natural amino acids on ethanol oxidation in isolated rat hepatocytes]

The effects of the various naturally occurring amino acids on ethanol oxidation in hepatocytes from starved rats was systematically studied. In order to minimize the non ADH pathways, the ethanol concentration used was 4 mmol/litre, the amino acids being added at the same concentration. In hepatocytes from fasted rats, alanine, arginine, asparagine, aspartate, citrulline, cysteine, glutamate, glutamine, glycine, histidine, hydroxyproline, ornithine and serine increase significantly ethanol consumption. The stimulatory effect of glutamine being much less pronounced than the asparagine one and proline being devoid of action, the influence of ammonium chloride addition on ethanol consumption in the presence of these amino acids was studied. Ammonium chloride determines an enhancement of ethanol oxidation in these conditions, the results showing no apparent correlation between intracellular glutamate concentration and ethanol oxidation rate, contrarily to previous data. In hepatocytes from fed rats, only alanine, asparagine, cysteine, glycine, hydroxyproline, ornithine and serine increase ethanol oxidation, although to a lesser extent than in cells from starved rats.     

Serine synthesis by an isolated perfused rat kidney preparation.

The isolated perfused rat kidney was shown to synthesize serine from aspartate or glutamate, both of which are also precursors of glucose. The major products of aspartate metabolism were ammonia, serine, glutamate, glucose, glutamine and CO2. Perfusion of kidneys with aspartate in the presence of amino-oxyacetate resulted in a near-complete inhibition of aspartate metabolism, illustrating the essential role of aspartate aminotransferase in the metabolism of this substrate. Radioactivity from 14C-labelled aspartate and from 14C-labelled glycerol was incorporated into serine and glucose. Production of both glucose and serine from aspartate was suppressed in the presence of 3-mercaptopicolinic acid. These data provide evidence for the operation of the phosphorylated and/or non-phosphorylated pathway for serine production to the presence of 3-mercaptopicolinic acid. This is explained by simultaneous glycolysis. The rate of glucose production, but not that of serine, was greater in kidneys perfused with glutamate or with aspartate plus glycerol than the rates obtained by perfusion with aspartate alone. These data are taken to suggest that serine synthesis occurred at a near-maximal rate, and that the capacity of the kidney for serine synthesis from glucose precursors is lower than that for glucose synthesis.     

Nitrogen metabolism in tumor bearing mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.     

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K⁺-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K⁺ stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K⁺ stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.     

Role of asparagine in the photorespiratory nitrogen metabolism of pea leaves

In pea leaves, much of the metabolism of imported asparagine is by transamination. This activity was previously shown to be localized in the peroxisomes, suggesting a possible connection between asparagine and photorespiratory nitrogen metabolism. This was investigated by examination of the transfer of ¹⁵N from the amino group of asparagine, supplied via the transpiration stream, in fully expanded pea leaves. Label was transferred to aspartate, glutamate, alanine, glycine, serine, ammonia, and glutamine (amide group). Under low oxygen (1.8%), or in the presence of α-hydroxy-2-pyridine methanesulfonic acid (an inhibitor of glycolate oxidase, a step in the photorespiratory formation of glyoxylate), there was a substantial (60-80%) decrease in transfer of label to glycine, serine, ammonia, and glutamine. Addition of isonicotinyl hydrazide (an inhibitor of formation of serine from glycine) caused a 70% decrease in transfer of asparagine amino nitrogen to serine, ammonia, and glutamine, while a 4-fold increase in labeling of glycine was observed. The results demonstrate the involvement of asparagine in photorespiration, and show that photorespiratory nitrogen metabolism is not a closed cyclic process.     

Effect of the antiepileptic drug sodium valproate on glutamine and glutamate metabolism in isolated human kidney tubules

We studied the effects of sodium valproate, a widely used antiepileptic drug and a hyperammonemic agent, on L-[1-14C]glutamine and L-[1-14C]glutamate metabolism in isolated human kidney-cortex tubules. Valproate markedly stimulated glutamine removal as well as the formation of ammonia, 14CO2, pyruvate, lactate and alanine, but it inhibited glucose synthesis; the increase in ammonia formation was explained by a stimulation by valproate mainly of flux through glutaminase (EC 3.5.1.2) and to a much lesser extent of flux through glutamate dehydrogenase (EC 1.4.1.3). By contrast, valproate did not stimulate glutamate removal or ammonia formation, suggesting that the increase in flux through glutamate dehydrogenase observed with glutamine as substrate was secondary to the increase in flux through glutaminase. Accumulation of pyruvate, alanine and lactate in the presence of valproate was less from glutamate than from glutamine. Inhibition by aminooxyacetate of accumulation of alanine from glutamine caused by valproate did not prevent the acceleration of glutamine utilization and the subsequent stimulation of ammonia formation. It is concluded from these data, which are the first concerning the in vitro metabolism of glutamine and glutamate in human kidney-cortex tubules, that the stimulatory effect of valproate is primarily exerted at the level of glutaminase in human renal cortex.     

Regional changes in amino acid levels of the neonate rat brain during anoxia and recovery

The aim of this study was to compare the changes in amino acids (alanine, aspartate, GABA, glutamate, glutamine, glycine, serine taurine) that are produced in different regions of the neonate brain (telencephalon, diencephalon cerebellum, brain stem) following a survivable period of anoxia and after the re-establishment of air respiration. Anoxia provoked different responses in the different regions. The changes during the anoxic period were as follows. In the brain stem there was a decrease in aspartate, in the telencephalon there was a significant increase in GABA and alanine and a decrease in aspartate, in the diencephalon, glutamate and GABA increased, and in the cerebellum, glycine and alanine levels were enhanced. The changes during recovery were even more dissimilar. Here the greatest shifts were seen in the brain stem with increases in glutamine, GABA, aspartate, glycine, serine, alanine, and taurine. In the telencephalon glutamate fell and alanine increased, in the diencephalon GABA increased, and in the cerebellum, glutamate fell while glycine and alanine increased. In none of the major brain regions did the pattern of changes in neurotransmitters correspond to that seen in anoxic tolerant species.     

The metabolism of amino acids in the bovine lens. Their oxidation as a source of energy

1. The metabolism by the bovine lens of nine (14)C-labelled l-amino acids was studied. These were: alanine, aspartate, glutamate, leucine, lysine, proline, serine, tyrosine and tryptophan. 2. All were taken up by the tissue and incorporated into protein. 3. Aspartate and glutamate, although poorly taken up, were readily metabolized to CO(2). Radioactivity from glutamate was also found in glutathione, glutamine, proline and ophthalmic acid. Aspartate was converted into glutamate, glutathione, proline, alanine and lactate. 4. Alanine was largely converted into lactate, which was released into the medium, but incorporation of radioactivity into CO(2), glutamate, glutathione, aspartate and lipids also occurred. 5. Radioactivity from leucine was detected in CO(2), lipids, glutamate, glutathione, proline and glutamine. 6. Lysine was only slightly broken down by the bovine lens; radioactivity was observed in CO(2), glutamate, glutathione, proline and two unidentified compounds. 7. Proline was metabolized to glutamate from which CO(2), glutathione and glutamine were formed. Hydroxyproline in the capsule collagen was labelled. 8. Radioactivity from serine was found in CO(2), lipids, glutathione, glycine, cystine, ATP, lactate and three unidentified compounds, one of which was probably taurine. 9. Neither tyrosine nor tryptophan were metabolized by the bovine lens. 10. The ability of the lens to metabolize amino acids was also shown by measurement of NH(3) production: more NH(3) was formed when glucose was absent from the incubation medium. 11. These experiments suggest that oxidation of amino acids is a source of energy for the lens.     

Changes in plasma amino acid concentrations with increasing age in patients with propionic acidemia

The objective of the study is to analyze plasma amino acid concentrations in propionic acidemia (PA) for the purpose of elucidating possible correlations between propionyl-CoA carboxylase deficiency and distinct amino acid behavior. Plasma concentrations of 19 amino acids were measured in 240 random samples from 11 patients (6 families) with enzymatically and/or genetically proven propionic acidemia (sampling period, January 2001–December 2007). They were compared with reference values from the literature and correlated with age using the Pearson correlation coefficient test. Decreased plasma concentrations were observed for glutamine, histidine, threonine, valine, isoleucine, leucine, phenylalanine and arginine. Levels of glycine, alanine and aspartate were elevated, while values of serine, asparagine, ornithine and glutamate were normal. For lysine, proline and methionine a clear association was not possible. Significant correlations with age were observed for 13 amino acids (positive correlation: asparagine, glutamine, proline, alanine, histidine, threonine, methionine, arginine; negative correlation: leucine, phenylalanine, ornithine, glutamate and aspartate). This study gives new insight over long-term changes in plasma amino acid concentrations and may provide options for future therapies (e.g., substitution of anaplerotic substances) in PA patients.     

Amino-acid metabolism enzyme activities in the liver, intestine and yolk sac membrane of developing domestic fowl

To contribute to our understanding of nitrogen metabolism in the developing chick we have studied in liver, intestine and yolk sac membrane the ontogeny of both aspartate- and alanine transaminases, glutamate dehydrogenase, adenylate deaminase, glutamine synthetase and xanthine dehydrogenase activities. Liver enzyme activities were much higher than those of the same enzymes in intestine and yolk sac membrane, the latter having the lowest activities. In the liver, both alanine transaminase and glutamate dehydrogenase increased their activity just before hatching, xanthine dehydrogenase and glutamine synthetase develop their highest activity just after hatching, while aspartate transaminase and adenylate deaminase attained the highest levels just with adulthood. From the pattern of enzyme activity in yolk sac membrane and intestine it can be inferred that after hatching, the amino-acid metabolism in these tissues is considerably enhanced, with higher production of ammonia from amino acids, as indicated by the rise in adenylate deaminase, as well as increased potentiality in production of both alanine and glutamine. It can be concluded that hatching coincides with a deep change of pace in amino-acid metabolism in the organs studied fully comparable with that observed in Mammals at the end of lactation, with the difference that the adaptation to the new diet in the case of the chick is much more sudden than weaning is for the rat.     

Ammonia production and amino acid metabolism by rat renal papillary epithelial cells in culture

A significant percentage of excreted ammonium is added to tubular fluid along the medullary collecting duct. However, it is not clear whether this ammonia is produced in the cortex and delivered into the medulla or is produced directly by medullary cells. To address this issue, rat epithelial cells derived from the renal papilla were grown in continuous culture and their ability to generate ammonia was examined. When grown in Dulbecco's modified Eagle's medium with 4 mM glutamine, these cells produced ammonia at a rate of approximately 27 nmol/10(6) cells/h. When these cells were grown in minimum essential medium without glutamine, ammonia production fell to 7 nmol/10(6) cells/ h. Increasing the glutamine concentrations of minimum essential medium to 4 mM increased ammonia production to slightly greater than 30 nmol/10(6) cells/ h. Increasing the media concentration of glutamate, glycine, or asparagine resulted in no significant increase in ammoniagenesis. Analysis of media amino acid concentration revealed that glutamine was the main amino acid consumed while alanine was the predominant amino acid produced. The glutaminase activity of these cells appears to be primarily phosphate-dependent, similar to that observed in vitro in papillary tubules. Alterations of K+ or H+ ion concentration did not alter ammoniagenesis, but addition of 2.5 mM ammonium chloride significantly reduced net ammonia production. It is concluded that rat papillary epithelial cells have the intrinsic ability to utilize glutamine to generate ammonia and alanine. In vivo ammonia produced locally in the medulla may contribute to final urinary ammonium excretion.     

Salmonella typhimurium LT-2 mutants with altered glutamine synthetase levels and amino acid uptake activities.

To determine whether Salmonella typhimurium has a nitrogen control response, we have examined the regulation of nitrogen utilization in two mutants with fivefold and threefold elevations in their glutamine synthetase activities. The mutants do not require glutamine for growth on glucose--ammonia medium but do have altered growth on other nitrogen sources. They grow better than an isogenic control on media containing arginine or asparate, but more slowly with proline or alanine as nitrogen sources. This unusual growth pattern is not due to altered regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase and glutamate synthase, or to changes in the enzymes for aspartate degradation. However, transport for several amino acids may be affected. Measurement of amino acid uptake show that the mutants with high glutamine synthetase levels have increased rates for glutamine, arginine, aspartate, and lysine, but a decreased rate for proline. The relationship between glutamine synthetase levels and uptake was examined in two mutants with reduced, rather than increased, glutamine synthetase production. The uptake rates for glutamine and lysine were lower in these two glutamine auxotrophs than in the Gln+ controls. These results show a correlation between the glutamine synthetase levels and the uptake rates for several amino acids. In addition, the pleiotropic growth of the mutants with elevated glutamine synthetase activities suggests that a nitrogen control response exists for S. typhimurium and that it can be altered by mutations affecting glutamine synthetase regulation.     

Formation of the intermediate products of the tricarboxylic acid cycle and ammonia from free amino acids in anoxic heart muscle

Glutamate and aspartate showed the highest rate of catabolism in oxygenated isolated rat heart with the formation of glutamine, asparagine and alanine. Under anoxia, the catabolism of branch chained amino acids and that of lysine, proline, arginine and methionine was inhibited. However, glutamate and aspartate catabolized at a higher rate as compared with oxygenation. Alanine was the product of their excessive degradation. During oxygenation, 70% of ammonia were produced via deamination of amino acids. Under anaerobic conditions the participation of amino acids in ammoniagenesis decreased to 4%; the principal source of ammonia was the adenine nucleotide pool. The total pool of the tricarboxylic acid cycle intermediates increased 2.5-fold due to accumulation of succinate. The data obtained suggest that the constant influx of intermediates into the cycle from amino acids is supported by coupled transamination of glutamate and aspartate. This leads to the formation of ATP and GTP in the tricarboxylic acid cycle during blocking of aerobic energy production.     

Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase

Five mutant lines of barley (Hordeum vulgare L.), which are only able to grow at elevated levels of CO₂, contain less than 5% of the wild-type activity of ferredoxin-dependent glutamate synthase (EC 1.4.7.1). Two of these lines (RPr 82/1 and RPr 82/9) have been studied in detail. Leaves and roots of both lines contain normal activities of NADH-dependent glutamate synthase (EC 1.4.1.14) and the other enzymes of ammonia assimilation. Under conditions that minimise photorespiration, both mutants fix CO₂ at normal rates; on transfer to air, the rates drop rapidly to 15% of the wild-type. Incorporation of ¹⁴CO₂ into sugar phosphates and glycollate is increased under such conditions, whilst incorporation of radioactivity into serine, glycine, glycerate and sucrose is decreased; continuous exposure to air leads to an accumulation of ¹⁴C in malate. The concentrations of malate, glutamine, asparagine and ammonia are all high in air, whilst aspartate, alanine, glutamate, glycine and serine are low, by comparison with the wild-type parent line (cv. Maris Mink), under the same conditions. The metabolism of [¹⁴C]glutamate and [¹⁴C]glutamine by leaves of the mutants indicates a very much reduced ability to convert glutamine to glutamate. Genetic analysis has shown that the mutation in RPr 82/9 segregates as a single recessive nuclear gene.Abbreviations GDH glutamate dehydrogenase (EC 1.4.1.2) - GS glutamine synthetase (EC 6.3.1.2) - RuBP ribulose 1,5-bisphosphate     

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.     

Effects of amino acids, adenine nucleotides and inorganic pyrophosphate on glutamine synthetase from Anabaena cylindrica.

Glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from Anabaena cylindrica was inhibited by alanine, glycine, serine and aspartate. The effects of alanine and serine were uncompetitive with respect to glutamate, while those of glycine and asparatate were uncompetitive with respect to glutamate, while those of glycine and aspartate were non-competitive and mixed type respectively. Different pairs of amino acids and their various combinations caused a cumulative inhibition of the enzyme activity. Glutamine synthetase was also inhibited by ADP and AMP and both nucleotides affected the enzyme competitively with respect to ATP and non-competitively for glutamate. Inorganic pyrophosphate, between 2 and 3 mM, produced a very pronounced inhibiton of enzyme activity. The inhibition by PPi was uncompetitive for ATP. Various combinations of the adenine nucleotides, PPi and Pi exerted a cumulative inhibitory effect on the enzyme activity, as did the amino acids, in different combinations with either adenine nucleotides, PPi or Pi. The effects of the adenine nucleotides and the amino acids were more pronounced at higher concentrations of ammonia. Except for serine similar responses of these effectors were obtained with increasing concentrations of Mg2+. It is proposed that changes in the free concentrations of Mg2+ are important in energy-dependent regulation of the enzyme activity in this alga.     

Turnover rates of amino acid neurotransmitters in regions of rat cerebellum

The turnover rates of aspartate, gamma-aminobutyric acid (GABA), glutamate, glutamine, alanine, serine, and glycine were measured in five regions of rat cerebellum. Turnover rates of the putative neurotransmitters (aspartate, glutamate, and GABA) were 2-20-fold higher than those of alanine and serine, and generally consistent with the proposed neurotransmitter functions for these amino acids. However, glutamate turnover was high and similar in magnitude in the deep nuclei and granule layer, suggesting possible release, not only from parallel fibers, but from mossy fibers as well. The differential distribution of turnover rates for GABA supports its neuronal release by Purkinje, stellate, basket, and Golgi cells, whereas aspartate may be released by both climbing and mossy fibers. The distribution of glycine turnover rates is consistent with release from Golgi cells, whereas alanine may be released from granule cell parallel fibers. Turnover rates measured in two other motor areas, the striatum and motor cortex, indicated that utilization of these amino acid neurotransmitters is differentially distributed in brain motor regions. The data indicate that turnover rate measurements may be useful in identifying neurotransmitter function where content measurements alone are insufficient.