Glutamine Transaminase K and ω-Amidase Activities in Primary Cultures of Astrocytes and Neurons and in Embryonic Chick Forebrain: Marked Induction of Brain Glutamine Transaminase K at Time of Hatching

Abstract: Glutamine transaminase K and ω-amidase activities are present in the chick brain and in the brains of adult mice, rats, and humans. However, the activity of gluta-mine transaminase K in adult mouse brain is relatively low. In the chick embryo, cerebral glutamine transaminase K activity is low between embryonic days 5 and 17, but by day 23 (day of hatching) activity rises dramatically (< 15-fold). Cerebral ω-amidase activity is relatively high at embryonic day 5 but lower between days 5 and 17; at embryonic day 23 the activity rises to a maximum. Both glutamine transaminase K and ω-amidase are present in cultured chick, rat, and mouse astrocytes and neurons. For each species, the activity of glutamine transaminase K is higher in the astrocytes than in the neurons. The activity of ω-amidase is about the same in the cultured chick astrocytes and neurons but significantly higher in rat astrocytes than in rat neurons. The data suggest that the rise in brain glutamine transaminase K activity in the chick embryo at hatching correlates with maturation of astrocytes. Glutamine transaminase K may be involved in glutamine cycling in astrocytes. Glutamine transaminase K appears to be a major cysteine S-conjugate β-lyase of the brain and may play a role in the neurotoxicity associated with exposure to dichloroacetylene and perhaps to other toxins.     

Decreased glucocorticoid induction of brain glycerol phosphate dehydrogenase following transplacental carcinogenesis with ethylnitrosourea

Abstract— Glutamine transaminase from rat brain was shown to occur in the mitochondrial fraction, whereas the ω-amidase was shown to occur in the soluble fraction. Both enzymes were also found to be widely distributed throughout human brain tissue. Specific activities for the glutamine transaminase and ω-amidase in the parietal cortex of one individual at 5 h post mortem were 8 and 53 μmol per hour per gram of tissue, respectively. Rat brain glutamine transaminase was shown to be identical to that of the liver mitochondrial enzyme. Improved assays for glutamine transaminase and ω-amidase activities in crude tissue homogenates are described. The possible physiological importance of glutamine transaminase and its potential role in the encephalopathy of hepatic disease are discussed.     

Structural insights into the catalytic active site and activity of human Nit2/ω-amidase: kinetic assay and molecular dynamics simulation

Human nitrilase-like protein 2 (hNit2) is a putative tumor suppressor, recently identified as ω-amidase. hNit2/ω-amidase plays a crucial metabolic role by catalyzing the hydrolysis of α-ketoglutaramate (the α-keto analog of glutamine) and α-ketosuccinamate (the α-keto analog of asparagine), yielding α-ketoglutarate and oxaloacetate, respectively. Transamination between glutamine and α-keto-γ-methiolbutyrate closes the methionine salvage pathway. Thus, hNit2/ω-amidase links sulfur metabolism to the tricarboxylic acid cycle. To elucidate the catalytic specificity of hNit2/ω-amidase, we performed molecular dynamics simulations on the wild type enzyme and its mutants to investigate enzyme-substrate interactions. Binding free energies were computed to characterize factors contributing to the substrate specificity. The predictions resulting from these computations were verified by kinetic analyses and mutational studies. The activity of hNit2/ω-amidase was determined with α-ketoglutaramate and succinamate as substrates. We constructed three catalytic triad mutants (E43A, K112A, and C153A) and a mutant with a loop 116-128 deletion to validate the role of key residues and the 116-128 loop region in substrate binding and turnover. The molecular dynamics simulations successfully verified the experimental trends in the binding specificity of hNit2/ω-amidase toward various substrates. Our findings have revealed novel structural insights into the binding of substrates to hNit2/ω-amidase. A catalytic triad and the loop residues 116-128 of hNit2 play an essential role in supporting the stability of the enzyme-substrate complex, resulting in the generation of the catalytic products. These observations are predicted to be of benefit in the design of new inhibitors or activators for research involving cancer and hyperammonemic diseases.     

CONTROL OF PYRUVATE AND β-HYDROXYBUTYRATE UTILIZATION IN RAT BRAIN MITOCHONDRIA AND ITS RELEVANCE TO PHENYLKETONURIA AND MAPLE SYRUP URINE DISEASE

—The effects of the amino acids (phenylalanine, valine, leucine and isoleucine) which accumulate in phenylketonuria (PKU) and maple syrup urine disease (MSUD), and their analogue α-keto acids (phenylpyruvate, α-keto isovalerate, α-keto isocaproate, α-keto-β-Me valerate) have been studied on rat brain mitochondrial respiration. Both phenylpyruvate and α-keto isocaproate specifically inhibited the oxidation of pyruvate plus malate and β-hydroxybutyrate plus malate by rat brain mitochondria in the presence of ADP. However, no inhibitory effects of similar concentrations of phenylpyruvate or α-keto isocaproate were observed on the isolated semipurified pyruvate or β-hydroxybutyrate dehydrogenases from rat brain mitochondria. The transport of pyruvate and β-hydroxybutyrate across the brain mitochondrial membrane was studied by both uptake and exchange of radioactively labelled substrates. Both these processes were inhibited by phenylpyruvate and α-ketoisocaproate. The results are interpreted as providing evidence for both pyruvate and β-hydroxybutyrate translocases across the brain mitochondrial membrane, and that the inhibition of these systems by phenylpyruvate and α-keto isocaproate may be important lesions in phenylketonuria and maple syrup urine disease respectively.     

MECHANISM OF ACTION OF β-N-OXALYL-L-α, β-DIAMINOPROPIONIC ACID, THE LATHYRUS SATIVUS NEUROTOXIN

The source of ammonia in the brain tissue of young rats treated with β-N-oxalyl-l -α, β-diaminopropionic acid (ODAP) has been studied. ODAP administration to 12-day-old rats causes a significant increase in the levels of adenylic acid deaminase in the brain. Glutaminase activity also shows an increase under these conditions. An increase in the levels of acid protease and transglutaminase is also observed in the brain of ODAP-treated animals. Glutamate dehydrogenase activity is decreased slightly. Glutamine synthetase enzyme is not affected. Aspartate-α-ketoglutarate transaminase and aspartate-pyruvate transaminase activities are enhanced in the brain tissue of ODAP-treated rats. It is held that protein degradation, especially the cleavage of free and protein-bound amide bonds, may be responsible for excess ammonia liberation in the brain of ODAP-treated young rats.     

Glutamine transport at the blood-brain and blood-cerebrospinal fluid barriers

Glutamine has multiple physiological and pathophysiological roles in the brain. Because of their position at the interface between blood and brain, the cerebral capillaries and the choroid plexuses that form the blood-brain barriers (BBB) and blood-cerebrospinal fluid (CSF) barriers, have the potential to influence brain glutamine concentrations. Despite this, there has been a paucity of data on the mechanisms and polarity of glutamine transport at these barrier tissues. In situ brain perfusion in the rat, indicates that blood to brain L-[14C]glutamine transport at the blood-brain barrier is primarily mediated by a pH-dependent, Na(+)-dependent, System N transporter, but that blood to choroid plexus transport is primarily via a pH-independent System N transporter and a Na(+)-independent carrier that is not System L. Transport studies in isolated rat choroid plexuses and primary cultures of choroid plexus epithelial cells indicate that epithelial L-[14C]glutamine transport is polarized (apical uptake>basolateral) and that uptake at the apical membrane is mediated by pH dependent System N transporters (identified as SN1 and SN2 by polymerase chain reaction) and the Na(+)-independent System L. Blood-brain barrier System N transport is markedly effected by cerebral ischemia and may be a good marker of endothelial cell dysfunction. The multiple glutamine transporters at the blood-brain and blood-CSF barriers may have role in meeting the metabolic needs of the brain and the barrier tissues themselves. However, it is likely that the main role of these transporters is removing glutamine, and thus nitrogen, from the brain.     

Molecular identification of ω-amidase, the enzyme that is functionally coupled with glutamine transaminases, as the putative tumor suppressor Nit2

Our purpose was to identify the sequence of ω-amidase, which hydrolyses the amide group of α-ketoglutaramate, a product formed by glutamine transaminases. In the Bacillus subtilis genome, the gene encoding a glutamine transaminase (mtnV) is flanked by a gene encoding a putative ‘carbon-nitrogen hydrolase’. The closest mammalian homolog of this putative bacterial ω-amidase is ‘nitrilase 2’, whose size and amino acid composition were in good agreement with those reported for purified rat liver ω-amidase. Mouse nitrilase 2 was expressed in Escherichia coli, purified and shown to catalyse the hydrolysis of α-ketoglutaramate and other known substrates of ω-amidase. No such activity was observed with mouse nitrilase 1. We conclude that mammalian nitrilase 2 is ω-amidase.     

Regulation of Transmitter γ-Aminobutyric Acid (GABA) Synthesis and Metabolism Illustrated by the Effect of γ-Vinyl GABA and Hypoglycemia

The effect of different treatments on amino acid levels in neostriatum was studied to throw some light on the synthesis and metabolism of gamma-aminobutyric acid (GABA). Irreversible inhibition of GABA transaminase by microinjection of gamma-vinyl GABA (GVG) led to a decrease in aspartate, glutamate, and glutamine levels and an increase in the GABA level, such that the nitrogen pool remained constant. The results indicate that a large part of brain glutamine is derived from GABA. Hypoglycemia led to an increase in the aspartate level and a decrease in glutamate, glutamine, and GABA levels. The total amino acid pool was decreased compared with amino acid levels in normoglycemic rats. GVG treatment of hypoglycemic rats led to a decrease in the aspartate level and a further reduction in glutamate and glutamine levels. In this case, GABA accumulation continued, although the glutamine pool was almost depleted. The GABA level increased postmortem, but there were no detectable changes in levels of the other amino acids. Pretreatment of the rats with hypoglycemia reduced both glutamate and glutamine levels with a subsequent decreased postmortem GABA accumulation. The half-maximal GABA synthesis rate was obtained when the glutamate level was reduced by 50% and the glutamine level was reduced by 80%.     

Glutamine transaminase K and cysteine S-conjugate beta-lyase activity stains.

An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing L-phenyl-alanine, alpha-keto-gamma-methiolbutyrate (alpha KMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and alpha KMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate beta-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing alpha KMB (to ensure maintenance of the enzyme in the pyridoxal 5'-phosphate form), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate beta-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent Mr approximately 95,000). This band coincides with both the cysteine S-conjugate beta-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate beta-lyase activity with an Rf value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repeated Administration of γ-VinylGABA Reduces Rat Brain Glutamine Synthetase Activity

Abstract: The glutamine cycle has been proposed as a pathway in which glutamine synthesized in glia provides substrate for synthesis of the neurotransmitters glutamate and GABA as they are lost from neurons. To test whether GABA may regulate this pathway, the effect of elevated GABA on the glial enzyme glutamine synthetase was examined in rat brain. Repeated subcutaneous injections of the antiepileptic GABA transaminase inhibitor γ-vinylGABA at a dose of 150 mg/kg per day for 21 days reduced glutamine synthetase activity by 36% in the cortex and 22% in the cerebellum. At 30 mg/kg per day, glutamine synthetase activity was reduced by 9.5% in the cortex but unchanged in the cerebellum. The reductions were brain specific because the skeletal muscle and liver enzymes were unaffected by γ-vinylGABA administration. Amino acid analysis of the cortex from γ-vinylGABA-treated rats demonstrated a 270% increase in GABA levels after 150 mg/kg but no change after 30 mg/kg. GABA levels and glutamine synthetase activity were inversely correlated. The 150 mg/kg dose significantly lowered cortical glutamine and glutamate levels. The decline in brain glutamine synthetase activity with chronic γ-vinylGABA administration developed gradually over time and may be due to the slow turnover of this enzyme in vivo.     

ENZYMES OF THE γ-GLUTAMYL CYCLE IN THE CHOROID PLEXUS AND BRAIN

—The presence of enzymes of the γ-glutamyl cycle in the bovine and rabbit brain and choroid plexus is described. The activities of γ-glutamyl transpeptidase, γ-glutamyl cyclotransferase and γ-glutamyl-cysteine synthetase in the choroid plexus were found to be higher than in the brain. The activity of γ-glutamyl transpeptidase in the choroid plexus was many times higher than the activity of the other enzymes. Brain and choroid plexus γ-glutamyl transpeptidase were activated by Na⁺ and K⁺. Both brain and choroid plexus showed only a very limited capacity to metabolize [¹⁴C]5-oxoproline to ¹⁴CO₂.     

Apparent Co-Sequestration of Immunoreactive Corticotropin, α-Melanot opin, and γ-Lipotropin in Hypothalamic Granules

Abstract: In the present study, the question of whether immunoreactive α-melanotropin (α-MSH₁), corticotropin (ACTH₁), and β-melanotropin (β-MSH₁) are co-sequestered in hypothalamic granules of adult male rats was addressed. When a 900 ×g supernatant fluid prepared from a hypothalamic homogenate was fractionated on continuous sucrose density gradients under non-equilibrium Conditions, two populations of particles containing α-MSH₁, ACTH₁, or β-MSH₁ Were observed. However, when fractionated under equilibrium conditions, the two populations of particles containing α-MSH₁ ACTH₁, or β-MSH₁ were recovered as a single band. This sedimentation characteristic indicates that the particles containing a given peptide differ in size but are similar in density. In their sedimentation, the small particles containing α-MSH₁, ACTH₁, and β-MSH₁ are indistinguishable from granules containing α-MSH₁, whereas the large particles containing α-MSH₁ (ACTH₁, and β-MSH₁ are indistinguishable from synaptosomes containing α-MSH₁, β-MSH₁ had an apparent molecular weight (M.W.) of about 5,000, which is similar to that of γ-lipotropin. ACTH₁ was comprised of three species of molecules: big (M.W. ≥ 10,000), 5.7K (M.W. ≌ 5,700), and 4.5K (M.W. ≌ 4,500). Big ACTH was the predominant and 5.7K ACTH the minor component of ACTH₁ present in granules as well as in synaptosomes. These results are suggestive that α-MSH, ACTH and its precursors, and γ-lipotropin are co-sequestered in hypothalamic granules.     

Expression of the β-subunit isoforms of the Na,K-ATpase in rat embryo tissues,inner ear and choroid plexus

Summary— We report evidence of the apical localization of the two Na, K-ATPase β-subunit isoforms in cells of the inner ear and of the choroid plexus of the rat. To this end, we generated isoform-specific antisera against the human Na, K-ATPase β1 and β2 subunits. These polyclonal rabbit antisera were raised against truncated β-isoform proteins that were made in E coli with pET expression vectors. Deglycosylation of the native antigen with N-endoglycosidase F shows four bands in the β1 isoform and five bands in the β2 iso-form immunoblots. In E15 rat embryos, the β1 isoform was detected in brain, heart and kidney and the β2 isoform only in brain. While β-subunit mRNA expression (Watts AG, Sanchéz-Watts G, Emanuel JR, Levenson R 1991 Proc Natl Acad Sci USA 88, 7425–7429), and immunoblotting and enzymatic activity have been determined (Zlokovic BV, Mackic JB, Wang L, McComb JG, McDonough A 1993 J Biol Chem 268, 8019–8025), very little is known about the specific localization of each β-isoform in the epithelia of choroid plexus and inner ear. Immunocytochemical preparations of 15-day-old whole rat embryos and adult rat brain showed an enhanced staining for the β1 and β2 isoforms in the apical membrane of the ampullary crests of the inner ear's semicircular ducts and in the cuboidal cells of the choroid plexus     

Similarity Between Rat Brain Nicotinic α-Bungarotoxin Receptors and Stably Expressed α-Bungarotoxin Binding Sites

Abstract: The present results demonstrate stable expression of α-bungarotoxin (α-BGT) binding sites by cells of the GH₄C₁ rat pituitary clonal line. Wild-type GH₄C₁ cells do not express α-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH₄C₁ cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH₄C₁ cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH₄C₁ cells also express saturable, high-affinity binding sites for ¹²⁵I-labeled α-BGT, with a K_D of 0.4 nM and B_max of 3.2 fmol/10⁶ intact cells. ¹²⁵I-α-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH₄C₁ cells. Furthermore, K_D and K_i values for ¹²⁵I-α-BGT binding sites on intact α7/GH₄C₁ cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α-BGT binding sites expressed in α7/GH₄C₁ cells was similar to that of the native brain α-BGT receptor. Chronic exposure of α7/GH₄C₁ cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α-BGT binding sites in transfected α7/GH₄C₁ cells resemble those for brain nicotinic α-BGT receptors. If the heterologously expressed α-BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α-BGT receptor has a similar homooligomeric structure. Alternatively, if α-BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α-BGT receptor.     

N-System Amino Acid Transport at the Blood-CSF Barrier

Abstract: Despite l -glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L-, A-, ASC-, and N-system amino acid transporters in l -[¹⁴C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na⁺-dependent mechanism. The Na⁺-independent uptake was inhibited by 2-amino-2-norbornane carboxylic acid, indicating that it is probably via an L-system transporter. Na⁺-dependent uptake was inhibited neither by the A-system substrate α-(methylamino)isobutyric acid nor by the ASC-system substrate cysteine. It was inhibited by histidine, asparagine, and l -glutamate γ-hydroxamate, three N-system substrates. Replacement of Na⁺ with Li⁺ had little effect on uptake, another feature of N-system amino acid transport. These data therefore indicate that N-system amino acid transport is present at the choroid plexus. The V _max and K _max for glutamine transport by this system were 8.1 ± 0.3 nmol/mg/min and 3.3 ± 0.4 m M , respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.     

Molecular cloning and expression of a cDNA for human kidney cysteine conjugate β-lyase

Kidney cysteine conjugate β-lyase (glutamine transaminase K, kyneurenine aminotransferase, EC 2.6.1.64) metabolises the cysteine conjugates of certain halogenated alkenes and alkanes to form reactive metabolites which can produce nephrotoxicicity and neurotoxicicity in experimental animals and man. Using a combination of hybridisation screening and PCR techniques we have isolated a full-length cDNA for human kidney cysteine conjugate β-lyase. Comparison of the deduced amino acid sequence with that of the rat enzyme indicated an 82% overall similarity, with 90% similarity around the pyridoxal phosphate binding site, many of the changes being conservative in nature. Expression of the cDNA in Cos-1 cells resulted in the production of a cytosolic enzyme which showed both cysteine conjugate β-lyase and glutamine transaminase K activity. Preliminary mapping of the gene for human cysteine conjugate β-lyase by PCR analysis of genomic DNA from human-rodent hybrid cells indicated that it is located on human chromosome 9.     

γ-Hydroxybutyric Acid in Subcellular Fractions of Rat Brain

The presence of gamma-hydroxybutyric acid (GHB) in synaptosome-enriched fractions of rat brain was ascertained using a GLC technique. The stability of GHB in synaptosomes was evaluated by addition of various gamma-aminobutyric acid (GABA) transaminase (GABA-T) inhibitors, GHB, or ethosuximide to the homogenizing medium. Furthermore, changes in whole brain GHB levels were compared with those in the synaptosomal fraction in animals treated with GABA-T inhibitors, GABA, or ethosuximide. GHB was present in synaptosome-enriched fractions in concentrations ranging from 40 to 70 pmol/mg of protein. There was no evidence for redistribution, leakage, or metabolism of GHB during the preparation of synaptosomes. The elevations of whole brain GHB level associated with GABA-T or ethosuximide treatment were reflected by a parallel increase in synaptosomal GHB content. These data add to the growing evidence that GHB may have neurotransmitter or neuromodulator function.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate beta-lyase

Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. The β-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. S-(1,1,2,2-Tetrafluoroethyl)-l-cysteine is an excellent aminotransferase and β-lyase substrate of rhGTK. Moderate aminotransferase and β-lyase activities occur with the chemopreventive agent Se-methyl-l-selenocysteine. l-3-(2-Naphthyl)alanine, l-3-(1-naphthyl)alanine, 5-S-l-cysteinyldopamine and 5-S-l-cysteinyl-l-DOPA are measurable aminotransferase substrates, indicating that the active site can accommodate large aromatic amino acids. The α-keto acids generated by transamination/l-amino acid oxidase activity of the two catechol cysteine S-conjugates are unstable. A slow rhGTK-catalyzed β-elimination reaction, as measured by pyruvate formation, was demonstrated with 5-S-l-cysteinyldopamine, but not with 5-S-l-cysteinyl-l-DOPA. The importance of transamination, oxidation and β-elimination reactions involving 5-S-l-cysteinyldopamine, 5-S-l-cysteinyl-l-DOPA and Se-methyl-l-selenocysteine in human tissues and their biological relevance are discussed.     

Glutamine synthetase, glutaminase and phosphodiesterase activities in brain under hypoxia: in vitro effect of cortisol, GABA and serotonin on glutamine synthetase.

The effect of hypobaric hypoxia on the activities of glutamine synthetase, glutaminase and cyclic 3'5' AMP phosphodiesterase in rat brain was studied after exposure to 25,000' for 6 h. Glutamine synthetase activity was increased in all the regions of brain studied, and addition of gamma amino butyric acid, serotonin and cortisol in vitro produced a differential response. Glutaminase activity decreased in the whole brain. Cyclic 3'5' AMP phosphodiesterase activity decreased in cerebellum, medulla, hypothalamus and pituitary showing an accumulation of cyclic 3'5' AMP in these regions. The results suggest that glutamine synthesis and degradation are regulated in the central nervous system by cyclic AMP and cortisol: Gamma aminoburyric acid and other compounds can modulate the activity of glutamine synthetase and glutaminase.