Regulation of Neurotransmitter Aspartate Metabolism by Glial Glutamine Synthetase

Aspartate levels and release from rat striatal slices following the inhibition of glutamine synthetase (GS) by methionine sulfoximine (MSO) were studied. Striatal levels of aspartate and glutamine were decreased over time in a manner that correlated with GS inhibition. Ca2+-dependent, K+-stimulated aspartate release was diminished in striatal tissue slices from animals pretreated with MSO. The decreased release of aspartate correlated over time with the inhibition of GS. The addition of glutamine to the perfusion medium completely reversed the effects of MSO on calcium-dependent aspartate release. It is suggested that glutamine is a major precursor for transmitter aspartate.     

Alteration of Striatal Glutamate Release After Glutamine Synthetase Inhibition

The effect of the glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSO), on glutamate levels in, and glutamate release from, rat striatal tissue was examined. Tissue levels of glutamate were unchanged 24 h after an intraventricular injection of MSO, but tissue glutamine levels were decreased 50%. Calcium-dependent, potassium-stimulated glutamate release was diminished in tissue prisms from animals pretreated with MSO compared to controls. The decreased release of glutamate correlated over time with the inhibition of GS following an intraventricular injection of MSO. The maximum diminution of calcium-dependent, potassium-stimulated glutamate release (50%) and the maximum inhibition of GS activity (51%) were observed 24 h after MSO. The addition of 0.5 mM glutamine to the perfusion medium completely reversed the effects of MSO pretreatment on calcium-dependent, potassium-stimulated glutamate release. Since GS is localized in glial cells and the measured glutamate release is presumed to occur from neurons, the data support the contention that astroglial glutamine synthesis is an important contributor to normal neuronal neurotransmitter release.     

Effect of Hyperammonemia and Methionine Sulfoximine on the Kinetic Parameters of Blood-Brain Transport of Leucine and Phenylalanine

The activity of the blood-brain neutral amino acid transport system is increased in rats infused with ammonium salts or rendered hyperammonemic by a portacaval anastomosis. This effect may be due to a direct action of ammonia or to some metabolic consequence of high ammonia levels, such as increased brain glutamine synthesis. To test these possibilities we evaluated the kinetic parameters of blood-brain transport of leucine and phenylalanine in control rats, in rats after continuous 24 h infusion of ammonium salts (NH4+ = 2.5 mmol X kg-1 X h-1), and in rats treated with methionine sulfoximine, an inhibitor of glutamine synthetase, before infusion of ammonium salts. In ammonia-infused rats without methionine sulfoximine treatment, the KD and Vmax of phenylalanine transport were increased, respectively, about 170% and 80% compared to controls, whereas the Km and Vmax of leucine transport were increased, respectively, about 100% and 200%. Electron microscopy demonstrated marked swelling of astrocytic processes around brain capillaries of ammonia-infused rats; however, capillary permeability to horseradish peroxidase apparently was not increased by ammonia infusion. Administration of methionine sulfoximine before ammonia infusion inhibited glutamine synthesis and prevented the changes in transport of leucine and phenylalanine, but apparently did not reverse the perivascular swelling. These results suggest that the ammonia-induced increase in the activity of transport of large neutral amino acids across the blood-brain barrier requires glutamine synthesis in brain, and is not a direct effect of ammonia.     

Biosynthesis of Polyamines in Mouse Brain: Effects of Methionine Sulfoximine and Adenosylhomocysteine

Abstract: This study examines the consequences on cerebral polyamine biosynthesis of increases and decreases in cerebral methylation. Increases were elicited by administering the convulsant agent methionine sulfoximine (MSO) and decreases by elevating in vivo the cerebral levels of the methylation inhibitor S -adenosyl-homocysteine. Following the intraventricular (i.vt.) administration of one of the two possible polyamine precursors, [1,4-¹⁴C]putrescine, the specific radioactivity (sra) of the newly formed [¹⁴C]spermidine remained unchanged. Conversely, after i.vt. l -[3,4-¹⁴C]methionine, the other polyamine precursor, significantly higher sra values for [¹⁴C]spermidine and [¹⁴C]spermine were recorded in the brains of the MSO-treated animals. [¹⁴C] S - adenosylmethionine in the brain of the MSO-treated animals was also more highly labeled following [1-¹⁴C]-methionine, indicating its accelerated formation relative to controls. We also investigated the effect of the administration of adenosine + homocysteine, a treatment that results in elevated brain adenosylhomocysteine levels, on polyamine biosynthesis from [3,4-¹⁴C]-methionine. The results of these experiments show both significantly lower sra values for [¹⁴C]spermidine and [¹⁴C]spermine and significantly higher than control endogenous methionine levels, a clear sign of the existence of a retardation in the conversion of methionine to polyamines under these conditions. In conclusion, the present study demonstrates that while interference with cerebral methylation results in significant alterations of the rate of formation of the methionine moiety of spermidine and spermine, it has no effect on the entry of the putrescine moiety into the two polyamine molecules.     

Stimulation of Fructose-1,6-Biphosphatase Activity and Synthesis in the Cerebral Cortex of Rats Submitted to the Convulsant Methionine Sulfoximine

Purification of rat cerebral cortex fructose-1,6-biphosphatase (FBPase) was performed by substrate elution from phosphocellulose, followed by Sephadex G-200 column filtration. The purified enzyme exhibited an optimum at pH 7.5, and its catalytic properties were very similar to those of the purified whole-brain enzyme previously prepared by Majumder and Eisenberg in 1977. The isolated preparation was electrophoretically homogeneous. The molecular weight of the enzyme subunit was 40,000; the hydrophobic amino acids predominated with 592 residues, and tryptophan was not detected. Expressed as mumol fructose-1,6-biphosphate hydrolysed per g brain tissue wet weight per min, FBPase activity increased twofold 24 h after an intraperitoneal injection of 100 mg per kg body weight of the convulsant methionine sulfoximine (MSO); the increase of the rate of incorporation of [1-14C]valine into brain FBPase was 2.8-fold under the same experimental conditions. A rabbit specific antiserum against rat cerebral cortex FBPase was prepared, and immunotitration studies confirmed both an increase in the number of molecules and the activation of brain FBPase, 24 h after administration of MSO. The increase of the number of brain FBPase molecules, induced by MSO, was due to an increase in synthesis of the enzyme, as shown by a double-label valine incorporation study.     

Methionine Sulfoximine In Vivo Modifies the tRNALys Pool of Developing Rat Brain

Abstract: tRNA was extracted from brains of 3-, 8-, and 18-day-old rats that were injected intracerebrally, 45 min before death, with [³H]methyl methionine or [8-³H]guanosine, and intraperitoneally, 3 h before death, with l -methionine-dl-sulfoximine (MSO), a methylation-activating convulsant agent. Although there was no effect of age or of MSO on the per gram yield of tRNA, its specific radioactivity (dpm/A₂₆₀) was highest at 3 days in both the control and the MSO groups. Age- and MSO-related changes in the tRNA^Lys content of the brain tRNA pool were investigated by means of benzoylated DEAE- cellulose (BDC) and reverse-phase chromatography (RPC). BDC chromatography revealed tRNA^Lys species in the brains of the MSO-treated animals that were absent in control brains. Of particular interest was the finding that differences in RPC-5 chromatographic mobility between control and MSO-tRNA^Lys species were abolished by conversion to lysyl-tRNA, suggesting that the MSO-elicited change(s) in tRNAL^Lys structure involved the binding site(s) for lysine. Two additional findings were made: (a) lysine acceptance by the [³H]methyl-labeled tRNA^Lys purified from brains of the MSO-treated animals was higher than that of controls at 18 days; and (b) omission of the BDC chromatographic step accentuated the differences in mobility on RPC-5 columns between tRNA^Lys species of control and MSO-treated brains. Lastly, we found that some tRNA^Lys species present in the MSO-treated brains contained significantly different proportions of N²-methyl guanine and 1-methyl adenine, relative to controls. These MSO-elicited changes in the methyl base content of tRNA^Lys of immature rat brain are the first evidence of an alteration of brain tRNA structure by a centrally acting excitatory agent.     

Effects of Glutamate Antagonists on Methamphetamine and 3,4-Methylenedioxymethamphetamine-Induced Striatal Dopamine Release In Vivo

Abstract: Several amphetamine analogues are reported to increase striatal glutamate efflux in vivo, whereas other data indicate that glutamate is capable of stimulating the efflux of dopamine (DA) in the striatum via a glutamate receptor-dependent mechanism. Based on these findings, it has been proposed that the ability of glutamate receptor-blocking drugs to antagonize the effects of amphetamine may be explained by their capacity to inhibit DA release induced by glutamate. To examine this possibility further, we investigated in vivo the ability of glutamate antagonists to inhibit DA release induced by either methamphetamine (METH) or 3,4-methylenedioxymethamphetamine (MDMA). Both METH and MDMA increased DA efflux in the rat striatum and, in animals killed 1 week later, induced persistent depletions of DA and serotonin in tissue. Pretreatment with MK-801 or CGS 19755 blocked the neurotoxic effects of METH and MDMA but, did not significantly alter striatal DA efflux induced by either stimulant. Infusion of 6-cyano-7-nitroquinoxaline-2,3-dione into the striatum likewise did not alter METH-induced DA overflow, and none of the glutamatergic antagonists affected the basal release of DA when given alone. The findings suggest that the neuroprotective effects of NMDA antagonists do not involve an inhibition of DA release, nor do the data support the proposal that glutamate tonically stimulates striatal DA efflux in vivo. Whether phasic increases in glutamate content might stimulate DA release, however, remains to be determined.     

Regulation of Glycogen Content in Primary Astrocyte Culture: Effects of Glucose Analogues, Phenobarbital, and Methionine Sulfoximine

Compounds known to affect glycogen metabolism in vivo or in cell-free preparations were used to investigate the regulation of glycogen content in intact astrocytes cultured from newborn rat cortex. Compounds were added with fresh medium to culture dishes, and astrocyte glucose and glycogen content determined 24 h later. Increasing the medium glucose concentration from 7.5 mM to 30 mM increased cell glycogen content 80%. Addition of 2-deoxyglucose or 3-O-methyl glucose (2.5-10 mM) also increased cell glycogen content, 50-100%, suggesting a regulatory rather than mass action effect of glucose on astrocyte glycogen content. The phosphorylase b inhibitors 2,2',4,4',5,5'-hexabromobiphenyl and riboflavin had no effect on astrocyte glycogen content, consistent with negligible phosphorylase b activity in normal astrocytes. Phenobarbital and L-methionine-DL-sulfoximine (MSO) are both known to induce astrocyte glycogen accumulation in vivo. The addition of phenobarbital (2 mM) had no effect on the glycogen content of cultured astrocytes, suggesting an indirect mechanism for the in vivo effect. MSO at 1 mM, however, induced a 300% increase in glycogen content. The time course of glucose and glycogen content after MSO administration suggests this increase to be the result of slowed glycogenolysis rather than accelerated glycogen synthesis.     

Differential Effects of Tachykinins Injected Intranigrally on Striatal Dopamine Metabolism

The effects of intranigral injection of kassinin, eledoisin, and substance P on striatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) contents as well as circling behavior were studied in rats. Kassinin and eledoisin produced a marked dose-dependent increase of DOPAC concentrations in the ipsilateral striatum, as well as in the number of contralateral circlings. Substance P produced a similar but weaker effect. At the larger dose (5 nmol), the three tachykinins also induced an increase of DA concentrations in the ipsilateral striatum. The rank order of activity was kassinin greater than eledoisin greater than substance P. These results suggest that tachykinins stimulated the nigro-striatal dopaminergic system by accelerating the dopamine metabolism in striatum.     

Importance of Glutamine for γ-Aminobutyric Acid Synthesis in Rat Neostriatum In Vivo

This work was carried out to evaluate the importance of glial cells in providing precursors for the in vivo synthesis of gamma-aminobutyric acid (GABA). Fluorocitrate, which selectively inhibits the tricarboxylic acid cycle in glial cells, was administered locally in rat neostriatum. Inhibition of the glial cell tricarboxylic acid cycle led to a decrease both in glutamine level and in gamma-vinyl GABA (GVG)-induced GABA accumulation, an observation indicating reduced GABA synthesis. The role of glutamine, which is synthesized in glial cells as a precursor for GABA, was further investigated by inhibition of glutamine synthetase with intrastriatally administered methionine sulfoximine. In this case, the glutamine level was reduced to near zero values, and the GVG-induced GABA accumulation was only half that of normal. The results show that glutamine is an important precursor for GABA synthesis, but it cannot be the sole precursor because it was not possible to depress the GVG-induced GABA accumulation completely.     

Methionine Sulfoximine Prevents the Accumulation of Large Neutral Amino Acids in Brain of Portacaval-Shunted Rats

Portal-systemic shunting and hyperammonemia lead to an accumulation of the large neutral amino acids in brain and apparently alter transport of neutral amino acids across the blood-brain barrier. It has been proposed that portal-systemic shunting leads to a high brain concentration of glutamine, a product of cerebral ammonia detoxification, and thereby affects the transport of other neutral amino acids across the blood-brain barrier. To test this hypothesis, rats with a portacaval shunt were treated with L-methionine-dl-sulfoximine (MSO), an inhibitor of glutamine synthesis. Treatment with MSO resulted in lower concentrations of the neutral amino acids in brain of portacaval-shunted rats and a higher brain ammonia concentration, compared with untreated shunted rats. These results suggest that the accumulation of neutral amino acids in brain after portacaval shunt depends on the increased synthesis of glutamine in brain.     

Effect of glutamine on glutamine-synthetase regulation in the green alga Monoraphidium braunii

Glutamine-synthetase (GS; EC 6.3.1.2) activity and protein levels were measured in crude extracts from Monoraphidium braunii Näegeli, strain 202-7d, cultures grown under different nitrogen sources. Only ammonium and l-glutamine promoted a partial enzyme inactivation, which, in the case of l-glutamine, was accompanied by a significant repression of GS. Methionine sulfoximine (MSX), a strong inhibitor of GS, produced a drastic inactivation of GS which was concomitant with a marked increase in GS protein as measured by rocket immunoelectrophoresis. Such an increase was prevented in the presence of cycloheximide. The effect of the l-glutamine analog on GS activity and protein was partially inhibited if l-glutamine was also added to cell cultures, possibly indicating competition in the transport of these two substances. In addition, the effects of MSX were reversed after longer times when cultures were treated with smaller concentrations of inhibitor. Treatment of cell cultures with azaserine, a specific inhibitor of glutamate synthase, the second enzyme acting in the ammonium assimilation pathway, promoted a strong GS inactivation and a partial repression of this enzyme, which paralleled a specific increase in the intracellular pools of glutamine High-performance liquid chromatography measurements of intracellular amino-acid concentrations showed that glutamine levels correlated negatively with GS concentration. A role for glutamine as a negative effector of GS synthesis is proposed.Abbreviations GS l-glutamine synthetase - GOGAT l-glu-tamine:2-oxoglutarate amidotransferase - MSX methionine sulfoximine During the course of this work, J.A. was supported by a fellowship from Junta de Andalucía, and J.M. G-F. by a fellowship from the Spanish Ministerio de Educatión y Ciencia. This work was supported by the Junta de Andalucía.     

Effects of amino acids on methionine-sulfoximine-induced heterocyst formation in Anabaena

Heterocyst development in ammonia-grown cultures of Anabaena variabilis and Anabaena 7120 was fully induced by the amino-acid analog methionine sulfoximine (MSO) at concentrations of 0.5–1.0 M. Glutamine, glutamate, aspartate, and alanine at 0.5 mM blocked the induction of heterocysts by MSO in A. variabilis. With Anabaena 7120, glutamine and glutamate were fully effective and alanine partially effective in preventing MSO-induced heterocyst formation. In MSO-treated algae, glutamine synthetase activity was reduced to less than 15% of control values within 4–6 h. Inactivation of the enzyme was prevented by all four amino acids tested.     

Glutamate Uptake and Metabolism in C-6 Glioma Cells: Alterations by Potassium Ion and Dibutyryl cAMP

Abstract: Uptake and metabolism of glutamate was studied in the C-6 glioma cell line grown in the absence or presence of dibutyryl cyclic AMP (dbcAMP). Glutamate and aspartate uptake were competitive in cells grown under both conditions. Increased [K⁺] in the medium caused a significant decrease in the uptake of both amino acids. A small part of this decrease (<25%) was due to an enhanced efflux of tissue amino acid. The effects of increased [K⁺] were observed whether or not the [Na⁺] in the medium was concomitantly decreased. In cells grown in the presence of 1 mM dbcAMP for 48 h, glutamate uptake and metabolism were altered. Tissue levels of glutamate, aspartate, glutamine, GABA, and alanine were generally less in treated than in naive cells. When incubated with 50 μM [U-¹⁴C]glutamate, there was significantly less incorporation of radioactivity into treated cells with time, resulting in greatly lowered specific radioactivities of glutamate, aspartate, and GABA. However, the rate of labeling of glutamine was greatly increased; this was consistent with the previously observed doubling in glutamine synthetase activity in dbcAMP-treated C-6 cells. Tissue glutamate decarboxylase activity was halved in treated cells, accounting for the large decrease in GABA labeling. The metabolic data suggested a decreased uptake of exogenous glutamate; in studies on initial rates of uptake, the V_max of high-affinity glutamate uptake was decreased by 40%. This decrease was of the same order of magnitude as that observed in the metabolic experiments. Thus, in this glial model, both rapid, acute changes and slower, long-term changes in neuroactive amino acid metabolism were observed. Each of these conditions mimics a stimulus of neuronal origin, and the resulting changes could modulate extrasynaptic activity of neuroactive amino acids.     

In vivo Effects of the Anatoxin-a on Striatal Dopamine Release

Anatoxin-a is an important neurotoxin that acts a potent nicotinic acetylcholine receptor agonist. This characteristic makes anatoxin-a an important tool for the study of nicotinic receptors. Anatoxin-a has been used extensively in vitro experiments, however anatoxin-a has never been studied by in vivo microdialysis studies. This study test the effect of anatoxin-a on striatal in vivo dopamine release by microdialysis.The results of this work show that anatoxin-a evoked dopamine release in a concentration-dependent way. Atropine had not any effect on dopamine release evoked by 3.5 mM anatoxin-a. However, perfusion of nicotinic antagonists mecamylamine and α-bungarotoxin induced a total inhibition of the striatal dopamine release. Perfusion of α7*-receptors antagonists, metillycaconitine or α-bungarotoxin, partially inhibits the release of dopamine stimulated by anatoxin-a. These results show that anatoxin-a can be used as an important nicotinic agonist in the study of nicotinic receptor by in vivo microdialysis technique and also support further in vivo evidences that α7*nicotinic AChRs are implicated in the regulation of striatal dopamine release.     

Role of Glial Cells for the Basal and Ca2+-Dependent K+-Evoked Release of Transmitter Amino Acids Investigated by Microdialysis

The role of glial cells for the inactivation and synthesis of precursors for amino acid transmitters was studied in the brains of anesthetized rats in vivo using the microdialysis technique. The dialysis probes were inserted stereotactically into each neostriatum. One neostriatum was treated with the gliotoxin fluorocitrate, whereas the contralateral side served as a control. The basal efflux of amino acids, reflecting the extracellular level, was measured as well as the efflux during depolarization with 100 mM K+ in the dialysis stream. The potassium-evoked efflux of transmitter amino acids was calcium dependent and thus considered to reflect release from the transmitter pool. gamma-Aminobutyric acid (GABA) and glutamate release from the treated side was higher than the control value during the first 2-3 h, a result indicating an important role of glial cells in the inactivation of released transmitter. After 6-7 h with fluorocitrate, the release of glutamate was lower than the control value, a result indicating an important role of glial cells in the synthesis of precursors for the releasable pool of glutamate. The role of glutamine for the production of transmitter glutamate and GABA in vivo was further investigated by inhibiting glutamine synthetase with intrastriatally administered methionine sulfoximine. The release of gluatamate into the dialysis probe decreased to 54% of the control value, whereas the release of GABA decreased to 22% of the control value, a result indicating that glutamine may be more important for transmitter GABA than for transmitter glutamate.     

Effects of Phenylalanine on the Release of Endogenous Dopamine from Rat Striatal Slices

We examined the effect of phenylalanine (50-400 microM) on the electrically stimulated release of endogenous 3,4-dihydroxyphenylethylamine (dopamine or DA) from superfused rat striatal slices. In the absence of tyrosine, phenylalanine (25 microM) partially sustained DA release, but less well than an equimolar concentration of tyrosine. In the presence of tyrosine (50 microM), phenylalanine (in concentrations of greater than or equal to 200 microM) inhibited DA release into the superfusate. This inhibition was not associated with changes in tissue levels of tyrosine or DA, nor was it mimicked by addition of high concentrations of tyrosine or leucine to the medium. We conclude that phenylalanine is a less effective precursor of DA in rat striatum than tyrosine and that it can also act to inhibit DA synthesis, depending on its concentration.     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.