Decreased glutamate, glutamine and citrulline concentrations in plasma and muscle in endotoxemia cannot be reversed by glutamate or glutamine supplementation: a primary intestinal defect?

Endotoxemia affects intestinal physiology. A decrease of circulating citrulline concentration is considered as a reflection of the intestinal function. Citrulline can be produced in enterocytes notably from glutamate and glutamine. The aim of this work was to determine if glutamate, glutamine and citrulline concentrations in blood, intestine and muscle are decreased by endotoxemia, and if supplementation with glutamate or glutamine can restore normal concentrations. We induced endotoxemia in rats by an intraperitoneal injection of 0.3?mg?kg^?1 lipopolysaccharide (LPS). This led to a rapid anorexia, negative nitrogen balance and a transient increase of the circulating level of IL-6 and TNF-α. When compared with the values measured in pair fed (PF) animals, almost all circulating amino acids (AA) including citrulline decreased, suggesting a decrease of intestinal function. However, at D2 after LPS injection, most circulating AA concentrations were closed to the values recorded in the PF group. At that time, among AA, only glutamate, glutamine and citrulline were decreased in gastrocnemius muscle without change in intestinal mucosa. A supplementation with 4% monosodium glutamate (MSG) or an isomolar amount of glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscle. However, MSG supplementation led to an accumulation of glutamate in the intestinal mucosa. In conclusion, endotoxemia rapidly but transiently decreased the circulating concentrations of almost all AA and more durably of glutamate, glutamine and citrulline in muscle. Supplementation with glutamate or glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscles. The implication of a loss of the intestinal capacity for AA absorption and/or metabolism in endotoxemia (as judged from decreased citrulline plasma concentration) for explaining such results are discussed.     

Role of Glutamate and GABA Transporters in Development of Pentylenetetrazol-Kindling

Kindling is a form of epileptogenesis that can be induced with pentylenetetrazol (PTZ). We undertook this study to evaluate the contribution of glutamate and GABA transporters to the process of PTZ kindling. Rats were injected i.p. three times per week with PTZ (40 mg/kg) until they were fully kindled. In rats who achieved full kindling, measurement of hippocampal glutamate and GABA transporters within 24 h by western blot showed that GLAST, GLT-1, and EAAC1 were elevated significantly. However, fully kindled rats at 30 days after their last seizure had no change in either glutamate or GABA transporters proteins. These sequential observations suggest that glutamate transporters may contribute to the occurrence of seizures, but were not associated with maintenance of epileptogenesis. During this experiment, we collected data from animals that had kindled easily and animals who were resistant to kindling. Easily-kindled rats reached full kindling with less than five injections of PTZ. Kindling resistant animals failed to achieve full kindling even after administration of 12 consecutive injections of PTZ. Levels of EAAC1 and GAT-1 in easily-kindled rats were decreased by 30% when compared to kindling resistant animals at 30 days after the last PTZ injection. Since decreased EAAC1 and GAT-1 would diminish GABA function, less quantity of these proteins would appear to be associated with the convulsive threshold at the beginning of kindling development. We wonder if glutamate and GABA transporters might be operant in a convulsion threshold set factor or as a pace factor for kindling.     

Can a glutamate-enriched diet counteract glutamine depletion in endotoxemic rats?

The study evaluated whether a glutamate-enriched diet would restore glutamine tissue pools and maintain tissue trophicity in endotoxemic rats. For this purpose, young male Sprague-Dawley rats received an intraperitoneal injection of lipopolysaccharide (LPS) from Escherichia coli at 3 mg/kg body weight. After 24 hours of food deprivation, the rats were enterally refed for 48 hours using Osmolite enriched with glutamate at 4 g/kg/d (LPS-Glu group, n = 7) or glycine isonitrogenous to glutamate (LPS-Gly group, n = 7). A control group (healthy group, n = 7) had free access to a standard rodent diet. Tissue weights and protein contents were significantly lower in both LPS-treated groups than in the healthy group. No plasma or tissue accumulation of glutamate was observed except in the liver. Glutamine concentrations were increased in the jejunum, liver, and plasma in the LPS-Glu group versus the other two groups (P < 0.05). Conversely, they were depleted in muscles of the endotoxemic groups versus the healthy group (P < 0.05). Villus height was significantly greater in the LPS-Glu group than in the LPS-Gly group in the jejunum (P < 0.05), but not in the ileum. In conclusion, a glutamate-enriched diet administered enterally to endotoxemic rats can counteract glutamine depletion in the splanchnic area but not in muscles. In addition, glutamate displayed a trophic effect restricted to the jejunum.     

THE EFFECTS OF HYPERKETONEMIA ON GLUTAMATE AND GLUTAMINE METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The effect of increased exposure to ketone bodies in the developing rat brain suggest that intrauterine and postnatal hyperketonemia lead to an altered metabolism of glutamine and glutamate. It is postulated that this effect is related to the delayed development of glutaminase ( l -glutamine amido-hydrolase EC 3.5.1.2) and glutamate dehydrogenase ( l -glutamate: NAD oxidoreductase EC 1.4.1.2).
The specific activities of glutamate dehydrogenase (GDH), glutaminase and glutamine synthetase ( l -glutamate: ammonia ligase EC 6.3.1.2) in the brains of newborn rats increased during early development. A positive correlation was observed between the specific activity of glutaminase and the concentration of glutamate in the brain as well as between the concentrations of blood and brain glutamine and glutamate in both control and hyperketonemic pups. This indicates a different degree of permeability and metabolism for glutamine and glutamate in the brain during the neonatal period, as compared to adulthood.
In hyperketonemic pups, glutamine and glutamate metabolism were found to differ from that in control animals. The concentrations of glutamate were higher, and glutamine lower, in both the blood and brain as compared to that in controls. The concentrations of α-ketoglutarate were also lower in their brain. In the brains of hyperketonemic and control pups, the concentration of malate was the same. During the first 3 weeks of life the increase of spec. act. of GDH and glutaminase was found to be suppressed in the brains of hyperketonemic pups. However, the spec. act. of glutamine synthetase was similar to that of the control pups.     

In vitro 1H NMR spectroscopy shows an increase in N-acetylaspartylglutamate and glutamine content in the hippocampus of amygdaloid-kindled rats

We examined energy metabolism and amino acid content in the hippocampus of amygdaloid-kindled rats using (1)H NMR spectroscopy. Three weeks after the last stage 5 seizure, kindled rats were killed by microwave irradiation. The hippocampus was dissected out and subjected to MeOH/CHCl(3) extraction. All (1)H spectra were analyzed to quantify absolute concentrations using a non-linear least squares method, combined with a prior knowledge of chemical shifts. Saturation effects were compensated for by the T1 measurement of each component. Levels of energy metabolism-related compounds, phosphocreatine, creatine, glucose and succinate were the same in both kindled rats and sham controls. Lactate concentration had a tendency to increase, although this was not statistically significant. When compared with sham controls, levels of aspartate, glutamate, glycine and glutamine, as well as GABA and inositol, were increased in the ipsilateral but not the contralateral hippocampus. In contrast, levels of taurine, alanine and threonine were unchanged. Finally, N-acetylaspartylglutamate content was elevated, whereas N-acetyl-l-aspartate content was unaltered in the ipsilateral hippocampus of kindled animals. Our results suggest that amygdala kindling may affects amino acid metabolism, but not energy metabolism.     

单侧迷路破坏后大鼠前庭神经内侧核区氨基酸含量的变化

本实验用脑部微量透析法和高效液相色谱法观察单侧迷路破坏(unilateral labyrinthectomy,经利多卡因或对氨基苯胂酸盐预处理以阻断单侧外周前庭器官)后大鼠同侧及对侧前庭神经内侧核(medial vestibular nucleus,MVN)区部分氨基酸(天冬氨酸、谷氨酸、谷氨酰胺、甘氨酸、牛磺酸和丙氨酸)含量的变化,以了解前庭代偿的部分神经化学机制.实验观察到,对照组大鼠MVN区天冬氨酸、谷氨酸、谷氨酰胺、甘氨酸、牛磺酸和丙氨酸浓度分别为(6.15±0.59),(18.13±1.21),(33.73±1.67),(9.26±0.65),(9.56±0.77)和(10.07±0.83)pmol/8 μL透析样本.左侧中耳内灌注2%利多卡因后10 min,同侧MVN区天冬氨酸、谷氨酸含量立即减少(P＜0.05),牛磺酸含量增加(P＜0.05);对侧MVN区谷氨酸含量立即增加(P＜0.05),甘氨酸和丙氨酸含量减少;双侧核团间谷氨酸、甘氨酸和丙氨酸含量失衡.而用对氨基苯胂酸盐永久阻断单侧前庭器官2周后,同侧MVN区谷氨酸和丙氨酸含量减少,谷氨酰胺含量增高;对侧MVN区谷氨酸含量也减少;同侧MVN区谷氨酰胺含量明显高于对侧MVN区.结果提示,单侧迷路破坏后双侧MVN区氨基酸含量立即失去平衡,随着前庭代偿的进展,此差异减少,但是在前庭代偿后却出现双侧前庭核区谷氨酰氨的含量失衡,说明在前庭代偿过程中氨基酸含量变化起着重要作用.     

Metabolism of glutamine and glutamic acid by isolated perfused kidneys of normal and acidotic rats

1. When isolated kidneys from fed rats were perfused with glutamine the rate of ammonia release at pH7.4 (110–360μmol/h per g dry wt.) was one to two times that of glutamine removal. Glucose formation from 5mm-glutamine was 16μmol/h per g. If kidneys were perfused with glutamine at pH7.1 (10–13mm-sodium bicarbonate) there was no increase in glutamine removal or in the formation of ammonia or glucose. 2. When isolated kidneys from fed rats were perfused with glutamate at pH7.4, glucose formation was 59μmol/h per g, glutamine formation was 182μmol/h per g and ammonia release was negligible. At pH7.1 glutamine synthesis was inhibited and formation of ammonia and glucose were increased. 3. In perfused kidneys from acidotic rats, which had received 1.5% (w/v) NH₄Cl to drink for 7–10 days, gluconeogenesis from glutamine was enhanced (101μmol/h per g). Glutamine removal and ammonia formation were also increased, compared with the rates in perfused kidney from normal rats. The extra glutamine consumed was equivalent to the extra glucose formed. 4. When the kidney from the 7–10-day-acidotic rat was perfused with glutamate gluconeogenesis was increased (113μmol/h per g). Synthesis of glutamine was decreased, and ammonia release was approximately equal to the rate of glutamate removal. 5. The time-course of these metabolic alterations was investigated after the rapid induction of acidosis by infusion of 0.25m-HCl into the right side of the heart. The increase in gluconeogenesis from glutamine developed gradually over several hours. When kidneys from 6h-acidotic rats were perfused with glutamate, formation of glucose and glutamine were both rapid. 6. In acidotic rat kidneys perfused with glutamine, tissue concentrations of glutamate and glucose 6-phosphate were increased compared with those in control perfused kidneys from non-acidotic rats. 7. The results are discussed in terms of control of the renal metabolism of glutamine. In particular, it is suggested that in acidotic rats glucose formation is the major fate of the carbon of the extra glutamine utilized by the kidney, and that inhibition of glutamine synthetase could contribute to the increase in intracellular ammonia concentration in the kidney.     

Intrauterine Malnutrition and the Brain: Effects on Enzymes and Free Amino Acids Related to Glutamate Metabolism

Abstract: The effect of feeding pregnant rats with wheat and Bengal gram (black chick pea) diets during the later part of pregnancy on brain growth, enzymes, and free amino acids of glutamate metabolism in 1-day-old rats was investigated. These diets did not induce growth dissociation, and the body and brain weights were equally affected. The concentrations of DNA, RNA, protein, and free α-amino nitrogen in brain decreased significantly and the activities of glutamine synthetase, glutamine transferase, glutaminase 1, glutaminase 11, and glutamate decarboxylase and the concentrations of free amino acids, glutamic acid, glutamine, alanine, and GABA were also decreased. The concentration of aspartic acid, however, was increased. Wheat and Bengal gram diets fortified with lysine and with methionine, cystine, and tryptophan respectively showed various beneficial effects on the changes observed in the brain. A 20% casein diet induced higher body and brain weights and better brain protein and free α-amino nitrogen concentrations than those observed on a 10% casein diet.     

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%.     

Glutamate-Glutamine Cycle and Aging in Striatum of the Awake Rat: Effects of a Glutamate Transporter Blocker

This study investigated the effects of aging on the actions of a specific glutamate reuptake blocker, L-trans-pyrrolidine-2, 4-dicarboxylic acid (PDC), in extracellular glutamate and glutamine in striatum of the awake rat. Microdialysis experiments were performed on young (2–3 months), middle-aged (12–14 months), aged (27–32 months) and very aged (37 months) male Wistar rats. Local infusion of PDC (1–4 mM) in striatum increased the dialysate concentration of glutamate and decreased dialysate concentration of glutamine in all the age-groups. In young rats, decreases of dialysate glutamine were correlated with increases of dialysate glutamate. The same profile glutamine/glutamate as in young rats was found in middle-aged, aged and very aged rats, which suggests that the action of glutamate on the glutamate-glutamine cycle in striatum of the awake rat is not modified as a consequence of aging. We also found a significant correlation between the increases of glutamate produced by PDC and the basal dialysate concentration of glutamine, a relationship that did show a significant change with age. Although the significance of this latter finding remains to be elucidated, it may be important to understand the changes in glutamate-glutamine cycle during aging.     

Cerebral Cortex Ammonia and Glutamine Metabolism in Two Rat Models of Chronic Liver Insufficiency-Induced Hyperammonemia: Influence of Pair-Feeding

Abstract: Enhanced cerebral cortex ammonia uptake, subsequent glutamine synthesis, and glutamine release into the bloodstream have been hypothesized to deplete cerebral cortex glutamate pools. We investigated this hypothesis in rats with chronic liver insufficiency-induced hyperammonemia and in pair-fed controls to rule out effects of differences in food intake. Cerebral cortex plasma flow and venous-arterial concentration differences of ammonia and amino acids, as well as cerebral cortex tissue concentrations, were studied 7 and 14 days after surgery in portacaval-shunted/bile duct-ligated, portacaval-shunted, and sham-operated rats, while the latter two were pair-fed to the first group, and in normal unoperated ad libitum-fed control rats. At both time points, arterial ammonia was elevated in the chronic liver insufficiency groups and arterial glutamine was elevated in portacaval shunt/biliary obstruction rats compared to the other groups. In the chronic liver insufficiency groups net cerebral cortex ammonia uptake was observed at both time points and was accompanied by net glutamine release. Also in these groups, cerebral cortex tissue glutamine, many other amino acid, and ammonia levels were elevated. Tissue glutamate levels were decreased to a similar level in all operated groups compared with normal unoperated rats, irrespective of plasma and tissue ammonia and glutamine levels. These results demonstrate that during chronic liver insufficiency-induced hyperammonemia, the rat cerebral cortex enhances net ammonia uptake and glutamine release. However, the decrease in tissue glutamate concentrations in these chronic liver insufficiency models seems to be related primarily to nutritional status and/or surgical trauma.     

The Acute Effect of Ammonium Acetate on Levels of Amino Acids in the Intact and Decorticated Rat Neostriatum

Unilateral frontal cortex ablations were performed in rats so that the glutamate terminals in the ipsilateral rostral neostriatum were removed. At 1 or 7 days later, intraperitoneal injections of ammonium acetate induced different changes in amino acid concentrations in the intact and deafferentated neostriatum. After 1 day, the level of glutamate decreased only in the intact side, whereas that of glutamine increased and that of aspartate decreased to the same extent on both sides following ammonia injection. After 7 days, the glutamate level decreased more in the intact than the decorticated side in both nonconvulsing and convulsing rats. The concentration of alanine increased most in the intact neostriatum, whereas glutamine levels increased and aspartate levels decreased to the same extent on both sides in nonconvulsing and convulsing rats. The results indicate that ammonia has a more pronounced effect on neuronal than glial glutamate pools.     

Effects of supplementation with free glutamine and the dipeptide alanyl‐glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise

In this study, we investigated the effect of the supplementation with the dipeptide L ‐alanyl‐L ‐glutamine (DIP) and a solution containing L ‐glutamine and L ‐alanine on plasma levels markers of muscle damage and levels of pro‐inflammatory cytokines and glutamine metabolism in rats submitted to prolonged exercise. Rats were submitted to sessions of swim training for 6 weeks. Twenty‐one days prior to euthanasia, the animals were supplemented with DIP (n = 8) (1.5 g.kg^?1), a solution of free L ‐glutamine (1 g.kg^?1) and free L ‐alanine (0.61 g.kg^?1) (G&A, n = 8) or water (control (CON), n = 8). Animals were killed at rest before (R), after prolonged exercise (PE—2 h of exercise). Plasma concentrations of glutamine, glutamate, tumour necrosis factor‐α (TNF‐α), prostaglandin E2 (PGE2) and activity of creatine kinase (CK), lactate dehydrogenase (LDH) and muscle concentrations of glutamine and glutamate were measured. The concentrations of plasma TNF‐α, PGE2 and the activity of CK were lower in the G&A‐R and DIP‐R groups, compared to the CON‐R. Glutamine in plasma (p < 0.04) and soleus muscle (p < 0.001) was higher in the DIP‐R and G&A‐R groups relative to the CON‐R group. G&A‐PE and DIP‐PE groups exhibited lower concentrations of plasma PGE2 (p < 0.05) and TNF‐α (p < 0.05), and higher concentrations of glutamine and glutamate in soleus (p < 0.001) and gastrocnemius muscles (p < 0.05) relative to the CON‐PE group. We concluded that supplementation with free L ‐glutamine and the dipeptide LL ‐alanyl‐LL ‐glutamine represents an effective source of glutamine, which may attenuate inflammation biomarkers after periods of training and plasma levels of CK and the inflammatory response induced by prolonged exercise. Copyright © 2009 John Wiley & Sons, Ltd.     

The transport and metabolism of glutamine by kidney-cortex mitochondria from normal and acidotic rats.

1. The oxidation of glutamine by kidney-cortex mitochondria from normal and acidotic rats was not inhibited by avenaciolide, which did inhibit glutamate uptake and oxidation. The oxidation of glutamine by these mitochondria was always greater than that of glutamate. Direct measurements of the metabolism of [1-14C]glutamine in the presence of glutamate, and of [1-14C]glutamate in the presence of glutamine, demonstrated that the uptake and metabolism of external glutamate is insufficient to account for the observed rate of glutamine uptake and metabolism. Thus the postulated glutamine/glutamate antiport does not play a quantitatively important role in the metabolism of glutamine by renal mitochondria. 2. Rapid swelling of these mitochondria was observed in iso-osmotic solutions of L-glutamine and L-glutamyl-gamma-monohydroxamate but not in D-glutamine or L-isoglutamine (1-amido-2-aminoglutaric acid). Thus a relatively specific glutamine uniport exists in these mitochondria. 3. The utilization of glutamine was increased about 3-fold in mitochondria from chronically acidotic rats. Thus mitochondrial adaptations play an important part in the renal response to metabolic acidosis.     

Acute Alterations of Glutamate,Glutamine, GABA,and Other Amino Acids After Spinal Cord Contusion in Rats

Spinal cord injury (SCI) leads to an alteration of energetic metabolism. As a consequence, glutamate, glutamine, aspartate and other important amino acids are altered after damage, leading to important disregulation of the neurochemical pathways. In the present study, we characterized the acute-phase changes in tissue concentration of amino acids involved in neurotransmitter and non-neurotransmitter actions after SCI by contusion in rats. Animals were submitted to either laminectomy or SCI by contusion and sacrificed at 2, 4, 8, and 12 h after lesion, for the analysis of tissue amino acids by HPLC. Results showed that both aspartate and glutamate contents diminished after SCI, while glutamine concentrations raised, however, the sum of molar concentrations of glutamate plus glutamine remained unchanged at all time points. GABA concentrations increased versus control group, while glycine remained unchanged. Finally, citrulline levels increased by effect of SCI, while taurine-increased only 4 h after lesion. Results indicate complex acute-phase changes in amino acids concentrations after SCI, reflecting the different damaging processes unchained after lesion.     

Metabolism of glutamine and glutamate by rat renal tubules. Study with 15N and gas chromatography-mass spectrometry

Gas chromatography-mass spectrometry was utilized to study the metabolism of [15N]glutamate, [2-15N]glutamine, and [5-15N]glutamine in isolated renal tubules prepared from control and chronically acidotic rats. The main purpose was to determine the nitrogen sources utilized by the kidney in various acid-base states for ammoniagenesis. Incubations were performed in the presence of 2.5 mM 15N-labeled glutamine or glutamate. Experiments with [5-15N]glutamine showed that in control animals approximately 90% of ammonia nitrogen was derived from 5-N of glutamine versus 60% in renal tubules from acidotic rats. Experiments with [2-15N]glutamine or [15N]glutamate indicated that in chronic acidosis approximately 30% of ammonia nitrogen was derived either from 2-N of glutamine or glutamate-N by the activity of glutamate dehydrogenase. Flux through glutamate dehydrogenase was 6-fold higher in chronic acidosis versus control. No 15NH3 could be detected in renal tubules from control rats when [2-15N]glutamine was the substrate. The rates of 15N transfer to other amino acids and to the 6-amino groups of the adenine nucleotides were significantly higher in normal renal tubules versus those from chronically acidotic rats. In tubules from chronically acidotic rats, 15N abundance in 15NH3 and the rate of 15NH3 appearance were significantly higher than that of either the 6-amino group of adenine nucleotides or the 15N-amino acids studied. The data indicate that glutamate dehydrogenase activity rather than glutamate transamination is primarily responsible for augmented ammoniagenesis in chronic acidosis. The contribution of the purine nucleotide cycle to ammonia formation appears to be unimportant in renal tubules from chronically acidotic rats.     

Excess Extracellular and Low Intracellular Glutamate in Poorly Differentiating Wobbler Astrocytes and Astrocyte Recovery in Glutamine-Depleted Culture Medium

Abstract: The wobbler mouse develops an inherited motoneuronal degeneration of unknown origin in the spinal cord. Primary cultures of adult wobbler spinal cord astrocytes display abnormal morphological characteristics with fewer processes and paucity of cell-cell contacts. We have searched for a possible involvement of glutamate and glutamine intra- and extracellular accumulations in vitro in the abnormal differentiation of mutant astrocytes. We have found significantly higher glutamate and glutamine concentrations in the culture media of mutant astrocytes over a 3-day period compared with normal control astrocytes. Moreover, intracellular glutamate concentrations decreased substantially in mutant astrocytes, but intracellular glutamine concentrations remained unchanged. Furthermore, decreasing initial glutamine concentrations in the culture medium (glutamine-depleted medium) led to the recovery of normal extra- and intracellular concentrations of glutamate and recovery of quasi-normal morphological differentiation and increased cell-cell contacts, leading to an essentially normal looking astrocyte network after 3 days of culture. Under these conditions, which lead to recovery, the only remaining abnormality was the higher glutamine extracellular concentration attained in the originally depleted glutamine media. These findings suggest that mechanisms regulating glutamate/glutamine synthesis and/or influx/efflux are defective in wobbler astrocytes, leading to metabolic imbalance and possible cytotoxic effects characterized by disturbed intercellular networks and poor differentiation.     

Glucocorticoids Exacerbate Kainic Acid–Induced Extracellular Accumulation of Excitatory Amino Acids in the Rat Hippocampus

Glucocorticoids (GCs) compromise the ability of hippocampal neurons to survive various insults, and do so, at least in part, by exacerbating steps in the glutamate/N-methyl-D-aspartate (NMDA)/calcium cascade of damage. As evidence, GCs impair uptake of glutamate by hippocampal astrocytes, the GC endangerment of the hippocampus is NMDA receptor dependent, and GCs exacerbate kainic acid (KA)-induced calcium mobilization. These observations predict that GCs should also exacerbate KA-induced accumulation of extracellular glutamate and aspartate. To test this, adrenalectomized rats were given replacement GCs in either the low or high physiological range. Three days later, rats were anesthetized and 1 mM KA was infused through a dialysis probe placed in the dorsal hippocampus. Extracellular amino acid concentrations in the dialysate were then assessed by HPLC. After KA infusion, high-GC rats (30 +/- 3 micrograms/dl) had significantly elevated concentrations of glutamate and aspartate compared with low-GC rats (all less than 0.95 micrograms/dl). The glutamate accumulation was due to GCs raising pre-KA concentrations, whereas the aspartate accumulation was due to GCs exacerbating the KA-induced rise. Glutamine concentrations were unaffected by KA, whereas the high-GC regimen elevated glutamine concentrations both before and after KA. Taurine concentrations rose after infusion of KA, but were unaffected by GC regime, whereas alanine concentrations were unaffected by either manipulation. Serine concentrations were unaffected by KA, but were depressed both before and after KA in high-GC rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Short-term metabolic fate of 13N-labeled glutamate, alanine, and glutamine(amide) in rat liver

Tracer quantities (in 0.2 ml) of 13N-labeled glutamate, alanine, or glutamine(amide) were administered rapidly (less than or equal to 2 s) via the portal vein of anesthetized adult male rats. Liver content of tracer at 5 s was 57 +/- 6 (n = 6), 24 +/- 1 (n = 3), and 69 +/- 7 (n = 3)% of the injected dose, respectively. Portal-hepatic vein differences for the corresponding amino acids were 17 +/- 6, 26 +/- 8, and 19 +/- 9% (n = 4), respectively, suggesting some export of glutamate and glutamine, but not of alanine, to the hepatic vein. Following L-[13N]glutamate administration, label rapidly appeared in liver alanine and aspartate (within seconds). The data emphasize the rapidity of nitrogen exchange via linked transaminases. By 30 s following administration of either L-[13N]glutamate or L-[13N]alanine, label in liver glutamate was comparable; yet, by 1 min greater than or equal to 9 times as much label was present in liver glutamine(amine) following L-[13N]glutamate administration than following L-[13N]alanine administration. Conversely, label in liver urea at 1 min was more pronounced in the latter case despite: (a) comparable total pool sizes of glutamate and alanine in liver; and (b) label incorporation from alanine into urea must occur via prior transfer of alanine nitrogen to glutamate. The data provide evidence for zonal differences in uptake of alanine and glutamate from the portal vein in vivo. The rate of turnover of L-[amide-13N]glutamine was considerably slower than that of L-[13N]alanine or of L-[13N]glutamate, presumably due in part to the higher concentration of glutamine in that organ. Nevertheless, it was possible to show that despite occasional suggestions to the contrary, glutamine(amide) is a source of urea nitrogen in vivo. The present findings continue to emphasize the rapidity of nitrogen exchange reactions in vivo.     

The metabolic fate of 13N-labeled ammonia in rat brain.

13N-labeled ammonia was used to study the cerebral uptake and metabolism of ammonia in conscious rats. After infusion of physiological concentrations of [13N]ammonia for 10 min via one internal carotid artery, the relative specific activities of glutamate, glutamine (alpha-amino), and glutamine (amide) in brain were approximately 1:5:400, respectively. The data are consistent with the concept that ammonia, entering the brain from the blood, is metabolized in a small pool of glutamate that is both rapidly turning over and distinct from a larger tissue glutamate pool (Berl, S., Takagaki, G., Clarke, D.D., and Waelsch, H. (1962) J. Biol. Chem. 237, 2562-2569). Analysis of 13N-metabolites, after infusion of [13N]ammonia into one lateral cerebral ventricle, indicated that ammonia entering the brain from the cerebrospinal fluid is also metabolized in a small glutamate pool. Pretreatment of rats with methionine sulfoximine led to a decrease in the label present in brain glutamine (amide) following carotid artery infusion of [13N]ammonia. On the other hand, 13N activity in brain glutamate was greater than that in the alpha-amino group of glutamine, i.e. following methionine sulfoximine treatment the expected precursor-product relationship was observed, indicating that the two pools of glutamate in the brain were no longer metabolically distinct. The amount of label recovered in the right cerebral hemisphere, 5 s after a rapid bolus injection of [13N]ammonia via the right common carotid artery, was found to be independent of ammonia concentration within the bolus over a 1000-fold range. This finding indicates that ammonia enters the brain from the blood largely by diffusion. In normal rats that were killed by a freeze-blowing technique 5 s after injection of an [13N]ammonia bolus, approximately 60% of the label recovered in brain had already been incorporated into glutamine, indicating that the t1/2 for conversion of ammonia to glutamine in the small pool is in the range of 1 to 3 s or less. The data emphasize the importance of the small pool glutamine synthetase as a metabolic trap for the detoxification of blood-borne and endogenously produced brain ammonia. The possibility that the astrocytes represent the anatomical site of the small pool is considered.