Different Concentrations Of Glutamine Research Articles

Abstract 3161: PCTK1 driven glutamine metabolic adaptation in brain metastasis

Abstract Brain metastasis (BrM) is the most common malignancy of the central nervous system and the median survival time of patients with BrM is less than 1 year. Although recent impressive advances in targeted therapy and immunotherapy enable more effective control of the systemic diseases, the incidence of BrM upon disease recurrence is steadily increasing. Unfortunately, there is no effective treatments for BrM. To surmount this challenge, we have performed an in vivo human kinome screening to uncover novel kinases that promote breast cancer (BC) BrMs in mice and may serve as therapeutic targets. We identified >20 different kinases enriched in BrM lesions of our mouse models and PCTK1 is one of the top “hits”. We transduced PCTK1 expression vector and the control vector into MDA-MB-231 human BC cells to generate 231.PCTK1 and 231.vector cells, respectively. Intra-carotid injection of the 231.PCTK1 cells into mice induced BrM significantly faster with shortened mouse survival than that of the control 231.vector cells, confirming PCTK1 as a BrM-promoting kinase. However, when 231.vector or 231.PCTK1 cells were injected into the mouse’ mammary glands, PCTK1 overexpression did not lead to enhanced primary tumor growth, suggesting that its pro-tumoral functions are BrM-specific. To uncover how PCTK1 promotes BrM but not primary tumor, we examined whether PCTK1 may confer growth advantage in BrM by utilizing the unique metabolic microenvironment of the brain. We analyzed the relationship between PCTK1 gene expression and gene expression of members of the glutamine metabolism pathway in TCGA database. We found that in the brain tumors, such as low-grade glioma, PCTK1 positively correlated with the expression of glutamine metabolizing genes, including glutaminase 1 and 2 (GLS and GLS2). To evaluate whether PCTK1-overexpressing cells can uptake glutamine from the media directly, we cultured 231.PCTK1 and 231.vector cells in low-nutrient BME media plus 5% d.FBS, with addition of different concentrations of glutamine. The PCTK1-overexpressing cells showed a clear growth advantage at glutamine concentrations ranging from 400 μM to 2 mM. Then, we knocked down PCTK1 in MDA-MB-231 cells by shRNAs and found that PCTK1 knocking down reduced the cell proliferation in 5% d.FBS.BME with 500 µM glutamine, a physiological glutamine level in the brain. We measured the expression of GLS, an enzyme that converts glutamine to glutamate, one of the key steps in the glutamine metabolism, and found that knockdown PCTK1 in MDA-MB-231 cells dramatically reduced the GLS expression, which indicates that PCTK1 can modulate glutamine metabolism in cancer cells. In summary, our data suggest that PCTK1 promotes BC BrM, at least partly, through increasing glutamine metabolism. Currently, we are exploring whether targeting PCTK1 or inhibiting glutamine metabolism can deter BrM progression. Our studies may reveal novel therapeutic targets for BrM inhibition. Citation Format: Lin Zhang, Frank Lowery, Chenyu Zhang, Yu-Wen Huang, Ya-Jing Jiang, Yimin Duan, Dihua Yu. PCTK1 driven glutamine metabolic adaptation in brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3161.

English

Jatropha is a drought-tolerant plant producing seed oil for the biodiesel. Limitation to the development of jatropha is unavailability of high-yielding varieties and efficient in vitro regeneration system which is required for micropropagation. In this study, in vitro regeneration system from jatropha juvenile cotyledon was established. Firstly, concentrations of hormones in the MS medium were optimized and it was found that 1.5 mgL-1 benzyl adenine (BA) + 0.05 mgL-1 indole-3-butyric acid (IBA) + 0.5 mgL-1 thidiazuron (TDZ) turned out to be the best for shoot induction (3.82 ± 0.18 shoots/explant). Secondly, shoot induction medium was fortified with different concentrations of glutamine and adenine sulfate. It was found that 25 mgL-1 each of glutamine and adenine sulfate was the most effective, resulting to 9.09 ± 0.37 shoots/explant and 93.0% regeneration frequency. Regenerated shoots were cultured on medium containing 0.5 mgL-1 BA and different concentrations of gibberellic acid (GA3), 0.5 mgL-1 GA3 with 0.5 mgL-1 BA  were found to be the best for shoot elongation (2.13 ± 0.18 cm). The highest frequency of root (40%) was observed on the medium with 0.5 mgL-1 IBA. The established procedures will be useful for the mass propagation and genetic transformation of elite jatropha genotypes.   Key words: Adenine sulfate (Ads), benzyl adenine (BA), indole-3-butyric acid (IBA), in vitro regeneration, glutamine (Gln).

Effect of glutamine and cysteine supplementation on quality of cryopreserved sperm of South American silver catfish

The aim of this study was to investigate the effects of different concentrations of glutamine (experiment 1) and glutamine + cysteine (experiment 2) on post-thaw sperm quality of South American silver catfish (Rhamdia quelen). The sperm was cryopreserved in cryoprotectant solution (fructose 50 g/L, powdered milk 50 g/L and methanol 100 ml/L) containing different glutamine concentrations (0, 2.5 and 5 mM; experiment 1) and a combination of glutamine (2.5 and 5 mM) and cysteine (2.5 and 5 mM; experiment 2). In experiment 1: glutamine supplementation groups increased DNA fragmentation; the concentration of carbonyl groups was lower and the concentration of sulfhydryl groups was higher in control; a higher activity of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and glutathione peroxidase (GPx) enzymes was observed in the 2.5 and 5 mM groups. In experiment 2: the group with 5 mM of cysteine +5 mM of glutamine presented the higher DNA damage; treatments with 5 mM of glutamine presented the higher concentration of carbonyl groups and a higher activity of CAT and GST. The findings of this study show that glutamine and cysteine, at the concentrations evaluated in this study, does not present satisfactory results but rather damaging effects on sperm quality of R. quelen.

Effects of various glutamine concentrations on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is one of the most important biologically sourced and low-calorie sweeteners that contains a lots of Steviol glycosides. Tissue culture is the best method for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. In the present study, we investigated the effect of different concentrations of glutamine (10, 20, 30 and 40 g/l) on expression of UGT74G1 and UGT76G1 genes and stevioside and rebaudioside A accumulation in the leaves of stevia under in vitro conditions. The highest level of expression for UGT74G1 (1.000 Total lab unit) was seen at plants grown in MS media without glutamine and the highest gene expression level for UGT76G1 (1.321 Total lab unit) was observed at plants grown in 2% glutamine. Based on HPLC results, the highest amount of stevioside (22.74) was accumulated in plants which were under 3% glutamine treatment and the lowest production level of stevioside (16.19) was resulted under MS (0 glutamine) medium. The highest rebaudioside A (12.19) accumulation was observed under 2% glutamine treatment and the lowest accumulation of rebaudioside A (8.41) was seen at plants grown in MS medium.

Glutamine modulates lipopolysaccharide-induced activation of NF-κB via the Akt/mTOR pathway in lung epithelial cells

Lung epithelial cells are important barriers in the respiratory system that provoke inflammatory responses through nuclear factor (NF)-κB activation to prevent pathogens from invading the body. Lipopolysaccharide (LPS) is a common pathogen-associated stimulus that activates IκB kinase (IKK) to regulate NF-κB-mediated inflammation through modulating nuclear translocation and phosphorylation of NF-κB. Previously, it was shown that Akt and the mammalian target of rapamycin (mTOR) are involved in the phosphorylation of IKK to activate NF-κB. Herein, we demonstrate that glutamine (GLN) modulated LPS-induced activation of NF-κB through the Akt/mTOR/IKK pathway in BEAS-2B cells. BEAS-2B cells in submerged culture were placed in medium containing different concentrations of GLN (0, 0.5, 1, and 2.5 mM) with 1 μg/ml LPS. Results showed that GLN deprivation induced phosphorylation of Akt/mTOR/IKK signaling, increased levels of NF-κB nuclear translocation and phosphorylated NF-κB, and upregulated NF-κB-dependent transcriptional activity, which was suppressed by GLN administration. Expressions of NF-κB-targeted genes were also reduced by supplemental GLN. GLN administration improved cell viability, whereas 0.5 mM GLN had a greater extent of inhibition on the Akt/mTOR/IKK/NF-κB signaling cascade. The inhibitory effects of GLN on NF-κB activation were also observed in cells cultured under air-liquid interface condition. These results indicate that GLN deprivation increased LPS-induced NF-κB activation and transcriptional activity, which was reversed by GLN administration. The findings provide potential mechanisms of GLN's modulation of LPS-induced NF-κB activation in lung epithelial cells and imply that maintaining a physiological concentration of GLN is essential in preventing LPS-induced lung inflammation.

Glutamine depletion induces murine neonatal melena with increased apoptosis of the intestinal epithelium

To investigate the possible biological outcome and effect of glutamine depletion in neonatal mice and rodent intestinal epithelial cells. We developed three kinds of artificial milk with different amounts of glutamine; Complete amino acid milk (CAM), which is based on maternal mouse milk, glutamine-depleted milk (GDM), and glutamine-rich milk (GRM). GRM contains three-fold more glutamine than CAM. Eighty-seven newborn mice were divided into three groups and were fed with either of CAM, GDM, or GRM via a recently improved nipple-bottle system for seven days. After the feeding period, the mice were subjected to macroscopic and microscopic observations by immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) and Ki-67 as markers of cell proliferation, and for cleaved-caspase-3 as a marker of apoptosis. Moreover, IEC6 rat intestinal epithelial cells were cultured in different concentrations of glutamine and were subject to a 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate cell proliferation assay, flow cytometry, and western blotting to examine the biological effect of glutamine on cell growth and apoptosis. During the feeding period, we found colonic hemorrhage in six of 28 GDM-fed mice (21.4%), but not in the GRM-fed mice, with no differences in body weight gain between each group. Microscopic examination showed destruction of microvilli and the disappearance of glycocalyx of the intestinal wall in the colon epithelial tissues taken from GDM-fed mice. Intake of GDM reduced BrdU incorporation (the average percentage of BrdU-positive staining; GRM: 13.8%, CAM: 10.7%, GDM: 1.14%, GRM vs GDM: P < 0.001, CAM vs GDM: P < 0.001) and Ki-67 labeling index (the average percentage of Ki-67-positive staining; GRM: 24.5%, CAM: 22.4% GDM: 19.4%, GRM vs GDM: P = 0.001, CAM vs GDM: P = 0.049), suggesting that glutamine depletion inhibited cell proliferation of intestinal epithelial cells. Glutamine deprivation further caused the deformation of the nuclear membrane and the plasma membrane, accompanied by chromatin degeneration and an absence of fat droplets from the colonic epithelia, indicating that the cells underwent apoptosis. Moreover, immunohistochemical analysis revealed the appearance of cleaved caspase-3 in colonic epithelial cells of GDM-fed mice. Finally, when IEC6 rat intestinal epithelial cells were cultured without glutamine, cell proliferation was significantly suppressed after 24 h (relative cell growth; 4 mmol/L: 100.0% ± 36.1%, 0 mmol/L: 25.3% ± 25.0%, P < 0.05), with severe cellular damage. The cells underwent apoptosis, accompanied by increased cell population in sub-G0 phase (4 mmol/L: 1.68%, 0.4 mmol/L: 1.35%, 0 mmol/L: 5.21%), where dying cells are supposed to accumulate. Glutamine is an important alimentary component for the maintenance of intestinal mucosa. Glutamine deprivation can cause instability of the intestinal epithelial alignment by increased apoptosis.

Effects of glucose and glutamine concentration in the formulation of the artificial cerebrospinal fluid (ACSF)

The composition of the ACSF is fundamental in controlling the extracellular environment of the brain slice preparation. ‘Typical’ formulations lack amino acids and contain a higher concentration of glucose (10 mM) than in the cerebrospinal fluid (0.47 – 4.4 mM). We examined the effects of different concentrations of glutamine, the most abundant amino acid in the CSF, and glucose on rat hippocampal slice physiology. Bipolar paired-pulse stimulation was applied to the Schaffer collaterals and population spikes were monitored in the CA1 pyramidal layer for ~1 hour. Addition of glutamine (0.5 mM) to slices superfused with 10 mM of glucose did not enhance population spike amplitude. Higher concentration of glutamine (2-5 mM) resulted in spreading-depression. Decreasing glucose concentration from 10 mM to 5 mM, in the absence of glutamine, attenuated population spikes. Decreasing glucose to 2 mM, in the absence of glutamine, suppressed evoked population spikes. Superfusing brain slices with ACSF containing ‘physiological’ concentrations of both glucose (2 mM) and glutamine (0.5 mM) similarly suppressed population spikes. In separate experiments, during high-K + induced epileptiform activity, glutamine (0.5 mM) did not affect the burst duration, frequency or waveform. These results suggest that the concentration of glucose in ACSF should conservatively be 10 mM in order to maximize paired-pulse population responses while the presence of physiological concentration of glutamine (0.5 mM) has minimal effects on paired-pulse responses and high-K + induced epileptiform activity. These results are discussed in the context of fundamental differences between in vitro brain slice superfusion and in vivo brain perfusion.

Increased chemosensitivity and elevated reactive oxygen species are mediated by glutathione reduction in glutamine deprived neuroblastoma cells

Glutamine is an essential amino acid for the synthesis of glutathione (GSH), the major endogenous antioxidant which protects cells from oxidative injury. To evaluate the effects of glutamine concentrations, cell growth, GSH levels, oxidative stress, and chemosensitivity were evaluated in neuroblastoma cell lines. Three human neuroblastoma cell lines (SMS-KCNR, SMS-KANR, SMS-LHN) were cultured with different concentrations of glutamine (2, 0.2 and 0 mM) under hypoxic (5% O(2)) or normoxic (20% O(2)) condition. Cell proliferation and chemosensitivity were determined by MTT assay, and the levels of intracellular GSH were measured by DTNB-GSSG reductase method. Cellular reactive oxidative species (ROS) were quantified by flow cytometry. There was a significant decrease of cell growth in low glutamine (0.2 and 0 mM) compared with control (2 mM) in all three cell lines (P < 0.01), while adding GSH partially restored the reduced cell proliferation by low glutamine. The levels of GSH in neuroblastoma cells decreased significantly in low glutamine compared with the levels of control cells cultured in 2 mM glutamine (P < 0.05), and the accumulation of cellular ROS was significantly higher in 0 mM glutamine compared to the control. Moreover, glutamine deprivation significantly enhanced cytotoxicity of L-PAM in all three cell lines, which was abolished after addition of GSH. Glutamine deprivation decreased cell proliferation and enhances cell chemosensitivity in neuroblastoma, which is presumably associated with GSH depletion.

Effect of Low Glutamine/Glucose on Hypoxia-Induced Elevation of Hypoxia-Inducible Factor-1α in Human Pancreatic Cancer MiaPaCa-2 and Human Prostatic Cancer DU-145 Cells

Tumor microenvironment is characterized by regions of fluctuating and chronic hypoxia, low extracellular pH, and nutrient depletion. Although it is well known that hypoxia stimulates the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha), the role of low extracellular pH and nutrient depletion on hypoxia up-regulation of HIF-1alpha is not well known. In this study, human pancreatic cancer MiaPaCa-2 and human prostatic cancer DU-145 cells were exposed to hypoxia in the presence or absence of glucose, glutamine, and/or pyruvate. We observed that low glucose and low glutamine, but not low pyruvate, effectively suppressed the elevation of HIF-1alpha level during hypoxia (0.1-1% oxygen). Deprivation of glutamine or glucose inhibited the accumulation of HIF-1alpha in the presence of MG-132, a protease inhibitor, regardless of oxygen tensions. Data from reverse transcription-PCR analysis revealed that the levels of HIF-1alpha mRNA were not significantly changed at different concentrations of glutamine or glucose under hypoxia. The amount of HIF-1alpha suppression was proportional to protein synthesis inhibition. Our data suggest that glutamine or glucose deprivation inhibits the accumulation of HIF-1alpha under hypoxic conditions by disrupting translational processes rather than transcriptional or proteasomal degradation processes.

Glutamine Concentration May Limit Glutathione Synthesis in the Presence of α-Adrenoceptor Agonists and Glucagon

ABSTRACTGlutathione (GSH) is an ubiquits tripeptide which is composed of glutamine-cysteine-glycine. In general, methionine is the preffered precursor of cysteine in cell culture. It has been indicated that glucagon, adrenaline and phenylephrine inhibit hepatocellular glutathione synthesis in the presence of methionine. Therefore, cysteine is considered rate-limiting for GSH synthesis. To examine whether changes in glutamine concentration in the medium are also rate-limiting for GSH synthesis, short term cultured hepatocytes were treated with different concentrations of glutamine in the presence of the other two precursors. The inhibitory effects of glucagon were observed when lower concentrations of glutamine (0.2 or 0.5mM) were used. However, this effect was attenuated when hepatocytes were incubated with 2mM concentrations of glutamine. The effect of phenylephrine on GSH synthesis was measured in the presence of different concentrations of methionine (2, 0.5, 0.2 and 0.1mM), given as a precursor of cysteine, with 2mM glutamine and 2mM glycine. The inhibitory effect of phenylephrine was not apparent at any concentration of methionine. Our results suggest that, under physiological conditions, the availability of glutamine in the plasma, and possible changes in L-glutamate concentration, appear to be important for maintaining GSH synthesis in the presence of glucagon and the α-adrenergic agents, adrenaline and phenylephrine.

Role of glutamine on the de novo purine nucleotide synthesis in Caco-2 cells.

The body's nucleotide pool derives from three potential sources: de novo synthesis, salvage of preformed-nucleosides/bases or the diet. The relative contributions of these pathways of assimilation are poorly understood in vivo. Dietary nucleotides have been suggested to have beneficial effects an the development and repair of the gastrointestinal tract. Tissues with a rapid turnover, such as the gut and the immune system cells, may utilise preformed nucleotides (coming from the diet), in situations in which there is a high demand of nucleotides for nucleic acid synthesis. Therefore, nucleotides could be considered as conditionally essential nutrients. Development of a method to measure synthesis de novo of RNA-purine nucleotides in Caco-2 cells, relying an the incorporation of 14C-glycine into the purine ring of the nucleotide. To establish the fractional synthesis rate of RNA purine nucleotides in Caco-2 cells, grown in culture medium containing different concentrations of glutamine, in the presence or absence of added nucleotides. To investigate the degree to which tissue ribonucleosides are derived from the culture medium or from de novo synthesis in the presence of different concentrations of glutamine, using undifferentiated Caco-2 cells, stressed or not by the addition of IL-1 beta to the medium. The presence of high levels of glutamine in the culture medium is essential for cell proliferation (estimated by measurement of the fractional synthesis rate of purine nucleotides) and the presence of nucleotides cannot replace the glutamine dependence of Caco-2 cell proliferation. The incorporation of exogenous purine nucleotides into RNA of Caco-2 cells is rather limited, and it becomes important when cells are stressed by glutamine deprivation. Stress by addition of interleukin-1 beta resulted in the maintenance or the increase in de novo synthesised RNA-purine nucleotides, even in the presence of exogenous nucleotides. However, the addition of interleukin-1 beta to the culture medium led to an enhanced salvage of preformed pyrimidine nucleotides for nucleic acid synthesis when glutamine was present in the medium at a concentration of 0.5 mmol/L.

Analysis of the ammonia metabolism of rat primary hepatocytes and a human hepatocyte cell line Huh 7.

Ammonia metabolism of ratprimary hepatocytes and a human hepatocyte cell line,Huh 7, at different concentrations of glutamine,glucose and ammonia was examined. During theincubation of the primary hepatocyte cells, glutamineand ammonia concentrations decreased, that of ureaincreased, and that of glucose remained the same. Inthe case of Huh 7 cells, glucose was consumed rapidly,the concentration of ammonia increased and that of urearemained the same. The major energy sources amongmedium components were glutamine for the primary cellsand glucose for Huh 7 cells, although the primaryhepatocytes may utilize intracellular glycogen asenergy source. As the glutamine concentration in theincubation medium increased, the specific rates of notonly glutamine consumption, but also ammonia productionby the primary cells and Huh 7 cells increased. Besides, specific urea production rate by the primarycells increased then. Increase of glucoseconcentration had no effect on glutamine and ammoniametabolism by both cells, although it increased glucoseconsumption by Huh 7 cells. The incubation of theprimary cells with higher ammonia concentrationincreased all specific rates of glutamine consumption,ammonia consumption and urea production. An increasein the ammonia concentration to 5 mM changed theammonia metabolism from production to consumption andincreased the specific glucose consumption rate. Consequently, increases in the glutamine and ammoniaconcentrations were revealed to have negative andpositive effects, respectively, on decreasing ammoniaconcentration by both of rat primary hepatocytes andHuh 7 cells.

The effects of cell number, concentrations of mitogen and glutamine and time of culture on [ 3H]thymidine incorporation into cervical lymph node lymphocytes stimulated by Concanavalin-A

The amount of [ 3H]thymidine incorporated into DNA in lymphocytes stimulated with Concanavalin-A increases exponentially with time at different concentrations of glutamine, reaches a peak value, then gradually decreases. When the value (log 10 thymidine incorporation glutamine present−log 10 thymidine incorporation glutamine absent) obtained from the exponential phase is plotted against time, a linear plot is obtained for each glutamine concentration. When these linear rates of incorporation are plotted against glutamine concentration, hyperbolic curves are obtained for different times of culture. The peak value of incorporation (which reflects the final number of cells which entered the cell cycle) is determined by the concentration of mitogen and occurs at an earlier time as the number of cells in culture is increased and as the concentration of glutamine is increased. These findings suggest that increasing the plasma glutamine concentration above the normal physiological level may be of value in increasing the proliferation of lymphocytes in conditions of lymphopenia. Adenosine, a fuel of purine nucleotide synthesis, which may affect the lymphoproliferative response also via specific adenosine receptors, increases the rate of incorporation of [ 3H]thymidine but this effect depends upon the concentration of glutamine; at low concentrations of glutamine, the stimulation by adenosine is apparent whereas at high concentrations of glutamine adenosine appeared to inhibit proliferation. Our data presented in this paper suggest that not only the number of cells and the concentration of mitogen, but also the concentration of glutamine, may play a role in determining the proliferative response of mitogenic stimulated peripheral lymphocytes. This emphasises that care must be taken to carry out a full kinetic study, including varying the concentration of glutamine to assess in these types of experiments whether a particular compound or condition causes immunostimulation or immunoinhibition.

Glutamine requirements in the generation of lymphokine-activated killer cells

The role of glutamine (GLN) in the generation of lymphokine-activated killer (LAK) cell activity was investigated. LAK cells were derived from healthy donors and peripheral blood mononuclear cells (PBMC) were obtained using either unseparated PMBC or DR − CD3 − CD16 + CD56 + enriched cells. PBMC were cultured for 6 or 10 days in medium supplemented with recombinant interleukin-2 (rlL-2; 100 U/ml) in the presence of different concentrations of GLN. K562 (natural killer-NK-sensitive targets), 1301 and U-937 (NK-resistant targets) cells were used as targets in the cytotoxic assays. Furthermore, the limiting dilution (LD) culture system was applied as an alternative to the bulk cell culture system. It was found that GLN affects the lytic potential of cultured cells while the frequency of responding cells did not significantly differ between the compared cell cultures performed in the presence of different amounts of GLN. Data on cell proliferation with IL-2 stimulation showed significant differences in cultures performed in the presence or absence of GLN. The results of present investigation suggest a supportive role of GLN in the generation of LAK cells. GLN deficit affects LAK cell killing activity by limiting the number of generated effector cells while acquisition of broad-range killing capacity was not affected.

Effect of Glutamine on Glutamate Release from Hippocampal Slices Induced by High K+ or by Electrical Stimulation: Interaction with Different Ca2+ Concentrations

To characterize the effect of glutamine on the release of glutamate, aspartate, and gamma-aminobutyric acid (GABA), rat hippocampal slices were superfused with different concentrations of glutamine or Ca2+. Amino acids released and retained were analyzed by HPLC. Glutamine (0.5 mmol/L) increased more than threefold the release of glutamate evoked by 50 mmol/L K+ in the presence of 2.6 mmol/L Ca2+ without a corresponding increase in glutamate content, while the release of aspartate was increased less and that of GABA not at all by glutamine. The evoked release of all three amino acids, including the enhanced release of glutamate in the presence of glutamine, was strongly dependent on Ca2+ concentrations between 0.1 and 2.6 mmol/L. The potentiation of glutamate release by glutamine reached a plateau at 0.25 mmol/L glutamine. Intermittent electrical field stimulation increased the release of only glutamate and this release was nearly doubled by glutamine. The increased release was Ca2+ dependent and tetrodotoxin (TTX) sensitive. Results suggest that extracellular glutamine promotes primarily the formation of releasable glutamate and this enhancement is dependent on extracellular Ca2+.