L-Glutamate, L-glutamine, 6-diazo-5-oxo-L-norleucine (DON), and L-methionine sulfoximine (MSO) were purchased from Sigma-Aldrich (St. Louis, MO). 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB) was obtained from Tocris Cookson (Ellisville, MO). D-[3H]aspartate, L-[3H]glutamate and L-[3H]glutamine were acquired from PerkinElmer-New England Nuclear (Waltham, MA). All cell culture media and sera were obtained from Invitrogen (Carlsbad, CA). All salts, buffers, solvents, and other reagents, were from Sigma-Aldrich, unless otherwise specified.
Primary astrocyte cultures
Confluent primary astrocyte cultures were prepared from the cerebral cortex of newborn Sprague-Dawley rats. These and other animal procedures were approved by the Albany Medical Center Institutional Animal Use and Care Committee and adhered to the NIH guidelines for care and use of laboratory animals. One-day old rat pups were euthanized by rapid decapitation. The cerebral cortices were separated from the meninges, hippocampi, and basal ganglia. Cortical tissue combined from 4 animals was minced and transferred into a solution of the recombinant protease TrypLE (Invitrogen), which was diluted 1:1 (v:v) with OptiMEM (Invitrogen). Cells were extracted using three 10-min incubations with TrypLE additionally supplemented with bovine pancreatic DNase I (Sigma). The first extraction was discarded, while the second and the third extractions were combined with Minimal Essential Medium (MEM) containing 10% heat inactivated horse serum (HIHS) and 50 U/ml penicillin plus 50 μg/ml streptomycin (Pen/Strep). Cells were sedimented by a brief centrifugation (1,000 g × 1.5 min) and then resuspended in MEM/HIHS. Dissociated cells were seeded on poly-L-lysine coated T75 culture flasks (Techno Plastic Products, TPP, Trasadingen, Switzerland) at the density of 250,000 cells/flask. Cultures were grown for two-three weeks in MEM/HIHS +Pen/Strep at 37°C in a humidified atmosphere of 5% CO2/95% air. The culture medium was changed twice a week. Culture purity was routinely confirmed by staining with antibody recognizing specific astrocytic marker, glial fibrillary acidic protein, (Sigma); ≥98% of cells were GFAP-positive. Confluent cells were re-plated as necessary on 6- or 12-well tissue culture plates (TPP), or 18-mm square coverslips (Carolina Biological, Burlington, NC).
Animal surgery and microdialysis procedures
Microdialysis procedures were performed in male Sprague-Dawley rats (Taconic Farms), weighing between 325 and 425 g. Rats were given atropine sulfate (0.05 mg/kg, i.m.) to reduce fluid secretion in the respiratory tract and initially anesthetized with 5% isoflurane. Animals were then intubated and mechanically ventilated with a gas mixture of 2.25% isoflurane in 30% O2/balance N2. Saline drip (0.9% NaCl) was administered intraperitoneally throughout the procedure to prevent dehydration. Body temperature was monitored throughout the experiment with a rectal probe and was maintained between 36°C and 36.5°C with a heating pad. Animals were placed in a stereotaxic frame and microdialysis probes (2 mm tip, 20 kD cutoff, CMA Microdialysis, North Chelmsford, MA) were slowly lowered through burr holes into the frontoparietal cortex (from bregma, 1 mm anterior; ±4 mm lateral; 2.6 mm down from the dura). Cortical tissue was perfused via the microdialysis probes with the artificial cerebral spinal fluid (aSCF) at a rate of 2 μL/min. aCSF contained (in mM): 120 NaCl, 25 NaHCO3, 2.7 KCl, 1 MgSO4, 1.2 CaCl2, and 0.05 ascorbic acid. The medium was bubbled with 5% CO2 to maintain pH=7.3. After two hours of probe stabilization, at least two 20-minute perfusate samples were collected by a CMA-170 refrigerated fraction collector (CMA Microdialysis) to determine baseline amino acid levels before the application of drug or hypoosmotic medium. Hypoosmotic aCSF contained (in mM): 25 NaCl, 25 NaHCO3, 2.7 KCl, 1 MgSO4, 1.2 CaCl2, and 0.05 ascorbic acid (pH=7.3). Hypoosmotic medium was perfused at the same rate (2 μL/min) for one hour but perfusate samples were collected more frequently: every 5 minutes. Each rat was implanted with two microdialysis probes placed bilaterally in the cortex, with one probe serving as a control (hypoosmotic solution only) and the probe on the other side (chosen at random) serving as the experimental condition (hypoosmotic solution plus drug).
Determination of the intracellular amino acid content in primary astrocyte cultures
Confluent astrocyte cultures grown in 6 well plates were preincubated for 40 min in basal HEPES-buffered medium containing (in mM): 135 NaCl, 3.8 KCl, 1.2 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 D-glucose, 10 HEPES (pH=7.4). Basal medium was then aspirated and replaced with fresh basal, or hypoosmotic, or isoosmotic low [NaCl] HEPES-buffered media. In hypoosmotic medium (HYPO), [NaCl] was reduced to 77 mM. In isoosmotic low [NaCl] medium (Low-Na): [NaCl] was also reduced to 77 mM but osmolarity was corrected by adding 116 mM mannitol. Cells were incubated in the experimental media for 30 min at 37°C. After completion of incubation, experimental media were aspirated and 1 mL of solution containing 5 mM Hepes and 1 mM EDTA was added to each well. Cells were scraped and sonicated for 4 minutes at room temperature. 100 μL aliquots of cell lysates were taken for protein assays, and the remaining lysates were clarified by rapid centrifugation (4 min × 12,100 g, MiniSpin centrifuge, Eppendorf, Hauppauge, NY). Supernatants were taken for HPLC analysis of amino acid content.
HPLC analysis of amino acid content in microdialysate samples and lysed cells
Amino acid levels in microdialysate samples and cell lysates were determined by a reverse-phase high performance liquid chromatography (HPLC) using an Agilent 1200 HPLC setup and Eclipse XDB-C18 column (4.6×150 mm, 5 μm particle diameter). Pre-column derivatization of amino acids was performed with freshly prepared mix of o-phthaldialdehyde and 2-mercaptoethanol in 0.4 M sodium tetraborate buffer (pH=9.5). The amino acid derivatives were eluted with solvent containing 30 mM NaH2PO4, 1% tetrahydrofuran, 30 mM sodium acetate, 0.05% sodium azide, and increasing concentration of the HPLC grade methanol (10–30%). Fluorescence signals were registered using a programmable 1200 series fluorescence detector (Agilent). Amino acid standards (L-aspartate, L-glutamate, L-glutamine, taurine, L-alanine), which were processed in the same fashion, were used to locate amino acid peaks and calculate concentrations of individual amino acids in the samples.
L-[3H]glutamine and L-glutamate (D-[3H]aspartate) release assay
To measure L-glutamine release, cells grown on 18-mm square glass coverslips were initially preincubated for 40 minutes with the glutaminase inhibitor 1 mM DON (Willis and Seegmiller 1977
). This was necessary to prevent conversion of radiolabeled L-glutamine into L-glutamate by glutaminase. DON was washed out by transferring cells into basal medium, and cells were preloaded for 30 min with 4 μCi/mL of L[3
H]glutamine plus 2 μM of unlabeled glutamine at 37°C. Coverslips were washed in 2 mL of basal medium to remove excess L-[3
H]glutamine and transferred into a Lucite perfusion chamber. This chamber had a depression precisely cut in the bottom to accommodate the coverslip and a Teflon screw top leaving space above the cells of around 150–200 μm in height. Cells were superfused at a flow rate of 1.2 mL/min in an incubator set at 37°C with Basal, Low-Na, or HYPO media as indicated in figures (for media composition, see above). One-min perfusate fractions were collected and analyzed for [3
H] content in a TriCarb 1900TR Liquid Scintillation Analyzer (PerkinElmer, Boston, MA) after adding 4 mLs of Ecoscint A scintillation cocktail (National Diagnostics, Atlanta, GA). At the end of each experiment, astrocytes on coverslips were lysed with 1 mL of 2% sodium dodecyl sulfate (SDS) plus 8 mM EDTA to calculate remaining isotope content. Fractional isotope release for each time point was calculated by dividing radioactivity released in each one min interval by the radioactivity left in the cells.
To determine the L-glutamate release rate we used its nonmetabolizable analogue D-[3H]aspartate, which is taken inside the cell by L-glutamate transporters and can be used as a tracer of L-glutamate fluxes. Cells were loaded for 30 min with 2 μCi/mL of D[3H]aspartate plus 2 μM of unlabeled L-glutamate at 37°C. The remaining procedures were identical to those described above.
L-[3H]glutamine uptake assay
Cells grown in 12-well plates were washed from serum-containing media with HEPES buffered Basal medium and pre incubated for 40 minutes in Basal medium with or without the irreversible glutaminase inhibitor 1 mM DON. They were then washed twice with 1 mL of Basal medium and incubated for 5–40 minutes at 37°C with either Basal, Low-Na or HYPO media containing additionally 4 μCi/mL of L-[3H]glutamine and 2 μM of unlabeled L-glutamine. Amino acid uptake was terminated by 3 washes with 1 mL of ice-cold Basal medium. Cells were then lysed in 1 mL of 2% SDS plus 8 mM EDTA. Lysates was transferred into scintillation vials and [3H] content was determined as described above.
Enzymatic assays of glutamine synthetase and glutaminase activity
We measured glutamine synthetase and glutaminase activity inside intact cells by quantifying enzymatic conversions of L-[3
H]glutamate and L-[3
H]glutamine after the separation of radiolabeled amino acids on anion exchange columns as originally proposed by Prusiner and Milner (1970)
For glutaminase activity assays, astrocytes grown in 6-well plates were preincubated for 40 minutes with the irreversible glutamine synthetase inhibitor 1 mM MSO (Ronzio and Meister 1968
). Cells were washed twice with 2 mL of Basal solution and incubated for 30 minutes in Basal, Low-Na, or HYPO media containing 4 μCi/mL of L-[3
H]glutamine plus 2 μM of unlabeled glutamine and 20 μM of DCPIB. The concentrations of labeled and unlabeled substrates were selected empirically to achieve high intracellular labeling levels. DCIPB was used to prevent loss of L-[3
H]glutamate via volume-regulated anion channel (VRAC) in swollen cells (Abdullaev et al. 2006
). After the completion of this 30-min reaction, the extracellular isotope was removed by 3 consecutive washes with 2 mL of ice-cold Basal solution. One mL of milliQ H2
O was added to each well to lyse astrocytes; cells were scraped and sonicated for 4 min. Lysates were clarified by rapid centrifugation (4 min × 12,100 g). Each cell lysate (1 mL) was added onto AG 1-X8 Polyprep column (BioRad, Hercules, CA) for anion exchange separation of L-[3
H]glutamate and L-[3
H]glutamine. Column content was eluted with three 2-mL volumes of milliQ H2
O, followed by three volumes of 0.1 M HCl. Water elution removes uncharged L-[3
H]glutamine, while subsequent acid elution extracts negatively charged L-[3
H]glutamate. Eluent fractions were transferred into scintillation vials and 4 mL of Ecoscint scintillation cocktail (National Diagnostics, Atlanta, GA) were added to each vial. [3
H] content was calculated in a Tri-Carb 1900TR Liquid Scintillation Analyzer. The glutaminase activity was calculated as % conversion of L[3
H]glutamine to L-[3
H]glutamate normalized to the total [3
H] recovered from each sample (L-[3
H]glutamate plus L-[3
Intracellular activity of glutamine synthetase was assayed in a similar fashion but in this case astrocytes we preincubated for 40 min with the irreversible glutaminase inhibitor 1 mM DON (Willis and Seegmiller 1977
). Cells were washed twice with 2 mL of Basal solution and then incubated for 30 minutes in Basal, Low-Na or HYPO media containing 2 μCi/mL of L-[3
H]glutamate plus 2 μM of unlabeled glutamate and 20 μM of ammonium sulfate to provide sufficient levels of NH4+
for the GS reaction. 20 μM DCPIB was also added to prevent L-[3
H]glutamate loss via VRAC in swollen cells. L[3
H]glutamine and L-[3
H]glutamate were then separated using AG 1-X8 columns as described above. The glutamine synthetase activity was calculated as a percent conversion of L-[3
H]glutamate to L-[3
H]glutamine normalized to the total [3
H] content recovered from each sample (L-[3
H]glutamine plus L-[3
The majority of data in this study are presented as mean values ±SE. Statistical differences between groups were determined using one-way ANOVA or repeated measures ANOVA and post hoc Tukey test for multiple comparisons. For enzymatic assays, the experimental data were normalized to controls in each experiment. In these latter experiments statistical differences were determined using one population t-test with post hoc Bonferroni correction if multiple comparisons were made. Origin 8.1 (Origin Labs, Northampton, MA) and Prism 5 (GraphPad Software, San Diego, CA) were used for statistical analyses.