L 3h glutamate

Mouse hippocampal tissues were homogenized in ice-cold 0.32M sucrose solution. The tissue lysate was first centrifuged at 800 x g for 10min, followed by a centrifugation at 20,000 x g for 20min at 4°C. Cell pellets were washed in 0.32M sucrose, re-centrifuged and re-suspended in ice-cold 0.32M sucrose. Glutamate uptake was initiated by adding synaptosomes in Na+ buffer (5mM Tris-HCl, pH 7.2, 10mM HEPES, 140mM NaCl, 2.5mM KCl, 1.2mM CaCl2, 1.2mM MgCl2, 1.2mM K2HPO4, and 10mM D-glucose) containing 0.5 μM L-glutamate and 0.075 μCi L-[3H]glutamate (PerkinElmer; glutamate ratio cold:radioactive=99:1) per sample and incubated for 3 minutes at 37°C. The uptake was stopped by quickly adding four volumes of ice-cold glutamate-free Na+-free buffer (5mM Tris-HCl, pH 7.2, 10mM HEPES, 140mM Choline-Cl, 2.5mM KCl, 1.2mM CaCl2, 1.2mM MgCl2, 1.2mM K2HPO4, and 10mM D-glucose). The synaptosome lysate was filtered through a Brandel Harvester and washed with Na+-free assay buffer. The filter paper (containing the labeled synaptosomes) was transferred into scintillation vials and radioactivity was measured using a scintillation counter. Radioactive counts were normalized to total protein levels as determined by Bradford method.

After treated with VPA and Mn for designated time periods, H4 astrocytes were washed twice with prewarmed glutamate uptake buffer (122 mM NaCl, 3.3 mM KCl, 0.4 mM MgSO₄, 1.3 mM CaCl₂, 1.2 mM KH₂PO₄, 25 mM HEPES, and 10 mM D-(+)-glucose, pH 7.4). Five μl of prewarmed uptake buffer containing 0.25 μCi/ml L-[³H] glutamate (specific activity, 49.0 Ci/mmol; PerkinElmer Life Sciences) and 100 nM unlabeled glutamate was added to each well. The reactions were terminated by washing three times with ice-cold PBS, immediately followed by cell lysis in 1 ml of 1 N NaOH. An aliquot of 750 μl of each sample was transferred into scintillation vials and neutralized with 75 μl of 10 N HCl. The radioactivity was measured in liquid scintillation counter (LS 6500; Beckman Coulter).

Cell culture media and reagents were purchased from Invitrogen (Carlsbad, CA) unless stated otherwise. Luciferase reporter assay kit was obtained from Promega (Madison, WI). Manganese chloride (MnCl₂) and valproic acid (VPA) were obtained from Sigma-Aldrich (St. Louis, MO). EAAT1 and EAAT2 antibodies were from Abcam (Cambridge, MA). Antibodies for β-actin, acetylated histone H3 (Ac-histone H3), acetylated histone H4 (Ac-histone H4), TH and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA). L-[³H] glutamate was purchased from PerkinElmer (Waltham, MA).

Sodium-dependent transport of L-[³H]-glutamate was measured as previously described (Robinson et al. 1991 (link); Whitelaw and Robinson 2013 (link)). HKII or control treated synaptosomes (10 µL, containing ~40 µg of protein) were incubated with 0.5 µM L-[³H]-glutamate (PerkinElmer, Waltham, MA USA) in uptake buffer, containing 5 mM Tris base, 10 mM HEPES, 140 mM NaCl, 2.5 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM K₂HPO₄, 10 mM glucose (pH=7.2) at 37°C for 3 min. We have previously determined that ³H-glutamate uptake is linear until at least 5 min in this system (Robinson 1991 (link)). Uptake was measured in the absence and presence of sodium by substituting equimolar amounts of choline chloride for NaCl. The assay was terminated by the addition of 2 ml of ice-cold choline-containing buffer. Following termination, the suspensions were filtered onto glass filter paper (FP-100; Brandel, Gaithersburg, MD USA) using a Brandel cell harvester and rinsed three times with 2 ml of cold choline-containing buffer. Radioactivity was solubilized with 5 ml of Cytoscint ES (MP Biochemicals, Solon, OH USA) and measured using scintillation spectrometry (Beckman-Coulter Instruments LS 6500). Sodium dependent uptake was calculated as the difference between the signal in the sodium-containing buffer from the signal in the choline-containing buffer.

The uptake of [³H] glutamic acid was determined as previously described (Mutkus et al., 2005 (link)). Briefly, cells were treated with compounds in Opti-MEM for the indicated times and then washed twice with pre-warmed uptake buffer (122 mM NaCl, 3.3 mM KCl, 0.4 mM MgSO₄, 1.3 mM CaCl₂, 1.2 mM KH₂PO₄, 25 mM HEPES, and 10 mM D-(+)-glucose, pH 7.4). The glutamate uptake assay was initiated by adding a mixture of non-radioactive glutamate and 0.25 μCi/mL L-[³H] glutamate (specific activity, 49.0 Ci/mmol; PerkinElmer, Waltham, MA) to the reaction media to provide a final concentration of 100 nM glutamate. The reaction was continued for 10 min at 37°C and terminated by washing 3 times with ice-cold PBS, immediately followed by cell lysis in 1 mL of 1 N NaOH. An aliquot of 750 μL was taken out in scintillation vials and neutralized with 75 μL of 10 N HCl. After adding 5 mL of liquid scintillation fluid, the radioactivity was measured using a liquid scintillation counter (LS 6500; Beckman Coulter). The uptake activity was calculated as nmol glutamate/mg protein/min after correcting for protein levels.

The uptake reaction was started by the addition of 25 μl of the corresponding tissue fraction in test tubes containing L-[³H]-glutamate (100 nM; specific activity 49.0 Ci/mmol; Perkin Elmer, MA) and 0.1-100 μM of unlabeled glutamate (for saturation analysis) in 200 μl of uptake buffer containing (in mM) 140 NaCl, 5 KCl, 1.3 CaCl₂, 1 MgCl₂, 0.4 KH₂PO₄, 10 glucose, and 20 HEPES (pH 7.4) and incubated at 37°C for 2 min. System x^-_c uptake was measured similarly except by replacing sodium chloride with equal concentrations of choline chloride. Under this condition, the system mediates the uptake of cystine along with tritiated glutamate in exchange of unlabeled intracellular glutamate, as described previously (e.g., Patel et al., 2004 ; Melendez et al., 2005 ). Hence, this system utilizes normally high intracellular levels of glutamate to drive the import of cystine (Patel et al., 2004 ). Uptake was terminated by rapid filtration with ice-cold sodium-free buffer onto pre-wetted Whatman GF/B filter paper using a tissue harvester (Brandel, MD). Filters were dissolved overnight in scintillation fluid (EcoLite, MP Biomedicals, CA) and processed for scintillation counting (Beckman Instruments, USA).