A simple method for measuring intracellular activities of glutamine synthetase and glutaminase in glial cells

Abstract

Mongin AA, Hyzinski-García MC, Vincent MY, Keller RW Jr. A simple method for measuring intracellular activities of glutamine synthetase and glutaminase in glial cells. Am J Physiol Cell Physiol 301: C814-C822, 2011. First published July 6, 2011; doi:10.1152/ajpcell.00035.2011.-Here we report and validate a simple method for measuring intracellular activities of glial glutamine synthetase (GS) and glutaminase (GLNase) in intact glial cells. These enzymes are responsible for glutamate and glutamine recycling in the brain, where glutamate and glutamine transport from the blood stream is strongly limited by the blood-brain barrier. The intracellular levels of glutamate and glutamine are dependent on activities of numerous enzymatic processes, including 1) cytosolic production of glutamine from glutamate by GS, 2) production of glutamate from glutamine by GLNase that is primarily localized between mitochondrial membranes, and 3) mitochondrial conversion of glutamate to the tricarboxylic cycle intermediate ␣-ketoglutarate in the reactions of oxidative deamination and transamination. We measured intracellular activities of GS and GLNase by quantifying enzymatic interconversions of L-[ 3 H]glutamate and L-[ 3 H]glutamine in cultured rat astrocytes. The intracellular substrate and the products of enzymatic reactions were separated in one step using commercially available anion exchange columns and quantified using a scintillation counter. The involvement of GS and GLNase in the conversion of 3 H-labeled substrates was verified using irreversible pharmacological inhibitors for each of the enzymes and additionally validated by measuring intracellular amino acid levels using an HPLC. Overall, this paper describes optimized conditions and pharmacological controls for measuring GS and GLNase activities in intact glial cells.glutamate-glutamine cycle; metabolism; astrocytes L-GLUTAMATE and L-glutamine are two major intermediates of nitrogen metabolism. These two amino acids are present at high micromolar (ϳ30 -500 M) levels in the circulatory system. They diffuse freely from capillaries to the interstitial liquids and extracellular space and are taken inside animal cells by several types of amino acid transporters (24). In cytosol and mitochondrial matrix, concentrations of glutamate and glutamine are in the millimolar range. These two amino acids exist in equilibrium because they are consumed and produced in numerous interrelated biochemical reactions. The abundance and the generic roles of glutamate and glutamine in metabolism and protein synthesis interfere with the signaling properties of glutamate in the central nervous system (CNS). In the brain, glutamate serves as the main excitatory neurotransmitter. Therefore, extracellular levels of glutamate have to be tightly regulated and kept at low levels (reviewed in Refs. 9 and 14). This is possible due to two major factors: 1) limited transport of glutamate and glutamine via the endothelial blood-brain barrier (33), and 2) continuous recycling of glutamate and glutamine in t...

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“…Within 30 minutes, ∼60% of L-[ 3 H]glutamate, which was accumulated inside astrocytes, was converted to L-[ 3 H]glutamine. Our previous study established that such conversion is completely mediated by astrocytic GS [40]. However, we observed no significant changes in GS activity between experimental groups (Fig.…”

“…Primary astrocyte cultures were prepared from the cerebral cortex of newborn Sprague-Dawley rats as previously described [40]. All animal procedures in this study strictly adhered to the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of Albany Medical College and Rensselaer Polytechnic Institute.…”

“…Glutamine synthetase activity was determined as described elsewhere [40]. Briefly, cells grown on 15×15 mm coverslips covered with various substrates were washed with basal HEPES-buffered medium (for composition see above).…”

“…They were then transferred into 60-mm dishes, five coverslips per dish, and pre-incubated in the basal medium additionally containing 1 mM of the irreversible glutaminase inhibitor 6-diazo-5-oxo- l -norleucine (DON, Sigma-Aldrich) for 40 min. This treatment prevents hydrolysis of newly formed glutamine by intracellular glutaminases [40]. Cells were then washed two times with basal medium, and incubated with glutamine synthetase substrates 2 µCi/mL l -[ 3 H]glutamate, 2 µM unlabeled L-glutamate, and 100 µM (NH 4 ) 2 SO 4 in the basal medium for 30 minutes at 37°C.…”

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“…Primary astrocyte cultures from the cerebral cortex of P2 Sprague–Dawley rat pups were isolated as previously described [33] and in accordance with protocols approved by Rensselaer Polytechnic Institute's Institutional Animal Care and Use Committee (IACUC). Briefly, P2 rat pups were euthanized by rapid decapitation.…”