Glutamine uptake and expression of mRNA’s of glutamine transporting proteins in mouse cerebellar and cerebral cortical astrocytes and neurons

Neurochemistry International 44 (2004) 75–81

Glutamine uptake and expression of mRNA’s of glutamine transporting proteins in mouse cerebellar and cerebral cortical astrocytes and neurons Monika Doli´nska a , Barbara Zabłocka b , Ursula Sonnewald c , Jan Albrecht a,∗ a b

Department of Neurotoxicology, Medical Research Centre, Polish Academy of Sciences, Pawiñskiego St. 5, Warsaw 02-106, Poland Laboratory of Molecular Biology, Medical Research Centre, Polish Academy of Sciences, Pawiñskiego St. 5, Warsaw 02-106, Poland c Department of Neuroscience and Locomotion, Norwegian University of Science and Technology, Trondheim, Norway

Abstract The relative roles of the three sodium-dependent transport systems: A, ASC and N in the uptake of [3 H]Gln, and the compatibility of the uptake characteristics with the expression of mRNAs coding for the Gln transporting molecules, were examined in primary cultures of astrocytes and neurons derived from mouse cerebellum, a glutaminergic system-enriched structure, and in cerebral cortex. Gln uptake activity (Vmax ) was higher in cerebellar astrocytes or neurons than in their cerebral cortical counterparts. The N-methylamino-isobutyric acid (MeAiB)- and pH-sensitive, system A-mediated component of the uptake, and the uptake of [14 C]MeAiB itself, was much more active in neurons than in astrocytes derived from either region. Also, the expression of mRNA for GlnT (SAT1), a system A isoform specific for Gln, was only expressed in neurons derived from both structures, while an alanine (Ala)-preferring system A transporter, SAT2, was expressed in neurons and astrocytes from either region. System ASC-mediated Gln uptake and expression of ASCT2 mRNA were in both structures more pronounced in astrocytes than in neurons, consistent with the postulated role of ASCT2 in the efflux of de novo synthesized Gln from astrocytes. System N-mediated (threonine + MeAiB-inhibitable) Gln uptake showed comparable activities in all four types of cells, which is compatible with the ubiquitous expression of NAT2 mRNA—a mouse brain-specific N-system isoform. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Mouse; Cerebral cortex; Cerebellum; Astrocytes; Neurons; Glutamine uptake; MeAiB uptake; System A; System N; System ASC

1. Introduction Active, sodium-dependent transport of glutamine (Gln) in astrocytes and neurons appears to be a critical step in the glutamate (Glu)–Gln cycle in the CNS. The major direction of Gln flux is from astrocytes where its synthesis occurs, to neurons where it is converted to neurotransmitter amino acids Glu and GABA, albeit astrocytes are also capable of Gln reuptake (reviewed by Broër, 2002). Studies with cells cultured in vitro have demonstrated that Gln transport in astrocytes and neurons mainly involves three sodium-dependent systems: A, ASC and N (Broër et al., 1999; Nagaraja and Brookes, 1996; Su et al., 1997; Tamarappoo et al., 1997). Gln efflux from cultured rat astrocytes appears to be carried out by ASCT2, an ASC system isoform strongly expressed in these cells (Broër et al., 1999), and in a rat astrocytoma-derived C6 cell line (Albrecht et al., 2001; Doli´nska et al., 2000, 2003), while systems A and N mediate Gln uptake to both astrocytes and neurons (Broër et al., 1999; Nagaraja and Brookes, 1996; Su et al., 1997; ∗

Corresponding author. Tel.: +48-22-668-5323; fax: +48-22-668-5532. E-mail address: [email protected] (J. Albrecht).

Tamarappoo et al., 1997). Recently, a plethora of system A and N isoforms differing in substrate- and cell-type specificity have been cloned (reviewed by Broër, 2002). Analysis of Gln transport in oocytes or cell lines transfected with the newly cloned transporters, combined with their immunocytochemical localization in situ, have shed some light on their roles. Thus, the A system variant, GlnT/SAT1 appears to be mainly engaged in Gln uptake to neurons (Varoqui et al., 2000; Armano et al., 2002). With regard to system N, the roles of brain-specific isoforms appear different in rat and mouse. The rat isoform, SN1 shows preferential astrocytic localization and is thought to mediate active Gln efflux from astrocytes (Chaudhry et al., 1999; Boulland et al., 2002). The mouse isoform, NAT2 is expressed in the retina, where it was localized to ganglion cells and axons, and in brain, where its cellular localization has not been specified (Gu et al., 2001). Several questions pertinent to regulation of Gln transport in CNS cells can be conveniently addressed using cultured CNS cells, a model which reflects at least some aspects of the native intracellular millieu. Firstly, since mRNAs coding for different Gln transport systems are frequently expressed in one and the same cell type, it will be of interest

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M. Doli´nska et al. / Neurochemistry International 44 (2004) 75–81

to delineate their relative contribution to the transport. Secondly, if active Gln transport is to play a regulatory role in the synthesis of neurotransmitters Glu and GABA, one may expect differences of its characteristics between brain regions showing mainly enrichment in the glutamatergic system. Whereas glutamate is taken up mostly into the surrounding astrocytes, GABA re-enters the neurons that were responsible for its release (Schousboe et al., 1977; Ereci´nska and Silver, 1990; Gegelashvili and Schousboe, 1998). The predicted complimentary roles of astrocytes (efflux) and neurons (uptake) in shuttling Gln are likely to be reflected in different characteristics of the transport in the two cell types derived from the same brain region. In this study, the above questions were addressed by comparing the characteristics of Gln uptake and expression of mRNA’s in cultured astrocytes and neurons derived from two mouse brain structures: cerebellum, in which glutamatergic neurotransmission is richly represented, and cerebral cortex, a GABAergic structure. Gln uptake was characterized with regard to its compatibility with systems A, ASCT (1 or 2) and N. Messenger RNA expression studies have taken into account the following transporters: ASCT2, GlnT/SAT1 and NAT2, but also SAT2, the ubiquitous A system variant showing no specificity for Gln (Yao et al., 2000).

2. Material and methods Plastic tissue culture dishes were purchased from Nunc A/S (Roskilde, Denmark), fetal bovine serum (FBS) from Seralab Ltd. (Sussex, UK) and culture medium from GIBCO BRL, Life Technologies (Roskilde, Denmark). NMRI mice were purchased from Møllegaard Breeding Center (Copenhagen, Denmark). 2.1. Cell cultures Cortex neurons were isolated from 16-day-old mouse fetuses (Hertz et al., 1989), cerebellar granule cells were cultured from cerebellum of 7-day-old mice (Schousboe et al., 1989), after mild trypsinization of the tissue followed by trituration in a DNase solution containing a trypsin inhibitor from soybeans. Cells were suspended ((2–3) × 106 cells/ml) in a slightly modified Dulbecco’s minimum essential medium (DMEM) containing 50 ␮M kainic acid (cerebellar granule neurons only) and 10% (v/v) fetal bovine serum. Cells were cultured in poly-l-lysine coated Petri dishes. Cytosine arabinoside (20 ␮M) was added after 48 h to prevent astrocyte proliferation. Cells were used for experiments after 1 week in culture. Cerebellar astrocytes were prepared from 7-day-old mice, cortical astrocytes from 1-day-old mice and cultured as described earlier (Hertz et al., 1989). Briefly, cerebella or cortex was passed through Nitex nylon netting (80 ␮m pore size) into Dulbecco’s minimum essential medium containing 20% (v/v) FBS. Medium was changed 2 days after plating

and subsequently twice a week gradually changing to 10% FBS. From the second week, dibutyryl-cAMP was added to the medium to promote the morphological differentiation of astrocytes for 1 week. Experiments were performed on 3-week-old astrocytes. 2.2. Amino acid uptake assays The uptake of radiolabeled Gln, or MeAiB (Glu) to the cultured cells was measured following a 2 min incubation of the radiolabeled amino acids with cultures grown in 24-well plates exactly as described previously for Gln uptake in C6 cells (Doli´nska et al., 2000; Albrecht et al., 2001). Briefly, incubation media (IM) contained 150 mM NaCl, 3 mM KCl, 2 mM CaCl2 , 0.8 mM MgCl2 , 5 mM glucose, 10 mM HEPES, pH 7.4 and either 0.25 ␮Ci of l-[G-3 H]Gln (Amersham, UK), or 0.2 ␮Ci of 2-[1-14 C]MeAiB (American Radiolabeled Chemicals, USA) supplemented with unlabeled Gln or MeAiB, respectively. For competition experiments, the concentration of unlabeled Gln was 0.078 mM and competing amino acids were added at 64-fold excess, together with radiolabeled amino acids. The concentrations of unlabeled Gln or MeAiB for kinetic analysis of the uptake were as described in the figures. The reaction was stopped with 2 ml of ice-cold choline chloride-containing SIM and the cells were lysed by incubation in 1N NaOH at 37 ◦ C for 30 min. 2.3. RT-PCR analysis Total RNA was isolated using TRIzol Reagent (Life Technologies) and total RNA (5 ␮g) was reverse transcribed using the Superscript II and oligo (dT)12–18 primers (Gibco/BRL). Each cDNA sample (2 ␮l) was then amplified by PCR using the following primers for mouse amino acid transporters; ASCT2: 5 -accatggtcctggtctcctg-3 and 5 -gccagtccacggccaagatc-3 (Broër et al., 1999), GlnT: 5 -cgcccagtcttacagaccag-3 and 5 -cagtgaatagagatgcccgag-3 (Varoqui et al., 2000), NAT2: 5 -ttcagcctggtacgtcgatg-3 and 5 -tgacatactttggtgtgcacg-3 (Gu et al., 2001), SAT2: 5 -cccattgtcactgctgagaaa-3 and 5 -tccctgatagtggggacaaa-3 (Yao et al., 2000). GAPDH, for which the primers used were: 5 -tgaaggtcggagtcaacggatttgg-3 and 5 -catgtaggcatgaggtccaccac-3 (Schreiber and Durre, 1999) served as a constitutive marker. The primers were obtained from TIB Biomol (Poznañ, Poland). Following 30 cycles of amplification (1 min at 94 ◦ C, 1 min at 55 ◦ C for ASCT2 and GlnT, 56 ◦ C for SAT2 or 58 ◦ C for NAT2, and 1 min at 72 ◦ C using the MJ Research thermal cycler PTC-100) the PCR products were resolved on 1.2% agarose gel in an ethidium bromide-containing buffer, with 588 bp ASCT2, 151 bp GlnT, 241 bp NAT2 and 679 bp SAT2 run as standards. The bands were recorded using the Nucleovision system (Nucleotech) and densitometric analysis was carried out using the GelExpert 4.0 program. For sequence analysis, the bands were extracted from agarose using DNA clean-up

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kit (Akor Laboratories). The analysis was performed using Perkin-Elmer ABI type 383 sequencer. 2.4. Protein assay Protein content was determined in the NaOH lysates. Protein in the cells used for uptake experiments was assayed directly in the plates by the method of Bradford (1976).

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Table 1 Kinetic parameters of l-[3 H]glutamine uptake to the different cell cultures measured in the presence of sodium chloride calculated from the Michaelis–Menten plots Vmax (nmole/(min mg protein)) Cerebral cortical astrocytes Cerebellar astrocytes Cerebral cortical neurons Cerebellar granule cells

5.19 16.67 10.79 14.05

± ± ± ±

0.21 0.71 0.63 0.67

Km (mM) 0.51 0.72 1.16 0.93

± ± ± ±

0.07 0.09 0.17 0.13

2.5. Statistical analysis

Results are mean ± S.D. for 4–6 independent experiments.

Statistical significance of the differences between the results was computed using one way ANOVA test followed by Dunnet’s test, either from Sigma Plot 5.0 or GraphPad Prism 3.02.

astrocytes from a given brain structure more than to neurons (Fig. 2). However, the system A substrates MeAiB, Pro and Gly almost failed to inhibit Gln uptake in cerebral cortical astrocytes, and were only weakly inhibitory in cerebellar astrocytes (Fig. 2). The relatively strong representation of the system A-mediated uptake in neurons in both structures as compared to astrocytes is best illustrated by the stronger pH dependence of the MeAiB-sensitive Gln uptake (Fig. 3), and more pronounced inhibition of the total uptake by ouabain (Fig. 4); the latter two features are the hallmarks of system A (Christensen, 1990; McGivan and Pastor-Anglada, 1994; Sugawara et al., 2000). Accordingly, the uptake of radiolabelled MeAiB showed more than an order of magnitude higher Vmax in neurons from both structures than in their astrocytic counterparts (Table 2). The mRNA for the Gln specific A-system carrier, GlnT/SAT1 was expressed in neurons, but not astrocytes in both structures (Fig. 5A and B), whereas the Ala-preferring system A carrier, SAT2 was expressed in all four types of cells (Fig. 5B). The identity of mouse and rat GlnT mRNA deserves a comment. Comparison of the rat GlnT (GI: 6978015) and mouse NAT2 sequence (GI: 13124896) reveals that only the first 161 bp of Gln do not match NAT2.

3. Results Gln uptake in either of the cell types studied was strongly inhibited by replacement of sodium chloride in the incubation medium with choline chloride or sucrose, pointing to the dominating role of the sodium-dependent systems (Fig. 1). Therefore, further experiments have concentrated on the characterization of the sodium-dependent uptake. In all the cell types, the sodium-dependent Gln uptake showed a one-component kinetics (not shown), and kinetic analysis revealed Vmax of the uptake to be significantly higher in cerebellar astrocytes or neurons than in the same cell types derived from the cerebral cortex (Table 1). Competition experiments revealed the Gln uptake in all the cell types cultured to be sensitive to the ASC system substrates Ala, Ser, Thr and Cys, whereby Thr, which is the most selective ASC substrate of all, showed a tendency to inhibit the uptake to

Fig. 1. l-[3 H]glutamine uptake to the different cell cultures in the presence of sodium chloride (“NaCl”), and with sodium chloride isotonically replaced with choline chloride (CholCl) and sucrose (“sucrose”). Results are mean ± S.D. for 5–7 independent experiments. ∗ P < 0.05 vs. uptake to the same preparation measured in the presence of NaCl.

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Fig. 2. Effects of system ASC (Gln, Thr, Ser, Cys) system ASC/A (Ala) and system A substrates (MeAiB, Pro, Gly) on l-[3 H]glutamine uptake to the different cell cultures in the presence of sodium chloride. Results are mean ± S.D. for 5–7 independent experiments.

Fig. 3. Effect of pH on the system A-mediated (MeAiB-sensitive) l-[3 H]glutamine uptake to the different cell cultures in the presence of sodium chloride. Values represent difference in the uptake measured in the presence or absence of 64-fold excess (5 mM) MeAiB, and are mean ± S.D. for 5–7 independent experiments. ∗ P < 0.05 vs. uptake to the same preparation measured at pH 6.0.

Accordingly, both PCR primers were designed to obtain the 151 bp product which shows no homology to any so far described mouse sequence. Both rat and mouse RT-PCR rendered a product of an identical size. The products were sequenced and analyzed at NCBI (program blast) and both Table 2 Kinetic parameters of 14 C MeAiB uptake to the different cell cultures measured in the presence of sodium chloride calculated from the Michaelis–Menten plots Vmax (nmole/(min mg protein)) Cerebral cortical astrocytes Cerebellar astrocytes Cerebral cortical neurons Cerebellar granule cells

0.24 0.26 8.06 6.69

± ± ± ±

0.01 0.02 0.25 0.37

Results are mean ± S.D. for 4–6 independent experiments.

Km (mM) 0.53 0.15 0.41 1.06

± ± ± ±

0.09 0.07 0.05 0.18

showed homology to rat solute carrier family 38, member 1 (GI: 20301959), rat GlnT (GI: 6978015), mouse solute carrier family 38, member 1 (GI: 20987930), and a number of mouse NAT2 homologues (GI: 26341589; 26325173; 26100836; 26093361). On the basis of the above analysis, we define the mRNA as mouse GlnT mRNA that, like the rat GlnT mRNA described by Varoqui et al. (2000) differs from mouse NAT2 mRNA by being absent in astrocytes (Fig. 5A and B). Expression of ASCT2 mRNA was likewise ubiquitous, albeit in either region it was more pronounced in astrocytes than in neurons (Fig. 5B). Activities of the MeAiB + Thr-insensitive uptake corresponding to system N in the different preparations are compared in Table 3. Except for cortical neurons, where the N-system-mediated uptake amounted to about 36% of the total uptake, in other cells these values were close to 50%. Also in cortical neurons, the uptake was relatively less sensitive to histidine, but more to glutamate than in other cells, a feature resembling system Nb earlier described in rat cortical neurons (Tamarappoo et al., 1997). The mRNA of the N-system carrier, NAT2 showed a similar degree of expression in all the structures.

4. Discussion There is a consensus that active cell membrane transport of glutamine plays an important role in the execution of the Gln–Glu cycle, a pathway which couples Gln production in astrocytes to the synthesis of neurotransmitter glutamate and GABA in neurons. It should be noted that no net synthesis of these amino acids can occur in the neurons from glucose due to the lack of the anaplerotic enzyme, pyruvate carboxylase, which is localized in astrocytes (Yu et al., 1983). Of the two brain structures from which the cell cultures have been isolated, cerebellum is the one relatively enriched in the glutamatergic system. Therefore, the observation that the total Gln uptake activity (Vmax )

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Fig. 4. Effect of 1 mM ouabain on l-[3 H]glutamine uptake to the different cell cultures in the presence of sodium chloride. Results are mean ± S.D. for five independent experiments. ∗ P < 0.05 vs. uptake to the same preparation measured in NaCl.

Fig. 5. (A) Expression of GlnT mRNA in the different cell cultures. CCA: cerebral cortical astrocytes, CA: cerebellar astrocytes, CCN: cerebral cortical neurons, CGC: cerebellar granule cells. Agarose gel electrophoresis of RT-PCR products. Product size of GlnT: 151 bp, M-bp marker. (B) Relative expression of mRNA’s coding for system ASC, A and N carriers. Results are expressed as fraction of GAPDH mRNA and are mean ± S.D. for 4–6 independent experiments. ∗ GlnT was not detected in cerebral cortical astrocytes and cerebellar astrocytes (bars absent).

Table 3 MeAiB + Thr-resistant glutamine uptake to the different cell cultures measured in the presence of sodium chloride and its inhibition by amino acids which are substrates for different N-system variants Cerebral cortical astrocytes MeAiB MeAiB MeAiB MeAiB

+ Thr + His + Gln + Glu

100 47 49 66

(49 ± 1) ±2 ±7 ±5

Cerebellar astrocytes 100 48 44 61

(47 ± 4) ±6 ±8 ±9

Cerebral cortical neurons 100 75 73 39

(36 ± 9) ±5 ±5 ±4

Cerebellar granule cells 100 34 66 59

(49 ± 8) ±5 ±3 ±8

MeAiB + Thr-resistant uptake (row “MeAiB + Thr”) is expressed in parentheses as percent of the total uptake. Uptake in the presence of the competing amino acids is expressed as percentage of MeAiB + Thr resistant uptake set at 100%. Results are mean ± S.D. for 5–6 independent experiments.

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was higher in cerebellar astrocytes or neurons than in their cerebral cortical counterparts is consistent with the role of Gln transport in regulating the glutamatergic system. The regional distribution pattern of Gln uptake activity resembles that previously observed with Glu uptake (Drejer et al., 1983a,b, see Anderson and Swanson, 2000 for a review). Recently a number of amino acid carriers have been cloned which show considerable specificity for Gln and preferential localization in the CNS, including cell type specificity (Broër, 2002). The newly cloned carriers mediate Gln transport that is mostly sodium-dependent and whose characteristics largely conform to the transport systems originally defined as systems A and N. The GlnT/SAT1 system A isoform is preferentially expressed on the plasma membranes of glutamatergic neurons and its mode of action in transfected oocytes suggests that it controls Gln uptake to neurons (Varoqui et al., 2000; Armano et al., 2002). System N isoform SN1 shows mostly astrocytic localization (Boulland et al., 2002), and because it behaves as a Gln-gated Na+ /H+ antiporter, it can mediate either Gln uptake to, or efflux from astrocytes, the direction of transport depending on the transmembrane pH gradient (Chaudhry et al., 1999; Broër et al., 2002). NAT2 is a mouse specific isoform of system N located exclusively in retina and brain. Although the characteristics of NAT2-mediated transport in a transfected cell line largely overlaps with those of SN1, in the retina it has been localized in ganglion cells and axons, whereas its cellular location in the brain has not been reported (Gu et al., 2001). The experimental strategy employed to characterize the newly cloned Gln transporters included RT-PCR analysis of mRNA expression and/or immunochemical localization of a protein in the native tissue, combined with Gln transport characteristics in oocytes or cell lines transfected with the respective transporter cDNA’s. We were interested to see whether and to what degree the major Gln transport characteristics are reflected in cultured astrocytes and neurons derived from different brain regions and how do these characteristics are correlated with the expression of the different transporter isoforms. The cell culture model more closely reflects the natural intracellular milieu than do transfected cells of different origin. This strategy has earlier been successfully used in studies that have led to detailed characterization of cell type- and brain region-specific aspects of cell membrane transport of the neurotransmitter amino acids Glu and GABA (Drejer et al., 1983a,b). The most unambiguous finding of the study was that in the cerebral cortex and cerebellum alike, system A-mediated Gln uptake was very active in neurons, showing at least an order of magnitude lower activity in astrocytes. As opposed to astrocytes, Gln uptake in neurons was inhibited by MeAiB, Pro and Gly (Fig. 2), which are system A substrates (Christensen, 1990; McGivan and Pastor-Anglada, 1994; Sugawara et al., 2000). The MeAiB-sensitive component of neuronal Gln uptake was not only more active than astrocytic uptake, but also strongly pH dependent (Fig. 3).

These observations, supported by the exclusive expression of SAT1 mRNA in neurons, are fully consistent with the view that a brain-specific system A isoform is a major mediator of Gln uptake in neurons. The uptake of the model system A substrate, MeAiB, was 30–40 times more active in neurons than in astrocytes from both brain regions, which is likewise consistent with the neuron-specific functioning of system A. However, the difference in the MeAiB uptake activity between astrocytes and neurons appears to be contradictory to the uniformly strong expression in both cell types of SAT2 mRNA, a system A isoform which prefers Ala to Gln as a substrate, but actively transports MeAiB in a transfected cell line (Yao et al., 2000). This indicates that SAT2, though actively expressed, may not be functional in cultured astrocytes. It is difficult to tell whether the incompatibility of the absence of SAT2 function with active expression of its mRNA reflects the situation occurring in the brain in situ or is due to dedifferentiation of native astrocytes in culture. Experiments using astrocytes acutely isolated from the brain may help to clarify this point. Of note, several observations point to crucial differences between native and cultured astrocytes with regard to the expression and function of different neurotransmitter receptors in astrocytes (reviewed by Kimelberg et al., 2000), and culturing conditions were found to affect the expression of glutamate transporters (Gegelashvili et al., 1997). System N-mediated Gln transport is functionally distinguished as the MeAiB + Thr-inhibitable portion of sodium-dependent transport (Tamarappoo et al., 1997, and references therein). The so defined component of Gln uptake was detected in astrocytes and neurons of both structures. As judged from relatively strong inhibition by Glu and relative resistance to His, the uptake in cerebral cortical neurons, but not in any of the remaining three cell types, resembled the Nb variant described in rat cerebral cortical neurons (Tamarappoo et al., 1997). Of the two N-system isoforms so far described in mouse: NAT (Gu et al., 2000), and NAT2, we focused on the latter as being specific for the brain and retina (Gu et al., 2001). In the retina, NAT2 has been immunocytochemically localized in ganglion cells and axons, but not in glia; its distribution between the cell types in the brain has not been analyzed, however (Gu et al., 2001). The present study suggests that, in contrast to the rat brain, in the mouse brain system N is not exclusively responsible for Gln efflux from astrocytes. ASCT2 is a variant of the ASC system that is strongly expressed in cultured rat astrocytes (Broër et al., 1999) and in a rat astroglioma cell line (Albrecht et al., 2001; Doli´nska et al., 2003), where it mediates Gln efflux in exchange with other amino acids. The present study shows that ASCT2 mRNA is also expressed in cultured mouse astrocytes. The fact that of all the cell types examined the expression was the strongest in astrocytes of the cerebellum, bespeaks the postulated role of ASCT2 in regulating the aspect of the Gln–Glu cycle engaged in replenishing the neurotransmitter pool of Glu.

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Acknowledgements We thank Bente Urfjell for excellent technical assistance. The study was supported by the Blix Foundation, SCSR grant no. 6P05A00321 and by a statutory grant to the Medical Research Centre (project no. 8). References Albrecht, J., Doli´nska, M., Dybel, A., Hilgier, W., Zieli´nska, M., Zabłocka, B., Bu˙za´nska, L., 2001. Glutamine transport in C6 glioma cells: substrate specificity and modulation in a glutamine deprived culture medium. J. Neurosci. Res. 66, 59–66. Anderson, C.M., Swanson, R.A., 2000. Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 95, 1–14. Armano, S., Coco, S., Bacci, A., Pravettoni, E., Schenk, U., Verderio, C., Varoqui, H., Erickson, J.D., Matteoli, M., 2002. Localization and functional relevance of system A neutral amino acid transporters in cultured hippocampal neurons. J. Biol. Chem. 277, 10467– 10473. Boulland, J., Osen, K.K., Levy, L.M., Danbolt, N.C., Edwards, R.H., Storm-Mathisen, J., Chaudhry, F.A., 2002. Cell-specific expression of the glutamine transporter SN1 suggests differences in dependence on the glutamine cycle. Eur. J. Neurosci. 15, 1615–1631. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Broër, S., 2002. Adaptation of plasma membrane amino acid transport mechanisms to physiological demands. Pflügers Arch. 444, 457– 466. Broër, A., Brookes, N., Ganapathy, V., Dimmer, K.S., Wagner, C.A., Lang, F., Broër, S., 1999. The astroglial ASCT2 amino acid transporter as a mediator of glutamine efflux. J. Neurochem. 73, 2184–2194. Broër, A., Albers, A., Sewatian, I., Edwards, R.E., Chaudhry, F.A., Lang, F., Wagner, C., Broër, S., 2002. Regulation of the glutamine transporter SN1 by extracellular pH and intracellular sodium ions. J. Physiol. 539, 3–14. Chaudhry, F.A., Reimer, R.J., Krizaj, D., Barber, D., Storm-Mathisen, J., Copenhagen, D.R., Edwards, R.H., 1999. Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission. Cell 99, 769–780. Christensen, H.N., 1990. Role of amino acid transport and countertransport in nutrition and metabolism. Physiol. Rev. 70, 3–77. Doli´nska, M., Dybel, A., Albrecht, J., 2000. Glutamine transport in C6 glioma cells. Neurochem. Int. 37, 139–146. Doli´nska, M., Dybel, A., Zabłocka, B., Albrecht, J., 2003. Glutamine transport in C6 glioma cells shows ASCT2 system characteristics, Neurochem. Int. 43, 501–507. Drejer, J., Larsson, O.M., Schousboe, A., 1983a. Characterization of uptake and release processes for d- and l-aspartate in primary cultures of astrocytes and cerebellar granule cells. Neurochem. Res. 8, 231– 243. Drejer, J., Meier, E., Schousboe, A., 1983b. Novel neuron-related regulatory mechanisms for astrocytic glutamate and GABA high affinity uptake. Neurosci. Lett. 30, 301–306.

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Ereci´nska, M., Silver, I.A., 1990. Metabolism and role of glutamate in mammalian brain. Prog. Neurobiol. 35, 245–296. Gegelashvili, G., Schousboe, A., 1998. Cellular distribution and kinetic properties of high-affinity glutamate transporters. Brain Res. Bull. 45, 233–238. Gegelashvili, G., Danbolt, N.C., Schousboe, A., 1997. Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia. J. Neurochem. 69, 2612– 2615. Gu, S., Roderick, L., Camacho, P., Jiang, X., 2000. Identification and characterization of an amino acid transporter expressed differentially in liver. Proc. Nat. Academy Sci. U.S.A. 97, 320–3235. Gu, S., Roderick, L., Camacho, P., Jiang, X., 2001. Characterization of an N-system amino acid transporter expressed in retina and its involvement in glutamine transport. J. Biol. Chem. 276, 24137–24144. Hertz, L., Juurlink, B.H.J., Hertz, E., Fosmark, H., Schousboe, A.S., 1989. Preparation of primary cultures of mouse (rat) astrocytes. In: Shahar, A., DeVellis, J., Vernadakis, A., Haber, B. (Eds.), A Dissection and Tissue Culture Manual for the Nervous System. Alan R. Liss, New York, pp. 105–108. Kimelberg, H.K., Schools, G.P., Cai, Z., Zhou, M., 2000. Freshly isolated astrocyte (FIA) preparations: a useful single cell system for studying astrocyte properties. J. Neurosci. Res. 61, 577–587. McGivan, J., Pastor-Anglada, M., 1994. Regulatory and molecular aspects of mammalian amino acid transport. Biochem. J. 299, 321–334. Nagaraja, T.N., Brookes, N., 1996. Glutamine transport in mouse cerebral astrocytes. J. Neurochem. 66, 1665–1674. Schousboe, A., Svenneby, G., Hertz, L., 1977. Uptake and metabolism of glutamate in astrocytes cultured from dissociated mouse brain hemispheres. J. Neurochem. 29, 999–1005. Schousboe, A., Meier, E., Drejer, J., Hertz, L., 1989. Preparation of primary cultures of mouse (rat) cerebellar granule cells. In: Shahar, A., DeVellis, J., Vernadakis, A., Haber, B. (Eds.), A Dissection and Tissue Culture Manual for the Nervous System. Alan R. Liss, New York, pp. 203–206. Sugawara, M., Nakanishi, T., Fei, Y., Huang, W., Ganapathy, M.E., Leibach, F.H., Ganapathy, V., 2000. Cloning of an amino acid transporter with functional characteristics and tissue expression pattern identical to that of system A. J. Biol. Chem. 275, 16473–16477. Su, T., Campbell, G.W., Oxender, D.L., 1997. Glutamine transport in cerebellar granule cells. Brain Res. 757, 69–78. Schreiber, W., Durre, P., 1999. The glyceraldehyde-3-phosphate dehydrogenase of Clostridium acetobutylicum: isolation and purification of the enzyme, and sequencing and localization of the gap gene within a cluster of other glycolytic genes. Microbiology 145, 1839–1847. Tamarappoo, B.K., Raizada, M.K., Kilberg, M.S., 1997. Identification of a system N-like Na+ -dependent glutamine transport activity in rat brain neurons. J. Neurochem. 68, 954–960. Varoqui, H., Zhu, H., Yao, D., Ming, H., Erickson, J.D., 2000. Cloning and functional identification of a neuronal glutamine transporter. J. Biol. Chem. 275, 4049–4054. Yao, D., Mckenzie, B., Ming, H., Varoqui, H., Zhu, H., Hediger, M.A., Erickson, J.D., 2000. A novel system A isoform mediating Na+ /neutral amino acid cotransport. J. Biol. Chem. 275, 22790–22797. Yu, A.C.H., Drejer, J., Hertz, L., Schousboe, A., 1983. Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. J. Neurochem. 41, 1484–1487.