Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons

Neurochemistry International 47 (2005) 92–102 www.elsevier.com/locate/neuint

Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons Helle S. Waagepetersen a, Hong Qu b, Ursula Sonnewald b, Keiko Shimamoto c, Arne Schousboe a,* a

Department of Pharmacology, The Danish University of Pharmaceutical Sciences, 2 Universitetsparken, DK-2100 Copenhagen, Denmark b Department of Neuroscience, Norwegian University of Science and Technology (NTNU), N-7489 Trondheim, Norway c Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto-cho, Mshima-gun, Osaka 618 8503, Japan Available online 25 May 2005

Abstract Glutamate exists in a vesicular as well as a cytoplasmic pool and is metabolically closely related to the tricarboxylic acid (TCA) cycle. Glutamate released during neuronal activity is most likely to a large extent accumulated by astrocytes surrounding the synapse. A compensatory flux from astrocytes to neurons of suitable precursors is obligatory as neurons are incapable of performing a net synthesis of glutamate from glucose. Glutamine appears to play a major role in this context. Employing cultured cerebellar granule cells, as a model system for glutamatergic neurons, details of the biosynthetic machinery have been investigated during depolarizing conditions inducing vesicular release. [U-13C]Glucose and [U-13C]glutamine were used as labeled precursors for monitoring metabolic pathways by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–mass spectrometry (LC-MS) technologies. To characterize release mechanisms and influence of glutamate transporters on maintenance of homeostasis in the glutamatergic synapse, a quantification was performed by HPLC analysis of the amounts of glutamate and aspartate released in response to depolarization by potassium (55 mM) in the absence and presence of DL-threo-b-benzyloxyaspartate (TBOA) and in response to L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC), a substrate for the glutamate transporter. Based on labeling patterns of glutamate the biosynthesis of the intracellular pool of glutamate from glutamine was found to involve the TCA cycle to a considerable extent (approximately 50%). Due to the mitochondrial localization of PAG this is unlikely only to reflect amino acid exchange via the cytosolic aspartate aminotransferase reaction. The involvement of the TCA cycle was significantly lower in the synthesis of the released vesicular pool of glutamate. However, in the presence of TBOA, inhibiting glutamate uptake, the difference between the intracellular and the vesicular pool with regard to the extent of involvement of the TCA cycle in glutamate synthesis from glutamine was eliminated. Surprisingly, the intracellular pool of glutamate was decreased after repetitive release from the vesicular pool in the presence of TBOA indicating that neuronal reuptake of released glutamate is involved in the maintenance of the neurotransmitter pool and that 0.5 mM glutamine exogenously supplied is inadequate to sustain this pool. # 2005 Elsevier Ltd. All rights reserved. Keywords: Depolarization; Glutamate transport; Threo-b-benzyloxyaspartate; Glutaminase

1. Introduction Abbreviations: DMEM, dulbeccos minimum essential medium; HPLC, high performance liquid chromatography; LC-MS, liquid chromatography mass spectrometry; NMR, nuclear magnetic resonance; PAG, phosphate activated glutaminase; PBS, phosphate buffered saline; TCA, tricarboxylic acid; TBOA, DL-threo-b-benzyloxyaspartate; t-2,4-PDCL, L-trans-pyrrolidine-2,4-dicarboxylate * Corresponding author. Tel.: +45 3530 6330; fax: +45 3530 6021. E-mail address: [email protected] (A. Schousboe). 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.04.012

Neuron-glia interactions are a necessity in the glutamatergic synapse in order to maintain an equilibrated homeostasis of glutamate metabolism. Glutamate neurotransmission is terminated by cellular uptake of the neurotransmitter (Danbolt, 2001). High affinity glutamate transporters localized in the glial cell membrane have been cloned and characterized and have been shown to be of

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major importance in this process (Danbolt, 2001). Due to the lack of quantitatively important anaplerotic enzymes in glutamatergic neurons, these cells are dependent on precursor input from surrounding cells to compensate for the glutamate taken up into the glial compartment (Yu et al., 1983; Shank et al., 1985; Waagepetersen et al., 2001a). It is experimentally well documented that the non-neuroactive amino acid glutamine, which is synthesized exclusively in astrocytes (Norenberg and Martinez-Hernandez, 1979) serves as an important precursor for biosynthesis of neurotransmitter glutamate (Hamberger et al., 1979a,b; Ward et al., 1983; Palaiologos et al., 1988, 1989). The malate–aspartate shuttle has been shown to play a major role in the synthesis of neurotransmitter glutamate (Palaiologos et al., 1988, 1989), which is in agreement with the classical finding that radiolabeled glucose is an extremely efficient substrate for production of labeled glutamate, a process requiring tricarboxylic acid (TCA) cycle activity and a high level of mitochondrial aspartate aminotransferase (AAT) activity (Howse and Duffy, 1975; Ward et al., 1983; Hamberger et al., 1979b). In spite of this, little is known about the specific role of the TCA cycle with regard to the biosynthesis of glutamate from glutamine in the different pools of glutamate, i.e. metabolic cytosolic and the vesicular pool. This question may be of interest since it has recently been reported that the TCA cycle may not be involved to the same extent in the biochemical reactions involved in the biosynthesis of cytoplasmic and vesicular GABA from glutamine (Waagepetersen et al., 2001b). Thus, it has been shown that the TCA cycle plays a more prominent role for the conversion of glutamine to GABA during release from the vesicular pool in comparison to that observed during release from the cytoplasmic pool (Waagepetersen et al., 1999, 2001b). The present study was undertaken in order to obtain analogous information regarding possible differences in the specific biosynthetic pathways involved in the synthesis of glutamate in these two pools. This was achieved using [U-13C]glucose and [U-13C]glutamine as precursors to label glutamate and related metabolites in cultured cerebellar granule cells. In order to study the dynamics of the cytosolic and vesicular glutamate pools repetitive exposure to a depolarizing concentration of potassium was used to induce the selective release of glutamate from the vesicular pool (Bak et al., 2004). It should be kept in mind that using neuronal monocultures the dynamics of glutamate homeostasis is restricted to neuronal capabilities with regard to transporters and metabolic machinery. Detailed analysis of the labeling of carbon atoms in glutamate and other intracellular metabolites with the aid of nuclear magnetic resonance (NMR) spectroscopy provides information about the biosynthetic pathways involved (Sonnewald and Kondziell, 2003). Moreover, metabolic information about the vesicular pool of glutamate was obtained from the depolarizing medium by liquid chromatography-mass spectrometry (LC-MS) analysis which has a higher sensitivity compared to 13C NMR spectroscopy.

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The functionality of a transporter in the glutamatergic nerve terminal has been demonstrated but its existence is still somehow mysterious since the molecular nature and characterization is missing (Gundersen et al., 1993, 1996; Danbolt, 2001). In order to further investigate the functional importance of neuronal uptake of transmitter glutamate for glutamate metabolism and homeostasis in cultured cerebellar granule cells, these cells were depolarized in the absence or presence of DL-threo-b-benzyloxyaspartate (TBOA), a non-transportable blocker of glutamate transporters (Waagepetersen et al., 2001c; Bak et al., 2003). To characterize the release mechanisms and possible influence by the glutamate transporters, a quantification was performed by HPLC analysis of the amounts of glutamate and aspartate released in response to depolarization by potassium (55 mM) in the absence and presence of TBOA or in response to L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4PDC), a substrate for the glutamate transporter.

2. Experimental 2.1. Materials Seven-day-old mice were obtained from the animal facility at Department of Pharmacology, The Danish University of Pharmaceutical Sciences. Plastic tissue culture flasks were purchased from Nunc A/S (Roskilde, Denmark), fetal calf serum from Seralab Ltd. (Sussex, UK) and culture medium from Gibco BRL, Life Technologies A/S (Roskilde, Denmark). Amino acids were from Sigma Chemical Co. (St. Louis, Mo, USA) and penicillin from Leo Pharmaceutical Products Ltd. (Ballerup, Denmark). DL-TBOA was prepared as described previously (Shimamoto et al., 1998). [U-13C]Glutamine and [U-13C]glucose were from Cambridge Isotope Laboratories (Woburn, Mass, USA). [3H]DAspartate (12.8 Ci/mmol) was from Amersham Biosciences (Hørsholm, Denmark). EZ-faast amino acid kit (KH0-7337) was obtained from Phenomenex (CA, USA) and a high performance liquid chromatography (HPLC) column Dynamax (150 mm  4.6 mm, length  i.d.) from Rainin Instr. Co. Inc. (MA, USA). All other chemicals were of the purest grade available from regular commercial sources. 2.2. Cell cultures Cerebellar granule cells were isolated and cultured from cerebellum of 7-day-old mice, after mild trypsinization of the tissue followed by trituration in a DNase solution containing a trypsin inhibitor from soybeans (Schousboe et al., 1989). The cells were suspended at a concentration of 3  106 cells/ml in a modified Dulbecco’s minimum essential medium (Hertz et al., 1982) containing 24.5 mM KCl, 6 mM glucose, 7 mM p-aminobenzoic acid, 50 mM kainic acid and 10% fetal calf serum, and seeded in poly-Dlysine coated flasks (20 ml/80 cm2) or Petri dishes (2 ml/

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35 mm). Kainic acid (50 mM) was present in the culture medium to eliminate GABA release from GABAergic neurons in these cultures (Drejer and Schousboe, 1989). After 48 h in culture, 20 mM (final concentration) cytosine arabinoside was added to the medium to prevent astrocytic proliferation. However, the cultures contain a small metabolically active population of astrocytes as judged from a low activity of glutamine synthetase (Drejer et al., 1985). These cultures, maintained for 7–8 days, constitute a valid experimental model system to study glutamatergic neurons (Drejer and Schousboe, 1989; Damgaard et al., 1996). 2.3. Experiments using [U-13C]glucose and [U-13C]glutamine for NMR and LC-MS analysis The culture medium was removed and the cells were washed twice with 5 ml phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 7.3 mM Na2HPO4, 1.5 mM KH2PO4, 0.9 mM CaCl2, 0.5 mM MgCl2) and incubated for 4 h at 37 8C in 12 ml serum-free DMEM (prepared without glutamine) containing either 2.5 mM [U-13C]glucose plus glutamine (0.5 mM) or 0.5 mM [U-13C]glutamine plus glucose (2.5 mM). After the incubation period, the cells were rinsed in 6 ml PBS/flask for 4 min. Thereafter, three different series of cell cultures were repetitively (8 times) exposed for 1 min periods to PBS containing 5 mM or 55 mM KCl (85 mM NaCl, 55 mM KCl, 0.6 mM MgSO4, 1 mM CaCl2, 2.5 mM glucose, pH 7.4) in either the absence or presence of TBOA (100 mM). The 1 min periods were accompanied by 4 min exposure to PBS containing 5 mM KCl plus glutamine, i.e. the media contained labeled glutamine during the 4 min periods for cultures preincubated in a medium containing [U-13C]glutamine. Glucose was labeled during the entire period of repetitive exposures to PBS containing 5 or 55 mM K+ after incubation in a medium containing [U-13C]glucose. Subsequent to the 1 h period of repetitive exposures, the cells were washed twice with 0.9% (w/v) NaCl and extracted with 70% (v/v) ethanol. Another set of cultures was extracted immediately after the incubation period. The cell extracts were scraped off the flasks and centrifuged at 17,000  g for 20 min to separate the metabolites from the insoluble proteins. The supernatants were lyophilized for subsequent NMR spectroscopy after being redissolved in D2O. Protein was determined in the dissolved pellets (1 M KOH at 20 8C for 24 h) according to Lowry et al. (1951) using bovine serum albumin as the standard. The series of fractions of PBS containing unlabeled glucose and 55 mM K+ in the absence and presence of TBOA were pooled and lyophilized for subsequent analysis by LC-MS to determine percentage of labeling of glutamate. 2.4. Experiments using [3H]D-aspartate After 7 days in culture [3H]D-aspartate (1 mCi; 5 mM, final concentration) was added to one of four cultures of

cerebellar granule cells and incubated for 30 min at 37 8C. The [3H]D-aspartate was present in one of four cultures to be able to track the fractions of glutamate release, since it has been shown that the non-metabolizable amino acid [3H]D-aspartate can be used as a marker of the vesicular as well as cytoplasmic pool of glutamate (Levi et al., 1984; Palaiologos et al., 1989; Belhage et al., 1992; Cousin and Nicholls, 1997; Cousin et al., 1997). After this incubation period, the culture medium of the four cultures was replaced with PBS containing 6 mM glucose and the cultures were placed in a superfusion system (Drejer et al., 1987). The cell monolayer was covered with a nylon mesh (80 mm) and the dishes were superfused (2 ml/min) with PBS for 20 min in order to remove excess radioactivity. Subsequently, the cell cultures were exposed to t-2,4-PDC (100 mM), 55 mM K+ (with an isomolar reduction in Na+) or 55 mM K+ plus TBOA (100 mM) in 1 min pulses with 4 min intervals. The superfusion media were collected in a fraction collector (30 s intervals, 1 ml) and fractions related to the cell culture preincubated with [3H]Daspartate were analyzed using a scintillation counter. The fractions that were associated with phases of release of [3H]D-aspartate above baseline were identified and corresponding fractions of the remaining three cultures were pooled. The pooled fractions of medium were lyophilized and redissolved in a small volume of H2O and analyzed by HPLC (see below) for the total amount of glutamate and aspartate in each phase of release induced by 30 s exposure to t-2,4-PDC, 55 or 55 mM K+ in the presence of TBOA. 2.5.

13

C NMR spectroscopy

Proton decoupled 125.5 MHz 13C NMR spectra were obtained on a Bruker DRX-500 spectrometer. Samples were re-dissolved in D2O containing 0.10% ethyleneglycol as an internal standard. Spectra were accumulated using a 308 pulse angle, 25 kHz spectral width with 64 K data points. The acquisition time was 1.3 s, and a 2.5 s relaxation delay was used. The number of scans was typically 10,000 for each cell extract. Some spectra were also broad band decoupled only during acquisition to avoid nuclear Overhauser effects. From several sets of spectra, factors for this effect were obtained and applied to all spectra. 2.6. LC-MS The lyophilized samples of depolarizing buffer were redissolved in an aliquot of water and derivatized using EZfaast amino acid kit for LC-MS from Phenomenex. The derivatized samples were analyzed on a Shimadzu LC-MS 2010. The percentage of 13C labeling, i.e. single, double, triple labeling, etc., was quantified with regard to glutamate after correction of natural abundance determined in a standard solution of glutamate as described by Biemann (1962).

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2.7. HPLC HPLC was used for quantitative analysis of the amino acids; aspartate, glutamate, glutamine and alanine, in cell extracts and superfusion buffer. Standard solutions of the amino acids were analyzed in every series of samples to perform a standard curve. The analyses were performed using a Varian solvent delivery system 9010, autosampler 9100 and computer system 4.02. The amino acids were precolumn derivatized with o-phthaldialdehyde and subsequently separated on a C18 column in accordance with the principles of reverse phase separation and essentially as described by Geddes and Wood (1984). The mobile phase consisted of a phosphate buffer (50 mM, pH = 5.9) (A) and a solution of methanol (98.75%) and tetrahydrofurane (1.25%) (B). The elution started at 70% of phosphate buffer, which for 20 min gradually decreased to 46%, and the amino acids were eluted within 25 min. The separated derivatized amino acids were detected using a fluorescence detector (Jasco 820 FP).

3. Results Fig. 1 shows typical spectra of cerebellar granule neurons incubated in medium containing [U-13C]glucose in the presence of unlabeled glutamine (Fig. 1A) or [U-13C]glutamine in the presence of unlabeled glucose (Fig. 1B) and subsequently repetitively exposed to medium containing 55 mM K+ plus [U-13C]glucose and [U-13C]glutamine, respectively. Labeling in glutamate and aspartate is evident. The labeling patterns of these two amino acids were clearly different depending upon the precursor. In addition, labeled

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lactate and alanine are seen in the spectra of cells exposed to [U-13C]glucose (Fig. 1A). A low labeling in glutamine (Fig. 1A) was observed probably reflecting the presence in the cultures of a small but metabolically active population of astrocytes (see Section 2.2). Fig. 2 shows schematically the distribution of label from [U-13C]glucose (Fig. 2A) and [U-13C]glutamine (Fig. 2B) into glutamate and aspartate. [U-13C]glucose is metabolized via glycolysis to [U-13C]pyruvate. [1,2-13C]Acetyl CoA is subsequently formed via the pyruvate dehydrogenase complex and enters the TCA cycle via condensation with unlabeled oxaloacetate. After two steps in the TCA cycle [4,5-13C]a-ketoglutarate can be converted to [4,5-13C]glutamate either by reductive amination or transamination. Due to the symmetrical succinate molecule, the 13C label is either in C-1 and C-2 or C-3 and C4 position of oxaloacetate and subsequently in aspartate as indicated in Fig. 2A. The double labeling from the first turn of the TCA cycle is seen as large doublets particularly in C-4 of glutamate but also in C-2/C-3 of aspartate in the NMR spectra (Fig. 1A). The portion of [1,2/3,4-13C]oxaloacetate not transaminated to aspartate may be condensed with another molecule of [1,2-13C]acetyl CoA. This can give rise to [1,2,4,5]- or [3,4,5-13C]glutamate, respectively. [3,4,5-13C]Glutamate is observed as a doublet in the C-3 position of the NMR spectrum. In the subsequent turn of the TCA cycle after repeated metabolism of [1,2-13C]acetyl CoA [1,2,4,5/1,3,4,5/1,2,3,4,5/2,3,4,5-13C]glutamate isotopomers are formed (for details see Waagepetersen et al., 1998a). This can to a certain extent be quantified in the NMR spectrum by the triplet observed in the C-3 position. On the basis of these quantifications, a cycling ratio was calculated, i.e. the total amount of labeling in C-3 divided by the amount of C-4 doublet representing the cycling of intermediates in

Fig. 1. 13C NMR spectrum of metabolites from cell extracts of cultured cerebellar granule neurons (see Section 2) incubated (4 h) and subsequently repetitively exposed (1 h) to medium containing [U-13C]glucose (A) or [U-13C]glutamine (B) (for details see Section 2). 1, lactate C-3; 2, glutamate C-3; 3, glutamate C-4; 4, aspartate C-3; 5, aspartate C-2; 6, glutamate C-2; 7, glutamine C-3; 8, glutamine C-4.

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Fig. 2. A simplified scheme of metabolism of [U-13C]glucose (A) and [U-13C]glutamine (B), showing the isotopomers which appear when these labeled precursors are metabolized. Glutamine (GLN) is converted to the amino acids aspartate (ASP) and glutamate (GLU). Additionally, the labeling patterns appearing after involvement of TCA cycle metabolism are shown (i.e. condensation of unlabeled acetyl CoA in one, two and three turns). The inter conversions of TCA cycle intermediates and amino acids as well as the reactions involving pyruvate and alanine or lactate are reversible, but due to simplicity the reactions are depicted by one headed arrow showing the primary route for the labeling. TCA, tricarboxylic acid.

the TCA cycle involved in synthesis of glutamate. The values are presented in Table 1. It should be mentioned that the presence of unlabeled glutamine increases the probability of [1,2-13C]acetyl CoA condensing with an unlabeled molecule of oxaloacetate (see Fig. 2A) thus increasing the synthesis of [4,5-13C]glutamate. In the case of using [U-13C]glutamine as the labeled precursor [U-13C]glutamate is formed after deamidation by phosphate-activated glutaminase (PAG). Glutamate may subsequently enter the TCA cycle via a-ketoglutarate as schematically presented in Fig. 2B. [U-13C]oxaloacetate will be generated after several reactions and transamination forms [U-13C]aspartate. Alternatively [U-13C]oxaloacetate condenses with unlabeled acetyl CoA which eventually leads to the formation of

[1,2,3-13C]glutamate and [1,2/3,4-13C]aspartate (Fig. 2B). For quantification of the extent to which the glutamine carbon skeleton was metabolized in the TCA cycle prior to synthesis of glutamate, the amount of doublet in C-3 glutamate was divided by the triplet of C-3 and the values are presented in Table 2. The cellular contents of glutamate, aspartate, alanine and glutamine of cells which were incubated for 4 h in medium containing glucose (2.5 mM) plus glutamine (0.5 mM) as well as of cells that were subsequently repetitively exposed to medium containing 5, 55, or 55 mM K+ plus TBOA are presented in Table 3. The contents of all amino acids measured were higher in cells repetitively exposed to medium containing 5 and 55 mM K+ compared to cells

Table 1 13 C labeling from [U-13C]glucose

Only incubation 5 mM K+ 55 mM K+ 55 mM K+ + TBOA

Glutamate [4,5-13C] (nmol/mg protein)a

Aspartate [3,4-13C] [2,3,4-13C] (nmol/mg protein)b

Lactate [U-13C] (nmol/mg protein)

Alanine [U-13C] (nmol/mg protein)

Cycling of glutamate (ratio)

13  1 17  1 22  1 * 21  2 *

10  1 11  1 12  2 10  0.2

12  1 5  1* 5  1* 6  0.1 *

2  0.1 4  0.5 41 31

1.7  0.2 0.9  0.01* 0.8  0.1* 1.0  0.02*

Amount of 13C labeling in glutamate, aspartate, lactate and alanine in cultured cerebellar granule neurons subsequent to incubation (4 h) in medium containing 0.5 mM glutamine and 2.5 mM [U-13C]glucose (only incubation) and repetitive exposure to medium containing [U-13C]glucose and glutamine plus 5 or 55 mM K+ in absence or presence of TBOA for 1 h. The cultured cells were extracted and analyzed by 13C NMR. The cycling ratio is the total amount of labeling in C-3 divided by the amount of C-4 doublet representing the cycling of intermediates in the TCA cycle involved in synthesis of glutamate. Results are averages  S.E.M. of 3–4 individual cultures. Statistically significant differences (P < 0.05) between the different groups were analyzed by ANOVA and Tukey post hoc test. An asterisk indicates statistically significant difference from ‘only incubation’. a It cannot be excluded that some labeling is present in position one or two. b The numbers includes the isotopomers [3-13C]aspartate and [U-13C]aspartate, as well. For details with regard to labeling of aspartate and glutamate from [U-13C]glucose see Waagepetersen et al. (1998a).

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Table 2 13 C labeling from [U-13C]glutamine and percent glutamate synthesis via the TCA cycle

Only incubation 5 mM K+ 55 mM K+ 55 mM K+ + TBOA

Glutamate [U-13C] (nmol/mg protein)

Glutamate [1,2,3-13C] (nmol/mg protein)

Glutamate synthesis via the TCA cycle (%)

Aspartate C-3 total [U-13C] (nmol/mg protein)

41 23  3 43  4*# 24  6*

3  0.4 10  2 19  2*# 12  3 *

66  3 43  4* 44  2* 51  4*

21 13  2* 24  3*# 15  2*

Amount of 13C labeling in glutamate and aspartate in cultured cerebellar granule neurons subsequent to incubation (4 h) in medium containing 0.5 mM [U-13C]glutamine and 2.5 mM glucose (only incubation) and repetitive exposure to medium containing [U-13C]glutamine and glucose plus either 5 or 55 mM K+ in absence or presence of TBOA for 1 h. The cultured cells were extracted and analyzed by 13C NMR. For quantification of the extent of glutamate synthesis from glutamine via the TCA cycle the amount of doublet was divided by the amount of triplet of C-3 glutamate. Results are averages  S.E.M. of 3–4 individual cultures. Statistically significant differences (P < 0.05) between the different groups were analyzed by ANOVA and Tukey post hoc test. An asterisk indicates statistically significant difference from ‘only incubation’. A number sign indicates statistically significant difference from cells repetitively exposed to 5 mM K+ and a currency sign indicates statistically significant difference from cell repetitively exposed to medium containing 55 mM K+.

Table 3 Cellular content of amino acids in cultured cerebellar granule cells

Only incubation 5 mM K+ 55 mM K+ 55 mM K+ + TBOA

Glutamate (nmol/mg protein)

Aspartate (nmol/mg protein)

Glutamine (nmol/mg protein)

Alanine (nmol/mg protein)

77  6 114  9* 124  5* 95  8

30  2 51  4* 55  2* 37  3 #

44  5 72  7 * 78  6 * 64  7

51 12  1 * 9  1* 7  1#

Cellular content (nmol/mg protein) of glutamate, aspartate, glutamine and alanine in cultured cerebellar granule neurons subsequent to incubation (4 h) in medium containing 0.5 mM glutamine and 2.5 mM glucose (only incubation) and repetitive exposure to either 5 or 55 mM K+ in absence or presence of TBOA for 1 h. The cultured cells (see Section 2) were extracted and analyzed by HPLC. Results are averages  S.E.M. of 11–13 individual cultures. Statistically significant differences (P < 0.05) between the different groups were analyzed by ANOVA and Tukey post hoc test. An asterisk indicates statistically significant difference from ‘only incubation’, a number sign indicates statistically significant difference from cells repetitively exposed to 5 mM K+ and a currency sign indicates statistically significant difference from cells repetitively exposed to a medium containing 55 mM K+.

extracted following the incubation period. The cellular content of glutamate was not different comparing the situation after the incubation and subsequent to repetitive exposure to 55 mM K+ in the presence of TBOA. Moreover, this latter value was significantly lower than that obtained after repetitive exposure to 55 mM K+. The same pattern of differences was observed with regard to the cellular content of aspartate. However, the content of aspartate observed subsequent to exposure to 55 mM K+ and TBOA was lower than that obtained in cells exposed to 5 as well as 55 mM K+ in the absence of TBOA. The cellular content of glutamine was not affected by repetitive exposure to 55 mM K+ in the presence of TBOA, while the content of alanine was lower compared to cells repetitively exposed to only 5 mM K+. The cycling ratio of glutamate, the amount of [4,5-13C]glutamate (C-4 doublet) and the total amounts of label in C-3 of aspartate, which is predominantly [3,4-13C] plus [2,3,4-13C], [U-13C]lactate and [U-13C]alanine in cerebellar neurons incubated and repetitively exposed to medium containing [U-13C]glucose and unlabeled glutamine are shown in Table 1. The amount of [4,5-13C]glutamate in cells repetitively exposed to a medium containing 55 mM K+ in the absence and presence of TBOA, was elevated compared to that measured in cells extracted immediately after the incubation. However, the repetitive exposure to 5 mM K+ did not lead to a significant change in the amount of this particular isotopomer. The cycling ratio of

glutamate was decreased in cells repetitively exposed to a medium containing either 5 or 55 mM K+ or 55 mM K+ plus TBOA. Increasing the potassium concentration and addition of TBOA did not have any further effects on the calculated cycling ratios. The amounts of label in C-3 of aspartate and alanine were not affected by any of the treatments. Lactate labeling, on the other hand, was decreased in cells repetitively exposed to a medium compared to cells extracted directly after the incubation period. The amounts of labeling from [U-13C]glutamine into [U-13C]glutamate and [1,2,3-13C]glutamate and the total amount of labeling of aspartate, including [U-13C]aspartate and [3,4-13C]aspartate, are listed in Table 2. Repetitive exposure of the cells to a medium containing 5 mM K+ had no significant effect on the values of [U-13C]glutamate and [1,2,3-13C]glutamate. However, the calculated percentage of glutamate synthesis via the TCA cycle was decreased by approximately 30%. [U-13C]Glutamate formation as calculated from the amount of label in the triplet of C-3 was increased in the cells after repetitive exposure to 55 mM K+ as was the doublet in C-3 of glutamate. The presence of TBOA in addition to 55 mM K+ eliminated these augmentations. In contrast to the differences observed in the individual amounts of glutamate isotopomers by repetitive exposure to 55 mM K+ and 55 mM K+ plus TBOA, no differences were obtained in the percentage synthesis of glutamate via the TCA cycle. The total amount

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Fig. 3. Single, double, triple, quadruple and uniform 13C labeling of glutamate in depolarizing medium containing and 55 mM K+ (open) or 55 mM K+ plus TBOA (filled). Cultured cerebellar granule neurons were incubated (4 h) in medium containing 0.5 mM [U-13C]glutamine and subsequently repetitively exposed to a medium containing and 55 mM K+ in absence or presence of TBOA (100 mM) for 1 h. Results are averages with error bars representing S.E.M. of 4–5 individual cultures. Statistically significant differences between the two groups were analyzed by one way ANOVA, but no differences were observed.

of labeling in C-3 of aspartate was increased by 1 h of repetitive exposure to a medium containing 5 mM K+ compared to cells extracted immediately after the incubation. Exposure to 55 mM K+ further increased the total amount of aspartate labeling. However, the presence of TBOA decreased the amount to a level similar to that obtained after exposure to only 5 mM K+. The cerebellar granule cells were repetitively exposed to medium containing 55 mM K+ in the absence or presence of TBOA. This medium was subsequently analyzed by LC-MS to determine the percentages of glutamate molecules containing single, double, triple, quadruple or uniform labeling (Fig. 3). No significant differences in the labeling of glutamate were observed between depolarizing medium containing 55 mM K+ in the absence and presence of TBOA. On the basis of the values obtained for triple and uniform labeling (see Fig. 2B), a percentage synthesis of glutamate via the TCA cycle was calculated, which is comparable to the values obtained from the corresponding cell extracts using 13C NMR (Table 2). These values were for the depolarizing medium containing 55 mM K+ and 55 mM K+ plus TBOA (25  4) and (37  6)%, respectively. These values were tested statistically by one way ANOVA towards the values obtained from the corresponding extracts. The intracellular pool of glutamate of cells repetitively exposed to medium containing 55 mM K+ had a significantly (P = 0.008) higher percentage synthesis (44  2)% via the TCA cycle compared to the pool of glutamate which had been released. The presence of TBOA erased this difference between the labeling pattern of the intracellular pool of glutamate and the one analyzed in the medium.

Fig. 4. Quantification of percentage release above baseline (at 0%) of glutamate (filled) and aspartate (open) from cultures of cerebellar granule cells (see Section 2) evoked by 30 s stimulation pulses of 55 mM K+, 55 mM K+ plus TBOA (100 mM) or t-2,4-PDC (100 mM) (see Section 2). Results are averages of 5–23 stimulations with error bars representing S.E.M. Statistically significant differences (P < 0.05) between the different groups were analyzed by ANOVA and Tukey post hoc test. Statistically significant differences between the release of glutamate and aspartate are indicated by asterisks. The currency sign indicates statistically significant difference from 55 mM K+ and the number sign significant differences from 55 mM K+ plus TBOA.

The magnitude of glutamate and aspartate release induced by 55 mM K+, 55 mM K+ plus TBOA and t-2,4-PDC, respectively, for 30 s are presented in Fig. 4. Glutamate release was approximately 260% above baseline following depolarization by 55 mM K+. This value was considerably higher in the presence of TBOA. In contrast, with regard to aspartate the release was almost negligible compared to the baseline level and it was not increased by the presence of TBOA. During exposure to t-2,4-PDC, a substrate for the glutamate carriers, glutamate and aspartate were released to a similar extent and the level was twice that found for glutamate after a depolarization induced by 55 mM K+.

4. Discussion Glutamate release has been studied in a number of preparations ranging from in vivo, brain slices, synaptosomes to cultured neurons (Hamberger et al., 1979a,b; Ward et al., 1983; Palaiologos et al., 1988, 1989; Jabaudon et al., 1999; Petroff et al., 2002; Sherman, 1991). In the present study employing NMR spectroscopy and LC-MS, a preparation of cultured cerebellar glutamatergic neurons has been used to elucidate changes in metabolic events during neuronal depolarization and glutamate release. Depolarizing the cells using 55 mM K+ has previously been shown to represent release of glutamate from a vesicular pool (Bak et al., 2003, 2004). By including the glutamate transport inhibitor TBOA in the depolarizing medium, reuptake of the released glutamate by the transporters is abolished (Bak et al., 2003) thus eliminating the influence of this parameter on glutamate metabolism. It should be mentioned that although neuronal glutamate uptake seems to be of minor importance in vivo compared to

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the astroglial contribution in the clearance of glutamate from the synaptic cleft, the Km and Vmax for cultured cerebellar granule cells are at the same level as that found in primary cultures of astrocytes (Drejer et al., 1982; Danbolt, 2001). This is in keeping with the dramatic increase in the extracellular amount of glutamate in the presence of TBOA observed in the present study. It should be noted that the release of aspartate induced by 55 mM K+ was not significantly above baseline, indicating that this depolarizing stimulus is not sufficient to induce release via the electrogenic glutamate transporters. This might be explained by compensatory changes in intracellular [Na+] in the presence of a high extracellular potassium concentration preventing reversal of the electrogenic carrier as suggested by Longuemare et al. (1999). Moreover, it is compatible with the observation that the presence of TBOA, a nontransportable glutamate transport inhibitor (Waagepetersen et al., 2001c), did not affect the release of aspartate. The presence of functional transporters is substantiated by the finding that t-2,4-PDC stimulated the release of glutamate and aspartate by heteroexchange. It may be of interest that while t-2,4-PDC had a similar stimulatory effect on glutamate and aspartate release, the cellular content of glutamate was considerably higher than that of aspartate. Thus, a larger fraction of the cellular glutamate pool represents a non-cytosolic pool, most likely predominantly mitochondrial and vesicular, not available for carrier mediated exchange. This aspect might be of interest in relation to glutamate release mediated by reversal of the carrier as an essential part of the pathogenesis of ischemia (Seki et al., 1999; Phillis et al., 2000; Rossi et al., 2000; Gegelashvili et al., 2001; Bonde et al., 2003). Glutamine is efficiently taken up primarily by the system A transporter in cultured cerebellar granule cells (Su et al., 1997; Dolinska et al., 2004). The observed increase in the four amino acids determined in the cells after the period of repetitive exposure to a physiological medium containing 2.5 mM glucose and 0.5 mM glutamine suggests that during the incubation period (4 h) the glutamine content of the medium had decreased. This is confirmed by the low amount of label in glutamate and aspartate after the incubation period in a medium containing [U-13C]glutamine. It is evident that glutamine is efficiently taken up during the period of repetitive exposure to medium containing 0.5 mM glutamine, and subsequently efficiently metabolized to glutamate and aspartate. That this is indeed the case is confirmed by the increase in labeling of these amino acids and in particular aspartate when [U-13C]glutamine was present during the period of repetitive exposure to medium containing 5 or 55 mM K+. Glutamine is deamidated to glutamate by PAG in the inner mitochondrial membrane and matrix (Laake et al., 1999; Kvamme et al., 2001). Glutamate may either be directly available in the cytosol as suggested by results obtained by Kvamme et al. (2001). However, based on other studies (Ziemin´ ska et al., 2004) glutamate formed in the

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PAG-catalyzed reaction may be available for metabolism in the mitochondrial matrix and thus be metabolized to aketoglutarate and other TCA cycle metabolites as shown in Fig. 2A. In the present study these pathways are monitored analyzing the 13C distribution in glutamate and aspartate (Fig. 2). This is possible since glutamate and aspartate are at least in the mitochondrial pool in equilibrium with aketoglutarate and oxaloacetate, respectively (Mason et al., 1995). The finding that approximately 40–50% of the metabolism of glutamine involves TCA cycle reactions, as judged from the abundancy of triple-labeled glutamate, is compatible with the notion that neurotransmitter glutamate biosynthesis in glutamatergic neurons is dependent on AAT activity and the mitochondrial malate-a-ketoglutarate carrier (Palaiologos et al., 1988). It is, however, not in agreement with the proposal that essentially all newly synthesized glutamate generated in the PAG-catalyzed reaction leaves the mitochondria without getting access to the matrix (Kvamme et al., 2001), a notion which may be controversial (Ziemin´ ska et al., 2004). Employing [U-13C]glucose and [U-13C]glutamine in analogous experiments it is possible to follow the metabolic pathways in more detail and get a more complete picture of the overall metabolic events. In the present study, a decrease in the amount of [U-13C]glutamate labeling from [U-13C]glutamine was observed by the presence of TBOA. This is an observation which is not obvious looking at the results gained from cells exposed to [U-13C]glucose, but it is consistent with the fact that when glucose is labeled, glutamine is unlabeled and the decrease of [U-13C]glutamate is therefore not evident in the quantification of [4,5-13C]glutamate from [U-13C]glucose. However, TBOA did not affect the amount of [1,2,3-13C]glutamate generated from [U-13C]glutamine which corresponds to the [4,5-13C]glutamate from [U-13C]glucose where no change was observed either. The incorporation of label from glucose into glutamate was increased during vesicular release, but the incorporation into lactate was not affected by changing to depolarizing conditions. This would indicate a stimulation of aerobic glycolysis and TCA cycle activity as previously reported for both glutamatergic and GABAergic neurons (Peng and Hertz, 1993; Waagepetersen et al., 2000). It should be noted that aspartate labeling after the repetitive exposure to a physiological medium containing [U-13C]glutamine was dramatically increased. Moreover, the cellular content of aspartate was substantially increased by glutamine, indicating a net synthesis of aspartate via a ‘truncated TCA cycle’ (Hertz et al., 1992). That the carbon skeleton of glutamine to a considerable extent enters the TCA cycle and serves as a precursor for oxaloacetate is supported by the decreased cycling ratio seen for glutamate when glucose was used as the labeled substrate, analogous to what has been reported for GABAergic neurons (Waagepetersen et al., 1998b, 2001b). The underlying explanation for this ‘truncated TCA cycle’ is at present not understood.

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After repetitive exposure to a depolarizing concentration of potassium (55 mM) the labeling of glutamate was increased indicating an increase in its synthesis from glutamine. This is also confirmed by the finding that conversion of glutamine to glutamate to a large extent involved the TCA cycle regardless of the composition of the medium during the repetitive superfusion. Moreover, that the release via the vesicular pool (55 mM K) did not impose a significant change in the capacity for synthesis of glutamate via any of the two possible pathways (Fig. 2). However, the labeling pattern of released vesicular glutamate collected during a 1 h period of repetitive stimulations indicates that the synthesis of vesicular glutamate from glutamine involves TCA cycle metabolism to a somewhat lesser extent than the synthesis of the remaining intracellular pool of glutamate. When TBOA was included in the depolarizing medium, thereby preventing reuptake of released glutamate leading to a three-fold increase in its release, there was a considerable decrease in the cellular content of glutamate and its labeling via direct synthesis from glutamine. This likely indicates that reuptake of glutamate may play an important functional role for the vesicular neurotransmitter pool. Moreover, the involvement of the TCA cycle in the synthesis of the vesicular and the remaining intracellular pool of glutamate was not significantly different in the case where reuptake was blocked by TBOA. Thus, the possibility for recycling of glutamate as a neurotransmitter decreases the involvement of the TCA cycle in the conversion of glutamine to glutamate. The present study supports previous investigations suggesting that conversion of glutamine to glutamate plays a major role in the maintenance of the pool of neurotransmitter glutamate (Hamberger et al., 1979a,b; Ward et al., 1983). However, the present findings show that glutamine is not sufficient for maintaining the neurotransmitter pool of glutamate in the case reuptake is blocked. This may be surprising in light of the fact that the activity of PAG in cerebellar granule neurons more than adequately accounts for the demand of de novo glutamate synthesis (Drejer et al., 1985). However, it should be noted that since PAG is highly regulated (Kvamme et al., 2001), it is likely that the maximal activity may not reflect the activity in situ in the cells. It could also be argued that the external glutamine concentration is insufficient to maintain the intracellular glutamine concentration. However, it was recently shown that the glutamine concentration in the extracellular fluid of the corticostriatal region of awake rats is approximately 0.4 mM and that of the human cerebrospinal fluid is 0.5 mM (Kanamori and Ross, 2004; Garseth et al., 2000). Thus, even though the glutamine concentration (0.5 mM) used in the present experimental procedure is only half of the Km value of glutamine uptake into cultured cerebellar granule cells, the concentration is comparable to the in vivo situation (Dolinska et al., 2004). The lack of an astrocytic component in the present experimental system could be of importance in this context. In glutamatergic synapses glutamate reentry

into the neurotransmitter pool is likely to be prevented by uptake into surrounding astrocytes to some extent mimicking the present situation when TBOA is included in the superfusion media. However, it is possible that the synaptic spatial organization in vivo with astrocytes in close proximity to the nerve endings may facilitate transfer of glutamine from astrocytes to the nerve endings. Such a highly efficient glutamine transfer may be able to compensate for the drain on the glutamate pool caused by astrocytic glutamate uptake. Direct experimental proof of this is presently unavailable but further experimental studies might elucidate this question. Co-cultures of neurons and astrocytes from cerebellum may provide information about the role of glutamine produced by astrocytes located in the immediate proximity to glutamatergic neurons.

Acknowledgements The expert technical assistance by Lone Rosenquist and Kirsten Thuesen and the expert secretarial assistance by Hanne Danø are cordially acknowledged. This study was supported by grants from The Danish State Medical Research Council (22-00-0503, 22-00-1011 and 22-030250), the Novo Nordisk, the Hørslev, the Norwegian Epilepsy and the Lundbeck Foundations.

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