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First direct demonstration of preferential release of citrate from astrocytes using [13C]NMR spectroscopy of cultured neurons and astrocytes
235
Neuroscience Letters, 128 (1991) 235 239 Elsevier Scientific Publishers Ireland Ltd. ADONIS 0304394091003744 NSL 07885
First direct demonstration of preferential release of citrate from astrocytes using [13C]NMR spectroscopy of cultured neurons and astrocytes U. Sonnewald 1,2, N. Westergaard 3, J. Krane l, G. Unsg~rd 2, S.B. Petersen 1 and A. Schousboe 4 tMR-Center, SINTEF, Trondheim (Norway), 2Departmentsof Cancer Research and Neurosurgery, University of Trondheim, Faculty of Medicine, Trondheim (Norway), 3pharmaBiotec Research Center, Department of Biochemistry A, Panum Institute, University of Copenhagen, Copenhagen (Denmark) and 4PharmaBiotec Research Center, Department of Biological Sciences, Royal Danish School of Pharmacy, Copenhagen (Denmark) (Received 11 March 1991; Revised version received 15 April 1991; Accepted 17 April 1991)
Key words: Astrocyte; Neuron; Metabolic interaction; Citrate; NMR spectroscopy Primary cultures of cerebral cortical neurons or astrocytes or the two cell types together (co-cultures) were incubated with [1-~3C]glucose for 20 or 48 h. Subsequently, perchloric acid (PCA) extracts of the cells as well as redissolved lyophilyzed media were subjected to NMR spectroscopy in order to detect ~3C-labeled amino acids (glutamine, glutamate, ~-aminobutyrate (GABA)) and other metabolites (lactate, tricarboxylic acid cycle (TCA) constituents). N M R spectra of PCA extracts of neurons or co-cultures exhibited distinct peaks for glutamate and GABA whereas the PCA extracts of astrocytes and co-cultures showed peaks corresponding to glutamine and glutamate. This pattern is consistent with the neuronal location of the GABA synthesizing enzyme glutamate decarboxylase and the astrocytic localization of the glutamine synthesizing enzyme, glutamine synthetase. NMR spectra of the incubation media showed clearly that ~3C-labeled citrate, alanine and glutamine were synthesized and released from astrocytes since only media from the astrocyte cultures or co-cultures of neurons and astrocytes contained these metabolites in detectable amounts. It may be concluded that astrocytes play an important role supplying neurons with precursors for biosynthesis of glutamate and GABA such as glutamine and TCA cycle constituents. Since among the latter only citrate could be found in significant amounts it may be hypothesized that this may be the quantitatively most important TCA constituent to be released from astrocytes and subsequently utilized by neurons.
Numerous studies of the metabolism of neurotransmitter amino acids have shown that an active interplay between neurons and astrocytes is essential for the continuous replenishment of the neuronal neurotransmitter pools of glutamate and ~-aminobutyrate (GABA) [7, 8, 16, 17, 20]. This notion is based on the demonstration that some of the key enzymes in the metabolic conversions of glucose and glutamine to these amino acids are specifically located in one of the cell types. It has been suggested [7, 8, 19] that tricarboxylic cycle intermediates could play an important role as precursors for biosynthesis of transmitter glutamate and GABA. Due to the fact that pyruvate carboxylase which is essential for de novo synthesis of tricarboxylic acid (TCA) constituents from glucose via pyruvate is exclusively present in astrocytes [21, 24] it appears that astrocytes are crucial for the maintenance of the neuronal pools of TCA constituents. While it has been shown that neurons are able to accuCorrespondence: A. Schousboe, PharmaBiotec Research Center, The Neurobiology Unit, Depatrment of Biological Sciences, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark.
mulate TCA constituents such as ~-ketoglutarate [19] it has not been directly demonstrated that astrocytes produce and release such compounds which subsequently would be available for neurons. Using 13C-labeled glucose and NMR spectroscopy, the present study has demonstrated that astrocytes indeed produce and release [laC]citrate as well as [13C]glutamine to the extracellular environment while neurons do not. Astrocytes were cultured essentially as described by Hertz et al. [9]. Prefrontal cortex was taken from newborn mice. The tissue samples were passed through sterile 80 ¢tm pore size Nitex nylon sieves into a slightly modified Dulbeccos medium [9] containing 20% (v/v) fetal calf serum. After 14 days in culture, the medium was changed to an analogous medium (neuronal medium) containing 25 mM KCI, 7 pM paraaminobenzoic acid, 6 mM glucose and insulin (200 mU/l) supplemented with 10% (v/v) fetal calf serum [5]. This medium was also used for culturing co-cultures (cf. below). The first 14 days of culture the entire culture medium was changed twice a week. After 2 weeks, only half of the culture medium was changed twice a week. In order to maintain a glucose concentration of approximately 6
236
mM, the glucose concentration of fresh medium for these subsequent changes was adjusted to 12 mM. Cerebral cortex neurons were isolated and cultured from prefrontal cortex of 15-day-old mice embryos after mild trypsinization followed by trituration in a DNAase solution containing a trypsin inhibitor from soybeans as described by Hertz et al. [5]. Cells were suspended (3 × 106 cells/ml) in 4 m! neuronal medium containing 10% (v/v) fetal calf serum and plated in poly-L-lysinecoated [18] 60 mm Petri dishes. After 48 h, cytosine ara-
binoside was added to the culture medium to prevent astrocytic proliferation [I 1]. Half of the culture medium was changed twice a week and supplemented with glucose to a final concentration of 6 mM (cf. above). The cells were cultured for 7-8 days. The co-cultures were established essentially as previously described [22] by plating cerebral astrocytes on poly-L-lysine-coated 60 mm Petri dishes. Two weeks later, cerebral cortical neurons were plated on top of the preformed layer of astrocytes. After 48 h, cytosine arabi-
GLU
;LC
GLU
GLU LAC
~C
ASP
~
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GLN
C-2 GLN
,,, [J~J_ 9too
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~ ~, 510
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Fig. 1. [t3C]NMR spectra of PCA extracts of co-cultures of cerebral cortical neurons and astrocytes (top) and cultures of cortical astrocytes (middle) or neurons (bottom) after incubation of the cells for 48 h (top, middle) or 20 h (bottom) in culture media containing 6 mM [l-13C]glucose. The peak at 30.2 ppm (top) is due to a small amount of added acetone-d6. GLC, glucose; LAC, lactate: GLU, glutamate; ASP, aspartate; GABA, 7aminobutyrate; GLN, glutamine; ALA, alanine; SER, serine. For further experimental details, see text.
237
noside (20 pM) was added to the co-cultures. The medium was changed as described for the astrocytes. The astrocytes or co-cultures were cultured for a total of 3 weeks and the neurons for one week at which time the culture medium was exchanged with a corresponding medium containing 6 mM [1-13C]glucose. After incubation for 20 h (neurons) or 48 h (astrocytes and co-cultures), the culture media were collected and lyophilized. Cells were washed twice with ice cold PBS free of magnesium and calcium ions, and placed onto liquid nitrogen until frozen. This was followed by addition of 20/A 7%
(v/v) perchloric acid (PCA). Subsequently, the culture dishes were placed on ice, until the celI-PCA mixture could be scraped off using a teflon scraper and transferred to a centrifuge tube placed on ice. The extracts were centrifuged at 4,000 rpm for 10 min (4°C). The supernatants were neutralized with 9 M KOH and centrifuged at 4,000 rpm for 10 min at 4°C. Extracts corresponding to 10-30 culture dishes were pooled and lyophilized. Proton-decoupled 125 MHz [13C]NMR spectra were obtained on a Bruker WM-500 spectrometer operating
GLC
LAC
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IG L C I
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i SER
9=0
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6=0 PPM
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Fig. 2. [t3C]NMR spectra of lyophilized media collected from the cultures described in Fig. 1, i.e. co-cultures of neurons and astrocytes (top), astrocytes alone (middle) or neurons alone (bottom). Cells were incubated for 48 h (top, middle) or 20 h (bottom) with 6 m M [1J3C]glucose. CIT, citrate. For further details, see text.
238 in the Fourier transform mode with quadrature detection. Spectra were accumulated using a 30 ° tip angle, 24,000 Hz spectral width with 32,768 data points and an aquisition time of 0.688 s and a relaxation delay of 0.5 s. Before Fourier transformation the FID was zero filled to 64 K and a line broadening of 2-5 Hz was used. Spectra were recorded at room temperature. The samples were prepared in D20, pH varying between 8.5 and 8.9. The number of scans for PCA extracts was typically 40,000 and for media 4,000. Chemical shifts are reported relative to tetramethylsilane using the lactate C-3 resonance at 21.3 ppm. Assignments were made by comparing with extensive tabulations of chemical shifts for metabolic intermediates [4] and by recording spectra of glutamine, glutamate, GABA and lactate under similar conditions. Spectra were also obtained of extracts and media without ~3C enrichment and of fresh culture medium. Only few signals of low signal to noise level are visible and they do not interfere with the interpretation of the results. As can be seen in Fig. 1A,C both cultured cerebral cortical neurons and co-cultures of these neurons with cortical astrocytes contained [13C]labeled GABA after incubation with [1J3C]glucose for 20-48 h. In contrast to this, N M R Spectra of PCA extracts of pure cultures of astrocytes (Fig. 1B) did not exhibit any peaks corresponding to GABA. This is consistent with the repeated demonstration that these neuronal cultures consist primarily of GABAergic neurons [3, 10, 25] and that astrocytes do not produce significant amounts of GABA [15, 23]. Analogously, only astrocytes and co-cultures of neurons and astrocytes contained [J3C]glutamine (Fig. IA,B) in keeping with the notion that the enzyme responsible for glutamine synthesis, glutamine synthetase is only present in astrocytes and not in neurons [13]. While the N M R spectra of PCA extracts of the cultures did not exhibit significant peaks corresponding to [13C]labeled TCA constituents, the corresponding spectra of redissolved, lyophilized culture media showed a distinct peak corresponding to [13C]citrate in case of the co-cultures and the astrocytes (Fig. 2A, B). It should be emphasized, however, that media isolated from pure neuronal cultures did not exhibit this [13C]citrate peak (Fig. 1C). This finding underlines the previous suggestions (cf. [7, 19]) that astrocytes which in contrast to neurons express pyruvate carboxylase [21, 24] could play an important role feeding TCA constituents to neurons where these compounds might be used to synthesize neurotransmitter amino acids. The finding that only citrate could be detected in the N M R spectra might indicate that this TCA constituent is the major compound to function as a precursor of the carbon skeleton for glutamate and GABA. It should be noted (Fig. 2A,B) that
the media from the astrocytes and co-cultures of neurons and astrocytes also contained appreciable amounts of [~3C]glutamine indicating that astrocytes may also export this amino acid as previously suggested [7, 8]. In this context it may be of interest that also alanine was found in the media from astrocytes (Fig. 2B) since it has been reported that this amino acid may function as the amino group donor for biosynthesis of transmitter glutamate from ~-ketoglutarate [14]. It may also be noted that media from astrocytes contained much less glucose than the media from neurons. This probably reflects the previous finding that glucose consumption is much higher in astrocytes than in neurons [! 2]. It should be noted that labeling with ~3C was observed in several positions in glutamine, glutamate and GABA (Fig. 1). However, due to the long incubation period an analysis of the exact positions of [13C]labelling in the amino acids cannot give any definite indications concerning the biosynthetic route and the labeling pattern is most likely the result of scrambling in the TCA cycle (cf. [2]). Further studies of the time course of labelling of amino acids and citrate from [1-13C]glucose and other precursors such as acetate may, however, allow conclusions to be made concerning the biosynthetic pathways of glutamate and GABA. Classical compartmentation studies have shown that acetate preferentially labels the small glutamate pool [1] which may represent a mainly gtial compartment (cf. [6]). Therefore this compound may be particularly well suited for studies of this nature. Preliminary experiments in this laboratory have indeed indicated that [13C]acetate is metabolized preferentially in astrocytes. The expert technical assistance of Ms. Bente Jensen is gratefully acknowledged. The work has been supported by grants from NATO (200577), The Danish State Biotechnology Program (1987-90 and 1991-1995), the NOVO and Lundbeck Foundations, and an N T N F Deminex scholarship to U.S. 1 Berl,S. and Clarke, D.D., Compartmentation of amino acid metabolism. In A. Lajtha (Ed.), Handbook of Neurochemistry,Plenum, New York, 1969,pp. 447~472. 2 Brainard, J.R., Kyner, E. and Rosenberg, G.A., t3C nuclear magnetic resonance evidencefor gamma-aminobutyricacid formation via pyruvate carboxylasein rat brain: a metabolic basis for compartmentation, J. Neurochem.,53 (1989) 1285 1292. 3 Drejer, J., Honor& T. and Schousboe,A., Excitatoryamino acidinduced releaseof 3H-GABAfrom cultured mouse cerebral cortex interneurons, J. Neurosci., 7 (1987)2911~2916. 4 Halliday,K.R., Fenoglio-Preiser,C. and Sillerud, L.O., Differentiation of human tumors from nonmalignant tissue by natural-abundance ~3C-NMRspectroscopy,Magn. Res. Med., 7 (1988) 384~411. 5 Hertz, E., Yu, A.C.H., Hertz, L., Juurlink, B.H.J. and Schousboe, A., Preparation of primary cultures of mouse cortical neurons. In A. Shahar, J. De Vellis,A. Vernadakis, and B. Haber (Eds.), A Dis-
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section and Tissue Culture Manual of the Nervous System, Liss, New York, 1989, pp. 105-108. Hertz, L., Functional interactions between neurons and astrocytes. I. Turnover and metabolism of putative amino acid transmitters, Prog. Neurobiol., 13 (1979) 277-323. Hertz, L. and Schousboe, A., Role of astrocytes in compartmentation of amino acid and energy metabolism. In S. Fedoroff and A. Vernadakis (Eds.), Astrocytes, Biochemistry, Physiology, and Pharmacology of Astrocytes, Academic Press, New York, 1986, pp. 179-208. Hertz, L. and Schousboe, A., Metabolism of glutamate and glutamine in neurons and astrocytes in primary cultures. In E. Kvamme (Ed.), Glutamine and Glutamate in Mammals, CRC Press, Boca Raton, FL, 1988, pp. 39-55. Hertz, L., Juurlink, B.H.J., Fosmark, H. and Schousboe, A., Astrocytes in primary culture. In S.E. Pfeiffer (Ed.), Neuroscience Approached Through Cell Culture, CRC Press, Boca Raton, FL, 1982, pp. 175-186. Kuriyama, K. and Ohkuma, S., Development of cerebral cortical GABAergic neurons in vitro. In A. Vernadakis, A. Privat, J.M. Lauder, P.S. Timiras and E. Giacobini (Eds.), Model Systems of Development and Aging of the Nervous System, Martinus Nijhoff, Boston, 1987, pp. 43-56. Larsson, O.M., Drejer, J., Kvamme, E., Svenneby, G., Hertz, L. and Schousboe, A., Ontogenetic development of glutamate and GABA metabolizing enzymes in cultured cerebral cortex interneurons and in cerebral cortex in vivo, Int. J. Dev. Neurosci., 3 (1985) 177-185. Lopes-Cardozo, M., Larsson, O.M. and Schousboe, A., Acetoacetate and glucose as lipid precursors and energy substrates in primary cultures of astrocytes and neurons from mouse cerebral cortex, J. Neurochem., 46 (1986) 773-778. Norenberg, M.D. and Martinez-Hernandez, A., Fine structural localization of glutamine synthetase in astrocytes of rat brain, Brain Res., 161 (1979) 303-310. Peng, L., Schousboe, A. and Hertz, L., Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells, Neurochem. Res., 16 (1991) 29-34. Schousboe, A., Primary cultures of astrocytes from mammalian brain as a tool in neurochemical research, Cell. Mol. Biol., 26 (1980) 505-513.
16 Schousboe, A. and Hertz, L., Regulation of glutamatergic and GABAergic neuronal activity by astroglial ceils. In N.N. Osborne (Ed.), Dale's Principle and Communication between Neurones, Pergamon, Oxford, 1983, pp. 113-141. 17 Schousboe, A., Drejer, J. and Hertz, L., Uptake and release of glutamate and glutamine in neurons and astrocytes in primary cultures. In E. Kvamme (Ed.), Glutamine and Glutamate in Mammals, CRC Press, Boca Raton, FL, 1988, pp. 21-38. 18 Sensenbrenner, M., Maderspack, K., Latzkowitz, L. and Jar6s, G.G., Neuronal cells from chick embryo cerebral hemispheres cultivated on poly-lysine coated surfaces, Dev. Neurosci., 1 (1978) 91101. 19 Shank, R.P. and Campbell, G.LeM. ~t-Ketoglutarate and malate uptake and metabolism by synaptosomes: further evidence for an astrocyte-to-neuron metabolic shuttle, J. Neurochem., 42 (1984) 1153-1161. 20 Shank, R.P. and Aprison, M.H., Glutamate as a neurotransmitter. In E. Kvamme (Ed.), Glutamine and Glutamate in Mammals, CRC Press, Boca Raton, FL, 1988, pp. 3-19. 21 Shank, R.P., Bennett, G.S., Freytag, S.O. and Campbell, G.L., Pyruvate carboxylase: astrocyte-specifie enzyme implicated in the replenishment of amino acid neurotransmitter pools, Brain Res., 329 (1985) 364-367. 22 Westergaard, N., Fosmark, H. and Schousboe, A., Metabolism and release of glutamate in cerebellar granule ceils cocultured with astrocytes from cerebellum or cerebral cortex, J. Neurochem., 56 (1991) 59~i6. 23 Wu, P.H., Durden, D.A. and Hertz, L., Net production of gammaaminobutyric acid in astrocytes in primary cultures determined by a sensitive mass spectrometric method, J. Neurochem., 32 (1979) 379-390. 24 Yu, A.C.H., Drejer, J., Hertz, L. and Schousboe, A., Pyruvate carboxylase activity in primary cultures of astrocytes and neurons, J. Neurochem., 41 (1983) 1484-1487. 25 Yu, A.C.H., Hertz, E. and Hertz, L., Alterations in uptake and release rates for GABA, glutamate and glutamine during biochemical maturation of highly purified cerebral cortical neurons, a GABAergic preparation, J. Neurochem., 42 (I 984) 951-960.
Neuroscience Letters, 128 (1991) 235 239 Elsevier Scientific Publishers Ireland Ltd. ADONIS 0304394091003744 NSL 07885
First direct demonstration of preferential release of citrate from astrocytes using [13C]NMR spectroscopy of cultured neurons and astrocytes U. Sonnewald 1,2, N. Westergaard 3, J. Krane l, G. Unsg~rd 2, S.B. Petersen 1 and A. Schousboe 4 tMR-Center, SINTEF, Trondheim (Norway), 2Departmentsof Cancer Research and Neurosurgery, University of Trondheim, Faculty of Medicine, Trondheim (Norway), 3pharmaBiotec Research Center, Department of Biochemistry A, Panum Institute, University of Copenhagen, Copenhagen (Denmark) and 4PharmaBiotec Research Center, Department of Biological Sciences, Royal Danish School of Pharmacy, Copenhagen (Denmark) (Received 11 March 1991; Revised version received 15 April 1991; Accepted 17 April 1991)
Key words: Astrocyte; Neuron; Metabolic interaction; Citrate; NMR spectroscopy Primary cultures of cerebral cortical neurons or astrocytes or the two cell types together (co-cultures) were incubated with [1-~3C]glucose for 20 or 48 h. Subsequently, perchloric acid (PCA) extracts of the cells as well as redissolved lyophilyzed media were subjected to NMR spectroscopy in order to detect ~3C-labeled amino acids (glutamine, glutamate, ~-aminobutyrate (GABA)) and other metabolites (lactate, tricarboxylic acid cycle (TCA) constituents). N M R spectra of PCA extracts of neurons or co-cultures exhibited distinct peaks for glutamate and GABA whereas the PCA extracts of astrocytes and co-cultures showed peaks corresponding to glutamine and glutamate. This pattern is consistent with the neuronal location of the GABA synthesizing enzyme glutamate decarboxylase and the astrocytic localization of the glutamine synthesizing enzyme, glutamine synthetase. NMR spectra of the incubation media showed clearly that ~3C-labeled citrate, alanine and glutamine were synthesized and released from astrocytes since only media from the astrocyte cultures or co-cultures of neurons and astrocytes contained these metabolites in detectable amounts. It may be concluded that astrocytes play an important role supplying neurons with precursors for biosynthesis of glutamate and GABA such as glutamine and TCA cycle constituents. Since among the latter only citrate could be found in significant amounts it may be hypothesized that this may be the quantitatively most important TCA constituent to be released from astrocytes and subsequently utilized by neurons.
Numerous studies of the metabolism of neurotransmitter amino acids have shown that an active interplay between neurons and astrocytes is essential for the continuous replenishment of the neuronal neurotransmitter pools of glutamate and ~-aminobutyrate (GABA) [7, 8, 16, 17, 20]. This notion is based on the demonstration that some of the key enzymes in the metabolic conversions of glucose and glutamine to these amino acids are specifically located in one of the cell types. It has been suggested [7, 8, 19] that tricarboxylic cycle intermediates could play an important role as precursors for biosynthesis of transmitter glutamate and GABA. Due to the fact that pyruvate carboxylase which is essential for de novo synthesis of tricarboxylic acid (TCA) constituents from glucose via pyruvate is exclusively present in astrocytes [21, 24] it appears that astrocytes are crucial for the maintenance of the neuronal pools of TCA constituents. While it has been shown that neurons are able to accuCorrespondence: A. Schousboe, PharmaBiotec Research Center, The Neurobiology Unit, Depatrment of Biological Sciences, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark.
mulate TCA constituents such as ~-ketoglutarate [19] it has not been directly demonstrated that astrocytes produce and release such compounds which subsequently would be available for neurons. Using 13C-labeled glucose and NMR spectroscopy, the present study has demonstrated that astrocytes indeed produce and release [laC]citrate as well as [13C]glutamine to the extracellular environment while neurons do not. Astrocytes were cultured essentially as described by Hertz et al. [9]. Prefrontal cortex was taken from newborn mice. The tissue samples were passed through sterile 80 ¢tm pore size Nitex nylon sieves into a slightly modified Dulbeccos medium [9] containing 20% (v/v) fetal calf serum. After 14 days in culture, the medium was changed to an analogous medium (neuronal medium) containing 25 mM KCI, 7 pM paraaminobenzoic acid, 6 mM glucose and insulin (200 mU/l) supplemented with 10% (v/v) fetal calf serum [5]. This medium was also used for culturing co-cultures (cf. below). The first 14 days of culture the entire culture medium was changed twice a week. After 2 weeks, only half of the culture medium was changed twice a week. In order to maintain a glucose concentration of approximately 6
236
mM, the glucose concentration of fresh medium for these subsequent changes was adjusted to 12 mM. Cerebral cortex neurons were isolated and cultured from prefrontal cortex of 15-day-old mice embryos after mild trypsinization followed by trituration in a DNAase solution containing a trypsin inhibitor from soybeans as described by Hertz et al. [5]. Cells were suspended (3 × 106 cells/ml) in 4 m! neuronal medium containing 10% (v/v) fetal calf serum and plated in poly-L-lysinecoated [18] 60 mm Petri dishes. After 48 h, cytosine ara-
binoside was added to the culture medium to prevent astrocytic proliferation [I 1]. Half of the culture medium was changed twice a week and supplemented with glucose to a final concentration of 6 mM (cf. above). The cells were cultured for 7-8 days. The co-cultures were established essentially as previously described [22] by plating cerebral astrocytes on poly-L-lysine-coated 60 mm Petri dishes. Two weeks later, cerebral cortical neurons were plated on top of the preformed layer of astrocytes. After 48 h, cytosine arabi-
GLU
;LC
GLU
GLU LAC
~C
ASP
~
C C - 4
GLN
C-2 GLN
,,, [J~J_ 9too
~'0
7O
, 60
PPM
~ ~, 510
L l ,
"
, r
4o
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[ ~o
--
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I 20
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'
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Fig. 1. [t3C]NMR spectra of PCA extracts of co-cultures of cerebral cortical neurons and astrocytes (top) and cultures of cortical astrocytes (middle) or neurons (bottom) after incubation of the cells for 48 h (top, middle) or 20 h (bottom) in culture media containing 6 mM [l-13C]glucose. The peak at 30.2 ppm (top) is due to a small amount of added acetone-d6. GLC, glucose; LAC, lactate: GLU, glutamate; ASP, aspartate; GABA, 7aminobutyrate; GLN, glutamine; ALA, alanine; SER, serine. For further experimental details, see text.
237
noside (20 pM) was added to the co-cultures. The medium was changed as described for the astrocytes. The astrocytes or co-cultures were cultured for a total of 3 weeks and the neurons for one week at which time the culture medium was exchanged with a corresponding medium containing 6 mM [1-13C]glucose. After incubation for 20 h (neurons) or 48 h (astrocytes and co-cultures), the culture media were collected and lyophilized. Cells were washed twice with ice cold PBS free of magnesium and calcium ions, and placed onto liquid nitrogen until frozen. This was followed by addition of 20/A 7%
(v/v) perchloric acid (PCA). Subsequently, the culture dishes were placed on ice, until the celI-PCA mixture could be scraped off using a teflon scraper and transferred to a centrifuge tube placed on ice. The extracts were centrifuged at 4,000 rpm for 10 min (4°C). The supernatants were neutralized with 9 M KOH and centrifuged at 4,000 rpm for 10 min at 4°C. Extracts corresponding to 10-30 culture dishes were pooled and lyophilized. Proton-decoupled 125 MHz [13C]NMR spectra were obtained on a Bruker WM-500 spectrometer operating
GLC
LAC
I.~C
IG L C I
GI.~
i SER
9=0
a'O
70
6=0 PPM
CIT
50
4'0
3'0
2'0
Fig. 2. [t3C]NMR spectra of lyophilized media collected from the cultures described in Fig. 1, i.e. co-cultures of neurons and astrocytes (top), astrocytes alone (middle) or neurons alone (bottom). Cells were incubated for 48 h (top, middle) or 20 h (bottom) with 6 m M [1J3C]glucose. CIT, citrate. For further details, see text.
238 in the Fourier transform mode with quadrature detection. Spectra were accumulated using a 30 ° tip angle, 24,000 Hz spectral width with 32,768 data points and an aquisition time of 0.688 s and a relaxation delay of 0.5 s. Before Fourier transformation the FID was zero filled to 64 K and a line broadening of 2-5 Hz was used. Spectra were recorded at room temperature. The samples were prepared in D20, pH varying between 8.5 and 8.9. The number of scans for PCA extracts was typically 40,000 and for media 4,000. Chemical shifts are reported relative to tetramethylsilane using the lactate C-3 resonance at 21.3 ppm. Assignments were made by comparing with extensive tabulations of chemical shifts for metabolic intermediates [4] and by recording spectra of glutamine, glutamate, GABA and lactate under similar conditions. Spectra were also obtained of extracts and media without ~3C enrichment and of fresh culture medium. Only few signals of low signal to noise level are visible and they do not interfere with the interpretation of the results. As can be seen in Fig. 1A,C both cultured cerebral cortical neurons and co-cultures of these neurons with cortical astrocytes contained [13C]labeled GABA after incubation with [1J3C]glucose for 20-48 h. In contrast to this, N M R Spectra of PCA extracts of pure cultures of astrocytes (Fig. 1B) did not exhibit any peaks corresponding to GABA. This is consistent with the repeated demonstration that these neuronal cultures consist primarily of GABAergic neurons [3, 10, 25] and that astrocytes do not produce significant amounts of GABA [15, 23]. Analogously, only astrocytes and co-cultures of neurons and astrocytes contained [J3C]glutamine (Fig. IA,B) in keeping with the notion that the enzyme responsible for glutamine synthesis, glutamine synthetase is only present in astrocytes and not in neurons [13]. While the N M R spectra of PCA extracts of the cultures did not exhibit significant peaks corresponding to [13C]labeled TCA constituents, the corresponding spectra of redissolved, lyophilized culture media showed a distinct peak corresponding to [13C]citrate in case of the co-cultures and the astrocytes (Fig. 2A, B). It should be emphasized, however, that media isolated from pure neuronal cultures did not exhibit this [13C]citrate peak (Fig. 1C). This finding underlines the previous suggestions (cf. [7, 19]) that astrocytes which in contrast to neurons express pyruvate carboxylase [21, 24] could play an important role feeding TCA constituents to neurons where these compounds might be used to synthesize neurotransmitter amino acids. The finding that only citrate could be detected in the N M R spectra might indicate that this TCA constituent is the major compound to function as a precursor of the carbon skeleton for glutamate and GABA. It should be noted (Fig. 2A,B) that
the media from the astrocytes and co-cultures of neurons and astrocytes also contained appreciable amounts of [~3C]glutamine indicating that astrocytes may also export this amino acid as previously suggested [7, 8]. In this context it may be of interest that also alanine was found in the media from astrocytes (Fig. 2B) since it has been reported that this amino acid may function as the amino group donor for biosynthesis of transmitter glutamate from ~-ketoglutarate [14]. It may also be noted that media from astrocytes contained much less glucose than the media from neurons. This probably reflects the previous finding that glucose consumption is much higher in astrocytes than in neurons [! 2]. It should be noted that labeling with ~3C was observed in several positions in glutamine, glutamate and GABA (Fig. 1). However, due to the long incubation period an analysis of the exact positions of [13C]labelling in the amino acids cannot give any definite indications concerning the biosynthetic route and the labeling pattern is most likely the result of scrambling in the TCA cycle (cf. [2]). Further studies of the time course of labelling of amino acids and citrate from [1-13C]glucose and other precursors such as acetate may, however, allow conclusions to be made concerning the biosynthetic pathways of glutamate and GABA. Classical compartmentation studies have shown that acetate preferentially labels the small glutamate pool [1] which may represent a mainly gtial compartment (cf. [6]). Therefore this compound may be particularly well suited for studies of this nature. Preliminary experiments in this laboratory have indeed indicated that [13C]acetate is metabolized preferentially in astrocytes. The expert technical assistance of Ms. Bente Jensen is gratefully acknowledged. The work has been supported by grants from NATO (200577), The Danish State Biotechnology Program (1987-90 and 1991-1995), the NOVO and Lundbeck Foundations, and an N T N F Deminex scholarship to U.S. 1 Berl,S. and Clarke, D.D., Compartmentation of amino acid metabolism. In A. Lajtha (Ed.), Handbook of Neurochemistry,Plenum, New York, 1969,pp. 447~472. 2 Brainard, J.R., Kyner, E. and Rosenberg, G.A., t3C nuclear magnetic resonance evidencefor gamma-aminobutyricacid formation via pyruvate carboxylasein rat brain: a metabolic basis for compartmentation, J. Neurochem.,53 (1989) 1285 1292. 3 Drejer, J., Honor& T. and Schousboe,A., Excitatoryamino acidinduced releaseof 3H-GABAfrom cultured mouse cerebral cortex interneurons, J. Neurosci., 7 (1987)2911~2916. 4 Halliday,K.R., Fenoglio-Preiser,C. and Sillerud, L.O., Differentiation of human tumors from nonmalignant tissue by natural-abundance ~3C-NMRspectroscopy,Magn. Res. Med., 7 (1988) 384~411. 5 Hertz, E., Yu, A.C.H., Hertz, L., Juurlink, B.H.J. and Schousboe, A., Preparation of primary cultures of mouse cortical neurons. In A. Shahar, J. De Vellis,A. Vernadakis, and B. Haber (Eds.), A Dis-
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