In this multinuclear NMR study myo-inositol is identified as a glia-specific marker for in vivo NMR studies. The unusually high inositol concentration may participate in the osmoregulatory system in asupcytes. Primary astrocytes also synthesize and export high amounts of hypotaurine, an intermediate of taurine synthesis. Taurine – another osmolyte – is synthesized from cysteine by astrocytes but not by primary neurons. Taurine as well as hypotaurine is accumulated by neurons from the extracellular medium. ¹³C NMR labelling results with 2-¹³C pyruvate indicate a considerable contribution of the anaplerotic pathway in primary neurons from rat. The activity is only half of the activity in primary astrocytes. The ratio of pyruvate carboxy-lase/malic enzyme activity versus pyruvate dehydrogenase activity reflects the degree of maturation. The ¹³C isotopomer ratio of glutamate and glutamine is different for pure astrocyte cultures. Therefore, the different isotopomer ratios of glutamate to glutamine obtained from intact brain studies alone do not prove TCA cycle compartimentation in the brain. Finally, the PCr/ATP ratio in primary astrocytes is 3 times higher than in primary neurons. This has to be considered in case of recovery from ischemic insults.

Docosahexaenoic acid (DHA) (22:6) is a polyunsaturated fatty acid of the n - 3 series which is believed to be a molecular target for lipid peroxides (LPO) formation. Its ubiquitous nature in the nervous tissue renders it particularly vulnerable to oxidative stress, which is high in brain during normal activity because of high oxygen consumption and generation of reactive oxygen species (ROS). Under steady state conditions potentially harmful ROS and LPO are maintained at low levels due to a strong antioxidant defense mechanism, which involves several enzymes and low molecular weight reducing compounds. The present review emphasizes a paradox: a discrepancy between the expected high oxidability of the DHA molecule due to its high degree of unsaturation and certain experimental results which would indicate no change or even decreased lipid peroxidation when brain tissue is supplied or enriched with DHA. The following is a critical review of the experimental data relating DHA levels in the brain to lipid peroxidation and oxidative damage there. A neuroprotective role for DHA, possibly in association with the vinyl ether (VE) linkage of plasmalogens (pPLs) in combating free radicals is proposed.

Diffusion-weighted in vivo 1H-NMR spectroscopy of F98 glioma cells embedded in basement membrane gel threads showed that the initial cell swelling to about 180% of the original volume induced under hypotonic stress was followed by a regulatory volume decrease to nearly 100% of the control volume in Dulbecco's modified Eagle's medium (DMEM) but only to 130% in Krebs-Henseleit buffer (KHB, containing only glucose as a substrate) after 7 h. The initial cell shrinkage to approx. 70% induced by the hypertonic stress was compensated by a regulatory volume increase which after 7 h reached almost 100% of the control value in KHB and 75% in DMEM. 1H-, 13C- and 31P-NMR spectroscopy of perchloric acid extracts showed that these volume regulatory processes were accompanied by pronounced changes in the content of organic osmolytes. Adaptation of intra- to extracellular osmolarity was preferentially mediated by a decrease in the cytosolic taurine level under hypotonic stress and by an intracellular accumulation of amino acids under hypertonic stress. If these solutes were not available in sufficient quantities (as in KHB), the osmolarity of the cytosol was increasingly modified by biosynthesis of products and intermediates of essential metabolic pathways, such as alanine, glutamate and glycerophosphocholine in addition to ethanolamine. The cellular nucleoside triphosphate level measured by in vivo 31P-NMR spectroscopy indicated that the energy state of the cells was more easily sustained under hypotonic than hypertonic conditions.

Nuclear magnetic resonance (NMR) spectroscopy and biochemical assays were used to study the fate of [3‐13C]alanine in astrocytes, neurons, and cocultures. 1H‐ and 13C‐NMR analysis of the media demonstrated a high and comparable uptake of [3‐13C]alanine by the cells. Thereafter, alanine is transaminated predominantly to [3‐13C]pyruvate, from which the 13C‐label undergoes different metabolic pathways in astrocytes and neurons: Lactate is almost exclusively synthesized in astrocytes, while in neurons and cocultures labeled neurotransmitter amino acids are formed, i.e., glutamate and γ‐aminobutyric acid (GABA). A considerable contribution of the anaplerotic pathway is observed in cocultures, as concluded from the ratio (C‐2–C‐3)/C‐4 of labeled glutamine. Analysis of the multiplet pattern of glutamate isotopomers indicates carbon scrambling through the TCA cycle and the use of alanine also as energy substrate in neurons. In cocultures, astrocyte‐deduced lactate and unlabeled exogenous carbon substrates contribute to glutamate synthesis and dilute the [2‐13C]acetyl‐CoA pool by 30%. The coupling of neuronal activity with shuttling of tricarboxylic acid (TCA) cycle‐derived metabolites between astrocytes and neurons is concluded from the use of [4‐13C]‐monolabeled glutamate leaving the first TCA cycle turn already for glutamine and GABA synthesis, as well as from the labeling pattern of extracellular glutamine. Further evidence of a metabolic interaction between astrocytes and neurons is obtained, as alanine serves as a carbon and nitrogen carrier through the synthesis and regulated release of lactate from astrocytes for use by neurons. Complementary to the glutamine‐glutamate cycle in the brain, a lactate‐alanine shuttle between astrocytes and neurons would account for the nitrogen exchange of the glutamatergic neurotransmitter cycle in mammalian brain. GLIA 32:286–303, 2000. © 2000 Wiley‐Liss, Inc.

The metabolism of [2-13C]glycine in astrogliarich primary cultures obtained from brains of neonatal Wistar rats was investigated using 13c NMR spectroscopy.After a 24-h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2-13C]glycine, cell extracts and incubation media were analyzed for 13C-labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2-13C]glycine, the amount of de novo synthesized [2-13C]creatine was twofold increased, and in addition, 13C-labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2-13C]glycine was utilized for the synthesis of glutathione in astroglial cells. 13Clabeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2~13C] serine, [3-13C] serine, and [2,3-13C] serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons. Key Words: Creatine-Glial metabolism-Glutathione-Glycine-NM R spectroscopy-Serine. Abbreviations used: DMEM, Dulbecco's modified Eagle's medium; GSx, amount of GSH plus twice the amount of GSSG; HSQC, heteronuclear single quantum coherence; MM, minimal medium; SHMT, serine hydroxymethyltransferase.

Abstract:The synthesis of hypotaurine and taurine was investigated in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by 1H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3-13C]cysteinefor 24 or 72 h. Cell extracts and incubation media were then analyzed with 13C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3-13C] cysteine. The presence of [1-13C]hypotaurine and [1-13C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3-13C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3-13C]-lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.

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