13C enrichment of extracellular neurotransmitter glutamate in rat brain--combined mass spectrometry and NMR studies of neurotransmitter turnover and uptake into glia in vivo.
13C-enrichment analysis of glutamate in the extracellular fluid (GLU(ECF): 2-3 microM) by gas-chromatography/mass-spectrometry (GCMS) was combined with in vivo NMR observation of whole-brain GLU (approximately10 mM) to study neurotransmitter uptake. Brain GLU C5 was 13C-enriched by intravenous [2,5-13C]glucose infusion. GLU(ECF) was collected by microdialysis from the cortico-striatal region of awake rats. The 13C-enrichment of basal dialysate GLU C5 during 0.75-1.25 hr of infusion was 0.263 +/- 0.01, very close to the enrichment of whole-brain GLU C5. The result strongly suggests that dialysate GLU consists predominantly of neurotransmitter GLU. For selective 13C-enrichment of neurotransmitter GLU, the whole-brain 13C-enrichment was followed by [12C]glucose infusion to chase 13C from the small glial GLU pool. This leaves [5-13C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. The uptake of synaptic [5-13C]GLU(ECF) into glia and metabolism to glutamine (GLN) were monitored in vivo by NMR observation of [5-13C,15N]GLN formed during 15NH4Ac infusion. The rate of GLN synthesis, derived from neurotransmitter GLU(ECF) (which provided 80-90% of the substrate) was 6.4 +/- 0.44 micromol/g/hr. Hence, the observed rate represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.
Kanamori
K
K
Magnetic Resonance Spectroscopy Laboratory, Huntington Medical Research Institutes, 660 S. Fair Oaks, Pasadena, CA 91105, USA. mrs@hmri.org
Kondrat
R W
RW
Ross
B D
BD
D000081:Acetamides; D000085:Acetates / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D001923:Brain Chemistry; D002247:Carbon Isotopes / Q000032:analysis / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003998:Dicarboxylic Acids / Q000494:pharmacology; D045604:Extracellular Fluid / Q000737:chemistry / Q000378:metabolism; D005463:Fluoroacetates; D008401:Gas Chromatography-Mass Spectrometry; D005947:Glucose / Q000494:pharmacology; D018698:Glutamic Acid / Q000032:analysis / Q000378:metabolism* / Q000493:pharmacokinetics; D007262:Infusions, Intravenous; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D017551:Microdialysis; D008954:Models, Biological; D009457:Neuroglia / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000032:analysis / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000378:metabolism; D017646:Organosilicon Compounds / Q000737:chemistry; D011759:Pyrrolidines / Q000494:pharmacology; D051381:Rats; D014269:Trifluoroacetic Acid / Q000737:chemistry
D000081:Acetamides; D000085:Acetates; D002247:Carbon Isotopes; D003998:Dicarboxylic Acids; D005463:Fluoroacetates; D018377:Neurotransmitter Agents; D009587:Nitrogen Isotopes; D017646:Organosilicon Compounds; D011759:Pyrrolidines; D018698:Glutamic Acid; C059151:N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide; C083486:pyrrolidine-2,4-dicarboxylic acid; D014269:Trifluoroacetic Acid; D005947:Glucose; C018824:ammonium acetate