Activity of the lactate-alanine shuttle is independent of glutamate-glutamine cycle activity in cerebellar neuronal-astrocytic cultures.
The glutamate-glutamine cycle describes the neuronal release of glutamate into the synaptic cleft, astrocytic uptake, and conversion into glutamine, followed by release for use as a neuronal glutamate precursor. This only explains the fate of the carbon atoms, however, and not that of the ammonia. Recently, a role for alanine has been proposed in transfer of ammonia between glutamatergic neurons and astrocytes, denoted the lactate-alanine shuttle (Waagepetersen et al. [ 2000] J. Neurochem. 75:471-479). The role of alanine in this context has been studied further using cerebellar neuronal cultures and corresponding neuronal-astrocytic cocultures. A superfusion paradigm was used to induce repetitively vesicular glutamate release by N-methyl-D-aspartate (NMDA) in the neurons, allowing the relative activity dependency of the lactate-alanine shuttle to be assessed. [(15)N]Alanine (0.2 mM), [2-(15)N]/[5-(15)N]glutamine (0.25 mM), and [(15)N]ammonia (0.3 mM) were used as precursors and cell extracts were analyzed by mass spectrometry. Labeling from [(15)N]alanine in glutamine, aspartate, and glutamate in cerebellar cocultures was independent of depolarization of the neurons. Employing glutamine with the amino group labeled ([2-(15)N]glutamine) as the precursor, an activity-dependent increase in the labeling of both glutamate and aspartate (but not alanine) was observed in the cerebellar neurons. When the amide group of glutamine was labeled ([5-(15)N]glutamine), no labeling could be detected in the analyzed metabolites. Altogether, the results of this study support the existence of the lactate-alanine shuttle and the associated glutamate-glutamine cycle. No direct coupling of the two shuttles was observed, however, and only the glutamate-glutamine cycle seemed activity dependent.
Bak
Lasse K
LK
Department of Pharmacology, The Danish University of Pharmaceutical Sciences, 2 Universitetsparken, 2100 Copenhagen, Denmark.
Sickmann
Helle M
HM
Schousboe
Arne
A
Waagepetersen
Helle S
HS
D000409:Alanine / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism*; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002952:Citric Acid Cycle; D018920:Coculture Techniques / Q000379:methods; D005110:Extracellular Space / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D014316:Tritium / Q000378:metabolism
D000596:Amino Acids; D005973:Glutamine; D014316:Tritium; D019344:Lactic Acid; D018698:Glutamic Acid; D000641:Ammonia; D000409:Alanine