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Activity of the lactate–alanine shuttle is independent of glutamate–glutamine cycle activity in cerebellar neuronal–astrocytic cultures
Author(s) -
Bak Lasse K.,
Sickmann Helle M.,
Schousboe Arne,
Waagepetersen Helle S.
Publication year - 2004
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.20319
Subject(s) - glutamine , alanine , glutamate receptor , biochemistry , glutamatergic , biology , deep cerebellar nuclei , glutamic acid , chemistry , amino acid , cerebellum , neuroscience , cerebellar cortex , receptor
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. © 2004 Wiley‐Liss, Inc.