Exocytotic release of alanine from cultured cerebellar neurons.
Many neurons release a variety of amino acids in response to depolarizing stimuli. Although some of these amino acids, namely, glutamate, aspartate, and gamma-aminobutyric acid (GABA), have been qualified as neurotransmitters, functional roles of the other amino acids including alanine remain obscure. We investigated the mechanism and the origin of alanine release from cultured rat cerebellar cells. High-K(+)-induced depolarization produced a considerable amount (139+/-8 pmol/2 min/dish) of alanine release, comparable to that of glutamate (103+/-7 pmol/2 min/dish). Other depolarizing agents including veratridine or 4-aminopyridine also induced alanine release, suggesting that the major source is excitable neurons, rather than non-excitable glial cells. Depolarization-evoked alanine release was suppressed in the absence of extracellular Ca(2+), and was almost abolished by treating the cells with botulinum type B neurotoxin (BoNT/B), indicating that alanine is released by Ca(2+)-dependent exocytosis of vesicle-associated membrane protein-2 (VAMP-2)-containing vesicles. The properties of alanine release were different from those of glutamate and GABA in several aspects: (a) Depolarization-dependent alanine release appeared as early as 7 days in vitro, much earlier than that of GABA. (b) Fifty microM kainate, which causes selective cell death of GABAergic neurons in the culture, only partially reduced alanine release, whereas it had no effect on glutamate release. (c) Alanine release was not affected by phorbol ester, which enhanced glutamate and GABA release in a kinase-dependent manner. We therefore conclude that alanine release occurs via exocytosis of a pool of synaptic vesicles distinct from those containing glutamate or GABA.
Koga
Takeshi
T
Department of Life Sciences (Biology), Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan.
Kozaki
Shunji
S
Takahashi
Masami
M
D000409:Alanine / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism*; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D018690:Excitatory Amino Acid Agonists; D000409:Alanine