Characterization of depolarization-coupled release of glutamate from cultured mouse cerebellar granule cells using DL-threo-beta-benzyloxyaspartate (DL-TBOA) to distinguish between the vesicular and cytoplasmic pools.
Release of preloaded [3H]D-aspartate in response to depolarization induced by N-methyl-D-aspartate (NMDA) or the endogenous agonist glutamate was characterized using cultured glutamatergic cerebellar granule neurons. Release from the vesicular and the cytoplasmic glutamate pools, respectively, was distinguished employing the competitive, non-transportable glutamate transport inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA). NMDA (300 microM)-induced release was enhanced (50%) by a simultaneous elevation of the extracellular potassium concentration to 15 mM, which lifts the voltage-dependent magnesium block of the NMDA receptors. This NMDA/K(+)-induced release was not sensitive to DL-TBOA (100 microM) but was inhibited by 75% in the presence of the unspecific calcium channel antagonist La(3+) (100 microM). Glutamate (100 microM) induced a large fractional release of the preloaded [3H]D-aspartate and in the presence of DL-TBOA the release was reduced by approximately 50%. In contrast, release evoked by 25 microM glutamate was not inhibited by DL-TBOA. These results indicate that the release elicited by 100 microM glutamate is comprised of a significant glutamate transporter-mediated component in addition to the vesicular release while the NMDA/K(+)-induced release is vesicular in nature. It is likely that the high glutamate concentration (100 microM) may facilitate heteroexchange of the preloaded [3H]D-aspartate.
Department of Pharmacology, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark.
Lasse K
LK
Bak
Arne
A
Schousboe
Helle S
HS
Waagepetersen
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism / Q000494:pharmacology*; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects* / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000187:drug effects* / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D051379:Mice; D016202:N-Methylaspartate / Q000494:pharmacology
C120673:benzyloxyaspartate; D001224:Aspartic Acid; D018698:Glutamic Acid; D016202:N-Methylaspartate