Synaptosomal and vesicular accumulation of L-glutamate, L-aspartate and D-aspartate

We examined the vesicular accumulation of the excitatory amino-acid (EAA) neurotransmitters, L-glutamate and L-aspartate, together with the non-metabolisable EAA analogue D-aspartate. Synaptosomes derived from whole brain were incubated in various concentrations of [H-3]-amino acids under conditions to facilitate vesicular turnover. Synaptosomes were then lysed in hypotonic medium and vesicles immunoprecipitated with monoclonal anti-synaptophysin antibodies coupled to sepharose beads. Using this method, saturable vesicular accumulation was observed for [H-3]-L-glutamate, [H-3]-L-aspartate, and [H-3]-D-aspartate but not for the excitatory amino acid receptor ligands [H-3]-AMPA or [H-3]-kainate. Vesicular accumulation (t(1 2) = 7.45 min) was markedly slower than synaptosomal accumulation (t(1 2) = 1.03 min) and was substantially reduced at 4 degreesC. Maximal accumulation of [H-3]-L-glutamate, [H-3]-L-aspartate, and [H-3]-D-aspartate was estimated to be 98, 68, and 112 pmol/mg of synaptosomal protein, respectively, and uptake affinities 1.6, 3.4, and 2.1 mM, respectively. Maximal accumulation of [H-3]-L-glutamate was non-competitively inhibited by both 100 muM unlabeled L-aspartate and 100 muM D-aspartate, suggesting that all are accumulated into a common vesicular pool by different transporters. (C) 2001 Elsevier Science Ltd. All rights reserved.