Synaptosomal and vesicular accumulation of l-glutamate, l-aspartate and d-aspartate

Abstract

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 [ 3H]-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 [ 3H]- l-glutamate, [ 3H]- l-aspartate, and [ 3H]- d-aspartate but not for the excitatory amino acid receptor ligands [ 3H]-AMPA or [ 3H]-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°C. Maximal accumulation of [ 3H]- l-glutamate, [ 3H]- l-aspartate, and [ 3H]- 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 [ 3H]- l-glutamate was non-competitively inhibited by both 100 μM unlabeled l-aspartate and 100 μM d-aspartate, suggesting that all are accumulated into a common vesicular pool by different transporters.