title: 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 degrees 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 microM unlabeled L-aspartate and 100 microM D-aspartate, suggesting that all are accumulated into a common vesicular pool by different transporters.
authors: Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors: M W
authors: MW
authors: Fleck
authors: G
authors: G
authors: Barrionuevo
authors: A M
authors: AM
authors: Palmer
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D001667:Binding, Competitive; D018698:Glutamic Acid / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013237:Stereoisomerism; D013572:Synaptic Vesicles / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
chemical_list: D001224:Aspartic Acid; D018698:Glutamic Acid