A study in rat brain cortex synaptic vesicles of endogenous ligands for N-methyl-D-aspartate receptors.
The presence of endogenous ligands for the N-methyl-D-aspartate receptor was looked for in highly purified rat brain cortex synaptic vesicles, the contents of which were extracted and fractionated by gel filtration on Sephadex G-10, or by three different high-voltage electrophoresis procedures. The presence of endogenous ligands was detected by their ability to compete with 50 nM L-[3H]glutamate for binding to whole rat brain N-methyl-D-aspartate receptors. The receptor preparations used were those present in purified postsynaptic densities, in which the quisqualate receptors were blocked by 10 microM quisqualate. Synaptic vesicles had a high content of N-methyl-D-aspartate receptor ligands, which on fractionation always coincided with glutamate or aspartate. A variable and very small amount of a highly acidic endogenous ligand was also found. The latter substance did not coincide in the electrophoresis with homocysteic, cysteic, quinolinic, cysteine sulphinic or homocysteine sulphinic acids, or with N-acetyl-aspartyl-glutamic acid, S-sulphocysteine or sulphoserine. We also found that a single centrifugation, in 0.25 M sucrose, 25 mM Tris-citrate, pH 7.1, of purified synaptic vesicles, at 135,000 gmax for 45 min, led to a 51% loss of endogenous glutamate, but did not change their aspartate content. Thus, in uncentrifuged vesicles the glutamate/aspartate ratio was 9.4, while in centrifuged ones the ratio was 3.9 ATP markedly enhanced L-[3H]glutamate uptake into synaptic vesicles, but did not change the binding of L-[3H]aspartate. Differences in labelled aspartate and glutamate efflux from the vesicles were also found.(ABSTRACT TRUNCATED AT 250 WORDS)
Villanueva
S
S
Department of Physiology and Biophysics, Faculty of Medicine, Universidad de Chile, Santiago.
Fiedler
J
J
Orrego
F
F
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002540:Cerebral Cortex / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis; D005971:Glutamates / Q000378:metabolism; D066298:In Vitro Techniques; D007608:Kainic Acid / Q000378:metabolism; D008024:Ligands; D016202:N-Methylaspartate / Q000378:metabolism; D016318:Quisqualic Acid / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism*; D014020:Tissue Extracts / Q000494:pharmacology; D014461:Ultracentrifugation
D000596:Amino Acids; D005971:Glutamates; D008024:Ligands; D016194:Receptors, N-Methyl-D-Aspartate; D014020:Tissue Extracts; D016202:N-Methylaspartate; D016318:Quisqualic Acid; D007608:Kainic Acid