Autoradiographic characterization of putative excitatory amino acid transport sites

Removal of excitatory amino acids from the extracellular space is now postulated to occur through at least two distinct transport systems that are distinguished by their ionic dependency. Thus, both sodium-dependent and chloride-dependent systems have been described in the mammalian central nervous system. In this report we attempt to characterize these sites by autoradiography, using D-[3H]aspartate and L-[3H]glutamate as ligands. Previous studies have shown that sequestration of radioligand into membrane vesicles can be a potential artifact when examining transport sites. We have found that sequestration can be alleviated by incubation of tissue sections in xylenes prior to incubation with radioligand. Using in vitro autoradiography we have characterized the two binding sites with respect to their distribution, kinetics and pharmacology. Both appeared to have a single, saturable binding site with Kds in the low micromolar range. Sodium-dependent D-aspartate binding predominated, having a Bmax that was five times greater than chloride-dependent L-glutamate binding in whole brain. The levels of binding to the two sites varied between brain regions. Sodium-dependent D-aspartate binding was highest in the cerebellar molecular layer greater than dentate gyrus molecular layer greater than entorhinal cortex. Chloride-dependent L-glutamate binding was highest in the outer layers of cerebral cortex greater than dentate gyrus molecular layer greater than entorhinal cortex greater than striatum. Pharmacological characterization of these sites also showed major differences. Sodium-dependent D-aspartate binding was most potently inhibited by L-aspartate greater than threo-beta-hydroxyaspartate greater than L-cysteine sulfinic acid greater than L-cysteic acid. Chloride-dependent glutamate binding was most potently inhibited by L-glutamate greater than L-alpha-amino adipic acid greater than quisqualate greater than L-serine-o-sulfate. The differences in distribution, ligand binding properties and pharmacology of these sites suggest that a significant variable in excitatory amino acid circuitry may include heterogeneity in transporters associated with excitatory pathways.