Quantitative autoradiography of Na+-dependent [H-3]L-aspartate binding to L-glutamate transporters in rat brain: structure-activity studies using L-trans-pyrrolidine-2,4-dicarboxylate (L-t-PDC) and 2-(carboxycyclopropyl)-glycine (CCG)

Sodium-dependent binding of [H-3]L-aspartate was studied in thaw-mounted ho rizontal sections of fresh-frozen (i.e. not fixed) rat brain. After the inc ubation with [H-3]L-aspartate, the sections were exposed against a H-3-sens itive him and the resulting autoradiograms were evaluated by quantitative d ensitometry. Effects of several inhibitors were examined and their potency expressed as IC50 and n(H). Together with previously published data, the pr esent study supports the view that [H-3]L-aspartate binding to fresh-frozen sections of rat brain represents interaction of the radioligand with the s ubstrate-binding sites on glutamate transporters. The most potent inhibitor s were (2S,3S,4R)-2-(carboxycyclopropyl)-glycine (L-CCG III) and (2S,4R)-4- methylglutamate. In contrast, L-anti,endo-3,4-methanopyrrolidine dicarboxyl ate (L-a,e-MPDC) was about an order of magnitude less potent. Only subtle r egional variations in the characteristics of inhibitors of [H-3]L-aspartate binding were detected. It is not certain whether these differences reflect regional variations in the distribution of individual glutamate transporte rs or regional peculiarities in their pharmacological characteristics. In p articular, (2S,4R)-4-methylglutamate, shown previously to differentiate bet ween GLT-1 (principal glutamate transporter in the forebrain) and GLAST (ex pressed mainly in the cerebellum), did not strongly differentiate between t he binding of [H-3]L-aspartate in forebrain and cerebellum. Computer-assist ed molecular modelling using selected glutamate analogues with restricted c onformation (L-trans-pyrrolidine-2,4-dicarboxylate and four isomers of 2-(c arboxycyclopropyl)-glycine: L- and D-CCG I, L-CCG III and L-CCG IV) identif ied at least one area of unfavourable steric interaction. We conclude that the quantitative autoradiographic studies using [H-3]L-aspartate or other t ransporter-specific ligands, will be a useful tool to study the pharmacolog y of substrate binding sites on glutamate transporters in the mammalian bra in in situ. (C) 2000 Published by Elsevier Science Ltd. All rights reserved .