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Dissociation of [³H]L-Glutamate Uptake from L-Glutamate-Induced [³H]D-Aspartate release by 3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic Acid and 3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic Acid, Two Conformationally Constrained Aspartate and Glutamate Analogs

Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy (M.F., T.M., M.G.) and Istituto di Chimica Farmaceutica e Tossicologica, Università di Milano, Milano Italy (P.C., M.DeA., C.DeM.)

We characterized the interaction of two conformationally constrained aspartate and glutamate analogs, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid (HIP-A) and 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B), with excitatory amino acid transporters (EAATs) in rat brain cortex synaptosomes. HIP-A and HIP-B were potent and noncompetitive inhibitors of [³H]L-glutamate uptake, with IC₅₀ values (17–18 µM) very similar to that of the potent EAAT inhibitor DL-threo--benzyloxyaspartic acid (TBOA). The two compounds had little effect in inducing [³H]D-aspartate release from superfused synaptosomes but they potently inhibited L-glutamate–induced [³H]D-aspartate release, thus behaving as EAAT blockers, not substrates, in a manner similar to those of TBOA and dihydrokainate (DHK). HIP-A and HIP-B, but not TBOA and DHK, unexpectedly inhibited L-glutamate–induced [³H]D-aspartate release with IC₅₀ values (1.2–1.6 µM) 10 times lower than those required to inhibit [³H]L-glutamate uptake. There is therefore a concentration window (1–3 µM) in which the two compounds significantly inhibited L-glutamate–induced release with very little effect on L-glutamate uptake. This selective inhibitory effect required quite long preincubation (>5 min) of synaptosomes with the drugs. At these low concentrations, however, HIP-A and HIP-B had no effect on the EAAT-mediated [³H]D-aspartate release induced by altering the ion gradients, indicating that they specifically affect some L-glutamate-triggered process(es)—different from L-glutamate translocation itself—responsible for the induction of reverse transport. These data are inconsistent with the classic model of facilitated exchange-diffusion and provide the first evidence that EAAT-mediated substrate uptake and substrate-induced EAAT-mediated reverse transport are independent. Compounds such as HIP-A and HIP-B could be useful to further clarify the mechanisms underlying these operating modes of transporters.

Address correspondence to: Marco Gobbi, Istituto di Ricerche Farmacologiche "Mario Negri", 20157 Milano, Italy. E-mail: gobbi{at}marionegri.it

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