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

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 [(3)H]L-glutamate uptake, with IC(50) values (17-18 microM) very similar to that of the potent EAAT inhibitor dl-threo-beta-benzyloxyaspartic acid (TBOA). The two compounds had little effect in inducing [(3)H]D-aspartate release from superfused synaptosomes but they potently inhibited l-glutamate-induced [(3)H]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 [(3)H]D-aspartate release with IC(50) values (1.2-1.6 microM) 10 times lower than those required to inhibit [(3)H]L-glutamate uptake. There is therefore a concentration window (1-3 microM) 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 [(3)H]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.