Abstract
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-β-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.
- Received December 22, 2003.
- Accepted May 21, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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