We characterized the interaction of two conformationally constrained aspartate and glutamate analogs, 3-hydroxy-4,5,6,6atetrahydro- 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 M) 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 M) 10 times lower than those required to inhibit [3H]L-glutamate uptake. There is therefore a concentration window (1–3 M) 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.
|Titolo:||Dissociation of [3H]L-Glutamate Uptake from L-Glutamate-Induced [3H]D-Aspartate release by 3-Hydroxy-4,5,6,6atetrahydro-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 Conf|
CONTI, PAOLA (Secondo)
|Parole Chiave:||EAAT ; excitatory amino acid transporter ; HIP-A|
|Settore Scientifico Disciplinare:||Settore CHIM/08 - Chimica Farmaceutica|
|Data di pubblicazione:||2004|
|Appare nelle tipologie:||01 - Articolo su periodico|