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.
Istituto di Ricerche Farmacologiche "Mario Negri", 20157 Milano, Italy.
Marcella
M
Funicello
Paola
P
Conti
Marco
M
De Amici
Carlo
C
De Micheli
Tiziana
T
Mennini
Marco
M
Gobbi
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D002264:Carboxylic Acids / Q000494:pharmacology*; D002478:Cells, Cultured; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D007477:Ions / Q000378:metabolism; D008297:Male; D010080:Oxazoles / Q000494:pharmacology*; D051381:Rats; D013574:Synaptosomes / Q000187:drug effects* / Q000378:metabolism; D014316:Tritium; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
C492800:3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo(3,4-d)isoxazole-4-carboxylic acid; C492801:3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo(3,4-d)isoxazole-6-carboxylic acid; D002264:Carboxylic Acids; D007477:Ions; D010080:Oxazoles; D014316:Tritium; D001224:Aspartic Acid; D018698:Glutamic Acid; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid