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

Marcella Funicello, Paola Conti, Marco De Amici, Carlo De Micheli, Tiziana Mennini, Marco Gobbi

Research output: Contribution to journalArticle

Abstract

We characterized the interaction of two conformationally constrained aspartate and glutamate analogs, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4- c/]isoxazole-4-carboxylic acid (HIP-A) and 3-hydroxy-4,5,6,6a-tetrahydro-3aH- pyrrolo[3,4-c/]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.

Original languageEnglish
Pages (from-to)522-529
Number of pages8
JournalMolecular Pharmacology
Volume66
Issue number3
Publication statusPublished - Sep 2004

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D-Aspartic Acid
Aspartic Acid
Amino Acid Transport Systems
Glutamic Acid
Excitatory Amino Acids
Synaptosomes
Isoxazoles
Carboxylic Acids
Inhibitory Concentration 50
Acids
Facilitated Diffusion
3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo(3,4-d)isoxazole-6-carboxylic acid
3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo(3,4-d)isoxazole-4-carboxylic acid
Ions

ASJC Scopus subject areas

  • Pharmacology

Cite this

@article{11f0a90fb4ff4ff8be87f38840c546e7,
title = "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",
abstract = "We characterized the interaction of two conformationally constrained aspartate and glutamate analogs, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4- c/]isoxazole-4-carboxylic acid (HIP-A) and 3-hydroxy-4,5,6,6a-tetrahydro-3aH- pyrrolo[3,4-c/]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.",
author = "Marcella Funicello and Paola Conti and {De Amici}, Marco and {De Micheli}, Carlo and Tiziana Mennini and Marco Gobbi",
year = "2004",
month = "9",
language = "English",
volume = "66",
pages = "522--529",
journal = "Molecular Pharmacology",
issn = "0026-895X",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
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TY - JOUR

T1 - 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

AU - Funicello, Marcella

AU - Conti, Paola

AU - De Amici, Marco

AU - De Micheli, Carlo

AU - Mennini, Tiziana

AU - Gobbi, Marco

PY - 2004/9

Y1 - 2004/9

N2 - We characterized the interaction of two conformationally constrained aspartate and glutamate analogs, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4- c/]isoxazole-4-carboxylic acid (HIP-A) and 3-hydroxy-4,5,6,6a-tetrahydro-3aH- pyrrolo[3,4-c/]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.

AB - We characterized the interaction of two conformationally constrained aspartate and glutamate analogs, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4- c/]isoxazole-4-carboxylic acid (HIP-A) and 3-hydroxy-4,5,6,6a-tetrahydro-3aH- pyrrolo[3,4-c/]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.

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