Neuroprotective effects of the novel glutamate transporter inhibitor (-)-3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]-isoxazole-4-carboxylic acid, which preferentially inhibits reverse transport (glutamate release) compared with glutamate reuptake.
(+/-)-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) are selective inhibitors of excitatory amino acid transporters (EAATs), as potent as DL-threo-beta-benzyloxyaspartic acid (TBOA). We report here that the active isomers are (-)-HIP-A and (+)-HIP-B, being approximately 150- and 10-fold more potent than the corresponding enantiomers as inhibitors of [3H]aspartate uptake in rat brain synaptosomes and hEAAT1-3-expressing cells. Comparable IC(50) values were found on the three hEAAT subtypes. (-)-HIP-A maintained the remarkable property, previously reported with the racemates, of inhibiting synaptosomal glutamate-induced [3H]D-aspartate release (reverse transport) at concentrations significantly lower than those inhibiting [3H]L-glutamate uptake. New data suggest that the noncompetitive-like interaction described previously is probably the consequence of an insurmountable, long-lasting impairment of EAAT's function. Some minutes of preincubation are required to induce this impairment, the duration of preincubation having more effect on inhibition of glutamate-induced release than of glutamate uptake. In organotypic rat hippocampal slices and mixed mouse brain cortical cultures, TBOA, but not (-)-HIP-A, had toxic effects. Under ischemic conditions, a neuroprotective effect was found with 10 to 30 microM (-)-HIP-A, but not with 10 to 30 microM TBOA or 100 microM (-)-HIP-A. The effect of (-)-HIP-A suggests that, under ischemia, EAATs mediate both release (reverse transport) and uptake of glutamate. The neuroprotection with the lower (-)-HIP-A concentrations may indicate a selective inhibition of the reverse transport confirming the data obtained in synaptosomes. The selective interference with glutamate-induced glutamate release might offer a new strategy for neuroprotective action.
Colleoni
Simona
S
Istituto di Ricerche Farmacologiche "Mario Negri," 20156 Milan, Italy.
Jensen
Anders A
AA
Landucci
Elisa
E
Fumagalli
Elena
E
Conti
Paola
P
Pinto
Andrea
A
De Amici
Marco
M
Pellegrini-Giampietro
Domenico E
DE
De Micheli
Carlo
C
Mennini
Tiziana
T
Gobbi
Marco
M
D000818:Animals; D001692:Biological Transport; D002264:Carboxylic Acids / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D027341:Excitatory Amino Acid Transporter 1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D015394:Molecular Structure; D018696:Neuroprotective Agents / Q000737:chemistry / Q000494:pharmacology*; D010080:Oxazoles / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013237:Stereoisomerism; D013574:Synaptosomes / Q000187:drug effects* / Q000378:metabolism
C492801:3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo(3,4-d)isoxazole-6-carboxylic acid; D002264:Carboxylic Acids; D003470:Culture Media; D027341:Excitatory Amino Acid Transporter 1; D018696:Neuroprotective Agents; D010080:Oxazoles; C493392:Slc1a3 protein, rat; D018698:Glutamic Acid