Abstract
(±)-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-β-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 IC50 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 μM (–)-HIP-A, but not with 10 to 30 μM TBOA or 100 μM (–)-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.
Footnotes
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A.A.J. was supported by The Lundbeck Foundation and by The Danish Medical Research Council.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.135251.
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ABBREVIATIONS: EAAT, excitatory amino acid transporter; HIP-A, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid; HIP-B, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid; TBOA, dl-threo-β-benzyloxyaspartic acid; OGD, oxygen-glucose deprivation; CI, confidence interval; FRR, fractional release rate; FMP, fluorometric imaging plate reader membrane potential; MS, medium stock; DIV, day(s) in vitro; LDH, lactate dehydrogenase; PI, propidium iodide; ER, eudismic ratio.
- Received December 13, 2007.
- Accepted April 30, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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