dl-threo-β-Benzyloxyaspartate, A Potent Blocker of Excitatory Amino Acid Transporters

Abstract

DL-threo-beta-Benzyloxyaspartate (DL-TBOA), a novel derivative of DL-threo-beta-hydroxyaspartate, was synthesized and examined as an inhibitor of sodium-dependent glutamate/aspartate (excitatory amino acid) transporters. DL-TBOA inhibited the uptake of [14C]glutamate in COS-1 cells expressing the human excitatory amino acid transporter-1 (EAAT1) (Ki = 42 microM) with almost the same potency as DL-threo-beta-hydroxyaspartate (Ki = 58 microM). With regard to the human excitatory amino acid transporter-2 (EAAT2), the inhibitory effect of DL-TBOA (Ki = 5.7 microM) was much more potent than that of dihydrokainate (Ki = 79 microM), which is well known as a selective blocker of this subtype. Electrophysiologically, DL-TBOA induced no detectable inward currents in Xenopus laevis oocytes expressing human EAAT1 or EAAT2. However, it significantly reduced the glutamate-induced currents, indicating the prevention of transport. The dose-response curve of glutamate was shifted by adding DL-TBOA without a significant change in the maximum current. The Kb values for human EAAT1 and EAAT2 expressed in X. laevis oocytes were 9.0 microM and 116 nM, respectively. These results demonstrated that DL-TBOA is, so far, the most potent competitive blocker of glutamate transporters. DL-TBOA did not show any significant effects on either the ionotropic or metabotropic glutamate receptors. Moreover, DL-TBOA is chemically much more stable than its benzoyl analog, a previously reported blocker of excitatory amino acid transporters; therefore, DL-TBOA should be a useful tool for investigating the physiological roles of transporters.

NAD(P)H Fluorescence Transients after Synaptic Activity in Brain Slices: Predominant Role of Mitochondrial Function

“…Regarding extended stimulus trails, the nontransportable inhibitor of glutamate uptake (TBOA; Jabaudon et al, 1999;Shimamoto et al, 1998) did not decrease the overshoot signal after extended stimulation (Figure 10). The concentration of TBOA used here (100 mmol/l) was the same as that used to block glial depolarizations evoked by synaptic stimulation in hippocampal slices (Kawamura et al, 2004).…”

Glucose is Necessary to Maintain Neurotransmitter Homeostasis during Synaptic Activity in Cultured Glutamatergic Neurons

“…The release of glutamate was monitored by the use of the nonmetabolizable glutamate analog D-[ 3 H]aspartate, which labels the vesicular as well as the cytoplasmic glutamate pools (Belhage et al, 1992;Cousin and Nicholls, 1997;Fleck et al, 2001). To examine a possible contribution of the transporters to the release of D-[ 3 H]aspartate, the nontransportable glutamate transport inhibitor DL-threo-b-benzyloxyaspartate (TBOA) was employed (Shimamoto et al, 1998;Waagepetersen et al, 2001;Bak et al, 2003Bak et al, , 2004. To assess the relative importance of glucose and lactate as substrates for neuronal metabolism, the cultures were superfused in media containing either [U-13 C]glucose (2.5 mmol/L) and lactate (1 or 5 mmol/L) or [U-13 C]lactate (1 mmol/L) and glucose (2.5 mmol/L).…”

Novel Animal Models of Pediatric Epilepsy

“…However, TSC2 GFAP CKO mice have an earlier onset (3 weeks in TSC2 GFAP CKO vs 4 weeks in TSC1 GFAP CKO) and higher frequency of seizures (30)(31)(32)(33)(34)(35) Sz/48 h at 7 weeks in TSC2 GFAP CKO vs less than 10Sz/ 48 h at 7 weeks in TSC1 GFAP CKO), and significantly more severe histological abnormalities. The differences between these 2 transgenic mice seem to be correlated with higher levels of mTOR activation in TSC2 GFAP CKO mice.…”

“…The authors concluded that partial seizures that are best characterized by rhythmic slow oscillations in 1 territory of the brain are most likely due to poor axonal myelination. The use of threo-beta-benzyloxyaspartate to modify glutamate transport has several advantages: threo-betabenzyloxyaspartate is a nontransportable inhibitor and, as such, does not induce artificial transmitter release through hetero exchange and does not act as a partial agonist of glutamate receptors (these properties were demonstrated with electrophysiological recordings and binding assays) [34]. They demonstrated the role of glutamate receptor activation in the generation of the EEG pattern by modifying the frequency and duration of activation by administering the N-Methyl-D-aspartate (NMDA) antagonist ketamine or the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist NBQX [33].…”