Excitatory amino acid transporter-1/2/3； EAAT-1/2/3

SN38-induced loss of viability is enhanced concentration-dependently by DL-TBOA ammonium treatment (70-350 μM; 48 hours). Oxaliplatin-induced loss of viability was restored by DL-TBOA[4]. A 24-hour supply of 350 μM DL-TBOA ammonium reduces the induction of p53 by SN38 and oxaliplatin[4].

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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.[1]

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We have used DL-threo-beta-benzyloxyaspartate (DL-TBOA), an inhibitor of glutamate uptake, to determine the role of glutamate transporters in the regulation of extracellular glutamate concentration. By using the N-methyl-D-aspartate receptors of patched CA3 hippocampal neurons as "glutamate sensors," we observed that application of TBOA onto organotypic hippocampal slices led to a rapid increase in extracellular glutamate concentration. This increase was Ca(2+)-independent and was observed in the presence of tetrodotoxin. Moreover, prevention of vesicular glutamate release with clostridial toxins did not affect the accumulation of glutamate when uptake was inhibited. Inhibition of glutamine synthase, however, increased the rate of accumulation of extracellular glutamate, indicating that glial glutamate stores can serve as a source in this process. TBOA blocked synaptically evoked transporter currents in astrocytes without inducing a current mediated by the glutamate transporter. This indicates that this inhibitor is not transportable and does not release glutamate by heteroexchange. These results show that under basal conditions, the activity of glutamate transporters compensates for the continuous, nonvesicular release of glutamate from the intracellular compartment. As a consequence, acute disruption of transporter activity immediately results in significant accumulation of extracellular glutamate.[2]

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D,L-threo-beta-Benzyloxyaspartate (DL-TBOA), an analog of threo-beta-hydroxyaspartate (THA) possessing a bulky substituent, is a potent non-transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while L-threo-beta-methoxyaspartate (L-TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4-expressing oocytes, D,L-TBOA acted as a non-transportable blocker, while L-TMOA like D,L-THA was a competitive substrate. In contrast, D,L-THA, D,L-TBOA and L-TMOA all strongly attenuated the glutamate-induced currents generated by EAAT5. Among them, L-TMOA showed the most potent inhibitory action. Moreover, D,L-THA, D,L-TBOA and L-TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that D,L-TBOA and L-TMOA, as well as D,L-THA, not only act as non-transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate-gated chloride channel[3].

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In SN38-resistant HCT116 and LoVo cells, SLC1A1 expression was down-regulated ~60 % and up-regulated ~4-fold, respectively, at both mRNA and protein level, whereas SLC1A3 protein was undetectable. The changes in SLC1A1 expression were accompanied by parallel changes in DL-Threo-β-Benzyloxyaspartic acid (DL-TBOA)-sensitive, UCPH101-insensitive [(3)H]-D-Aspartate uptake, consistent with increased activity of SLC1A1 (or other family members), yet not of SLC1A3. DL-TBOA co-treatment concentration-dependently augmented loss of cell viability induced by SN38, while strongly counteracting that induced by oxaliplatin, in both HCT116 and LoVo cells. This reflected neither altered expression of the oxaliplatin transporter Cu(2+)-transporter-1 (CTR1), nor changes in cellular reduced glutathione (GSH), although HCT116 cell resistance per se correlated with increased cellular GSH. DL-TBOA did not significantly alter cellular levels of p21, cleaved PARP-1, or phospho-Retinoblastoma protein, yet altered SLC1A1 subcellular localization, and reduced chemotherapy-induced p53 induction. Conclusions: SLC1A1 expression and glutamate transporter activity are altered in SN38-resistant CRC cells. Importantly, the non-selective glutamate transporter inhibitor DL-TBOA reduces chemotherapy-induced p53 induction and augments CRC cell death induced by SN38, while attenuating that induced by oxaliplatin. These findings may point to novel treatment options in treatment-resistant CRC[4].

When morphine-dependent rats are given DL-TBOA ammonium (10 nmol; icv), it greatly promotes the expression of naloxone-precipitated somatic symptoms and conditioned place aversion[5].

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There is a body of evidence implying the involvement of the central glutamatergic system in morphine dependence. In this study, we examined the effect of intracerebroventricular (i.c.v.) administration of a potent glutamate transporter inhibitor, DL-threo-beta-benzyloxyaspartate (DL-TBOA), on acute morphine-induced antinociception, expression of somatic and negative affective components of morphine withdrawal, and acquisition of morphine-induced conditioned place preference in rats. I.c.v administration of DL-TBOA (10 nmol) to naive rats did not affect the acute antinociceptive effect of morphine. I.c.v. administration of DL-TBOA (10 nmol) to morphine-dependent rats significantly facilitated the expression of naloxone-precipitated somatic signs and conditioned place aversion. DL-TBOA (3 and 10 nmol) significantly facilitated acquisition of morphine-induced conditioned place preference. DL-TBOA itself produced neither conditioned place aversion nor place preference in naive rats. These results suggest that central glutamate transporters play inhibitory roles in the expression of somatic and negative affective components of morphine withdrawal and the reinforcing effect of morphine.[5]

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Effect of i.c.v. Administration of DL-TBOA on morphine antinociception [5]
The effects of i.c.v. administration of dl-TBOA on the acute antinociceptive effects of morphine were investigated in naive rats not implanted with any pellets (Fig. 1). In the i.c.v. vehicle-treated rats, s.c. administration of morphine at a dose of 3 mg/kg dramatically elevated the mechanical nociceptive threshold, which peaked 30 min and disappeared 150 min after s.c. administration, while a dose of 0.5 mg/kg produced no or a slight elevation of the nociceptive threshold. I.c.v. administration of dl-TBOA (10 nmol) did not alter the mechanical nociceptive threshold 10 min after i.c.v. administration (just before s.c. administration of morphine), or the acute antinociceptive effect of morphine at doses of both 0.5 mg/kg (F(1,117)=0.52, P=0.47) and 3 mg/kg (F(1,144)=0.10, P=0.75).

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Effect of i.c.v. administration of DL-TBOA on the expression of somatic signs induced by naloxone-precipitated morphine withdrawal [5]
In the placebo pellet-implanted naive rats i.c.v. injected with vehicle, i.p. injection of naloxone (0.1 mg/kg) did not precipitate any characteristic somatic signs. In the morphine pellet-implanted morphine-dependent rats i.c.v. injected with vehicle, i.p. injection of naloxone precipitated characteristic somatic signs. The one-way ANOVA showed that there were significant differences between naive and morphine-dependent rats in weight loss (F(1,12)=12.37, P<0.01) and teeth chattering (F(1,12)=9.20, P<0.05). I.c.v. administration of dl-TBOA (1, 3 and 10 nmol) dose dependently facilitated various somatic signs induced by naloxone-precipitated morphine withdrawal. The one-way ANOVA showed that dl-TBOA significantly increased stretching (F(3,24)=3.59, P<0.05), wet-dog shaking (F(3,24)=3.87, P<0.05), teeth chattering (F(3,24)=7.65, P<0.001), and ejaculation (F(3,24)=3.15, P<0.05), and the Chi-square test showed significant differences in salivation (df=3, χ2=10.58, P<0.05) and rhinorrhea (df=3, χ2=9.71, P<0.05). However, dl-TBOA had no significant effect on other somatic signs such as weight loss, jumping, paw shaking, head shaking, backwards walking, diarrhea, lacrimation and ptosis. The facilitative effects of dl-TBOA (10 nmol) were observed at 0–45 min after the i.p. injection of naloxone, and disappeared toward the end of the session (data not shown). In the morphine-dependent rats, i.p. injected with saline instead of naloxone and in the placebo pellet-implanted naive rats which were i.p. injected with naloxone, i.c.v. administration of dl-TBOA (10 nmol) did not produce somatic signs (Table 1).

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Effect of i.c.v. administration of DL-TBOA on naloxone-precipitated morphine withdrawal-induced conditioned place aversion [5]
In the i.c.v. vehicle-injected morphine-dependent rats, i.p. injection of naloxone (0.003 mg/kg) did not change the time spent in the naloxone-paired compartment in the test session compared with that in the pre-conditioning session, and the aversion score was −10±22 s. I.c.v. administration of dl-TBOA (3 and 10 nmol) to morphine-dependent rats lowered the aversion score (−17±18 and −92±25 s, respectively). The one-way ANOVA demonstrated a significant difference among groups (F(2,33)=4.32, P<0.05). Post hoc comparison by the Student–Newman–Keuls test revealed that dl-TBOA at a dose of 10 nmol, but not 3 nmol, produced a significant facilitation of the naloxone-precipitated morphine withdrawal-induced conditioned place aversion, compared with the effect of vehicle (P<0.05). However, in neither the morphine-dependent rats i.p. injected with saline instead of naloxone, nor the placebo pellet-implanted naive rats i.p. injected with naloxone, did i.c.v. administration of dl-TBOA (10 nmol) change the aversion score (−10±19 and −9±22 s, respectively) (Fig. 2).

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Effect of i.c.v. administration of DL-TBOA on morphine-induced conditioned place preference [5]
In the i.c.v. vehicle-injected rats, the preference score of the morphine (0.5 mg/kg)-paired group was 52±57 s, which was slightly higher than that of the saline-paired control group (−6±69 s), although there was no significant difference. I.c.v. administration of dl-TBOA (3 and 10 nmol) dose dependently increased the preference score (199±20 and 243±50 s, respectively). The one-way ANOVA demonstrated a significant difference among groups (F(2,19)=4.78, P<0.05). Post hoc comparison revealed that dl-TBOA at doses of 3 and 10 nmol produced a significant facilitation of morphine-induced conditioned place preference, compared with the effect of vehicle (P<0.05). However, in the saline-paired control group, dl-TBOA (10 nmol) did not change the preference score (−18±36 s) (Fig. 3).

Electrophysiological Recordings. [2]
After 12–25 days in vitro, the cultures were transferred to a recording chamber mounted onto the stage of an upright microscope and superfused with an external solution (31°C, pH 7.4) containing 137 mM Na+, 2.7 mM K+, 146.2 mM Cl−, 2.8 mM Ca2+, 0.5 mM Mg2+, 11.6 mM HCO3, 0.4 mM H2PO4, and 5.6 mM d-glucose. Patch-clamp recordings were obtained from CA3 pyramidal cells and CA3 stratum radiatum astrocytes with patch pipettes (2–5 MΩ) filled with 122.5 mM Cs gluconate/10 mM Hepes/8 mM NaCl/10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA). Cells were held at +40 mV and were considered acceptable if the holding current was stable and less than 600 pA, with access resistances from 8 to 16 MΩ. During the experiment, input resistance was assessed by applying 0.4-s voltage commands of +10 or –10 mV. l-Glutamate was applied locally by pressure-ejection as indicated. Cultures treated with clostridial toxins [botulinum A (BoNT A) and tetanus (TeNT) toxins; 100 ng/ml], as well as their respective controls, were incubated during 3 days in serum-free medium.

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Electrophysiological analysis [3]
Capped RNAs transcribed from linearized pOTV-EAAT4 or -EAAT5 using T7 RNA polymerase were microinjected into defolliculated stage IV or V oocytes (∼10 ng/oocyte) (Fairman et al. 1995; Arriza et al. 1997). The oocytes were kept at 17°C for 2–8 days. Two-microelectrode voltage-clamp recordings were performed using a GeneClamp 500 amplifier with a Digidata 1200 interface. The pClamp7 suite of programs (Axon Instruments) and Mac Lab were used to control stimulation parameters and data acquisition. Currents were low-pass filtered between 10Hz and 2 kHz and digitized at rates between 1 and 5 kHz. Microelectrodes were filled with 3 m KCl (resistance, < 1 MΩ). ND 96 recording solution contained 96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2 and 5 mm HEPES (pH 7.5). The voltage dependence of EAAT4 or EAAT5 currents were studied using the following protocol. Oocytes were held at −60 mV and stepped by 10 mV increments for 125 ms test potentials ranging from −90 to + 80 mV. Steady-state currents were measured at the end of each test potential jump. Drug-elicited currents were determined by subtracting control trials from the drug trials (Idrug − Icontrol).

Cell Viability Assay[4]
Cell Types: HCT116 cells, LoVo cells
Tested Concentrations: 70 μM, 350 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Enhanced SN38-induced, and counteracted Oxaliplatin-induced, cell death.

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Cell Viability Assay[4]
Cell Types: HCT116 cells, LoVo cells
Tested Concentrations: 350 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: diminished p53 induction by SN38 and oxaliplatin.

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Cell viability assays [4]
Cells were seeded at appropriate density (LoVo 10,000, and HCT116 5000 cells /100 μl) in growth medium in 96-well plates. Next day, cells were treated with inhibitors and/or chemotherapy in a total volume of 200 μl in growth medium and incubated for 48 h at 37 °C, 5 % CO2. The medium was replaced with 100 μl of 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in growth medium. The reaction was stopped 1.5-2.5 h later by addition of 100 μl 20 % SDS in 0.02 M HCl, and incubation overnight to allow complete lysis of cells and formazan crystals. Absorbance was measured in an ELISA plate reader at 550 nm. Data were background subtracted and % viability relative to controls calculated.

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Cell counting using the OPERA high throughput confocal [4]
Cells were seeded in 96-well plates one day prior to experiments. Cells were treated with chemotherapy and/or inhibitors as indicated and incubated for 48 h. Medium was aspirated, and cells were washed gently in ice cold PBS, fixed in 2 % paraformaldehyde, washed in PBS, and nuclei stained by 5 min incubation with DAPI. Plates were scanned using an OPERA high-throughput microscope. Nucleus counting was performed using proprietary OPERA software, based on 100 points per well, and cell counts from non-treated cells as controls.

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[3H]-D-Asp uptake assay [4]
The [3H]-D-Asp uptake assay was performed essentially. Briefly, cells were split into poly-D-lysine-coated white 96-well plates. A similar number of cells were seeded for HCT116 parental, HCT116-SN38 and HCT116-Oxa cell lines (7 × 104 cells/well) and LoVo parental, Lovo-SN38 and Lovo-Oxa cell lines (6 × 104 cells/well). 16–24 h later, culture medium was aspirated, and cells were washed once with 100 μl assay buffer (Hank’s Buffered Saline Solution supplemented with 20 mM HEPES, 1 mM CaCl2 and 1 mM MgCl2, pH 7.4). 50 μl assay buffer supplemented with 100 nM [3H]-D-Asp and test compounds as indicated was added, and the plate was incubated at 37 °C for 6 min. Non-specific [3H]-D-Asp uptake was determined in wells with 3 mM L-glutamate. The assay mixture was quickly removed, and wells were washed with 2 × 100 μl ice-cold assay buffer, followed by 150 μl Microscint™20 scintillation fluid. The plate was shaken for 1 h and counted in a Wallac 1450 MicroBeta Trilux scintillation counter.

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Measurement of cellular glutathione levels [4]
Cells were seeded in 24-well plates (104 cells per well), treated the next day with chemotherapy and/or DL-TBOA and incubated for 24 h. Medium was removed and cells were washed twice with ice-cold PBS, which was removed and 500 μl ice-cold 1 % Sulfosalicylic acid was added per well. Cells were incubated on ice for at least 10 min. After centrifugation (1 min, 15,000 g), 10 μl lysate was used to determine total GSx content. To measure GSSG content, 130 μl sample was mixed with 55 μl 0.2 M Tris (pH 9) and 5 μl 2-Vinylpyridine. Tubes were vortexed carefully and incubated for at least 1 h at room temperature. 10 μl of this mix was mixed first with 90 μl of water in a 96-well plate and then with a reaction mix (0.1 M sodium phosphate buffer pH 7.5 containing 1 mM EDTA, 10 mM NADPH, 10 mM DTNB and 0.05 μl Glutathione reductase, 2U/μl). Measurements were taken every 30 s for 10 min in an ELISA plate reader at 405 nm absorbance. GSH values were obtained by subtraction of GSSG values from GSx values.

Animal/Disease Models: Male SD (Sprague-Dawley) rats (180-250 g)[5]
Doses: 1 nmol, 3 nmol, 10 nmol
Route of Administration: Intracerebroventricular injection (icv)
Experimental Results: Dose dependently facilitated various somatic signs induced by Naloxone (0.1 mg/kg)-precipitated morphine withdrawal.

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Surgery and i.c.v. administration [5]
Under thiamylal sodium (50 mg/kg, intraperitoneally (i.p.)) anesthesia, a stainless steel guide cannula (o.d. 0.7 mm) was implanted on the right side at coordinates of 0.8 mm caudal to the bregma, 1.5 mm lateral to the midline, and 2.0 mm below the surface of the skull according to the atlas of Paxinos and Watson (1998). After surgery, the rats were individually returned to their cages and left to recover for at least 5 days before the experiments. DL-TBOA or vehicle was i.c.v. administered 10 min before i.p. injection of naloxone or subcutaneous (s.c.) injection of morphine. An injection cannula (o.d. 0.35 mm) was inserted into the right lateral ventricle just 5.0 mm below the surface of the skull when attached to the guide cannula. I.c.v. administration was carried out in a volume of 5 μl at a constant rate of 5 μl/30 s by a microinfusion pump. The injection cannula was left in place for an additional 30 s to prevent backflow. During the injection procedure, the experimenter loosely held the animals. Because, in preliminary experiments, i.c.v. administration of DL-TBOA at a dose of 30 nmol to naive rats elicited irritability, motor hyperexcitability and/or convulsion immediately or soon after i.c.v. administration, we used doses lower than 10 nmol. I.c.v. administration of dl-TBOA at a dose of 10 nmol, but not 1 or 3 nmol, elicited teeth chattering and/or passivity (loss of struggle responses from the holding) in some rats, although these behaviors disappeared within a few minutes (experimenter's observation).

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Measurement of nociceptive threshold [5]
Mechanical nociceptive threshold was evaluated by the paw pressure test using an analgesimeter with a cuneate piston. The piston was put on the ventral surface of the hind paw. The pressure was loaded at a rate of 32 g/s. The pressure that elicited paw withdrawal was determined as the nociceptive threshold. The procedures for the measurement were carried out three times a day to habituate the animals to the procedure. After 2 days of habituation, the threshold was measured following two additional habituation procedures, and the value was taken as a control. The control nociceptive threshold was 194.3±3.4 g (n=33). Soon after measuring the control value, vehicle or DL-TBOA (10 nmol) was i.c.v. administered. After 10 min, the nociceptive threshold was measured (time zero), and morphine (0.5 and 3 mg/kg) was s.c. administered. Then, the threshold was measured at 15, 30, 45, 60, 90, 120 and 150 min.

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Measurement of naloxone-precipitated morphine withdrawal-induced somatic signs [5]
Measurement of naloxone-precipitated morphine withdrawal-induced somatic signs was performed as previously described (Nakagawa et al., 2000) with slight modifications. On the first day (day 1), under light ether anesthesia, the rats had a morphine pellet implanted in the back of the neck. Twenty-four hours later (day 2), the rats received a second morphine pellet. After 72 h (day 5), each rat was placed in a Plexiglass cylinder to acclimatize it to the experimental environment. After the 30-min habituation period, DL-TBOA (1, 3 and 10 nmol) or vehicle was i.c.v. administered. Ten minutes after the i.c.v. administration, naloxone (0.1 mg/kg) was i.p. injected without removing the implanted pellets. This dose of naloxone is reported to moderately precipitate somatic signs in morphine-dependent rats Higgins and Sellers, 1994, Le Guen et al., 2001. Then, the rats were immediately returned to the cylinder and behavior was observed every 5 min for 1 h. Body weight was measured just before and 1 h after naloxone injection, and is presented as the means±S.E.M. of percentage body weight loss. The number of occurrences of stretching, wet dog shaking, teeth chattering, jumping, paw shaking, head shaking, ejaculation and backwards walking was counted, and data are presented as means±S.E.M. of total numbers. The occurrence of diarrhea, salivation, lacrimation, rhinorrhea and ptosis was monitored and is presented as the number of rats showing positive signs over the total number of rats tested.

[1]. DL-threo-beta-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters. Mol Pharmacol. 1998 Feb;53(2):195-201.

[2]. Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin. Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8733-8.

[3]. Effects of threo-beta-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5). J Neurochem. 2001 Oct;79(2):297-302.

[4]. The glutamate transport inhibitor DL-Threo-β-Benzyloxyaspartic acid (DL-TBOA) differentially affects SN38- and oxaliplatin-induced death of drug-resistant colorectal cancer cells. BMC Cancer. 2015 May 16;15:411.

[5]. Facilitation of morphine withdrawal symptoms and morphine-induced conditioned place preference by a glutamate transporter inhibitor DL-threo-beta-benzyloxyaspartate in rats. Eur J Pharmacol. 2004 Feb 6;485(1-3):201-10.

(3s)-3-(Benzyloxy)-L-Aspartic Acid is an aspartic acid derivative.

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In conclusion, we have established that TBOA is an appropriate tool to study glutamate transporter function in the central nervous system. Our data demonstrate that the low [glu]o present in the brain is the result of a dynamic equilibrium, in which glutamate is constantly being released from the intracellular compartment by a nonvesicular mechanism and is continuously being taken up by the membrane transporters.[2]

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We have studied the effects of three different THA derivatives, d,l-THA, d,l-TBOA, and l-TMOA on EAAT4 and EAAT5 using an electrophysiological analysis. d,l-TBOA itself did not elicit a detectable inward or outward current at −60 mV in EAAT4-expressing oocytes (n = 14) (Figs 2a and b). Recently, Mitrovic et al. reported that application of d,l-TBOA to EAAT4-expressing oocytes, at concentrations that inhibit the substrate-activated current, does not block the constitutive conductance (Mitrovic et al. 2001). These results indicate that d,l-TBOA is the only non-transportable blocker so far reported for EAAT4 without affecting the leak current. l-TMOA and d,l-THA act as competitive substrates for EAAT4. Km value of l-TMOA (1.2 ± 0.6 µm) is almost the same as that of d,l-THA (0.6 ± 0.3 µm). On the other hand, d,l-TBOA, l-TMOA and d,l-THA all act as non-transportable blockers for EAAT5 with Ki values of 3.2, 1.0, and 2.5 µm, respectively, and all of them block the EAAT5 leak currents. We have previously reported that d,l-TBOA is a potent non-transportable blocker for EAATs1–3 with IC50 values in the range of 7–48 µm in the uptake assay and that it shows no functional activity on the metabotropic glutamate receptors (mGluR1, mGluR2 and mGluR4) or ionotropic glutamate receptors [kainate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA), and NMDA] (Lebrun et al. 1997; Shimamoto et al. 1998; Shimamoto et al. 2000). Taken together, d,l-TBOA has proven to be a potent non-transportable blocker for all EAAT subtypes and a valuable pharmacological tool in the field of glutamate transporter studies.[3]

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In conclusion, SLC1A1 expression and glutamate transporter activity are altered in SN38-resistant CRC cells, and the glutamate transporter inhibitor DL-TBOA reduces chemotherapy-induced p53 induction and augments CRC cell death induced by SN38, while strongly attenuating that induced by oxaliplatin. Our findings indicate that changes in glutamate transporter expression and activity may be relevant in CRC, diagnostically and in the context of choice of treatment regimen.[4]

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In conclusion, we have shown that i.c.v. administration of dl-TBOA facilitates naloxone-precipitated morphine withdrawal-induced somatic signs and conditioned place aversion, and the acquisition of morphine-induced conditioned place preference, without affecting acute morphine-induced antinociception. These findings suggest that central glutamate transporters play inhibitory roles in the expression of somatic and negative affective components of morphine withdrawal and the reinforcing effect of morphine. The present study may provide a new strategy for preventing morphine dependence.[5]

C11H16N2O5

256.26

256.105

2093503-71-8

DL-TBOA;205309-81-5

91900718

Typically exists as solid at room temperature

4

7

6

18

275

2

C1=CC=C(C=C1)CO[C@@H]([C@@H](C(=O)O)N)C(=O)O.N

NWSNCKZFVJQJRK-OZZZDHQUSA-N

InChI=1S/C11H13NO5.H3N/c12-8(10(13)14)9(11(15)16)17-6-7-4-2-1-3-5-7;/h1-5,8-9H,6,12H2,(H,13,14)(H,15,16);1H3/t8-,9-;/m0./s1

(2S,3S)-2-amino-3-phenylmethoxybutanedioic acid;azane

DL-TBOA ammonium; L-TBOA ammonia salt; 2093503-71-8; TBOA; (3S)-3-(Phenylmethoxy)-L-aspartic acid ammonia salt; (2S,3S)-2-amino-3-phenylmethoxybutanedioic acid;azane;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)