Effects of pregabalin on the activity of glutamate transporter type 3.

BACKGROUND: Pregabalin, (S)-3-aminomethyl-5-methyl hexanoic acid, is a ligand for the α2δ subunit (a component of voltage-gated calcium channels) and has analgesic and anticonvulsant properties. Glutamate uptake by glutamate transporters may be a mechanism for these properties. We investigated the effects of pregabalin on the activity of the neuronal glutamate transporter type 3 (EAAT3). METHODS: EAAT3 was expressed in Xenopus laevis oocytes. Two-electrode voltage clamping was used to record membrane currents before, during, and after applying l-glutamate (30 µM) in the presence or absence of pregabalin. Currents were also measured in oocytes pretreated with a protein kinase C (PKC) activator (phorbol-12-myristate-13-acetate, PMA), PKC inhibitors (chelerythrine or staurosporine), or a phosphatidylinositol-3-kinase (PI3K) inhibitor wortmannin. RESULTS: The exposure of the oocytes injected with EAAT3 mRNA to serial concentrations of pregabalin (0.06-60 µM) significantly increased their responses to 30 µM l-glutamate. A kinetic study showed that pregabalin significantly increased V(max) without changing K(m). Treatment of oocytes with PMA, pregabalin, or pregabalin plus PMA significantly increased transporter currents vs controls, but treatment with PMA plus pregabalin did not increase the responses further vs PMA or pregabalin alone. In addition, pretreatment of oocytes with two PKC inhibitors (chelerythrine or staurosporine), or inhibitor wortmannin, significantly reduced basal and pregabalin-enhanced EAAT3 activity. CONCLUSIONS: Pregabalin increased EAAT3 activity and PKC and PI3K were involved. This may explain the analgesic effect of pregabalin in neuropathic pain.

Volatile anesthetics attenuate oxidative stress-reduced activity of glutamate transporter type 3.

BACKGROUND: Volatile anesthetics enhance the activity of glutamate transporter Type 3 (also called excitatory amino acid transporter Type 3, EAAT3), the major neuronal EAAT. In addition to glutamate, EAAT3 can also uptake L-cysteine, the rate-limiting substrate for the synthesis of glutathione. Our previous study showed that oxidative stress inhibited glutamate-induced EAAT3 activity. We determined whether oxidative stress would reduce L-cysteine-induced EAAT3 activity and whether this reduction would be attenuated by volatile anesthetics. METHODS: Rat EAAT3 was expressed in Xenopus oocytes. L-glutamate- and L-cysteine-induced membrane currents were recorded using the 2-electrode voltage clamp technique. The peak current was quantified to reflect the amount of transported substrates because transport of substrates via EAATs is electrogenic. RESULTS: Exposure of oocytes to 5 mM tert-butyl hydroperoxide, an organic oxidant, for 10 min reduced the V(max), but did not affect the K(m), of EAAT3 for L-cysteine. The volatile anesthetics isoflurane, sevoflurane, and desflurane at concentrations from 1% to 3% attenuated the tert-butyl hydroperoxide-reduced EAAT3 activity for L-glutamate and L-cysteine. CONCLUSIONS: Our results suggest that volatile anesthetics preserve EAAT3 function to transport L-glutamate and L-cysteine under oxidative stress, which may be a mechanism for the neuroprotective effects of volatile anesthetics.

Propofol reverses oxidative stress-attenuated glutamate transporter EAAT3 activity: evidence of protein kinase C involvement.

The authors investigated the effects of propofol on EAAT3 (excitatory amino acid transporter 3) activity under oxidative stress induced by tert-butyl hydroperoxide (t-BHP), and the mediation of these effects by protein kinase C (PKC). Rat EAAT3 was expressed in Xenopus oocytes and L-glutamate (30 microM)-induced membrane currents were measured using the two-electrode voltage clamp technique. Exposure of these oocytes to t-BHP (1-20 mM) for 10 min dose-dependently decreased EAAT3 activity, and t-BHP (5 mM) significantly decreased the Vmax, but not the Km of EAAT3 for glutamate, and propofol (1-100 microM) dose-dependently reversed this t-BHP-attenuated EAAT3 activity. Phorbol-12-myristate-13-acetate (a PKC activator), also abolished this t-BHP-induced reduction in EAAT3 activity, whereas staurosporine (a PKC inhibitor), significantly decreased EAAT3 activity. However, as compared with staurosporine or t-BHP alone, t-BHP and staurosporine in combination did not further reduce EAAT3 activity. A similar pattern was observed for chelerythrine (also a PKC inhibitor). In oocytes pretreated with combinations of t-BHP and PMA (or staurosporine), propofol failed to change EAAT3 activity. Our results suggest that propofol restores oxidative stress-reduced EAAT3 activity and that these effects of propofol may be PKC-mediated.

Stereoselective chemoenzymatic synthesis of the four stereoisomers of l-2-(2-carboxycyclobutyl)glycine and pharmacological characterization at human excitatory amino acid transporter subtypes 1, 2, and 3.

The four stereoisomers of l-2-(2-carboxycyclobutyl)glycine, l-CBG-I, l-CBG-II, l-CBG-III, and l-CBG-IV, were synthesized in good yield and high enantiomeric excess, from the corresponding cis and trans-2-oxalylcyclobutanecarboxylic acids 5 and 6 using the enzymes aspartate aminotransferase (AAT) and branched chain aminotransferase (BCAT) from Escherichia coli. The four stereoisomeric compounds were evaluated as potential ligands for the human excitatory amino acid transporters, subtypes 1, 2, and 3 (EAAT1, EAAT2, and EAAT3) in the FLIPR membrane potential assay. While the one trans-stereoisomer, l-CBG-I, displayed weak substrate activity at all three transporters, EAAT1-3, we found a particular pharmacological profile for the other trans-stereoisomer, l-CBG-II, which displayed EAAT1 substrate activity and inhibitory activity at EAAT2 and EAAT3. Whereas l-CBG-III was found to be a weak inhibitor at all three EAAT subtypes, the other cis-stereoisomer l-CBG-IV was a moderately potent inhibitor with 20-30-fold preference for EAAT2/3 over EAAT1.

Effects of chronic exposure to ethanol on glutamate transporter EAAT3 expressed in Xenopus oocytes: evidence for protein kinase C involvement.

BACKGROUND: Glutamate transporters (excitatory amino acid transporters, EAAT) regulate extracellular concentrations of glutamate, a major excitatory neurotransmitter. We reported that acute ethanol exposure increases the activity of a major neuronal EAAT, EAAT3. This effect is consistent with the general inhibitory effect of acute alcohol toxicity in the central nervous system (CNS). However, chronic ethanol exposure has CNS presentations different from acute alcohol toxicity. We hypothesize that chronic ethanol exposure will affect the EAAT3 activity differently from acute ethanol exposure. METHODS: EAAT3 was expressed in Xenopus oocytes by injection of EAAT3 mRNA. Oocytes were incubated with diluted ethanol for 24-96 hr. Using two-electrode voltage clamp, membrane currents were recorded after the application of L-glutamate. Responses were quantified by integration of the current trace and reported as microCoulombs (microC). RESULTS: Ethanol (10-100 mM) reduced EAAT3 activity in a time-dependent and reversible manner. After a 96 hr-incubation, the activity was significantly decreased compared to the control values at any concentrations tested in this study. Kinetic study demonstrated that a 96 hr-exposure to 50 mM ethanol significantly decreased Vmax (3.6 +/- 0.3 for control versus 2.6 +/- 0.3 microC for ethanol, n = 20, p < 0.05) but had no effect on Km (57.6 +/- 12.8 for control versus 51.8 +/- 16.3 microM for ethanol, n = 20, p > 0.05) of EAAT3 for glutamate. When ethanol (50 mM for 96 hr)-treated oocytes were incubated with phorbol-12-myrisate-13-acetate (50 or 100 nM for 10 min), ethanol-induced decrease in EAAT3 activity was abolished. Preincubation of the oocytes with 100 microM chelerythrine significantly decreased EAAT3 activity (1.00 +/- 0.08 for control versus 0.51 +/- 0.09 microC for chelerythrine group, n = 18-20, p < 0.05). However, there was no statistical difference among the chelerythrine, ethanol, or chelerythrine plus ethanol groups. Likewise, staurosporine (2 microM for 1 hr) significantly decreased EAAT3 activity and there was no statistical difference among the staurosporine, ethanol, or staurosporine plus ethanol groups. CONCLUSIONS: Our results show that chronic ethanol exposure decreases EAAT3 activity at clinically relevant concentrations and that this effect may be protein kinase C-dependent. Such an effect could be a neuroadaptive mechanism to overcome the inhibitory effect of ethanol on the excitatory neurotransmission.