Doxepin and imipramine but not fluoxetine reduce the activity of the rat glutamate transporter EAAT3 expressed in Xenopus oocytes
BACKGROUND Glutamate is a major excitatory neurotransmitter in the central nervous system. Glutamate transporters play a critical role in maintaining extracellular glutamate concentrations. We investigated the effects of ethanol on a neuronal glutamate transporter, excitatory amino acid transporter type 3 (EAAT3), and the role of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3 K) in mediating these effects. METHODS EAAT3 was expressed in Xenopus oocytes by injection of EAAT3 messenger RNA. By using a 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. Data are mean +/- SEM. RESULTS Ethanol enhanced EAAT3 activity in a concentration-dependent manner. At 25, 50, 100, and 200 mM of ethanol, the responses were significantly increased compared with control values. Kinetic study demonstrated that ethanol (50 mM) significantly increased Vmax (3.48 +/- 0.2 microC for control versus 4.16 +/- 0.24 microC for ethanol; n = 19; p < 0.05) without a significant change in the Km (65.6 +/- 11.1 microM for control versus 55.8 +/- 9.6 microM for ethanol; n = 19; p > 0.05) of EAAT3 for glutamate. Preincubation of the oocytes with phorbol-12-myristate-13-acetate (PMA) significantly increased EAAT3 activity (0.98 +/- 0.08 muC for control versus 1.28 +/- 0.09 microC for ethanol; n = 19; p < 0.05). However, there was no statistical difference among the responses of EAAT3 to PMA, ethanol, or PMA plus ethanol. Although the PKC inhibitors chelerythrine and staurosporine did not decrease the basal EAAT3 activity, they abolished the enhancement of EAAT3 activity by ethanol. Pretreatment with wortmannin, a PI3 K inhibitor, also abolished the ethanol-enhanced EAAT3 activity. CONCLUSIONS These results suggest that acute ethanol exposure increases EAAT3 activity at clinically relevant concentrations and that PKC and PI3 K may be involved in mediating these ethanol effects.