Effects of ethanol on the rat glutamate excitatory amino acid transporter type 3 expressed in Xenopus oocytes: role of protein kinase C and phosphatidylinositol 3-kinase.
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.
Department of Anesthesiology, Seoul City Boramae Hospital, Seoul, Korea.
Jin-Hee
JH
Kim
Young-Jin
YJ
Lim
Young-Jin
YJ
Ro
Seong-Won
SW
Min
Chong-Soo
CS
Kim
Sang-Hwan
SH
Do
Yong-Lak
YL
Kim
Zhiyi
Z
Zuo
D027322:Amino Acid Transport System X-AG / Q000096:biosynthesis*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D027981:Symporters / Q000096:biosynthesis*; D014982:Xenopus laevis
D027322:Amino Acid Transport System X-AG; D000431:Ethanol; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D027981:Symporters