Inhibition of the activity of excitatory amino acid transporter 4 expressed in Xenopus oocytes after chronic exposure to ethanol.
BACKGROUND
The extracellular glutamate concentration is tightly controlled by excitatory amino acid transporters (EAATs). EAAT4 is the predominant EAAT in the cerebellar Purkinje cells. Purkinje cells play a critical role in motor coordination and may be an important target for ethanol to cause motor impairments. We designed this study to determine the effects of chronic ethanol exposure on the activity of EAAT4 and evaluate the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in these effects.


METHODS
EAAT4 was expressed in Xenopus oocytes following injection of EAAT4 mRNA. Oocytes were incubated with ethanol-containing solution for 24 to 96 hours. Membrane currents induced by l-aspartate were recorded using 2-electrode voltage clamps. Responses were quantified by integration of the current trace and reported in microCoulombs (microC).


RESULTS
Ethanol dose- and time-dependently reduced EAAT4 activity. EAAT4 activity after a 96-hour exposure was significantly decreased compared to the control values at all concentrations tested (10 to 100 mM). Ethanol (50 mM) significantly decreased the Vmax (2.2 +/- 0.2 microC for control vs. 1.6 +/- 0.2 microC for ethanol, n = 18, p < 0.05) of EAAT4 for L-aspartate. Preincubation of ethanol-treated (50 mM for 96 hours) oocytes with phorbol-12-myrisate-13-acetate (100 nM for 10 minutes) abolished the ethanol-induced decrease in EAAT4 activity. While staurosporine (2 microM for 1 hour) or chelerythrine (100 microM for 1 hour) significantly decreased EAAT4 activity, no difference was observed in EAAT4 activity among the staurosporine, ethanol, or ethanol plus staurosporine groups. Similarly, EAAT4 activity did not differ among the chelerythrine, ethanol, or ethanol plus chelerythrine groups. Pretreatment of the oocytes with wortmannin (1 microM for 1 hour) also significantly decreased EAAT4 activity. However, no difference was observed in the wortmannin, ethanol, or ethanol plus wortmannin groups.


CONCLUSIONS
The results of this study suggest that chronic ethanol exposure decreases EAAT4 activity and that PKC and PI3K may be involved in these effects. These effects of ethanol on EAAT4 may cause an increase in peri-Purkinje cellular glutamate concentration, and may be involved in cerebellar dysfunction and motor impairment after chronic ethanol ingestion.
Department of Anesthesiology, Seoul National University College of Medicine, Seoul, Korea.
Seung-Yeon
SY
Yoo
Jin-Hee
JH
Kim
Sang-Hwan
SH
Do
Zhiyi
Z
Zuo
D000818:Animals; D002492:Central Nervous System Depressants / Q000008:administration & dosage / Q000494:pharmacology*; D000431:Ethanol / Q000008:administration & dosage / Q000494:pharmacology*; D050581:Excitatory Amino Acid Transporter 4 / Q000037:antagonists & inhibitors* / Q000235:genetics; D005260:Female; D008845:Microinjections; D009865:Oocytes; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D014982:Xenopus laevis
D002492:Central Nervous System Depressants; D050581:Excitatory Amino Acid Transporter 4; D000431:Ethanol; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C