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.
Kim
Jin-Hee
JH
Department of Anesthesiology, Seoul National University College of Medicine, Seoul, Korea.
Do
Sang-Hwan
SH
Kim
Yong-Lak
YL
Zuo
Zhiyi
Z
D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity*; D050580:Excitatory Amino Acid Transporter 3 / Q000187:drug effects / Q000378:metabolism*; D050491:Glutamate Plasma Membrane Transport Proteins / Q000187:drug effects / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D009865:Oocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010617:Phenanthridines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012333:RNA, Messenger / Q000008:administration & dosage / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D009435:Synaptic Transmission / Q000187:drug effects; D014981:Xenopus
D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D010617:Phenanthridines; D012333:RNA, Messenger; D000431:Ethanol; D018698:Glutamic Acid; C016299:chelerythrine; D011493:Protein Kinase C; D019311:Staurosporine