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

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Authors
Kim, Jin-Hee; Do, Sang-Hwan; Kim, Yong-Lak; Zuo, Zhiyi
Issue Date
2005-12-13
Publisher
Wiley-Blackwell
Citation
Alcohol Clin Exp Res. 2005 Nov;29(11):2046-52.
Keywords
AlkaloidsAnimalsBenzophenanthridinesDose-Response Relationship, DrugEnzyme Inhibitors/pharmacologyEthanol/metabolism/pharmacology/*toxicityExcitatory Amino Acid Transporter 3/drug effects/*metabolismGlutamate Plasma Membrane Transport Proteins/drug effects/*metabolismGlutamic Acid/pharmacologyMembrane Potentials/drug effectsOocytes/drug effects/enzymology/*metabolismPatch-Clamp TechniquesPhenanthridines/pharmacologyProtein Kinase C/antagonists & inhibitors/*metabolismRNA, Messenger/administration & dosage/metabolismStaurosporine/pharmacologySynaptic Transmission/drug effectsXenopus
Abstract
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.
ISSN
0145-6008 (Print)
Language
English
URI
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16340463

http://hdl.handle.net/10371/28925
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College of Medicine/School of Medicine (의과대학/대학원)Anesthesiology and Pain Medicine (마취통증의학전공)Journal Papers (저널논문_마취통증의학전공)
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