ENT1 Regulates Ethanol‐Sensitive EAAT2 Expression and Function in Astrocytes

Glutamate is the primary excitatory neurotransmitter in the brain (Monaghan et al., 1989), and alterations in glutamatergic neurotransmission are thought to be involved in several psychiatric disorders, including alcoholism (Spanagel et al., 2005; Vengeliene et al., 2008). One of the important roles of astrocytes, which are the most abundant cell type in the mammalian central nervous system, is to uptake glutamate in the synaptic cleft to prevent neurons from overexcitement by glutamatergic neurotransmission (Miguel-Hidalgo, 2009). Glutamate uptake is mainly mediated by excitatory amino acid transporters (EAATs; also known as glutamate transporters). EAATs are sodium- and potassium-dependent members of solute carrier family 1 (SLC1) and are found widely distributed throughout the mammalian brain (Amara and Fontana, 2002). Among the five mammalian EAATs, EAAT1 and EAAT2 are most abundantly expressed in astrocytes and are responsible for clearance of glutamate during neurotransmission (Amara and Fontana, 2002). While EAAT1 (GLAST) is expressed throughout the central nervous system (Danbolt, 2001), EAAT2 (GLT1) is expressed abundantly in the forebrain region with moderate expression throughout the remaining central nervous system (Gegelashvili and Schousboe, 1997). EAAT3 is a predominant subtype in peripheral tissues and found in both astroglia and neurons (Kanai and Hediger, 1992). EAAT4 is found predominantly in the Purkinje cells of the cerebellum (Fairman et al., 1995) while EAAT5 is primarily expressed in the retina (Arriza et al., 1997). Because extracellular adenosine levels in the brain are known to mediate the acute and chronic effects of ethanol, adenosine transportation across the plasma membrane is one of the main mechanisms in regulating the effects of alcohol (Diamond and Gordon, 1994; Dunwiddie and Masino, 2001). Nucleoside transport across the plasma membrane is one of several factors that regulate extracellular adenosine concentrations, which, under basal conditions, are in the range of 25 to 250 nM in the brain (Dunwiddie and Masino, 2001). In vitro, acute ethanol treatment increases extracellular adenosine because of selective inhibition of one subtype of nucleoside transporter, ENT1, in S49 mouse lymphoma and NG108-15 neuroblastoma × glioma cells (Nagy et al., 1989, 1990). Recent studies demonstrated that astrocytes regulate adenosine levels in the synapse (Boison, 2008a). Adenosine uptake into astrocytes is mainly mediated by nucleoside transporters (Boison, 2008b). Sodium-independent equilibrative nucleoside transporters (ENTs) include four ENT subtypes (ENT1-4). ENTs are one of two main membrane nucleoside transporter families, mediating nucleoside transport bidirectionally across the plasma membrane (Anderson et al., 1999). ENT1 is sensitive to nM concentrations of nitrobenzylmercaptopurine riboside (NBMPR; also known as nitrobenzylthioionosine, NBTI), whereas ENT2 is resistant to NBMPR even up to mM concentrations (Citron et al., 1997; Coe et al., 1997; Crawford et al., 1998). Both ENT1 and ENT2 are widely expressed in astrocytes (Peng et al., 2005). Previously, our laboratory reported that ENT1 null mice displayed a higher glutamate tone in striatal slices when compared to wild-type mice (Choi et al., 2004). Because ENT1 and EAAT2 are predominantly expressed in astrocytes, we hypothesized that the expression and function of EAAT2 may be regulated by ENT1 activity in astrocytes. Using a selective and water-soluble ENT1 inhibitor, NBMPR-P, and ENT1-selective siRNA, we investigated the effect of ENT1 inhibition in astrocytes. Our study provides a possible functional correlation between ENT1 and EAAT2 in response to ethanol in astrocytes.