Type 1 equilibrative nucleoside transporter regulates ethanol drinking through accumbal N-methyl-D-aspartate receptor signaling

Background: Mice lacking type 1 equilibrative nucleoside transporter (ENT1^-/-) exhibit increased ethanol-preferring behavior compared with wild-type littermates. This phenotype of ENT1^-/- mice appears to be correlated with increased glutamate levels in the nucleus accumbens (NAc). However, little is known about the downstream consequences of increased glutamate signaling in the NAc. Methods: To investigate the significance of the deletion of ENT1 and its effect on glutamate signaling in the NAc, we employed microdialysis and iTRAQ proteomics. We validated altered proteins using Western blot analysis. We then examined the pharmacological effects of the inhibition of the N-methyl-D-aspartate (NMDA) glutamate receptor and protein kinase Cγ (PKCγ) on alcohol drinking in wild-type mice. In addition, we investigated in vivo cyclic adenosine monophosphate response element binding activity using cyclic adenosine monophosphate response element-β-galactosidase mice in an ENT1^-/- background. Results: We identified that NMDA glutamate receptor-mediated downregulation of intracellular PKCγ-neurogranin- calcium-calmodulin dependent protein kinase type II signaling is correlated with reduced cyclic adenosine monophosphate response element binding activity in ENT1^-/- mice. Inhibition of PKCγ promotes ethanol drinking in wild-type mice to levels similar to those of ENT1^-/- mice. In contrast, an NMDA glutamate receptor antagonist reduces ethanol drinking of ENT1^-/- mice. Conclusions: These findings demonstrate that the genetic deletion or pharmacological inhibition of ENT1 regulates NMDA glutamate receptor-mediated signaling in the NAc, which provides a molecular basis that underlies the ethanol-preferring behavior of ENT1^-/- mice.