Acute ethanol treatment modulates delta opioid receptors in N18TG2 cells

Background: The in vitro adaptive responses of delta opiate receptors (DOR) to chronic ethanol treatment have been wed documented. The acute effects o f ethanol on these receptors are not well characterized beyond its effect o n ligand binding. The aim of this study was to evaluate the acute effects o f clinically relevant concentrations of ethanol (50-200 mM) on the saturati on binding kinetics, receptor/ligand internalization, and agonist stimulati on of G-protein coupling in N18TG2 cells expressing the Flag epitope-tagged mouse DOR Methods: Confocal microscopy was used to localize Flag epitope-tagged DOR i n N18TG2 cells. Saturation binding assays at 4 degrees C and 37 degrees C w ere conducted in the absence or presence of ethanol on cells not pretreated or pretreated with ethanol for 30 min at 37 degrees C. Highly specific del ta agonist, DPDPE ([D-Pen(2), D-Pen(5)]enkephalin), was used in these studi es. The effect of ethanol on agonist stimulation of G-protein coupling was examined using [S-35]GTP gamma S (guanosine-5'-O-(3-thio)triphosphate) bind ing to membranes. Agonist-mediated receptor internalization was examined us ing flow cytometry of cells labeled with the antiserum directed against the Flag epitope, and the ligand internalization was examined using [H-3]DPDPE . Results: Ethanol decreased the binding of the agonist [H-3]DPDPE, and not t he antagonist [H-3]diprenorphine, in a dose-dependent manner. These effects were temperature-dependent. Ethanol reversibly inhibited agonist stimulati on of [S-35]GTP gamma S binding. In non-pretreated cells, ethanol decreased the rate of receptor/ligand internalization, but this effect was not seen in ethanol pretreated cells. Taken together, these results suggest ;that pr etreatment of N18TG2 cells with ethanol Induces compensatory mechanisms tha t allow the receptor to function efficiently in its presence. Conclusion: Acute ethanol decreased the binding, agonist-mediated functiona l coupling and receptor/ligand internalization in N18TG2 cells expressing e pitope-tagged DOR. In these cells, 30-min pretreatment with ethanol was suf ficient to reverse these effects.