To explore if ethanol affects adenosine-activated ion transport function of airway epithelium, we employed air-liquid interface cultures of Calu-3 cells, a system widely used to investigate airway epithelial electrophysiological properties [27, 28] . The cultured epithelia were exposed basolaterally for 24 hours to different concentrations of ethanol (0, 25, 50 and 100 mM) and adenosineinduced transepithelial ion transport was assessed by measuring I SC with an Ussing chamber apparatus. Two buffers with asymmetric chloride were applied: apical low chloride (10.4 mM) and basolateral high chloride (139.8 mM). After voltage clamp, sodium channels were blocked by apical amiloride (100 mM) and I SC was stimulated by apical addition of adenosine (100 mM). As shown (Fig. 1) , the ethanol exposure decreased adenosine-activated I SC in a dose-dependent manner. Significant differences were detected by Figure 1 . Effect of ethanol pre-exposure on adenosine-induced epithelial ion transport. Calu-3 cells were cultured at an air-liquid interface on membrane filters and basolaterally exposed to 0, 25, 50 and 100 mM of ethanol for 24 hours. These Calu-3 epithelia were placed in an Ussing chamber with asymmetrical buffers of chloride (apical 10.4 mM and basolateral 139.8 mM). Following voltage clamp, stable short circuit baselines were attained in 15 to 20 min. I SC was measured after blocking Na + channels with 100 mM of apical amiloride and stimulated with 100 mM of apical adenosine. Alterations in ion transport are expressed as difference in I SC from their baseline. Ethanol preexposure decreased 100 mM adenosine mediated epithelial ion transport in a dose-dependent manner. Asterisks indicate significant differences between groups by One-way ANOVA test (p,0.05, n = 5 for each condition). doi:10.1371/journal.pone.0032112.g001