The aforementioned data suggest that the negative modulation of epithelial chloride secretion by ethanol is likely through the adenosine-adenosine receptor signaling pathway. Up to date, four different adenosine receptors have been identified: A1, A 2A , A 2B and A3 receptors [31] . In airway epithelial cells, A 2B AR predominantly regulate CFTR function [18] . Here, we wanted to examine if the same signaling pathway is involved in the alcohol-induced inhibition of CFTR-mediated chloride transport. To this end, alloxazine (50 mM), a commonly used A 2B AR specific blocker, was applied to the apical buffer after adenosine stimulation. The data ( Figure 4) demonstrate that ,75% adenosine-stimulated epithelial I SC was reduced by alloxazine, indicating that A 2B AR was largely responsible for the adenosinemediated chloride secretion. Figure 2 . Adenosine-stimulated short circuit current is chloride conductance. A) I SC of Calu-3 epithelia was measured by using the asymmetric chloride buffers. After stimulated with 100 mM of apical adenosine, the cells gave rise to I SC of ,75 mA/cm 2 . The I SC was blocked by ,58% by basolateral addition of bumetanide (100 mM), but not by acetazolamide (20 mM) or DNDS (100 mM). B) I SC of Calu-3 epithelia was measured with either asymmetric chloride buffers or symmetric chloride buffers. The adenosine-stimulated I SC with no chloride-gradient buffers was ,92% lower than that with chloride-gradient buffers. Significance of the difference was determined by Student's t-test (p,0.01, n = 5). doi:10.1371/journal.pone.0032112.g002 Figure 3 . Adenosine-stimulated transepithelial I SC was mediated by CFTR channel. Calu-3 epithelia were stimulated with 100 mM of apical adenosine, resulting in a significant increase in I SC above baseline. Such an adenosine-induced I SC was mostly absent in CFBE41o cells in which CFTR channel was dysfunctional or was significantly inhibited with 25 mM of CFTR inhibitor CFTR inh 172. Student's t-test was performed to determine the statistic significance (p,0.05, n = 5). doi:10.1371/journal.pone.0032112.g003