Calu-3 cells, cultured at the air-liquid interface for 2 weeks, were exposed to different concentrations of ethanol for 24 hours and mounted on an Ussing chamber apparatus (World Precision Instruments, Sarasota, FL). The cells were bathed with an apical low chloride buffer (135 mM sodium gluconate, 5.0 mM HEPES, 1.2 mM MgCl 2 , 0.6 mM KH 2 PO 4 , 4 mM CaCl 2 , 2.4 mM K 2 HPO 4 3H 2 O, and 10 mM Dextrose, pH 7.4) and with a basal high chloride buffer (135 mM NaCl, 5.0 mM HEPES, 1.2 mM MgCl 2 , 0.6 mM KH 2 PO 4 , 1.2 mM CaCl 2 , 2.4 mM, K 2 HPO 4 3H 2 O, and 10 mM Dextrose, pH 7.4). Both buffers were continuously stirred, gassed with 95% O 2 and 5% CO 2 and maintained at 37uC. These buffers were to maintain a chloride gradient across the epithelial monolayer. The TEER was measured with an open circuit by applying an electrical pulse across the epithelial monolayer. Short circuit currents (I SC ) were measured by applying an epithelial voltage clamp. For all the experiments, 100 mM of amiloride was added to the apical side to block sodium channels. Anion currents were induced by apical addition of 100 mM adenosine. The A 2B AR blockade was achieved by apical application of 50 mM alloxazine. To differentiate between chloride and bicarbonate currents, 100 mM bumetanide was used basally to block chloride transport. Acetazolamide (20 mM) or DNDS (4,49dinitro stilbene-2,29-disulfonate) (100 mM) was employed apically to block bicarbonate transport. Further, to inhibit epithelial phosphodiesterases, 100 mM of IBMX (3-isobutyl-1-methylxanthine) or 50 mM papaverine was added apically.