The purpose of this study was to identify Cl À channels in the basolateral membrane of airway epithelial cells at the molecular level. We have focused on a new family of Cl À channels, bestrophins, which have previously been identified in retinal pigment epithelium. RT-PCR, Western blot and confocal microscopy studies revealed the presence of bestrophin in airway epithelial cells. Decreasing bestrophin expression using siRNA resulted in diminished 36 Cl À flux. These studies also showed that bestrophin regulation is similar to that of native basolateral Cl À channels. The data indicate that the presence of a functional bestrophin may contribute to the basolateral cell conductance in airway epithelial cells.

The presence of basolateral Cl(-) channels in airway epithelium has been reported in several studies, but little is known about their role in the regulation of anion secretion. The purpose of this study was to characterize regulation of these channels by nitric oxide (NO) in Calu-3 cells. Transepithelial measurements revealed that NO donors activated a basolateral Cl(-) conductance sensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and anthracene-9-carboxylic acid. Apical membrane permeabilization studies confirmed the basolateral localization of NO-activated Cl(-) channels. Experiments using 8-bromo cyclic guanosine monophosphate (8Br-cGMP) and selective inhibitors of soluble guanylyl cyclase and inducible NO synthase (1H-[1, 2, 4] oxadiazolol-[4, 3-a] quinoxalin-1-one [ODQ] and 1400W [N-(3-Aminomethyl)benzyl)acetamidine], respectively) demonstrated that NO activated Cl(-) channels via a cGMP-dependent pathway. Anion replacement and (36)Cl(-) flux studies showed that NO affected both Cl(-) and HCO (3) (-) secretion. Two different types of Cl(-) channels are known to be present in the basolateral membrane of epithelial cells: Zn(2+)-sensitive ClC-2 and DIDS-sensitive bestrophin channels. S-Nitrosoglutathione (GSNO) activated Cl(-) conductance in the presence of Zn(2+) ions, indicating that ClC-2 channel function was not affected by GSNO. In contrast, DIDS completely inhibited GSNO-activated Cl(-) conductance. Bestrophin immunoprecipitation studies showed that under control conditions bestrophin channels were not phosphorylated but became phosphorylated after GSNO treatment. The presence of bestrophin in airway epithelia was confirmed using immunohistochemistry. We conclude that basolateral Cl(-) channels play a major role in the NO-dependent regulation of anion secretion in Calu-3 cells.

Our data suggest that, in addition to their role in IFN-gamma-mediated inhibition of allergic airway inflammation, products of tryptophan catabolism play an important role in the prevention of sensitization to potential allergens in the respiratory airway.

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