Because of the paucity of reliable antibodies that detect the cleaved forms of endogenously expressed ENaC, it is difficult to determine which subunits are processed, and to what extent cleavage occurs when the ASL is expanded. However, aprotinin appears to inhibit CAPs that activate ENaC via cleavage of the �� subunit, because elastase, which only cleaves http://www.selleckchem.com/products/Gefitinib.html the �� subunit (18, 24), is capable of fully activating the channel in aprotinin-treated cultures. When additional proteases were inhibited with FCI, the single cleavage event caused by elastase was insufficient to restore full channel activity. Restoration of normal channel activity in FCI-treated cultures required the addition of trypsin, and it is unclear if this additional activation is attributable to further processing of the �� or �� subunit.

Recent work from Carattino and colleagues suggests that the relevant activating proteolysis occurs with double cleavage of the �� subunit and removal of the intervening inhibitory peptide, regardless of the degree to which �� is processed (30). It may be possible to address these questions biochemically in the future, when validated high-quality ENaC antibodies become available. Several lines of evidence suggest that ENaC trafficking contributes to the increased INa with ASL expansion. First, the addition of elastase to the apical bathing solution from the beginning of recording in the Ussing chamber demonstrates that the rate of INa activation after ASL expansion is unaltered by the presence of exogenous protease or protease inhibitor.

Because the rate of channel activation by elastase is more rapid than the rate of INa increase with ASL expansion, the addition of elastase to the apical surface would be expected to accelerate the ISC increase, if unprocessed channels were present in the apical membrane. We observed a lack of effect, suggesting AV-951 that channels residing in the apical membrane under basal conditions are fully processed, and that activation by CAPs is not rate-limiting. Furthermore, when endogenous CAPs were inhibited by aprotinin and the HBE cultures were subsequently exposed to elastase, a pool of protease-susceptible channels is evident after 30 minutes. This finding suggests that these elastase-activated channels represent uncleaved ENaCs that were delivered from an elastase-inaccessible subapical pool. Otherwise, they would have been activated when elastase was included at the beginning of the trace. Second, blocking trafficking though a variety of different means significantly attenuated the increase in INa after ASL expansion, suggesting the either ENaC or an accessory protein is trafficked with ASL expansion.