The height of the ASL is determined by the net osmotic gradient established by Na+ absorption and Cl? secretion through apically located ion channels (1�C4). The epithelial sodium make it clear channel (ENaC), in conjunction with basolaterally located Na+/K+ ATPase, is thought to be the predominant means of Na+ absorption across the airway epithelium. ENaC activity in renal and colonic epithelia is dictated by both local and systemic stimuli, whereas ENaC activity in airway epithelium appears to be regulated primarily by local factors in the luminal environment (5). This localized regulation facilitates ENaC autoregulation to maintain an optimal ASL volume. Evidence to date indicates that proteolytic activation (6�C8), flow activation (9, 10), and cyclic nucleotides/purino-receptor regulation (11�C13) appear to be the predominant mechanisms that regulate airway ENaC under physiologic conditions, rather than responses to hormonal stimuli.

Previous work from our group and others indicates that a balance between the protease activity of membrane-tethered, channel-activating proteases (CAPs) and soluble protease inhibitors in the ASL modulates ENaC activity and therefore Na+ absorption across human bronchial epithelial (HBE) cells (6, 7). When the ASL volume is low, during steady-state and nonpathologic conditions, the concentration of soluble protease inhibitor is sufficient to minimize the constitutive activation of ENaC by CAPs. Conversely, when the ASL volume is expanded, the soluble protease inhibitors are diluted, allowing for CAP-mediated activation of ENaC.

In previous work, Na+ absorption in HBE cells under ASL volume expansion conditions was recognized to be greater than that found in control cultures with basal ASL volumes that were exposed to exogenous channel activating proteases (6). This suggested that alternative mechanisms, in addition to proteolytic activation, are present that significantly augment Na+ absorption in the airway in response to ASL expansion. Na+ absorption via the ENaC is enhanced through either an increase in the open probability (PO) of the channel or through an increase in channel number (n) (14). Although proteolytic processing, flow activation, and cyclic nucleotides/purino-receptor regulation are some mechanisms that alter the PO of the channel, the contribution Drug_discovery of ENaC trafficking on the regulation of Na+ absorption in the airway epithelium is unknown. Therefore, this study sought to determine the relative contributions of proteolytic processing and trafficking of ENaC in response to increases in the ASL volume in primary HBE cells.