Next, a series of experiments was performed to confirm that the increase in INa observed during ASL washout is attributable to an increase in conductive sodium always find useful information absorption though ENaC. To ensure that the increase in INa did not reflect an artifact introduced by voltage clamping, HBE cultures were placed into the Ussing chamber, and the transepithelial electric potential difference (VT) was recorded under open-circuit conditions (as shown in Figure E1 in the online supplement). Because the increase in VT is similar to the increase in ISC over the 30-minute interval, the increase in current after placement in the Ussing chamber was unlikely to be the result of voltage clamping.

To determine whether the increase in INa after ASL washout was attributable to a change in apical membrane conductance or changes in Na+/K+ ATPase activity, we performed apical and basolateral permeabilization experiments (Figure E2). Interestingly, when the basolateral membrane was permeabilized with nystatin and an apical to basolateral sodium gradient was established, the increase in INa during ASL washout was diminished, suggesting that either an intact plasma membrane is required for the response, or that a change occurred in Na+/K+ ATPase activity during the experiment. Therefore, we directly determined whether Na+/K+ ATPase activity changes during the course of the ASL washout. After apical permeabilization with nystatin, a large increase in ISC was observed, demonstrating that apical membrane conductance constitutes the rate-limiting step in transepithelial Na+ absorption.

Because the ISC was abolished with the addition of basolateral ouabain, the peak ISC observed after apical permeabilization was assumed to reflect ATPase activity. No change in ATPase activity was evident during the course of ISC recording and ASL washout. Therefore, the increase in ISC associated with ASL washout is likely attributable to a change in ENaC activity, and not an artifact of voltage clamping or changes in Na+/K+ ATPase activity. Proteolytic Activation of ENaC in Response to ASL Washout Previous work suggested that the increase in Na+ absorption after ASL volume expansion is attributable to the dilution of soluble protease inhibitors and a subsequent proteolytic activation of ENaC’s by CAPs (6, 7). Therefore, we investigated the degree to which the proteolytic activation of ENaC contributes to the observed increase in INa after ASL washout in an Ussing chamber.

Matched HBE cultures were mounted in the Ussing chamber containing Ringer’s solution, with or without 300 nM elastase or 10 ��M aprotinin. This combination of CAP and protease inhibitor was selected because aprotinin was previously shown to inhibit Na+ currents, and elastase is not inhibited AV-951 by aprotinin (16�C18), allowing for the sequential addition of elastase without concerns about protease inhibition.