Therefore, multiple cleavage events are required to activate the channel CHIR-258 fully when the endogenous pro-protein convertases are inhibited. Figure 3. Effect of aprotinin and a furin convertase inhibitor on the acute activation of ENaC in control and cystic fibrosis (CF) HBE cultures. HBE cells from control and CF tissue donors were cultured with apical Ringer’s solution and basolateral media, with … We and others previously reported that cystic fibrosis (CF) HBE cultures exhibit an imbalance between endogenous CAPs and protease inhibitors, leading to increased levels of proteolytic activation of ENaC in the CF airway (6, 7). Therefore, we tested the susceptibility of HBE cultures from CF tissue donors to protease inhibitors and exogenous CAPs.
As shown in Figures 3B and 3C, no differences were evident between CF and control HBE cultures in the response to protease inhibition or activation by aprotinin or FCI. Because these experiments were performed after overnight ASL washout, the lack of difference between CF and control HBE cultures suggests that the difference in proteolytic processing between CF and non-CF HBE cultures is only evident when cells are maintained on an air�Cliquid interface. ENaC Half-Life Is Similar between Non-CF and CF HBE Cultures Knight and colleagues previously showed that channels containing the Liddle’s mutations undergo increased proteolytic processing as the result of a prolonged membrane residency time (26). Therefore, we questioned whether mutant cystic fibrosis transmembrane conductance regulator (CFTR) prolongs the functional t1/2 of ENaC as a potential means by which CF HBE cells undergo excessive ENaC processing.
To determine the functional channel t1/2, CF and non-CF HBE cells were treated with CHX, and the ISC was monitored to determine the decay rate in channel activity, as shown in Figure 4. Interestingly, the increase in INa was not affected by treatment with CHX (Figure 5B), suggesting that new channel synthesis is not required for channel activation with ASL volume expansion. The ISC decreased after treatment with CHX, and the functional t1/2 was determined as described in Materials and Methods. Although variability was observed between tissue donors, no significant differences in ENaC t1/2 were observed between non-CF and CF HBE cultures (74.366 �� 8.539 minutes versus 87.442 �� 13.88 minutes, P = 0.82 according to Mann-Whitney rank sum test, n = 19�C22 cultures from five tissue donors). Based on these functional half-life decays, the CFTR genotype appears unlikely to affect the rate at which ENaC is degraded, and no differences Drug_discovery in ENaC activity were consistently observed between CF and non-CF HBE cultures when the ASL was expanded. Figure 4.