Acute regulation of the epithelial sodium channel in airway epithelia by proteases and trafficking

Abstract

Effective clearance of inhaled pathogens is the primary innate defense mechanism in the lung, and requires the maintenance of a proper airway surface liquid (ASL) volume to facilitate ciliary beat and optimize mucociliary clearance. Na(+) absorption via the epithelial sodium channel (ENaC) is tightly regulated and, together with chloride movement, provides the optimal osmotic gradients to absorb excessive fluid in the airway lumen while preventing excessive ASL dehydration, which would compromise mucus clearance from the lung. To absorb excessive fluid from the luminal surface, a local mechanism of ENaC activation allows for an increase in Na(+) absorption at times when the ASL volume is expanded. To help define these regulatory mechanisms, we examined the effects of ASL volume expansion on ENaC activity in primary human bronchial epithelial (HBE) cell cultures. We found that ENaC activity increases dramatically after rapid dilution of endogenous ASL. Approximately 35% of the increase in Na(+) absorption was attributable to activation of ENaC by proteases. The remainder of the increase in Na(+) current was prevented when membrane trafficking was disrupted with brefeldin A, nocodazole, or myosin light chain kinase inhibitors, demonstrating that trafficking is involved with ENaC regulation in the airway. These findings demonstrate that Na(+) absorption in the airway is acutely modulated by the coordinated trafficking of channels to the luminal surface and by the proteolytic activation of ENaC in response to ASL volume expansion.

Figure 1.

Na⁺ absorption increases after airway surface liquid (ASL) washout in an Ussing chamber. Human bronchial epithelial (HBE) cell cultures were cultured in DMEM/F12 ± bronchial epithelial growth medium (BEGM), 50 nM dexamethasone (Dex), or 50 nM aldosterone (Aldo) for 24 hours before short-circuit current (I_SC) recording. After a 30-minute equilibration, 10 μM of amiloride were added to the apical chamber to determine the amiloride-sensitive I_SC (I_Na). Subsequently, Cl⁻ secretion was assessed by adding 10 μM forskolin and then 100 μM bumetanide to the basolateral chamber. Data shown are (A) representative I_SC recordings, (B) mean amiloride-sensitive current (I_Na) ± SE, and (C) and mean t_1/2 ± SE of the I_Na increase from 0 to 30 minutes (n = 12 cultures from three tissue donors). USG, Ultroser G. *P < 0.001, different from control culture as determined by ANOVA with Bonferroni post hoc analysis.

Figure 2.

Effects of proteases and protease inhibitors on acute activation of epithelial sodium channel (ENaC) after ASL washout in an Ussing chamber. HBE cultures were mounted in an Ussing chamber containing apical Ringer's solution ± 300 nM elastase (Elast) or 10 μM aprotinin (Apro). After a 30-minute equilibration, 300 nM of elastase were added to the apical chamber. Data shown are (A) representative I_SC recordings, (B) mean amiloride-sensitive current normalized to the post-elastase amiloride-sensitive current (I_Na/I_Na(elastase)) ± SE, and (C) mean t_1/2 ± SE of the I_Na increase from 0 to 30 minutes (n = 8 cultures from two tissue donors). *P < 0.01, different from control culture as determined by ANOVA with Bonferroni post hoc analysis.

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 or without 10 μM aprotinin or 50 μM furin convertase inhibitor (FCI) for 16 hours before I_SC recording. After a 30-minute equilibration, 300 nM elastase and 10 μM trypsin were sequentially added apically. Data shown are (A) representative I_SC recordings, and (B and C) mean amiloride-sensitive current normalized to the post-trypsin amiloride-sensitive current (I_Na/I_Na(trypsin)) ± SE at 30 minutes, after elastase, and after trypsin for (B) control and (C) CF cultures (n = 12 cultures from three tissue donors). *P < 0.004, different from control culture as determined by ANOVA with Bonferroni post hoc analysis.

Figure 4.

Functional ENaC half-life in control and CF HBE cultures. (A) Differentiated non-CF and CF HBE cultures were treated with 200 μg/ml cycloheximide (as indicated by +), and I_SC was measured at 10-minute intervals. (B) Functional t_1/2 was calculated from the I_SC decay for each individual culture. Data shown are mean ± SE, n = 19–22 cultures from five tissue donors.

Figure 5.

Effect of trafficking inhibition, A2a receptor blockade, BAPTA-AM, cyclic adenosine monophosphate (cAMP), and cycloheximide (CHX) on acute activation of ENaC after ASL washout. Differentiated HBE cultures were exposed basolaterally to the indicated drug for 30 minutes before I_SC recording and in Ringer's solution bath during I_SC recording. (A) To determine the contribution of membrane trafficking during I_SC recording, 5 μg/ml brefeldin A (BFA), 10 μM nocodazole (Nocod), 100 μM ML-7, or 100 μM ML-9 were added to inhibit trafficking. (B) The effects of A2a (A2a), 10 μm forskolin (Forsk), 50 μM H-7, or 10 μM BAPTA-AM were also examined. After a 30-minute equilibration, 10 μM of amiloride were added to the apical chamber. Data shown are the mean amiloride-sensitive current normalized to the mean amiloride-sensitive current of matched control HBE cultures after the 30-minute equilibration (I_Na/I_Na(control)) ± SE at 0 minutes (black) and 30 minutes (gray) (n ≥ 6 cultures from ≥2 tissue donors). *P < 0.001, different from control culture as determined by ANOVA with Bonferroni post hoc analysis.

Figure 6.

Effect of apical osmolarity on I_Na. Representative I_SC tracings of differentiated HBE cultures during the addition (light gray bar) and removal of 100 mM (A) and 300 mM (B) mannitol. Amiloride was applied (black bar) at the end of the experiment to determine the I_Na. (C) I_Na relative to I_Na at 30 minutes during the various timeouts during the experiments with 100 mM mannitol (black bar) and 300 mM mannitol (gray bar). Data shown are mean ± SE amiloride-sensitive current normalized to the I_Na at 30 minutes (n = 5–11 HBE cultures). (D) Kinetics of I_Na changes associated with osmolarity shift induced by 100 mM mannitol (black bar) and 300 mM mannitol (gray bar). Data shown are mean ± SE t_1/2, as defined in Materials and Methods (n = 5–11 HBE cultures).

Figure 7.

Effect of hyperosmolarity and trafficking inhibitors on I_Na during ASL washout. Differentiated HBE cultures were incubated basolaterally, with and without 50 μg/mL vinblastine for 30 minutes before I_SC recording. The apical Ringer's solution during the initial portion of the I_SC trace was isotonic or contained an additional 100 μM mannitol. After the initial equilibration period, bathing solutions were exchanged with several washes of isotonic Ringer's solution. Amiloride was applied (black bar) at the end of the experiment to determine the I_Na. Data shown are (A) representative I_SC recordings, and (B) mean amiloride-sensitive current normalized to the amiloride-sensitive current of control HBE cells at 30 minutes (I_Na/I_Na(control)) ± SE at 0 minutes, at 30 minutes, and after the isotonic wash (n > 12 cultures from two tissue donors). *P < 0.01, different from control culture as determined by ANOVA with Bonferroni post hoc analysis.