No abstract

Many epithelia, including the superficial epithelia of the airways, are thought to secrete “volume sensors,” which regulate the volume of the mucosal lining fluid. The epithelial Na + channel (ENaC) is often the rate limiting factor in fluid absorption, and must be cleaved by extracellular and/or intracellular proteases before it can conduct Na + and absorb excess mucosal liquid, a process that can be blocked by proteases inhibitors. In the airways, airway surface liquid dilution or removal activates ENaC. Therefore, we hypothesized that endogenous proteases are membrane-anchored, whereas endogenous proteolysis inhibitors are soluble and can function as airway surface liquid volume sensors to inhibit ENaC activity. Using a proteomic approach, we identified short palate, lung, and nasal epithelial clone (SPLUNC)1 as a candidate volume sensor. Recombinant SPLUNC1 inhibited ENaC activity in both human bronchial epithelial cultures and Xenopus oocytes. Knockdown of SPLUNC1 by shRNA resulted in a failure of bronchial epithelial cultures to regulate ENaC activity and airway surface liquid volume, which was restored by adding recombinant SPLUNC1 to the airway surface liquid. Despite being able to inhibit ENaC, recombinant SPLUNC1 had little effect on extracellular serine protease activity. However, SPLUNC1 specifically bound to ENaC, preventing its cleavage and activation by serine proteases. SPLUNC1 is highly expressed in the airways, as well as in colon and kidney. Thus, we propose that SPLUNC1 is secreted onto mucosal surfaces as a soluble volume sensor whose concentration and dilution can regulate ENaC activity and mucosal volumes, including that of airway surface liquid.

Normal airways homeostatically regulate the volume of airway surface liquid (ASL) through both cAMP-and Ca 2+ -dependent regulation of ion and water transport. In cystic fibrosis (CF), a genetic defect causes a lack of cAMP-regulated CFTR activity, leading to diminished Cl -and water secretion from airway epithelial cells and subsequent mucus plugging, which serves as the focus for infections. Females with CF exhibit reduced survival compared with males with CF, although the mechanisms underlying this sex-related disadvantage are unknown. Despite the lack of CFTR, CF airways retain a limited capability to regulate ASL volume, as breathing-induced ATP release activates salvage purinergic pathways that raise intracellular Ca 2+ concentration to stimulate an alternate pathway to Cl -secretion. We hypothesized that estrogen might affect this pathway by reducing the ability of airway epithelia to respond appropriately to nucleotides. We found that uridine triphosphate-mediated (UTP-mediated) Cl -secretion was reduced during the periovulatory estrogen maxima in both women with CF and normal, healthy women. Estrogen also inhibited Ca 2+ signaling and ASL volume homeostasis in non-CF and CF airway epithelia by attenuating Ca 2+ influx. This inhibition of Ca 2+ signaling was prevented and even potentiated by estrogen antagonists such as tamoxifen, suggesting that antiestrogens may be beneficial in the treatment of CF lung disease because they increase Cl -secretion in the airways.

Background: COPD is the 4th leading cause of death worldwide, yet little is known about its pathogenesis. Results: Exposure to cigarette smoke elevated intracellular Ca 2ϩ levels, which triggered CFTR internalization/inhibition. Conclusion: Cigarette smoke-induced Ca 2ϩ release and CFTR inhibition may be involved in COPD pathogenesis. Significance: Our data provide novel information regarding the underlying mechanism of cigarette smoke-induced lung injury.

Adenosine (ADO) is an extracellular signaling molecule that is an important regulator of innate lung defense. On binding ADO, the A2B receptor (A2BR) stimulates cAMP production to activate the CFTR Cl(-) channel, increase ciliary beating, and initiate cytokine secretion. We tested the hypothesis that CFTR served as a positive regulator of the A2BRs. We found that A2BR and CFTR coimmunoprecipitated. They also underwent ADO-dependent Förster resonance energy transfer (FRET), which increased from 5% in the absence of agonist to 18% with 100 μM ADO (EC₅₀ 1.7 μM), suggesting that they dynamically associate in the plasma membrane. In contrast, despite colocalization, no FRET was observed between CFTR and GAP43. The interaction between A2BR and CFTR had some specificity: A2BR-stimulated but not forskolin-stimulated cAMP production was ~50% greater in the presence of CFTR, due to a CFTR-dependent increase in plasma membrane A2BR levels. These CFTR-dependent increases in A2BR levels and cAMP production resulted in significantly enhanced ciliary beating and increased cytokine secretion in normal compared to cystic fibrosis airway epithelia. Thus, we hypothesize that CFTR regulates A2BR levels in the plasma membrane to modulate cell signaling and to enhance selective components of the innate lung defense system.