Impaired PGE2-stimulated Cl- and HCO3- secretion contributes to cystic fibrosis airway disease

Abstract

Background: Airway mucociliary clearance (MCC) is an important defense mechanism against pulmonary infections and is compromised in cystic fibrosis (CF). Cl- and HCO3- epithelial transport are integral to MCC. During pulmonary infections prostaglandin E2 (PGE2) production is abundant.

Aim: To determine the effect of PGE2 on airway Cl- and HCO3- secretion and MCC in normal and CF airways.

Methods: We examined PGE2 stimulated MCC, Cl- and HCO3- secretion using ferret trachea, human bronchial epithelial cell cultures (CFBE41o- with wildtype CFTR (CFBE41 WT) or homozygous F508del CFTR (CFBE41 CF) and human normal bronchial submucosal gland cell line (Calu-3) in Ussing chambers with or without pH-stat.

Results: PGE2 stimulated MCC in a dose-dependent manner and was partially impaired by CFTRinh-172. PGE2-stimulated Cl- current in ferret trachea was partially inhibited by CFTRinh-172, with niflumic acid eliminating the residual current. CFBE41 WT cell monolayers produced a robust Cl- and HCO3- secretory response to PGE2, both of which were completely inhibited by CFTRinh-172. CFBE41 CF cells exhibited no response to PGE2. In Calu-3 cells, PGE2 stimulated Cl- and HCO3- secretion. Cl- secretion was partially inhibited by CFTRinh-172, with additional inhibition by niflumic acid. HCO3- secretion was completely inhibited by CFTRinh-172.

Conclusions: PGE2 stimulates bronchotracheal MCC and this response is decreased in CF. In CF airway, PGE2-stimulated Cl- and HCO3- conductance is impaired and may contribute to decreased MCC. There remains a CFTR-independent Cl- current in submucosal glands, which if exploited, could represent a means of improving airway Cl- secretion and MCC in CF.

Fig 1. PGE₂-stimulated mucociliary transport in ferret trachea.

A. PGE₂ stimulates a dose-dependent increase in MCC in ferret trachea. Each tissue was exposed to 2–3 doses of PGE₂ for 30 minutes each (n = 3 each dose). Data are shown as the mean PGE₂-stimulated increase in MCC over baseline ± SEM. The half-maximal effective concentration (EC₅₀) is noted in lower right corner. B. Timecourse of PGE₂-stimulated MCC with and without CFTR inhibition (n ≥ 6 each). For CFTR inhibition, tissues were bathed in apical and serosal solution for 30 minutes with CFTR_inh-172 (20 μM) prior to the 15-minute period and kept in the serosal bath for the length of the experiment. PGE₂ (1 μM) was added to the serosal bath. Circles represent means with bars indicating SEM. Asterisks represent P < 0.05 by ANOVA.

Fig 2. In ferret trachea, PGE₂ stimulated I_sc is mediated by CFTR and Ca²⁺-activated Cl^- channels.

A. Representative I_sc trace with vertical deflections indicating the change in I_sc after a 1 mV pulse was applied (every 1 minute). Ferret trachea was exposed to serosal to mucosal Cl^- gradient with equivalent bilateral HCO₃^-. PGE₂ (1 μM, serosal) was added to ferret trachea after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added after 30 minutes. B. Representative I_sc trace of ferret trachea incubated in CFTR_inh-172 (20 μM, mucosal) for at least 30 minutes prior to PGE₂ (1 μM, serosal) stimulation. After 30 minutes, niflumic acid (100 μM, mucosal) was added. C. Change in PGE₂-stimulated I_sc (mean ± SEM, n ≥ 5) in ferret trachea, with comparisons between no inhibition, CFTR inhibition, or CFTR and Ca²⁺-activated Cl^- inhibition. Asterisks denote significance by Student’s t-test (*, P < 0.05, **, P < 0.01). Mean percent inhibition compared to PGE₂ stimulation alone noted.

Fig 3. In human bronchial epithelial cells, PGE₂ stimulated Cl^- secretion is completely CFTR dependent.

A. Representative I_sc trace with vertical deflections indicating the change in I_sc after a 1 mV pulse was applied (every 1 minute). Bronchial epithelial cells were exposed to serosal to mucosal Cl^- gradient with equivalent bilateral HCO₃^-. PGE₂ (1 μM, serosal) was added to HBE41 WT cells after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added afterwards. To verify cell viability, ATP (500 μM, mucosal) was added. B. Representative I_sc trace from a similar experiment with CFBE41 CF cells. C. Change in PGE₂-stimulated I_sc (mean ± SEM, n ≥ 4) in CFBE41 WT and CF cells. Asterisks denote significance by Student’s t-test (***, P < 0.001). Mean percent inhibition compared to CFBE41 WT noted. D-F. PGE₂ stimulated Cl^- secretion in 16HBE14o- cells (D), primary cultures of human bronchial epithelial cells (E), and primary cultures from CF nasal polyp extract (F). Experiments were performed in the same manner as Fig 3A and representative I_sc traces are shown. N ≥ 3 experiments were performed for each set of cells with similar responses.

Fig 4. In Calu-3 cells, PGE₂ stimulated Cl^- secretion is mediated by CFTR and Ca²⁺-activated Cl^- channels.

A. Representative I_sc trace with vertical deflections indicating the change in I_sc after a 1 mV pulse was applied (every 1 minute). Calu-3 cells were exposed to serosal to mucosal Cl^- gradient with equivalent bilateral HCO₃^-. PGE₂ (1 μM, serosal) was added to Calu-3 cells after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added after 30 minutes. B. Representative I_sc trace of Calu-3 cells incubated in CFTR_inh-172 (20 μM, mucosal) for at least 30 minutes prior to PGE₂ (1 μM, serosal) stimulation. After 30 minutes, niflumic acid (100 μM, mucosal) was added. C. Change in PGE₂-stimulated I_sc (mean ± SEM, n ≥ 4) in Calu-3 cells, with comparisons between no inhibition, CFTR inhibition, or CFTR and Ca²⁺-activated Cl^- inhibition. Asterisks denote significance by Student’s t-test (*, P < 0.05). Mean percent inhibition compared to PGE₂ stimulation alone noted.

Fig 5. In CFBE41 cells, PGE₂ stimulated HCO₃^- secretion is completely CFTR dependent.

A. Representative I_sc trace with vertical deflections indicating the change in I_sc after a 1 mV pulse was applied (every 1 minute). CFBE41 WT cells were exposed to serosal to mucosal HCO₃^- gradient with equivalent bilateral Cl^-. PGE₂ (1 μM, serosal) was added to CFBE41 WT cells after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added afterwards. B. Representative I_sc trace from a similar experiment with CFBE41 CF cells. To verify cell viability, ATP (500 μM, mucosal) was added. C. Representative I_sc trace from a similar experiment as Fig 5A with CFBE41 WT cells, except experiments were performed in HCO₃^--free conditions. D. Change in PGE₂-stimulated I_sc (mean ± SEM, n = 3) in CFBE41 WT and CF cells in HCO₃^- containing and HCO₃^--free conditions. Asterisks denote significance by Student’s t-test (**, P < 0.01). Mean percent inhibition compared to CFBE41 WT noted.

Fig 6. In Calu-3 cells, PGE₂ stimulated HCO₃^- secretion is completely CFTR dependent.

A. Representative I_sc trace with vertical deflections indicating the change in I_sc after a 1 mV pulse was applied (every 1 minute). Calu-3 cells were exposed to serosal to mucosal HCO₃^- gradient with equivalent bilateral Cl^-. PGE₂ (1 μM, serosal) was added to Calu-3 cells after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added 30 minutes after. B. Representative I_sc trace from a similar experiment with Calu-3 cells in HCO₃^--free conditions. C. Change in PGE₂-stimulated I_sc (mean ± SEM, n = 3) in Calu-3 cells, with comparisons between no inhibition, CFTR inhibition, and HCO₃^--free conditions. Asterisks denote significance by Student’s t-test (**, P < 0.01, ***, P < 0.001). Mean percent inhibition compared to Calu-3 cells under control conditions. D. Timecourse of HCO₃^- secretion measured by pH-stat. The serosal solution was bathed with 95% O₂/5% CO₂ (similar to experiments in A-C), but the mucosal solution was bathed with 100% O₂ to prevent base formation from carbonhic anhydrase conversion of CO₂. Calu-3 cells were incubated in DMSO (5 μL; 1:1000 with bath; n = 10) or CFTR_inh-172 (20 μM, mucosal; n = 6) for 30–60 minutes prior to PGE₂ stimulation (1 μM, serosal). Circles represent means with bars indicating SEM. Asterisks represent P < 0.05 by ANOVA. E. Timecourse of I_sc measured by pH-stat measured simultaneously as pH-stat. Circles represent means with bars indicating SEM. Asterisks represent P < 0.05 by ANOVA.

Fig 7. In Calu-3 cells, PGE₂ stimulated HCO₃^- secretion is not affected by apical oubain, an inhibitor of the non-gastric H⁺/K⁺ ATPase.

Experiments were performed to determine the potential role of ATP12A in measured PGE₂-stimulated HCO₃^- secretion in normal and CF conditions. Calu-3 experiments were performed similar to that in Fig 6, with the exception that an additional set of experiments were done with oubain (10 μM, mucosal) pre-treatment for ≥ 40 minutes prior to PGE₂ stimulation. Bars represent change in PGE₂-stimulated I_sc (mean ± SEM, n ≥ 5) in Calu-3 cells. Statistical comparisons were done between PGE₂ with and without oubain and PGE₂ with CFTR inhibition with and without oubain. No statistical difference was noted in either case (P > 0.05 by Student’s t-test).

Fig 8. Simplified working model of PGE₂-stimulated Cl^- and HCO₃^- secretion and mucociliary clearance in non-CF and CF airway.

A. In the airway, microbial infections cause an increase in PGE₂ through release from infiltrating inflammatory cells (not pictured) and production by airway epithelia via COX-2 activation. H₂O₂ produced by DUOX activates COX-2 and HVCN1 channels provide the H⁺ shunt from H₂O₂ production. PGE₂ exits the cell and activates PGE₂ (EP) receptors. In the current study we did not examine specific EP receptor involvement, however, we propose that EP₄ is the predominant mediator of serosal PGE₂ stimulation in bronchial epithelial cells. Submucosal gland cells may also utilize the EP₃ receptor, or Ca²⁺-activated Cl^- channels (CaCC) may get activated via EP₄-mediated cAMP-Ca²⁺ crosstalk. In bronchial epithelial cells, PGE₂ stimulates Cl^- and HCO₃^- secretion via CFTR, whereas in submucosal glands, both CFTR and CaCC are activated. Cl^- and HCO₃^- secretion will then influence airway pH, mucus properties, hydration, and ultimately, mucociliary clearance. B. In CF airway, lack of CFTR-dependent Cl^- and HCO₃^- secretion in bronchial epithelial cells, coupled with no HCO₃^- secretion and decreased Cl^- secretion from submucosal glands, leads to an acidic airway pH, thick-adherent mucus, and decreased mucociliary clearance. This results in increased microbial infection and rampant inflammation, in part by increased PGE₂ production.