In ferret trachea, PGE₂ stimulated <i>I</i>_<i>sc</i> is mediated by CFTR and Ca²⁺-activated Cl^- channels.

<p><b>A.</b> Representative <i>I</i>_<i>sc</i> trace with vertical deflections indicating the change in <i>I</i>_<i>sc</i> 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>B.</b> Representative <i>I</i>_<i>sc</i> 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. <b>C.</b> Change in PGE₂-stimulated <i>I</i>_<i>sc</i> (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.</p

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

<p><b>A.</b> In the airway, microbial infections cause an increase in PGE₂ through release from infiltrating inflammatory cells (not pictured) and production by airway epithelia <i>via</i> 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 <i>via</i> EP₄-mediated cAMP-Ca²⁺ crosstalk. In bronchial epithelial cells, PGE₂ stimulates Cl^- and HCO₃^- secretion <i>via</i> 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>B.</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.</p

PGE₂-stimulated mucociliary transport in ferret trachea.

<p><b>A.</b> 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>B.</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.</p

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

<p><b>A.</b> Representative <i>I</i>_<i>sc</i> trace with vertical deflections indicating the change in <i>I</i>_<i>sc</i> 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>B.</b> Representative <i>I</i>_<i>sc</i> 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. <b>C.</b> Change in PGE₂-stimulated <i>I</i>_<i>sc</i> (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.</p

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

<p><b>A.</b> Representative <i>I</i>_<i>sc</i> trace with vertical deflections indicating the change in <i>I</i>_<i>sc</i> 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>B.</b> Representative <i>I</i>_<i>sc</i> trace from a similar experiment with CFBE41 CF cells. To verify cell viability, ATP (500 μM, mucosal) was added. <b>C.</b> Representative <i>I</i>_<i>sc</i> trace from a similar experiment as Fig 5A with CFBE41 WT cells, except experiments were performed in HCO₃^--free conditions. <b>D.</b> Change in PGE₂-stimulated <i>I</i>_<i>sc</i> (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.</p

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

<p><b>A.</b> Representative <i>I</i>_<i>sc</i> trace with vertical deflections indicating the change in <i>I</i>_<i>sc</i> 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>B.</b> Representative <i>I</i>_<i>sc</i> trace from a similar experiment with Calu-3 cells in HCO₃^--free conditions. <b>C.</b> Change in PGE₂-stimulated <i>I</i>_<i>sc</i> (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. <b>D.</b> 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. <b>E.</b> Timecourse of <i>I</i>_<i>sc</i> measured by pH-stat measured simultaneously as pH-stat. Circles represent means with bars indicating SEM. Asterisks represent P < 0.05 by ANOVA.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189894#pone.0189894.g006" target="_blank">Fig 6</a>, 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>I</i>_<i>sc</i> (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).</p