(452) Synergistic Mucociliary Clearance by β-adrenergic + Cholinergic Agonists Involves ENaC Inhibition and Bicarbonate Secretion

Background: Mucociliary clearance (MCC) is a vital innate defense mechanism that is impaired in CF patients and animal CF models. Dysfunctional MCC contributes to airway inflammation and infection, which hasten the rate of lung function decline. Most people with CF benefit from highly effective CFTR modulators, but some mutations are unresponsive to currently available modulators. Furthermore, even CF patients who benefit from modulator therapy may still be unable to clear chronic pulmonary infections. Accordingly, CFTR-independent methods to improve dysfunctional MCC are needed for these patients. We previously discovered that the combination of low dose cholinergic with β-adrenergic agonists synergistically increased MCC velocity (MCCV) in ex vivo tracheal preparations from both ferrets and newborn piglets. Of critical importance, MCC was also significantly increased in tracheas from CF ferrets to a value ~55% of that in WT animals. The MCCV increases were produced without inducing airway narrowing (Joo, Sci Rep, 2021, 11, 18828). To further our preclinical work, we tested three hypotheses. We hypothesized that synergistic increases in MCCV by the combined agonists involve 1. ENaC inhibition. 2. Increased secretion of bicarbonate. 3. Additivity with CFTR modulators.

Methods: To test these hypotheses, we measured MCCV in excised newborn piglet tracheas with 10 µM formoterol (b-adrenergic agonist) + 0.3 µM methacholine (cholinergic agonist) ± 10 µM benzamil (ENaC inhibitor) using particle tracking. Bicarbonate secretion rates were measured in tracheal mucosa of Yucatan minipigs mounted in Ussing chambers using a pH-stat method with pH electrodes and automated titrators (Metrohm Titrando 902). To assess if the synergy agonists produced further improvements on CF tissues exposed to CFTR modulators, we used high speed digital microscopy to measure the effective diffusivity (D_eff in µm²/msec) of ~2µm fluorescent polystyrene spheres (0.1%, ThermoFisher) added to the apical surface fluid layer of human CF primary nasal cell cultures (F508del homozygote) grown at air-liquid interface conditions ± ETI (3 μM elexacaftor + 3 μM tezacaftor + 10 μM ivacaftor).

Results: 1. Baseline MCCV was increased ~6-fold by benzamil inhibition of ENaC, from 0.5 ± 0.7 to 3.0 ± 0.7 mm/min (P = 0.02, 4 piglets). However, when benzamil was present during synergistically stimulated MCCV, no further increase was seen, consistent with the hypothesis that ENaC was already inhibited by the synergy agonists. MCCV (in mm/min): synergy agonists, 13.9 ± 1.6 vs synergy agonists + benzamil, 14.0 ± 1.6 (P = 0.97, n = 4 piglets, each condition). 2. The synergy agonists increased bicarbonate secretion rates by about 83% (in µmol/cm²×hr): 0.6 ± 0.2 at baseline vs 1.1 ± 0.3 (n = 5, 3 pigs) with synergy agonists. 3. Particle diffusivity in CF primary nasal cell cultures showed: synergy agonists + ETI > synergy agonists > ETI > no treatment.

Conclusions: Results were consistent with our hypotheses. The combination of β adrenergic + low dose cholinergic agonists produces synergistic increases in MCCV by inhibiting ENaC and increasing HCO₃^- secretion; and appears to be at least additive to the effects induced by ETI modulator therapy.

Acknowledgements: Supported by CFF, CFRI and CFFT.

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