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Chronic β2AR stimulation limits CFTR activation in human airway epithelia
John J. Brewington, … , L. Jason Lu, John P. Clancy
John J. Brewington, … , L. Jason Lu, John P. Clancy
Published February 22, 2018
Citation Information: JCI Insight. 2018;3(4):e93029. https://doi.org/10.1172/jci.insight.93029.
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Research Article Cell biology Pulmonology

Chronic β2AR stimulation limits CFTR activation in human airway epithelia

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Abstract

Traditional pulmonary therapies for cystic fibrosis (CF) target the downstream effects of CF transmembrane conductance regulator (CFTR) dysfunction (the cause of CF). Use of one such therapy, β-adrenergic bronchodilators (such as albuterol), is nearly universal for airway clearance. Conversely, novel modulator therapies restore function to select mutant CFTR proteins, offering a disease-modifying treatment. Recent trials of modulators targeting F508del-CFTR, the most common CFTR mutation, suggest that chronic β-agonist use may undermine clinical modulator benefits. We therefore sought to understand the impact of chronic or excess β-agonist exposure on CFTR activation in human airway epithelium. The present studies demonstrate a greater than 60% reduction in both wild-type and modulator-corrected F508del-CFTR activation following chronic exposure to short- and long-acting β-agonists. This reduction was due to reduced cellular generation of cAMP downstream of the β-2 adrenergic receptor–G protein complex. Our results point towards a posttranscriptional reduction in adenylyl cyclase function as the mechanism of impaired CFTR activation produced by prolonged β-agonist exposure. β-Agonist–induced CFTR dysfunction was sufficient to abrogate VX809/VX770 modulation of F508del-CFTR in vitro. Understanding the clinical relevance of our observations is critical for CF patients using these drugs, and for investigators to inform future CFTR modulator drug trials.

Authors

John J. Brewington, Jessica Backstrom, Amanda Feldman, Elizabeth L. Kramer, Jessica D. Moncivaiz, Alicia J. Ostmann, Xiaoting Zhu, L. Jason Lu, John P. Clancy

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Figure 8

Chronic albuterol-induced CFTR impairment is rescued with application of exogenous cAMP.

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Chronic albuterol-induced CFTR impairment is rescued with application of...
wtCFTR+ and VX809-corrected F508del-CFTR+ CFBE41o- cells were exposed to albuterol and/or VX809 in media for 72 hours, then mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. In contrast to previously demonstrated experiments, cell-permeant 8-bromoadenosine cAMP (8-Br cAMP) was used to stimulate CFTR function to bypass cellular mechanisms of cAMP generation. In wtCFTR+ cells (representative short-circuit current [Isc] tracing in A, aggregate data in B; n = 4 inserts/condition. Circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]), maximal stimulation with 8-Br cAMP in place of forskolin/IBMX rescues the previously demonstrated albuterol-induced CFTR dysfunction. Stimulation with increasing doses of 8-Br cAMP (C, n = 3 inserts/data point) revealed a small increase in CFTR function in albuterol-pretreated cells at low stimulation doses. Squares, control conditions; triangles, albuterol pretreated. Similarly, in VX809-corrected F508del-CFTR+ cells (representative Isc tracing in D, aggregate data in E; n = 4 inserts/condition), maximal stimulation with 8-Br cAMP rescues albuterol-induced CFTR dysfunction. Unlike wtCFTR+ cells, stimulation with increasing doses of 8-Br cAMP demonstrated no difference in VX809-corrected F508del-CFTR function following albuterol pretreatment at any stimulation dose (F, n = 3 inserts/data point. All indicators of statistical significance are comparisons with untreated cells; no difference was noted between VX809-corrected cells with or without albuterol). Circles, untreated; squares, VX809 alone; triangles, VX809 + albuterol. All studies are internally normalized as indicated to allow for comparisons. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (100 μM 8-Br cAMP; dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR+; 1 μM VX770 for F508del-CFTR+ cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). Both experiments are representative of studies repeated in duplicate with similar results. Data presented represent the mean ± SEM. *P < 0.05; ***P < 0.005; NS, non-significant by 2-way ANOVA with Tukey’s multiple comparisons test.

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