Abstract
BackgroundPhosphodiesterases (PDEs) break down cAMP, thereby regulating intracellular cAMP concentrations and diffusion. Since PDE4 predominates in airway epithelial cells, PDE4 inhibitors can stimulate Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by increasing cAMP. Tobacco smoking and COPD are associated with decreased CFTR function and impaired mucociliary clearance (MCC). However, the effects of the PDE4 inhibitor roflumilast on smoke-induced mucociliary dysfunction have not been fully explored.MethodsPrimary normal human bronchial epithelial cells (NHBE) from non-smokers, cultured at the air-liquid interface (ALI) were used for most experiments. Cultures were exposed to cigarette smoke in a Vitrocell VC-10 smoking robot. To evaluate the effect of roflumilast on intracellular cAMP concentrations, fluorescence resonance energy transfer (FRET) between CFP- and YFP-tagged protein kinase A (PKA) subunits was recorded. Airway surface liquid (ASL) was measured using light refraction scanning and ciliary beat frequency (CBF) employing infrared differential interference contrast microscopy. Chloride conductance was measured in Ussing chambers and CFTR expression was quantified with qPCR.ResultsWhile treatment with 100 nM roflumilast had little effect alone, it increased intracellular cAMP upon stimulation with forskolin and albuterol in cultures exposed to cigarette smoke and in control conditions. cAMP baselines were lower in smoke-exposed cells. Roflumilast prolonged cAMP increases in smoke-exposed and control cultures. Smoke-induced reduction in functional, albuterol-mediated chloride conductance through CFTR was improved by roflumilast. ASL volumes also increased in smoke-exposed cultures in the presence of roflumilast while it did not in its absence. Cigarette smoke exposure decreased CBF, an effect rescued with roflumilast, particularly when used together with the long-acting ß-mimetic formoterol. Roflumilast also enhanced forskolin-induced CBF stimulation in ASL volume supplemented smoked and control cells, confirming the direct stimulatory effect of rising cAMP on ciliary function. In active smokers, CFTR mRNA expression was increased compared to non-smokers and ex-smokers. Roflumilast also increased CFTR mRNA levels in cigarette-smoke exposed cell cultures.ConclusionsOur results show that roflumilast can rescue smoke-induced mucociliary dysfunction by reversing decreased CFTR activity, augmenting ASL volume, and stimulating CBF, the latter particularly in combination with formoterol. As expected, CFTR mRNA expression was not indicative of apical CFTR function.
Highlights
Phosphodiesterases (PDEs) break down cAMP, thereby regulating intracellular cAMP concentrations and diffusion
We found that roflumilast increased intracellular cAMP concentrations, improved cystic fibrosis transmembrane conductance regulator (CFTR) function and enhanced Ciliary beat frequency (CBF) in cigarette smoke-exposed normal human bronchial epithelial cells (NHBE) cells
Effect of roflumilast on intracellular cAMP To evaluate the effect of roflumilast on intracellular cAMP concentration ([cAMP]i), we used our previously described fluorescence resonance energy transfer (FRET) assay of fluorescently tagged protein kinase A (PKA) subunits [13] where changes in FRET ratio (ΔFRET ratio) are indicative of changes in [cAMP]i [13]
Summary
Phosphodiesterases (PDEs) break down cAMP, thereby regulating intracellular cAMP concentrations and diffusion. Since PDE4 predominates in airway epithelial cells, PDE4 inhibitors can stimulate Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by increasing cAMP. Tobacco smoking and COPD are associated with decreased CFTR function and impaired mucociliary clearance (MCC). The effects of the PDE4 inhibitor roflumilast on smoke-induced mucociliary dysfunction have not been fully explored. Two major determinants of effective MCC are directly regulated by cAMP: Ciliary beat frequency (CBF) [1] and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a major anion channel regulating airway surface liquid (ASL) volume [2]. The lack of effective drug therapy for chronic obstructive pulmonary disease (COPD) has increased the interest in PDE4 inhibition in recent years [5], as PDEs are highly expressed in airway epithelial cells, and in leukocytes and other inflammatory cells, which are involved in the pathogenesis of COPD [3]. Roflumilast, a PDE4 inhibitor with an IC50 of 0.6 nM [3], has been approved in the United States and Europe to reduce the risk of COPD exacerbations in patients with severe COPD associated with chronic bronchitis and a history of exacerbations [6]
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