Abstract
IntroductionWe have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl−) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.MethodsPrimary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl− transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca2+]i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.ResultsSinupret-mediated Cl− secretion [ΔISC(µA/cm2)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca2+ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl− secretion. Sinupret also enhanced CBF and ASL depth.ConclusionSinupret stimulates CBF, promotes transepithelial Cl− secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca2+-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl− secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.
Highlights
We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl2) secretion in vitro and in vivo
The purpose of the present study was to determine whether this bioflavonoid therapeutic activates ciliary beat frequency (CBF) and enhances airway surface liquid (ASL), and to investigate the underlying mechanism responsible for the Cl2 secretagogue activity of Sinupret
One of the goals of this study was to test whether Sinupret stimulated CFTR or calcium-activated Cl2 (TMEM16A) channels on the apical cell surface
Summary
We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl2) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, and investigate the mechanisms that underlie activity of this bioflavonoid. Dysfunctional mucociliary clearance (MCC) is a common pathophysiologic process that underlies inflammation and infection in many airway diseases, including chronic rhinosinusitis (CRS) [1,2]. Transport of the mucus layer within the airway surface liquid (ASL) covering respiratory epithelia is influenced by the transepithelial movement of ions, such as Cl2, and the rate at which cilia beat. Enhancing MCC represents an important therapeutic strategy for patients with active sinus disease (by accelerating clearance of bacterial pathogens), and as a preventative measure among individuals prone to developing recurrent infections (by minimizing the accumulation of inhaled particles, including allergens, pollutants, and debris) [4,5,6]. CRS is a prevalent respiratory illness affecting 16% of the U.S population and incurs an estimated aggregated cost of 8.6 billion dollars annually in healthcare expenditures [7,8,9]
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