title: Sinupret activates CFTR and TMEM16A-dependent transepithelial chloride transport and improves indicators of mucociliary clearance.
abstract: BACKGROUND
  We 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.
  
  
  METHODS
  Primary 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 [Ca(2+)]i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.
  
  
  RESULTS
  Sinupret-mediated Cl- secretion [ΔISC(µA/cm(2))] 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 Ca(2+) 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.
  
  
  CONCLUSIONS
  Sinupret 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 Ca(2+)-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.
authors.affiliation: Departments of Surgery/Division of Otolaryngology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Shaoyan
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Departments of Surgery/Division of Otolaryngology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Daniel
authors.initials: D
authors.lastname: Skinner
authors.affiliation: Departments of Surgery/Division of Otolaryngology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Stephen Bradley
authors.initials: SB
authors.lastname: Hicks
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Mark O
authors.initials: MO
authors.lastname: Bevensee
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Sorscher
authors.affiliation: Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Lazrak
authors.affiliation: Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Sadis
authors.initials: S
authors.lastname: Matalon
authors.affiliation: Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Carmel M
authors.initials: CM
authors.lastname: McNicholas
authors.affiliation: Departments of Surgery/Division of Otolaryngology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Bradford A
authors.initials: BA
authors.lastname: Woodworth
mesh_terms: D000818:Animals; D000075369:Anoctamin-1; D002118:Calcium / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D002923:Cilia / Q000187:drug effects / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D017136:Ion Transport / Q000187:drug effects*; D051379:Mice; D009079:Mucociliary Clearance / Q000187:drug effects*; D009363:Neoplasm Proteins / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010936:Plant Extracts / Q000494:pharmacology*; D054730:Protein Interaction Domains and Motifs; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C476213:ANO1 protein, human; C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D018118:Chloride Channels; D002712:Chlorides; D009363:Neoplasm Proteins; D010936:Plant Extracts; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C028353:Sinupret; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium