Airway surface liquid depth measured in ex vivo fragments of pig and human trachea: dependence on Na+ and Cl- channel function

• Published in: American journal of physiology. Lung cellular and molecular physiology Vol. 297; no. 6; pp. L1131 - L1140
• Main Authors: Song, Yuanlin, Namkung, Wan, Nielson, Dennis W, Lee, Jae-Woo, Finkbeiner, Walter E, Verkman, A. S
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 01.12.2009
• Subjects: Airway management; Amiloride - pharmacology; Animals; Cell Differentiation - drug effects; Cells; Cells, Cultured; Chloride Channels - metabolism; Cystic fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; Epithelial Cells - cytology; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Hogs; Human subjects; Humans; In Vitro Techniques; Nasal Mucosa - cytology; Nasal Mucosa - drug effects; Nasal Mucosa - metabolism; Perfluorocarbons; Porosity - drug effects; Reproducibility of Results; Sodium; Sodium Channels - metabolism; Surface Properties - drug effects; Sus scrofa; Swine; Trachea - cytology; Trachea - drug effects; Trachea - physiology
• ISSN: 1040-0605; 1522-1504; 1522-1504
• DOI: 10.1152/ajplung.00085.2009

Departments of 1 Anesthesia and Perioperative Care, ; 2 Medicine and Physiology, and ; 3 Pediatrics, University of California, San Francisco; and ; 4 Department of Pathology, San Francisco General Hospital, University of California, San Francisco, California Submitted 16 March 2009 ; accepted in final form 8 October 2009 The airway surface liquid (ASL) is the thin fluid layer lining the airways whose depth may be reduced in cystic fibrosis. Prior measurements of ASL depth have been made in airway epithelial cell cultures. Here, we established methodology to measure ASL depth to 1-µm accuracy in ex vivo fragments of freshly obtained human and pig tracheas. Airway fragments were mounted in chambers designed for perfusion of the basal surface and observation of the apical, fluorescently stained ASL by scanning confocal microscopy using a high numerical aperture lens immersed in perfluorocarbon. Measurement accuracy was verified using standards of specified fluid thickness. ASL depth in well-differentiated primary cultures of human nasal respiratory epithelium was 8.0 ± 0.5 µm (SE 10 cultures) under basal conditions, 8.4 ± 0.4 µm following ENaC inhibition by amiloride, and 14.5 ± 1.2 µm following CFTR stimulation by cAMP agonists. ASL depth in human trachea was 7.0 ± 0.7 µm under basal conditions, 11.0 ± 1.7 µm following amiloride, 17.0 ± 3.4 µm following cAMP agonists, and 7.1 ± 0.5 µm after CFTR inhibition. Similar results were found in pig trachea. This study provides the first direct measurements of ASL depth in intact human airways and indicates the involvement of ENaC sodium channels and CFTR chloride channels in determining ASL depth. We suggest that CF lung disease may be caused by the inability of CFTR-deficient airways to increase their ASL depth transiently following secretory stimuli that in non-CF airways produce transient increases in ASL depth. cystic fibrosis; fluorescence microscopy; cystic fibrosis transmembrane conductance regulator Address for reprint requests and other correspondence: A. S. Verkman, Univ. of California, San Francisco, Box 0521, HSE 1246, 513 Parnassus Ave., San Francisco, CA 94143-0521 (e-mail: alan.verkman{at}ucsf.edu ; http://www.ucsf.edu/verklab ).