Sensitivity of Chloride Efflux vs. Transepithelial Measurements in Mixed CF and Normal Airway Epithelial Cell PopulationsIllek B. · Lei D. · Fischer H. · Gruenert D.C.
1Children’s Hospital Oakland Research Institute, Oakland,2Novartis Corporation, Emeryville,3Departments of Otolaryngology-Head and Neck Surgery and Laboratory Medicine, Helen Diller Family Comprehensive Cancer Center, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco,4Department of Medicine, University of Vermont, Burlington
Background/Aims: While the Cl- efflux assays are relatively straightforward, their ability to assess the efficacy of phenotypic correction in cystic fibrosis (CF) tissue or cells may be limited. Accurate assessment of therapeutic efficacy, i.e., correlating wild type CF transmembrane conductance regulator (CFTR) levels with phenotypic correction in tissue or individual cells, requires a sensitive assay. Methods: Radioactive chloride (36Cl) efflux was compared to Ussing chamber analysis for measuring cAMP-dependent Cl- transport in mixtures of human normal (16HBE14o-) and cystic fibrosis (CF) (CFTE29o- or CFBE41o-, respectively) airway epithelial cells. Cell mixtures with decreasing amounts of 16HBE14o- cells were evaluated. Results: Efflux and Ussing chamber studies on mixed populations of normal and CF airway epithelial cells showed that, as the number of CF cells within the population was progressively increased, the cAMP-dependent Cl- decreased. The 36Cl efflux assay was effective for measuring Cl- transport when ≥ 25% of the cells were normal. If < 25% of the cells were phenotypically wild-type (wt), the 36Cl efflux assay was no longer reliable. Polarized CFBE41o- cells, also homozygous for the ΔF508 mutation, were used in the Ussing chamber studies. Ussing analysis detected cAMP-dependent Cl- currents in mixtures with ≥1% wild-type cells indicating that Ussing analysis is more sensitive than 36Cl efflux analysis for detection of functional CFTR. Conclusions: Assessment of CFTR function by Ussing analysis is more sensitive than 36Cl efflux analysis. Ussing analysis indicates that cell mixtures containing 10% 16HBE14o- cells showed 40-50% of normal cAMP-dependent Cl- transport that drops off exponentially between 10-1% wild-type cells.
Dieter C. Gruenert, PhD
Department of Otolaryngology-Head and Neck Surgery, Head and Neck Cancer Lab
Mt Zion Cancer Center, University of California, San Francisco
Box 1330, 2340 Sutter St, N319, San Francisco, CA 94115-1330 (USA)
Tel. +1 415-476-8586, Fax +1 415-476-8588, E-Mail email@example.com
Accepted: November 15, 2010
Published online: January 04, 2011
Number of Print Pages : 8
Cellular Physiology and Biochemistry (International Journal of Experimental Cellular Physiology, Biochemistry andPharmacology)
Vol. 26, No. 6, Year 2010 (Cover Date: 2010)
Journal Editor: F. Lang, Tübingen
ISSN: 1015–8987 (Print), eISSN: 1421–9778 (Online)
For additional information: http://www.karger.com/journals/cpb