X-ray Microanalysis of Apical Fluid in Cystic Fibrosis Airway Epithelial Cell Lines

Abstract

The ionic composition of the fluid lining the airways (airway surface liquid, ASL) in healthy subjects and patients with cystic fibrosis (CF) has been a matter of controversy. It has been attempted to resolve conflicting theories by using cell cultures, but published results show a wide variety of values for the ionic concentrations in the apical fluid in these cultures. To investigate CFTR-mediated HCO 3 -conductance and the role of HCO 3 - in regulating ASL pH we determined the pH of the fluid covering the apical surface of airway epithelial cells. A normal (16HBE14o - ) and a CF (CFBE41o - ) bronchial epithelial cell line were grown on membrane inserts in both a liquid-liquid interface culture system for 7 days, and in an air-liquid interface culture system for one month. The elemental composition of the fluid covering the apical surface was determined by X-ray microanalysis of frozen-hydrated specimens, or by X-ray microanalysis of Sephadex beads that had been equilibrated with the apical fluid. Analysis showed that the apical fluid had a Na + and Cl -concentration of about 80-100 mM and thus was slightly hypotonic. The ionic concentrations were somewhat higher in air-liquid interface than in liquid-liquid interface cultures. The apical fluid in CF cells had significantly higher concentrations of Na and Cl than that in control cultures. In control cultures, the concentrations of Na and Cl in the apical fluid increased if glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR) was added to the apical medium. Exposing the cells to the metabolic inhibitor NaCN also resulted in a significant increase of the Na and Cl concentrations in the apical fluid. The results agree with the notion that these cell cultures are mainly absorptive cells, and that ion absorption by the CF cells is reduced compared to that in normal cells. The pH measurements of the fluid covering the apical part of cell cultures support the notion that bicarbonate ions may be transported by CFTR, and that this can be inhibited by specific CFTR inhibitors.

Composition of nasal airway surface liquid in cystic fibrosis and other airway diseases determined by X-ray microanalysis

“…In contrast to earlier papers (Hull et al, 1998;Knowles et al, 1997) we find, however, that CF patients have higher salt levels in their ASL than healthy controls. We have found a similar increase in ion concentrations in the ASL from transgenic CF mice, compared with those in control mice (Kozlova et al, 2005b) and in the apical fluid covering cultured airway epithelial cells of CF origin compared to the control cells (Kozlova et al, 2005c).…”

Acidic pH increases airway surface liquid viscosity in cystic fibrosis

“…Finding that noncovalent interactions influence τ ASL /τ saline and previous evidence that Ca 2+ may cross-link and stabilize mucins (59)(60)(61) led us to test the effect of Ca 2+ concentration. The Ca 2+ concentrations of human and porcine ASL are approximately 4 mM and approximately 1 to 2 mM, and they are similar in CF and non-CF ASL (62)(63)(64)(65).…”

ENaC, iNOS, mucins, and wound healing in cystic fibrosis airway epithelial and submucosal cells

“…Under the culture conditions used, these cells are polarised. Under other culture conditions (air–liquid interface), the surface epithelial cells can develop into ciliated cells (Kozlova et al, ), but in the present study, the cells were cultured in liquid medium only and did not develop cilia. The CFBE41o‐cells have in our hands a very low cAMP‐induced Cl − efflux and a low CFTR expression compared to the 16HBE14o‐cells (Andersson et al, ; Servetnyk et al, ).…”