A Pharmacologic Approach to Acquired Cystic Fibrosis Transmembrane Conductance Regulator Dysfunction in Smoking Related Lung Disease
Figure 1
Cigarette smoke reduces CFTR-dependent activity and expression.
(A) Representative tracings from primary human bronchial epithelial cells were grown at air-liquid interface until fully differentiated, followed by exposure to cigarette smoke extract (CSE 2%) on the apical surfaces for 24 h. Cells were then mounted in modified Ussing chambers and sequentially exposed to forskolin (20 µM) and CFTRInh-172 (10 µM) in the setting of amiloride (100 µM) and Cl–free gluconate on the apical compartment. (B–C) Summary data of experiments shown outlined in (A). Change in short-circuit current (Isc) following addition of forskolin is shown as a percentage of wild-type non-CF current and absolute Isc (B). Change following 2% CSE is also shown for CFTRInh-172 (10 µM, C). *P<0.05, **P<0.005, n = 4 per concentration. (D) Western blot of cell lysates of primary HBE cells shown in (A) harvested immediately after treatment in the Ussing chamber. CFTR bands B and C are shown by the black and white arrows, respectively. All lanes were normalized for protein concentration and no difference was observed in protein levels at baseline: vehicle control treated wells (1.79±0.37 mg/ml) vs. CSE treated wells (1.92±0.40 mg/ml; P = 0.81); α-tubulin is also shown as a loading control. This blot is representative of 3 similar experiments. (E) Densitometry of CFTR band C shown in (D). **P<0.01. (F) Surface CFTR expression was quantified by a cell surface biotinylation assay following CSE (2%) or vehicle control treatment for 24 hrs. Blot is representative of 3 similar experiments. (G) Densitometry of surface CFTR band shown in (F). *P<0.05. (H) Primary HBE cells were exposed to CSE (2%) 24 h, then RNA isolated, and quantitative RT-PCR performed in comparison to 18S RNA. *P<0.05, n = 9/condition. (I) cAMP concentration in HBE cells exposed to CSE (2%) or vehicle control for 24 h prior to assay. Equivalent forskolin concentration was calculated based on a standard curve in the same cells (See Fig. S2). *P<0.05.