Exposure to cigarette smoke condensate reduces calcium activated chloride channel transport in primary sinonasal epithelial cultures

Abstract

Objectives/hypothesis: The cystic fibrosis transmembrane conductance regulator (CFTR) serves as a predominant Cl(-) transport conduit in airway epithelium and is inhibited by cigarette smoke in vitro and in vivo. Activation of secondary Cl(-) transport pathways through calcium-activated Cl(-) channels (CaCC) has been postulated as a mechanism to bypass defects in CFTR-mediated transport. Because it is not known whether CaCCs are also inhibited by tobacco exposure, the current study was designed to investigate the effect of cigarette smoke condensate (CSC) on CaCC transport.

Study design: In vitro study.

Methods: Well-characterized primary murine nasal septal epithelial (MNSE) and human sinonasal epithelial (HSNE) cultures were exposed to CSC in Ussing chambers. We monitored CaCC short-circuit current through stimulation of P2Y purinergic receptors with uridine triphosphate or adenosine triphosphate and selective inhibition of the CFTR-dependent secretory pathway. Characterization of CaCC current was also accomplished in primary airway cells derived from transgenic CFTR(-/-) (knockout) murine models.

Results: Change in CaCC-mediated current (DeltaI(SC) representing transepithelial Ca-mediated Cl(-) secretion in muA/cm(2)) was significantly decreased in CSC-exposed wild type MNSE when compared to controls (32.8 +/- 4.6 vs. 47.5 +/- 2.3; respectively; P < .02). A similar effect was demonstrated in CFTR(-/-) MNSE cultures (33.4 +/- 2.8 vs. 38.6 +/- 2.0; P < .05>. HSNE cultures also had a significant reduction in I(SC) (16.1 +/- 0.6 vs. 22.7 +/- 0; P = .008).

Conclusions: CSC affects multiple pathways fundamental to airway ion transport, including both cyclic adenosine monophosphate and calcium activated Cl(-) transport. Inhibition of Cl(-) transport may contribute to common diseases of the airways, such as chronic rhinosinusitis and chronic obstructive pulmonary disease.

Figure 1

The change in CaCC-stimulated current (ΔI_SC) in wild type MNSE, CFTR knockout MNSE, and HSNE cultures was significantly decreased when exposed to CSC in comparison to DMSO controls.

Figure 2

Representative I_SC tracing of wild type MNSE cultures. Wild type MNSE cells grown on transwell permeable supports were mounted in modified Ussing chambers under short-circuit conditions and sequentially exposed to amiloride, INH-172, CSC or DMSO control, ADA, ATP, and niflumic acid. Note no changes in Cl⁻ dependent I_SC (after amiloride) until the application of ATP. Either DMSO control (upper tracing) or 200 µg/ml of CSC was added to the apical surface at the start of the experiment. Decreased ATP-stimulated CaCC I_SC was observed in the upper tracing (arrows). Niflumic acid (NA) inhibition was considered unreliable due to non-specificity and variable results.

Figure 3

Representative I_SC tracing of CFTR^−/− MNSE cultures. Cultures were sequentially exposed to amiloride, CSC or DMSO control, UTP, niflumic acid, and forskolin. Decreased ATP-stimulated CaCC I_SC was observed in the upper tracing (arrows). The addition of forskolin confirms lack of CFTR in these transgenic mice.