Modelling Dysregulated Na⁺ Absorption in Airway Epithelial Cells with Mucosal Nystatin Treatment

Abstract

In cystic fibrosis (CF), the absence of functional CFTR leads to dysregulated Na 1 absorption across airway epithelia. We established an in vitro model of dysregulated Na 1 absorption by treating polarized normal human bronchial epithelial cells (HBEs) with nystatin (Nys), a polyene antibiotic that enables monovalent cations to permeate biological membranes. Acute mucosal Nys produced a rapid increase in short circuit current (I sc ) that reflected increased transepithelial Na 1 absorption and required Na 1 /K 1 ATPase activity. The acute increase in I sc was associated with increased mucosal liquid absorption. Prolonged mucosal Nys treatment resulted in sustained Na 1 hyperabsorption, associated with increased mucosal liquid absorption in comparison with naïve (nontreated, kept under airliquid interface conditions) or vehicle-treated cultures. Nys treatment was not toxic. Increased lactate accumulation in Nys-treated culture media suggested a higher metabolic rate associated with the higher energy demand for Na 1 transport. After chronic Nys treatment, the increased I sc was rapidly lost when the cultures were mounted in Ussing chambers, indicating that Nys could be rapidly removed from the apical membrane. Importantly, chronic Nys treatment promoted sustained mucosal liquid depletion and caused mucus dehydration, compaction, and adhesion to the apical surface of Nys-treated cultures. We conclude that mucosal Nys treatment of HBEs provides a simple in vitro model to recapitulate the Na 1 and volume hyperabsorptive features of CF airway epithelia.

Airway Surface Liquid Volume Regulation Determines Different Airway Phenotypes in Liddle Compared with βENaC-overexpressing Mice

“…This hypothesis is consistent with recent data that suggest that bypassing the normal regulation of ENaC at low ASL volumes with an unregulated "exogenous channel" (e.g. nystatin) can produce a phenotype of ASL depletion and mucus adhesion to airway surfaces (64). In the mouse experiments described here and in human bronchial epithelial cells treated with mucosal nystatin (64), the persistence of the capacity to secrete Cl Ϫ is not sufficient to protect the hydration status of the airway surface.…”

“…nystatin) can produce a phenotype of ASL depletion and mucus adhesion to airway surfaces (64). In the mouse experiments described here and in human bronchial epithelial cells treated with mucosal nystatin (64), the persistence of the capacity to secrete Cl Ϫ is not sufficient to protect the hydration status of the airway surface. This result probably reflects the inability of airway epithelia to generate driving forces sufficient to secrete Cl Ϫ in the absence of ENaC inhibition (65).…”

A Multifunctional Chemical Probe for the Measurement of Local Micropolarity and Microviscosity in Mitochondria

“…This is consistent with a mitochondrial accumulation of Na + and water molecules as a result of an increase in the Na + influx through the nystatin pores with a simultaneous osmotically driven H 2 O influx. 34 Similar results were obtained with monensin. Small variations were observed with rotenone, artesunate and artemisinin.…”

Potentiator ivacaftor abrogates pharmacological correction of ΔF508 CFTR in cystic fibrosis

“…Nystatin, a polyene antibiotic that enables monovalent cations to permeate biological membranes and raise Na + /K + ATPase activity, did not produce significantly different I SC responses when applied to vehicle- or VX-770-treated HBE cultures (Fig. 6C, Table S10), suggesting intact Na + /K + ATPase activity (37). Measurements of intracellular concentrations of VX-809 and VX-770 by mass spectrometry confirmed the presence of these compounds in treated HBE cultures and indicated that cellular VX-809 concentrations were not affected by the presence of VX-770 (Fig.…”