Abstract
The pH of airway epithelial secretions influences bacterial killing and mucus properties and is reduced by acidic pollutants, gastric reflux, and respiratory diseases such as cystic fibrosis (CF). The effect of acute acid loads depends on buffer capacity, however the buffering of airway secretions has not been well characterized. In this work we develop a method for titrating micro-scale (30 μl) volumes and use it to study fluid secreted by the human airway epithelial cell line Calu-3, a widely used model for submucosal gland serous cells. Microtitration curves revealed that HCO−3 is the major buffer. Peak buffer capacity (β) increased from 17 to 28 mM/pH during forskolin stimulation, and was reduced by >50% in fluid secreted by cystic fibrosis transmembrane conductance regulator (CFTR)-deficient Calu-3 monolayers, confirming an important role of CFTR in HCO−3 secretion. Back-titration with NaOH revealed non-volatile buffer capacity due to proteins synthesized and released by the epithelial cells. Lysozyme and mucin concentrations were too low to buffer Calu-3 fluid significantly, however model titrations of porcine gastric mucins at concentrations near the sol-gel transition suggest that mucins may contribute to the buffer capacity of ASL in vivo. We conclude that CFTR-dependent HCO−3 secretion and epithelially-derived proteins are the predominant buffers in Calu-3 secretions.
Highlights
Healthy airway epithelium is covered by a microscopic layer of airway surface liquid (ASL) that is overlaid with patches of mucus
Acidification of the ASL occurs in cystic fibrosis (CF) and other inflammatory diseases including asthma (Hunt et al, 2000), chronic obstructive pulmonary disease (COPD; Kostikas et al, 2002), and acute respiratory distress syndrome (Gessner et al, 2003)
Parental Calu-3 cells were cultured in Eagle’s minimum essential medium (EMEM) containing 15% fetal bovine serum (FBS), 1 mM sodium pyruvate and the non-essential amino acids glycine and L-isomers of alanine, asparagine aspartate, glutamate, proline, and serine at the concentrations normally used in MEM (Gibco, Burlington ON)
Summary
Healthy airway epithelium is covered by a microscopic layer of airway surface liquid (ASL) that is overlaid with patches of mucus. Acidification increases mucus viscosity (Holma, 1989), nitric oxide levels (Gaston et al, 2006) and sodium channel activity (Garland et al, 2013), reduces ciliary beat frequency (ClaryMeinesz et al, 1998) and bacterial killing (Pezzulo et al, 2012), and may trigger bronchoconstriction and cough (Kollarik et al, 2007). These derangements emphasize the importance of pH in this microscopic compartment
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