The buffer capacity of airway epithelial secretions

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.

Nominal carbonic anhydrase activity minimizes airway-surface liquid pH changes during breathing

“…The slow change in pH ASL was a result of low carbonic anhydrase activity of the ASL. These data are consistent with previous reports that ASL removed from epithelia has minimal carbonic anhydrase activity (Candiano et al 2007;Kim et al 2014). Thus, loss of carbonic anhydrase activity in the bulk ASL may represent an evolutionary selection to minimize dynamic changes in pH ASL in response to respiration.…”

“…The reaction CO 2 + H 2 O ↔ H + + HCO 3 – is catalyzed by carbonic anhydrase (Kim et al. ). Because the observed pH change is slow, we predicted that carbonic anhydrase activity is low within intact ASL.…”

“…; Kim et al. ). Thus, loss of carbonic anhydrase activity in the bulk ASL may represent an evolutionary selection to minimize dynamic changes in pH ASL in response to respiration.…”

CFTR, bicarbonate, and the pathophysiology of cystic fibrosis

“…Normal airway surface liquid (ASL) has a pH of around 7.0 (estimated bicarbonate concentration of approximately 10–20 mMol). A recent review describes the evidence that ASL pH is controlled by bicarbonate secretion via CFTR and other secreted proteins . CFTR in airway cells derived from a lung cancer cell line (Calu‐3) will secrete either bicarbonate or chloride, depending on the activity of basolateral membrane cotransporters .…”

Relationships among CFTR expression, HCO 3 − secretion, and host defense may inform gene- and cell-based cystic fibrosis therapies

“…The * indicates the difference from epithelia with 100% WT cells by repeated measures ANOVA, P < 0.05. epithelium are WT or overexpress CFTR, Cl − secretion rises to the level of WT epithelia (10)(11)(12)(13)(14). However, knowledge that CFTR conducts HCO 3 − (48), that HCO 3 − is the major pH buffer of ASL (49), and that HCO 3 − secretion plays a key role in airway host defense (31)(32)(33)(34)(35)50) led us to test the relationship between CFTR expression and HCO 3 − secretion. We found that, unlike Cl − secretion, HCO 3 − secretion was directly proportional to CFTR expression.…”