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.

Keywords: CFTR; airway submucosal glands; bicarbonate secretion; cystic fibrosis.

Figure 1

Chamber schematic and characterization of the preparation. (A) Top and side views of the polypropylene chamber. The sample (30 μl) was covered with a layer of FC-77 (10 μl) and stirred continuously. (B) Chamber arranged for microtitration. Micromanipulators holding the pH electrode and nanoinjector are not shown. (C) Measurement of CO₂ efflux from the chamber under different conditions. An aliquot of 25 mM NaHCO₃ solution (30 μl) that had been equilibrated with 5% CO₂ was placed in the chamber and pH was measured at 1 min intervals. The red symbols show the increase in pH (circles) and decline in calculated CO₂ concentration (squares) when sample was left open to the air. The green symbols show the pH (circles) and calculated [CO₂] when sample was covered with 10 μl perfluorocarbon (FC-77). The blue symbols show the pH (circles) and calculated [CO₂] when the chamber was completely immersed in a beaker of paraffin oil. (D) Time course of pH responses during a series of injections of 1N HCl using a Drummond Nanoject II (9.24 nl delivered per injection). pH stabilized within 1 min after adding titrant to the sample of Calu-3 secretions (30μl).

Figure 2

Confirmation that the chamber approximates a closed system for CO₂/HCO⁻₃. (A) Microtitration curves obtained for pure solutions having sodium HCO⁻₃ concentrations between 10 and 50 mM. Unbuffered water is shown for comparison (dashed red line). Means ± SD, n = 3–5. (B) Reproducibility of microtitrations. A sample of 20 mM HCO⁻₃ solution was microtitrated with HCl, neutralized using NaOH, supplemented with 20 mM HCO⁻₃ by injection of concentrated HCO⁻₃ solution, and re-titrated. The second titration curve (open circles) was within the standard deviation of the initial curve. Means ± SD, n = 3–5. (C) Comparison of (Δ) microtitration curve for 10 mM NaHCO₃ solution when samples are covered by perfluorocarbon and () macrotitration of the same solution performed in a closed under paraffin oil. Also shown is a titration curve for water titrated with NaOH in an open system (, i.e., vigorously bubbled with air). The microtitration results resemble those obtained by macrotitration under paraffin oil (i.e., a closed system). (D) equilibrium buffer capacity calculated over a similar pH range from macroscopic titrations under open (, bubbled with air) vs. closed conditions (, under paraffin oil).

Figure 3

Titration curves for Calu-3 secretions. (A) Microtitration of fluid produced by parental Calu-3 cell monolayers under basal conditions (, treated with DMSO vehicle) and during stimulation by 10 μM forskolin (). Dotted line shows the control curve obtained with distilled water. HCl and NaOH were the titrants and were added in increments of 0.4 mM final concentration. Means ± SD, n = 4–5. (B,C) Mean buffer capacity β for fluid from unstimulated (DMSO) and forskolin (FSK) stimulated Calu-3 secretions, respectively, calculated from (A). (D) Effect of CFTR knockdown on the titration curves obtained under basal conditions (DMSO) and during forskolin stimulation (FSK). Curved arrows show the impact of CFTR on buffering of Calu-3 secretions.

Figure 4

Evidence for a non-bicarbonate buffer in Calu-3 fluid. (A) Forward and back titration curves for Calu-3 secretions. Samples were equilibrated with air for 1 h at pH < 4 after the forward titration to drive off CO₂. (B) Buffer capacity as a function of pH calculated from the forward () and back () titrations. Means ± SD; n = 3–5.

Figure 5

Microtitration curves for phosphate and albumin at relevant concentrations. (A) Curves obtained with 1 and 10 mM phosphate solutions. Dashed red line shows results with distilled water. (B) Microtitration curves obtained with albumin. Means ± SD; n = 4.

Figure 6

Lysozyme expression and buffer capacity in pure solutions. (A) Immunoblot probed with anti-lysozyme antibody in duplicate samples from control Calu-3 and Calu-3 CFTR KD secretions under basal conditions and during forskolin stimulation. (B) Quantitation of lysozyme by ELISA under each condition shown in (A). Data are means ± SD; n = 3. (C) Titration curves for lysozyme solutions (n = 4). Dotted line also shows the pH of distilled water as a reference (unbuffered). Some aliquots of lysozyme were desalted by filtration to remove sodium acetate.

Figure 7

Microtitration curves for porcine gastric mucins and simulation of Calu-3 fluid buffer capacity using individual components. (A) Buffering by different concentrations of mucin. (B) Simulation of Calu-3 by mixtures of PO⁼₄, HCO⁻₃, and albumin. Curved arrows show the effect of adding 10 mM HCO⁻₃, 10 mg/ml albumin, and raising the concentration of HCO⁻₃ to 25 mM. The gray rectangle represents the region between the stationary points of the buffer.