A new role for bicarbonate in mucus formation

Abstract

The impact of small anions on the physical properties of gel-forming mucin has been almost overlooked relative to that of cations. Recently, based on the coincident abnormalities in HCO(3)(-) secretion and abnormal mucus formed in the hereditary disease cystic fibrosis (CF), HCO(3)(-) was hypothesized to be critical in the formation of normal mucus by virtue of its ability to sequester Ca(2+) from condensed mucins being discharged from cells. However, direct evidence of the impact of HCO(3)(-) on mucus properties is lacking. Herein, we demonstrate for the first time that mucin diffusivity (∼1/viscosity) increases as a function of [HCO(3)(-)]. Direct measurements of exocytosed mucin-swelling kinetics from airway cells showed that mucin diffusivity increases by ∼300% with 20 mM extracellular HCO(3)(-) concentration. Supporting data indicate that HCO(3)(-) reduces free Ca(2+) concentration and decreases the amount of Ca(2+) that remains associated with mucins. The results demonstrate that HCO(3)(-) enhances mucin swelling and hydration by reducing Ca(2+) cross-linking in mucins, thereby decreasing its viscosity and likely increasing its transportability. In addition, HCO(3)(-) can function as a Ca(2+) chelator like EGTA to disperse mucin aggregates. This study indicates that poor HCO(3)(-) availability in CF may explain why secreted mucus remains aggregated and more viscous in affected organs. These insights bear on not only the fundamental pathogenesis in CF, but also on the process of gel mucus formation and release in general.

Fig. 1.

Bicarbonate reduces the free Ca²⁺ ion concentration. A: incremental increases in HCO₃⁻ concentration apparently reduce free Ca²⁺ from HBSS medium (1.2 mM Ca²⁺). The data show 1) that free Ca²⁺ in normal isotonic extracellular media in the presence of physiological concentrations of HCO₃⁻ should be <0.1 mM, and 2) that the high concentrations of Ca²⁺ in the condensed mucin of exocytosed granules likely could be chelated by HCO₃⁻/CO₃²⁻ in the media. EGTA, used as a positive control, also decreases the level of free Ca²⁺ in normal HBSS medium (1.2 mM Ca²⁺; see inset). Each point corresponds to the mean ± SD (n ≥ 6). B: chloride ions have no effect on the free Ca²⁺ ion concentration in PBS solution. Each point corresponds to the mean ± SD (n = 5).

Fig. 2.

Bicarbonate can chelate the bound Ca²⁺ ions from mucus gels. Each data point corresponds to the mean ± SD (n ≥ 8). All bicarbonate treated groups are significantly different from the control (bicarbonate-free; **P < 0.005). The amount of bound Ca²⁺ ions was determined with inductively coupled plasma optical emission spectrometry (ICP-OES), and the total organic carbon was measured with a Total Organic Carbon Analyzer (TOC-V_CSH; Shimadzu).

Fig. 3.

Bicarbonate (20 and 50 mM) disperses aggregated mucus gels monitored with dynamic laser scattering. The average size of mucin gels at Ca²⁺ = 8.2 mM (●, n ≥ 4) increased to ∼9.4 μm within the initial 48 h of incubation and decreased on bicarbonate application (20 mM, ●, n ≥ 6; 50 mM, ◊, n ≥ 6). Addition of EGTA (positive control) also dispersed aggregated mucus gels (5 mM, ▵, n ≥ 7). The control sizes remained low (○, n ≥ 3) compared with treatment groups.

Fig. 4.

Mucus powders solubilized in 100 mM Cl⁻ or HCO₃⁻. The amount of filtered mucus retained on filters (0.45 μm) was more than double for the Cl⁻ solution (0.56 mg/ml) than the HCO₃⁻ solution (0.27 mg/ml; **P < 0.005).

Fig. 5.

Identification of secretory granules and measurement of mucin matrix expansion. A: a representative plot of the swelling kinetics (hydration) of mucin matrices secreted by A549 cells is shown. The data points were fitted with the characteristic first order kinetics equation (see materials and methods). The control (bicarbonate-free, ○) shows a lower rate of mucin matrix swelling compared with the swelling rate during bicarbonate exposure (140 mM HCO₃⁻, ●; 20 mM HCO₃⁻, ▴). B: digital images demonstrating mucin matrix expansion during exocytosis recorded by videomicroscopy are presented. Exocytosis was triggered by ionomycin. The arrows indicate the expanding mucin granule matrix for which radii are automatically measured by green circles generated by NIS-Elements (Nikon, Melville, NY). C: loading of quinacrine dye showed the presence of green secretory granules in the cytosol of A549 cells. The nucleus was stained blue with Hoechst dye.

Fig. 6.

Bicarbonate increases mucin diffusivity secreted from A549 cells. The increase of diffusivity suggests that HCO₃⁻ may chelate Ca²⁺ from 2 different pools (●). EGTA (5 and 10 mM, ○) also markedly increased the diffusivity and is shown as ○. Each point corresponds to the mean ± SD (n ≥ 6). D, diffusivity.