Abstract
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A new role for bicarbonate in mucus formation
1Abstract
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 HCO3− secretion and abnormal mucus formed in the hereditary disease cystic fibrosis (CF), HCO3− was hypothesized to be critical in the formation of normal mucus by virtue of its ability to sequester Ca2+ from condensed mucins being discharged from cells. However, direct evidence of the impact of HCO3− on mucus properties is lacking. Herein, we demonstrate for the first time that mucin diffusivity (~1/viscosity) increases as a function of [HCO3−]. Direct measurements of exocytosed mucin-swelling kinetics from airway cells showed that mucin diffusivity increases by ~300% with 20 mM extracellular HCO3− concentration. Supporting data indicate that HCO3− reduces free Ca2+ concentration and decreases the amount of Ca2+ that remains associated with mucins. The results demonstrate that HCO3− enhances mucin swelling and hydration by reducing Ca2+ cross-linking in mucins, thereby decreasing its viscosity and likely increasing its transportability. In addition, HCO3− can function as a Ca2+ chelator like EGTA to disperse mucin aggregates. This study indicates that poor HCO3− 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.
rarely do biology and medicine instruct the physical and chemical sciences, but in one instance a genetic disease may offer new insights into the physical chemistry of forming polymer gels. During gel mucin exocytosis, secreted mucin networks undergo a typical polymer gel phase transition in which the volume may change as much as 1,000-fold within seconds (46). Because of the polyanionic nature of the molecular network of mucins, this decondensation of the mucin matrix, driven by a Donnan potential, is considered to be triggered by extracellular Na+ exchanging for cross-linking Ca2+ ions (44, 46). Removing or chelating Ca2+ ions results in the rapid swelling, hydration, and dispersion of mucin networks into the extracellular space. Mobile anions in free solution have not been thought to play a significant role in the process. However, cystic fibrosis (CF), a life-shortening inherited disease of electrolyte transport, suggests a critical role for the free solution anion (HCO3−) as a Ca2+ chelator in the process of rapid mucin gel swelling (35, 37).
The pathogenesis in CF has been well-established to result from the formation of abnormal mucus that does not clear properly from the lungs, intestine, and most exocrine glands (6, 32). Various ion transport imbalances have been associated with CF pathology (8, 24, 25, 38, 52). Several cation-centered models (Na+, Ca2+, and H+) have been proposed to explain the pathogenic mucus (3, 24, 27, 48). Tangled Ca2+-cross-linked polyanionic polymer networks, like the matrix of mucus, exhibit unique Na+/Ca2+ ion exchange properties that can drastically control mucus swelling equilibrium (16, 48, 49) and thereby the viscoelastic properties and rheology of the mucus gel (14, 46). Thus abnormal fluid absorption, failure to reabsorb or chelate Ca2+, and low pH have been proposed to interfere with mucus swelling, leading to defective mucus associated with CF (3, 24, 27, 48).
The most commonly held notion maintains that CF mucus is thick and viscous due to an exaggerated Na+-dependent fluid absorption that dehydrates secreted mucus (7). Whereas this effect may seem consistent with manifestations of the disease in the lungs, it is very difficult to extend this rationale to other affected organs where fluid absorption is not present. Thus an explanation for the mucus pathology remains perplexing and controversial. Even though HCO3− secretion is apparently impaired in organs affected in CF, and the severity of the disease seems to correlate with the degree of this impairment (11, 35–37), the possible involvement of this anion with mucus formation has received little attention (35, 37). Evidence that defective HCO3− secretion is associated with abnormal mucus hydration has been recently reported (18, 29, 35). Furthermore, activating peroxisome proliferator-activated receptor-γ (PPAR-γ) in CF mice was found to ameliorate the disease by normalizing defects in both HCO3− secretion and mucus retention (19). Thus defective HCO3− secretion could be the possible etiological feature responsible for viscous, unswollen mucus in CF. Nonetheless, evidence that HCO3− directly affects mucin swelling and thereby its rheological properties by chelating Ca2+ ions has not been experimentally demonstrated until now.
The polymer network of mucus has a characteristic tangled topology (49). Here, we utilized the fact that rheological properties are governed mainly by the tangle density of mucin polymers, which decreases with the square of the volume of the mucin matrix, i.e., the degree of swelling (hydration) critically dictates mucus rheological properties (46). The diffusivity of mucin matrices, which is closely related to mucin viscosity (13, 14, 26, 40, 45, 46), can be calculated from polymer-swelling kinetics. The rapid swelling of a mucin matrix, such as that which occurs when a mucin granule is secreted and exocytosed, is described with first-order kinetics developed by Tanaka and Fillmore (45). As shown by previous reports based on polymer physics, polymer diffusivity is inversely proportional to its viscosity (13, 14, 26, 40). Low mucin diffusivity is associated with higher viscosity, less-dispersed, poorly hydrated, and probably less-transportable mucus that appears to characterize the thick, adhesive accumulations commonly noted in CF (15, 39). We now demonstrate for the first time that HCO3− modulates the rheological properties of mucins released from living cells. Our results show that HCO3− impedes mucus gel aggregation and increases the diffusivity of exocytosed mucin most likely by chelating Ca2+.
MATERIALS AND METHODS
Free calcium measurement.
The free calcium measurement was carried out using calcium-selective minielectrode with a Nernstian response down to a free [Ca2+] of ≤100 nM. The minielectrodes were prepared as previously described by Schefer et al. (42) and Baudet et al. (4). In brief, polyethylene tubes were dipped in a membrane solution containing 25 mg of Ca2+ ionophore ETH 129, 451.5 μl of N-phenyl-octyl ether, 12.9 mg of potassium tetrakis chlorophenyl borate, and 250 mg of polyvinyl chloride, dissolved in 5 ml of tetrahydrofuran. The membrane was allowed to air-dry, and the filling solution for the electrodes contained 100 mM CaCl2 to give a 1 × 10−1 M concentration at pH 7.4. Electrode calibration solutions (CALBUF-1) of 1 × 10−1 M through 1 × 10−8 M were purchased from World Precision Instruments (Sarasota, FL). Normal HBSS (buffered with 20 mM Tris·HCl, 10 mM MES, pH 7.4; Sigma, St. Louis, MO) containing 1.2 mM free [Ca2+] was measured as the control. The differences in the millivolts measured in the control and in subsequent titrations of HCO3− or EGTA (reconstituted in normal HBSS with Tris·HCl and MES, pH 7.4; Sigma) were used to calculate Ca2+ concentrations.
To test Cl− effects on free [Ca2+], PBS (pH 7.4)-based solution containing 1.2 mM Ca2+ and 400 mM Cl− was serially diluted to various concentrations ranging from approximately 6 to 400 mM Cl− with diluent (1.2 mM CaCl2 and 3 mM KCl, pH 7.4). The differences in millivolts that were measured in the control (1.2 mM Ca2+) and in serially diluted concentrations of Cl− were used to determine Ca2+ concentrations.
Bound Ca2+ measurement.
Bound Ca2+ concentration on mucus was measured by inductively coupled plasma optical emission spectrometry (ICP-OES; PerkinElmer Optima 5300 DV with concentric nebulizer and cyclonic spray chamber; PerkinElmer, Waltham, MA). Porcine gastric mucus (1 mg/l) was prepared by incubating with 4.2 mM Ca2+-containing HBSS buffer with Tris·HCl and MES at pH 7.4 for 48 h. This incubation period allowed the binding between mucins and Ca2+ to reach equilibrium. After 48 h, mucins were cross-linked by Ca2+ ions to form mucin aggregates of significant sizes >0.22 μm. This solution was then filtered through a 0.2-μm GTBP Millipore Isopore membrane (Thermo Fisher Scientific, Waltham, MA) to trap the mucin gels. Bicarbonate was then applied to assess its ability to remove mucin-bound Ca2+ on Isopore membrane. The Isopore filter was subsequently soaked in 5 ml of 1% HNO3 (Sigma) to redissolve the trapped mucus with bound Ca2+. The solution was filtered once more using a 0.22-μm Millipore PES membrane (Thermo Fisher Scientific). To eliminate background interference from the filter-bound Ca2+, an internal control was in place where an Isopore membrane was soaked in 4.2 mM CaCl2 for 48 h, and the amount of Ca2+ deposited in the membrane was measured. This basal value was subsequently subtracted from every treatment to ensure that only mucin-bound Ca2+ was quantified.
Calcium in filtrate was detected at wavelengths of 315.893 and 317.933 nm. Calcium inductively coupled plasma mass spectrometry (ICP-MS) calibration standards (Thermo Fisher Scientific) of 0.05 (1.25 μM), 0.1 (2.5 μM), 1 (25 μM), 10 (250 μM), and 25 (625 μM) mg/l were prepared for the element analyzed and were run at the beginning of the suite. Calcium concentrations were derived from an external standard calibration curve.
An internal control of the total organic carbon in the mucus samples was determined using a Total Organic Carbon Analyzer (TOC-VCSH; Shimadzu, Columbia, MD). The same filtrate used for ICP-OES analysis was first acidified to remove inorganic carbon, such as calcium carbonate and/or bicarbonate, and then diluted 4.2 times with deionized water before its measurement for total carbon content. Calibration standards in concentrations of 1, 10, and 50 mg organic carbon/l were prepared from the potassium hydrogen phthalate stock standard (Sigma). Carbon contents were measured by TOC-VCSH Analyzer and calculated based on a calibration standard curve. Final results were presented as a molar ratio between bound Ca2+ and total mucus organic carbon.
Particle sizing.
The aggregation of mucus was monitored by measuring particle size by homodyne dynamics laser scattering. Samples of porcine gastric mucus at 1 ng/l were prepared with HBSS buffer (Tris·HCl, MES, pH 7.4) and mixed until homogeneity was reached. Aliquots of mucus solution samples (10 ml) were immediately filtered through a 0.22-μm Millipore PES membrane (prewashed with 0.1 N HCl) into clean scintillating vials to remove dust particles and nonmucin particulates. The scintillating vials were positioned in the goniometer of a Brookhaven laser spectrometer (Brookhaven Instruments, Holtsville, NY). Mucus gel aggregation was allowed by equilibrating with filtered 8.2 mM CaCl2 for 48 h and was subsequently analyzed by detecting the scattering fluctuations at a 45° scattering angle. Filtered 20 or 50 mM bicarbonate and EGTA (5 mM) solution (buffered with Tris·HCl and MES at pH 7.4) was later added into scintillating vials to test for its ability in dispersing mucus aggregates and was monitored at 1, 2, 4, 6, 16, and 26 h. The pH was also monitored and maintained at ~7.4 during the experiments. The autocorrelation function of the scattering intensity fluctuations was averaged over a 3-min sampling time using a Brookhaven BI-9000AT autocorrelator. Particle size distribution was calculated by CONTIN (10). Control experiments were conducted by simultaneously adding 8.2 mM CaCl2 and 20 mM HCO3− to 1 ng/l mucus samples (as described above). Mucus aggregation in control samples were monitored throughout 74 h. Calibrations were conducted with standard monodisperse suspensions of latex microspheres ranging from 50 nm to 10 μm (Polysciences, Warrington, PA).
Mucus aggregation.
HCO3− was tested for its effect on reducing the amount of mucus aggregates. Porcine gastric mucus (5 μg/ml) was used as a model for gel-forming mucus. The dried mucus was added with 13 mM Ca(OH)2 to distilled water and agitated overnight until the mucus powder dissolved. This solution was divided into equal volumes of two aliquots: 100 mM NaCl was added to one, and 100 mM NaHCO3 (Sigma) was added to the other; both were stirred for 3 h. Each mucus solution was filtered through an Immobilon-P transfer membrane (cat. no. IPVH00010; 0.45-μm filter pore size; Millipore, Billerica, MA). The membrane filters were dried in a vacuum oven and weighed before and after filtration. The difference in weights of the dried membrane filter before and after filtering defined the amount of aggregated mucus retained on the filter. The amount of retained mucus was normalized to the volume of mucus solution filtered.
A549 cell culture.
The human lung carcinoma cell line A549 was obtained from American Type Culture Collection (ATCC, Manassas, VA). The A549 cell line is an airway alveolar epithelial cell line commonly used as a secretory model (5). Cells were cultured in 15-cm cell culture plates (VWR, Brisbane, CA) containing F-12 nutrient mixture medium (Invitrogen, Carlsbad, CA) supplemented with 100 U penicillin/streptomycin (Invitrogen) and 10% heat-inactivated FBS (Invitrogen). The A549 lung cells were cultured in 15-cm Falcon plates and incubated in a humidified incubator at 37°C, 5% CO2. Cell counts were performed using trypan blue (Sigma) exclusion and a Bright-Line Hemacytometer.
Secretory granule labeling.
The presence of secretory granules of A549 cells was identified by staining with quinacrine (10 μM; Sigma) for 15 min. The nucleus was counterstained with Hoechst (1:1,000; Sigma) followed by thorough rinsing and mounting of the sample. Expression of MUC5AC in A549 cells was confirmed by immunostaining (data not shown).
Swelling kinetics and A549 cell preparation.
The culture plates were rinsed with HBSS buffer twice. Nontrypsin dissociation buffer (Invitrogen) was added to detach cells from plates and subsequently incubated at 37°C for 15 min, centrifuged at 700 rpm for 5 min, and resuspended in HBSS buffer (Invitrogen). Resuspended cells were dispersed into MatTek glass bottom dishes (MatTek, Ashland, MA) and equilibrated in a 37°C incubator for 10 min before adding varying concentrations of HCO3− or EGTA. Both HBSS and HCO3− solutions were buffered with Tris·HCl and MES (Sigma) to pH 7.4. The pH was monitored and maintained at ~7.4 throughout the experiments.
A549 cells were viewed and video-recorded with phase-contrast lens using a Nikon Eclipse TE2000-U inverted fluorescence microscope (Nikon, Tokyo, Japan). Degranulation of A549 was induced by 1 μM ionomycin (Sigma) and was found to be a readily observable discrete quantal process. During exocytosis into extracellular HBSS, released granules undergo rapid swelling. Video-recordings of granular exocytosis and swelling were captured at 30 frames/s. The analysis of the changing mucin matrix dimension was assessed with NIS-Elements software (Nikon, Melville, NY).
Measurements of the radii of the released mucus matrices, as a function of time, were used to verify that the swelling of the secreted material followed the characteristic features of polymer gel-swelling kinetics (15, 45). The swelling of a polymer follows typical diffusive kinetics that is independent of the size, internal topology, or chemical composition of the gel (45). For spherical gels, as observed with the exocytosed mucin granules of A549 cells, the radial dimension increases following a characteristic first order kinetics of the form r(t) = rf − (rf − ri) e−t/τ, where ri and rf are the initial and final radii of the granule matrix, respectively, and τ is the characteristic relaxation time of the swelling process (46). The polymer network of gels diffuses into the solvent (HBSS) with diffusivity = (rf)2/τ (cm2/s). The diffusivity of polyionic gels varies with the concentration of counterions in the swelling medium. In this study, we measured the swelling kinetics of exocytosed mucin gels in HBSS buffer (Invitrogen) with HCO3− concentrations in the physiological range of 0–140 mM. Previous reports have predicted that polymer viscosity is proportional to the molecular weight of polymers and that polymer diffusivity is inversely proportional to the molecular weight. Therefore, polymer viscosity and diffusivity are inversely proportional to each other (13, 14, 26, 40). Higher polymer diffusivity indicates lower viscosity of polymers. Thus we can take direct measurements of changes in mucin diffusivity under different HCO3− concentrations as changes in mucin viscosity.
Statistical analysis.
Data were presented as means ± SD. Each experiment was performed independently at least three times. Statistical significance was determined using a Student's t-test analysis with P values of <0.05 (Microsoft Excel and GraphPad Prism 4.0, GraphPad Software, San Diego, CA).
RESULTS
HCO3− lowers free Ca2+ concentration by chelating Ca2+.
We directly measured the concentration of free Ca2+ in HBSS (1.2 mM Ca2+) with a Ca2+-selective minielectrode in increasing concentrations of HCO3−. As [HCO3−] increased from 0 to 50 mM, the concentration of free Ca2+ decreased exponentially from 1.2 mM to <50 μM (Fig. 1A), with most of the reduction occurring at <5 mM HCO3−. As [HCO3−] approaches 10 mM, the decrease in free [Ca2+] starts to plateau so that there was little change with 20–50 mM HCO3−. These results demonstrate that HCO3− removes most free Ca2+ from HBSS buffer solutions at relatively low concentrations (extracellular HCO3− concentration is normally ~24 mM). As a positive control, increasing concentrations of EGTA (1–5 mM) resulted in a subsequent decrease of free [Ca2+] from 1.2 to almost 0 mM in normal HBSS (Fig. 1A, inset). To confirm that Cl−, the most common physiologically abundant extracellular anion (normally ~110 mM), does not chelate free Ca2+ ions, we increased total [Cl−] up to 400 mM and found that the level of free [Ca2+] in HBSS buffer was not altered (Fig. 1B).
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).
HCO3− chelates mucus-bound Ca2+.
To investigate whether HCO3− ions are also able to chelate mucus-bound Ca2+, ICP-OES was used to quantify the relative amount of bound Ca2+ displaced from mucus after adding 20 mM HCO3− at increasing intervals of time. Mucus (1 mg/l) was prepared in HBSS (buffered with Tris·HCl and MES at pH 7.4) containing 4.2 mM Ca2+ since [Ca2+] found in CF mucus is approximately 2–4 mM (48). The mucus solution was combined with 20 mM HCO3− and assayed at 0, 5, 15, and 30 min (Fig. 2). No significant amount of CaCO3 precipitation (>200 nm) was observed during the sample preparation (data not shown). The mucus sample was filtered through a 0.2-μm Isopore membrane and redissolved in 1% HNO3 solution. The data show that as HCO3− incubation time increases, the amount of mucus-bound Ca2+ decreases (Fig. 2). Within 5 min of HCO3− exposure, bound Ca2+ dramatically dropped to 56% and continued to fall to 28% of the control after 30 min of incubation with HCO3−. These results show that HCO3− readily sequesters bound Ca2+ from mucus.
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-VCSH; Shimadzu).
HCO3− disperses aggregated mucus.
We then undertook showing that HCO3− can directly disperse aggregated mucus gels (Fig. 3). Porcine gastric mucus (1 ng/l) was added with 8.2 mM Ca2+ in HBSS (buffered with Tris·HCl and MES at pH 7.4) and was equilibrated for ≥48 h until significant aggregation of mucus masses of ~9 μm in diameter was attained. The 8.2 mM Ca2+ was used to gelate dilute mucus for studying the dispersion capacity of HCO3−. On addition of 20 or 50 mM HCO3− (buffered with Tris·HCl and MES at pH 7.4), aggregated mucus gel particles dispersed and decreased in size from ~9 to ~4.5 μm (Fig. 3). A time-dependent decrease in mucus gel size reached an apparent minimum in ~6 h. Addition of EGTA (Ca2+ chelator, 5 mM) also dispersed aggregated mucus gels to approximately the same size. Aggregated mucus gel size did not return to its original size (~0.2 μm) with either HCO3− or EGTA treatment (Fig. 3). These data indicate that HCO3− likely disperses Ca2+ cross-linked mucus gels (35) by sequestering free and bound Ca2+ ions from mucus networks as well as from free solutions.
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, ![[open triangle]](/corehtml/pmc/pmcents/utri.gif)
, n ≥ 7). The control sizes remained low (○, n ≥ 3) compared with treatment groups.
The result shown in Fig. 4 demonstrates on a gross level that HCO3− reduces apparent mucus aggregates. After filtering gastric mucus through a 0.45-μm pore Immobilon-P transfer membrane in the presence of NaHCO3 (100 mM), only 0.27 mg/ml was retained. However, in the presence of NaCl (100 mM), twice as much filtered mucus gel (0.56 mg/ml) was retained. This direct measurement of aggregated mucus confirms that HCO3− disperses aggregated mucus and that Cl− ions have little, if any, effect on mucus aggregation.
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).
HCO3− accelerates mucin matrix expansion.
A representative plot comparing the swelling kinetics of newly exocytosed mucin matrices between the control (HCO3−-free) and HCO3− treatment is shown in Fig. 5A. The rate of mucin network swelling (hydration) was significantly elevated in the presence of HCO3− (Fig. 5A). Converting swelling rate into diffusivity yielded similar results (Fig. 6). Digital images capturing the process of mucin matrix gel swelling were also presented (Fig. 5B). In addition, through the use of fluorescent dye, quinacrine, we confirmed the presence of mucin granules in A549 cells (23, 31) (Fig. 5C). The fluorescent secretory granules (green) were distributed in the cytosol surrounding the nucleus, which was stained blue with Hoechst dye.
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−, ![[filled triangle]](/corehtml/pmc/pmcents/utrif.gif)
). 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.
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.
HCO3− markedly enhances secreted mucin diffusivity.
Exocytosis of mucin granules from cultured A549 cells was stimulated with 1 μM ionomycin (1), and mucin diffusivity was calculated from the swelling kinetics as previously described (see materials and methods and Ref. 15). A faster mucin gel-swelling rate indicates greater mucin diffusivity and a less viscous gel (15). Mucin matrix swelling rates were accelerated by increasing bicarbonate concentrations (0–140 mM; Fig. 6). The increase in mucin-swelling rate was HCO3− concentration dependent. Compared with controls in HCO3−-free HBSS medium, in 1 mM HCO3−, there is a 160% increase in mucin diffusivity, whereas at 10 mM HCO3−, diffusivity was increased by 190%. Diffusivity further increased to 280% at 20 mM HCO3− and to 540% at 140 mM HCO3−, relative to the control (no bicarbonate). The concentration range of HCO3− applied was based on physiological concentrations (20, 21). Moreover, EGTA (5 and 10 mM) was used as a positive control for Ca2+ chelation, which increased diffusivity by 190 and 360% (Fig. 6, ○). These results confirm that HCO3− at physiological concentrations dramatically decreases mucin viscosity by sequestering Ca2+.
DISCUSSION
The formation of mucus is one of the most complex processes of physical chemistry in biology; fundamentally, it is accepted that before being secreted, highly negatively charged mucin polymer matrices are tightly packed within granules at a low pH in a very high concentration of intragranular Ca2+ (46). The negative charges of the mucin matrices are shielded and neutralized by protons and electrostatic divalent Ca2+ cross-links. To maintain proper and normal luminal and ductal transport of released mucin, adequate swelling and hydration of gel matrices are essential (39, 46). In order for the electrostatic forces to expand the matrix, the mucin anions must be unshielded and the inter- and intramolecular cross-links removed. Until now, it has been generally accepted that the swelling process depends essentially on exchanging monovalent cations (Na+ and K+) in the medium for protons and divalent Ca2+ in the matrices; however, recent recognition that the abnormally viscous mucus consistently found in CF may be the result of the absence of HCO3− in this disease (31) seems to provide additional insight into the physical/chemical process of mucus formation. Moreover, the chemical properties of HCO3−, per se, also suggest that among biological ions, it is ideally suited to support Ca2+ removal. That is, the poor solubility of CaCO3 [solubility product (K'sp) = 4.8 × 10−9] indicates strong binding between Ca2+ and CO32− ions (33), and the equilibrium between HCO3− and CO32− supports multiple complexes with Ca2+ ions (30, 35).
To confirm that bicarbonate sequesters Ca2+ at physiological fluid concentrations, we measured free Ca2+ concentration in HBSS after adding HCO3− and found that a steep drop in free Ca2+ occurred with low micromolar HCO3− concentrations (Fig. 1A) due to forming Ca2+ complexes, e.g., CaCO3 and CaHCO3+ (30). Further decreases in free Ca2+ were negligible even in the presence of higher HCO3− concentration (50 mM), indicating that nearly all Ca2+ available in solution can be sequestered by HCO3− concentrations that are easily achieved physiologically. The other major physiological anion, Cl−, had no effect on free Ca2+ as CaCl2 remains essentially dissociated in the HCO3−-free solution (Fig. 1B). To be effective in mucus expansion, HCO3− must remove not only free solution Ca2+, but also calcium bound in the mucin matrix. We used a commercial source of mucus, which is not pure mucin but may serve to represent mucus gel interactions. This approach is consistent with previous studies using this porcine gastric mucin to examine the viscoelastic properties and biological applications of mucin (12, 28, 41, 50). There is currently no commercial mucin sample available that has been perfectly preserved in the native state. After equilibrating with Ca2+, we found that adding 20 mM HCO3− to the mucus solution removed more than half of the bound Ca2+ within a few minutes and about three-fourths within 30 min (Fig. 2). Thus HCO3− is capable of chelating free Ca2+ from solution and sequestering bound Ca2+ from mucus gel matrices.
We then asked whether sequestering Ca2+ with HCO3− could enhance the dispersal of aggregated mucus. Both 20 and 50 mM HCO3− dispersed aggregated mucus gels and almost immediately reduced the size of consolidated aggregates by 50% (Fig. 3), but further disaggregation did not occur. Chelation of Ca2+ has been reported to disperse polymer gels (10). Adding EGTA, a highly efficient Ca2+ chelator, in place of HCO3− dispersed aggregated mucus very similarly (Fig. 3). This result probably indicates the importance of Ca2+ cross-linking in mucin aggregation and accumulation. However, the fact that EGTA produced results similar to HCO3− indicates that the failure to completely reverse the disaggregation is not due to incomplete sequestration of Ca2+ by HCO3− and may not be entirely due to Ca2+. Additional hydrophobic interactions may also be present within aggregated mucus (2, 9). The data in Fig. 4 corroborate these findings by showing that, compared with Cl−, HCO3− greatly reduced the amount of aggregated mucus gels retained on a microfilter (Fig. 4).
These combined results demonstrate that HCO3− can play a critical role in the decondensation of mucin granules, but proof awaits a direct demonstration of its effects on mucus properties in vivo. Hydration-induced swelling is the single most critical determinant of mucus rheological characteristics (15, 46, 47), and measurements of mucin-swelling kinetics (diffusivity) provides a unique method to directly assess changes in mucin rheological properties (viscosity) released from living cells under physiological conditions (46, 47). A549 cells were used as our representative model system for studying mucin-swelling kinetics because it produces both major respiratory MUC5AC and MUC5B gel-forming mucins (43, 51). Therefore, using videomicroscopy of living A549 cells in culture, we measured the rates of swelling in real-time of mucin granules released from cells that were stimulated to secrete into different concentrations of HCO3−. First, our data showed that at physiological HCO3− concentrations, a mucin network rapidly hydrates (Fig. 5A), which we surmise would render it more easily transportable. Second, a biphasic response of mucin-swelling rates to increasing bicarbonate concentrations is clearly demonstrated in Fig. 6. The two different slopes may suggest the existence of two different Ca2+ binding sites within mucins. This second phase might reflect removal of a more tightly bound source of Ca2+ in the mucin. That is, at lower HCO3− concentrations, the swelling may be dominated by repulsion of the highly anionic, highly glycosylated regions of the mucin freed on removing Ca2+ cross-linking as fixed negative charges are unshielded (22, 34, 35). Higher concentrations of HCO3− (and associated CO32−) might be “competing” tightly bound Ca2+ away from other binding sites such as those recently described as nodes of tightly bound protein domains in mucins (22, 34). Chelating Ca2+ with EGTA (5 and 10 mM) confirmed the notion that the increased rate of diffusivity is due to Ca2+ removal. EGTA (5 and 10 mM) sequestration of Ca2+ increased the mucin network diffusivity by 190 and 360% compared with the control (no EGTA), whereas 5, 20, and 140 mM HCO3− increased diffusivity by 140, 280, and 540% compared with the control (HCO3−-free; Fig. 6). Based on concentration, EGTA seems to be somewhat more effective than HCO3− likely due to the stronger chelation affinity of EGTA for Ca2+. These data provide the first direct experimental evidence that HCO3− modulates the mucin hydration rate and the viscoelastic properties (viscosity) of mucins in live cells.
Since the medium concentrations of Na+ and K+ were constant in all protocols, the increases in diffusivity cannot be solely due to the exchange of calcium with Na+ or K+ ions. The same consideration can be made in CF where the concentration of these monovalent cations is relatively normal and constant, but it is the HCO3− that is diminished. Clearly, Na+-Ca2+ exchange must occur to maintain electroneutrality within the matrix. However, HCO3− may be necessary to speed or facilitate the exchange by competing Ca2+ away from fixed anions on the mucin molecules so that the monovalent cations (Na+) can replace the Ca2+ that would otherwise remain bound and slow or impossible to remove solely by cation exchange. This would almost certainly be the case if fixed mucin anions have a higher affinity for Ca2+ than for Na+.
With respect to CF, which instigated this study, previous work recently demonstrated that the amount of mucus discharged from the intestine (17) and the uterine cervix (29) in vitro was significantly reduced when HCO3− secretion was impeded. The present work provides a likely basis for those findings and possibly for the characteristically aggregated and tenacious mucus abnormalities found in CF as well. That is, in CF where the genetic defect occurs in a gene that codes for an anion channel, it is now well-established that, like Cl−, HCO3− permeability and secretion are greatly reduced or absent (35, 37). Our studies here strongly indicate that in CF organs, when mucins are released into fluids without adequate HCO3−, their diffusivity would be decreased (viscosity increased); normal expansion of mucins would be retarded and impaired due to retained Ca2+ cross-linking, and the long observed pathogenic aggregation and stagnation of “sticky” mucus would tend to occur. A tentative model of this process was proposed recently (29). It should be noted that organ failure in CF is not immediate and generally occurs over many months or years, so pathogenic mucus aggregation is almost surely a multifactorial process.
In summary, we have demonstrated that HCO3− ions play a critical role in determining the viscosity of mucins and mucus by controlling swelling and dispersion most likely by competing with fixed anions in mucins for Ca2+, which condenses and aggregates mucus polymers via divalent cross-linking. These findings show that defects in bicarbonate ion transport such as those associated with CF can lead to pathologically thick and viscid mucus for which CF is known. These results may provide insights for new therapeutic strategies to reduce organ failure in CF due to mucus obstructions and to ameliorate problems in other diseases of mucus.
GRANTS
This work was supported by grants from National Heart, Lung, and Blood Institute (1R15-HL-095039), National Science Foundation (CBET-0932404), and University of California Center for Information Technology Research in the Interest of Society (UC CITRIS) Program to W.-C. Chin and grants from Cystic Fibrosis Research, Nancy Olmsted Trust, National Heart, Lung, and Blood Institute (HL-084042), and the U.S. Cystic Fibrosis Foundation to P. M. Quinton.
ACKNOWLEDGMENTS
We gratefully acknowledge help with ICP-OES analysis from Liying Zhao. We thank Ariel Escobar and Patricio Velez for assistance with free Ca2+ measurements and also gratefully acknowledge critical suggestions from Pedro Verdugo.
REFERENCES
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Funding
Funders who supported this work.
NHLBI NIH HHS (2)
Grant ID: HL-084042
Grant ID: 1R15-HL-095039