A new role for bicarbonate secretion in cervico-uterine mucus release

Abstract

Cervical mucus thinning and release during the female reproductive cycle is thought to rely mainly on fluid secretion. However, we now find that mucus released from the murine reproductive tract critically depends upon concurrent bicarbonate (HCO(3)(-)) secretion. Prostaglandin E(2) (PGE(2))- and carbachol-stimulated mucus release was severely inhibited in the absence of serosal HCO(3)(-), HCO(3)(-) transport, or functional cystic fibrosis transmembrane conductance regulator (CFTR). In contrast to mucus release, PGE(2)- and carbachol-stimulated fluid secretion was not dependent on bicarbonate or on CFTR, but was completely blocked by niflumic acid. We found stimulated mucus release was severely impaired in the cystic fibrosis F508 reproductive tract, even though stimulated fluid secretion was preserved. Thus, CFTR mutations and/or poor bicarbonate secretion may be associated with reduced female fertility associated with abnormal mucus and specifically, may account for the increased viscosity and lack of cyclical changes in cervical mucus long noted in women with cystic fibrosis.

Figure 1. Mucus collection perfusion system

The excised reproductive tract was mounted vertically in a custom-made water-jacketed chamber, with one uterine horn and the vaginal opening ligated to glass capillary tubes as illustrated. The tissue was maintained at 35 ± 2°C and bath solutions were constantly bubbled with 100% O₂ or 95% O₂–5% CO₂ and changed as needed.

Figure 2. PGE₂ and carbachol stimulate mucus release

A, an example of stimulated mucus release (μg ml⁻¹ g⁻¹ tissue) in response to PGE₂ (10⁻⁶m) and carbachol (10⁻⁵m) illustrating the spontaneous rate of release of mucus 10 min prior to stimulation and the peak rate of release that occurs within 5 min of stimulation at time = 0. B, mean rates of mucus release stimulated by the presence of PGE₂ or carbachol or both combined and normalized to percentage increases over pre-stimulation spontaneous rates. C, summary of means of normalized maximal mucus release within 5 min after stimulation with PGE₂ (n= 5), carbachol (n= 5) and both drugs combined (n= 6; ***P < 0.001, means ±s.e.m.). D, correlation of lectin binding with periodic acid Schiff (PAS) assay and MUC5B antibody binding to the same samples collected after stimulation with PGE₂ and carbachol.

Figure 3. Mucus release requires HCO₃⁻ and CFTR

A, in the absence of extracellular HCO₃⁻, PGE₂+ carbachol did not stimulate mucus release (n= 4). B, likewise in the presence of DIDS (10⁻⁴m, basolateral; n= 4) to inhibit HCO₃⁻ transport, mucus release was inhibited. C, stimulated mucus release was essentially absent in homozygous CFTR defective (ΔF508 mutation) mice (n= 4) or when GlyH-101 (2 × 10⁻⁵m) and MalH-1 (10⁻⁵m) were both added concurrently to luminal and basolateral solutions to inhibit CFTR function (n= 4). D, summary and comparison of maximal PGE₂+ carbachol-stimulated mucus release after 5 min for A–C compared to control (Fig. 1C) (***P < 0.001; means ±s.e.m.).

Figure 4. Mucus release requires fluid secretion

A, in the presence of HCO₃⁻, PGE₂ (n= 7), carbachol (n= 5), or both agonists combined (n= 10) stimulated fluid secretion. B and C, inhibitory effect of basolateral bumetanide (10⁻⁴m) on PGE₂+ carbachol-stimulated fluid secretion (n= 4). D, stimulated mucus release was significantly reduced in the presence of bumetanide (10⁻⁴m) to inhibit fluid secretion (n= 4).

Figure 5. Stimulated fluid secretion is independent of the presence of HCO₃⁻

A and B, stimulated fluid secretion was not significantly different in the absence of extracellular HCO₃⁻ (A; compare Fig. 4A, P= 0.84; n= 4), or in the presence of basolateral DIDS (10⁻⁴m) (B; P= 0.27; n= 4). C, summary of maximum PGE₂+ carbachol-stimulated fluid secretion rates after 5 min (ns, not significant vs. stimulation in HCO₃⁻). Data shown are means ±s.e.m.

Figure 6. Fluid secretion does not require CFTR and is inhibited by niflumic acid

A, in the presence of HCO₃⁻, stimulated fluid secretion was not significantly different in the presence of combined CFTR inhibitors GlyH-101 (2 × 10⁻⁵m) and MalH-1 (10⁻⁵m) (P= 0.68; n= 6) or in tissues from ΔF508 mice (P= 0.69; n= 4). B, in the presence of HCO₃⁻, niflumic acid (10⁻⁴m) in the luminal and basolateral solution abolished fluid secretion (P < 0.001; n= 4). C, summary of maximal PGE₂+ carbachol-stimulated fluid secretion rates after 5 min (ns, not significant; ***P < 0.01 compare to Fig. 3A). Data shown are means ±s.e.m.

Figure 7. Mucus is retained in the glands in the absence of HCO₃⁻

Uterine tissue was treated with PGE₂+ carbachol for 20 min before fixing. In the presence of serosal HCO₃⁻: A, PAS staining shows the uterine glands (arrow) do not contain a substantial amount of mucus (scale bar: 50 μm; 10× magnification) and B, MUC5B mucin staining (red) counterstained with DAPI (blue) to visualize the nuclei correlates to PAS staining (scale bar 25 μm; 40× magnification). In the absence of serosal HCO₃⁻: C, substantially more PAS positive material (pink) is present in the lumens of the uterine glands (arrow) (scale bar: 50 μm; 10× magnification) and D, MUC5B binding correlates to PAS staining demonstrating significant mucus in the glands (scale bar 25 μm; 40× magnification).

Figure 8. Proposed roles for bicarbonate in mucus release

Highly condensed polyanionic macromolecular mucins are packaged within granules by virtue of high intragranular concentrations of Ca²⁺ and H⁺ that shield negatively charged sites on mucins from electrostatic repulsion. Removing these cations from mucins immediately upon release from the cell (by sequestering Ca²⁺ or buffering H⁺) is essential to allow the negative electrostatic forces of the mucin anions to rapidly expand the condensed mucins into extremely long extended linear polymers that form mucus (right side). Extracellular HCO₃⁻ appears to be the simplest, most available means to efficiently remove the shielding cations from mucins for optimal expansion. In the absence of HCO₃⁻, as likely occurs in CF, inadequate unshielding apparently results in poorly expanded, tenacious and viscous mucus that is difficult to liberate from the epithelial surfaces (Quinton, 2008; Garcia et al. 2009). Still, HCO₃⁻ might also be critical in enhancing the initial expansion of the mucin molecule to allow other factors (enzymes) access to aggregated nodes of protein domains that must be freed to complete the process (left side) (Kesimer et al. 2009; Quinton, 2009), and may also play a role in creating a local environment that enhances expansion and unravelling by creating a pH for optimal enzymatic activities that cleave intramolecular covalent crosslinks (I. Carlstedt, personal communication).