The effect of ingredients commonly used in nasal and inhaled solutions on the secretion of mucus in vitro.

Hypersecretion of mucus is associated with impaired mucociliary clearance that can influence the retention of active pharmaceutical ingredients in the airway but is also linked with recurrent airway disease. Therefore, the effect on mucin secretion of a range of ingredients used in solutions delivered to the nose and lung was studied. Mucin secretion from explants of ovine epithelium was quantified using an enzyme-linked lectin assay (ELLA) or sandwich ELLA depending on the compatibility of the ingredients with the assay. Benzalkonium chloride (0.015% w/w), Methocel™ E50 premium LV (1.0% w/w), propylene glycol (1.5% w/w), potassium sorbate + propylene glycol (0.3% w/w + 1.5% w/w) and polysorbate 80 (0.025% w/w), used at common working concentrations, all increased the secretion of mucin from the explants (P < 0.05). Ethylenediamine tetraacetic acid-disodium salt (EDTA) (0.015% w/w), Avicel® RC591 (1.5% w/w), fluticasone furoate (0.0004% w/w, concentration in solution) and dimethyl sulfoxide (DMSO) (0.2% w/w) did not affect mucin secretion. Compounds increasing mucin secretion could alter the rate of mucociliary clearance and the mucus could provide a barrier to drug absorption. This could predispose patients to disease and affect the activity of delivered drugs, decreasing or increasing their clinical efficacy.

A comparison of three mucus-secreting airway cell lines (Calu-3, SPOC1 and UNCN3T) for use as biopharmaceutical models of the nose and lung.

The aim of this work was to compare three existing mucus-secreting airway cell lines for use as models of the airways to study drug transport in the presence of mucus. Each cell line secreted mature, glycosylated mucins, evidenced by the enzyme-linked lectin assay. The secretagogue, adenylyl-imidodiphosphate, increased mucin secretion in SPOC1 (3.5-fold) and UNCN3T (1.5-fold) cells but not in Calu-3 cells. In a novel mucus-depleted (MD) model the amount of mucus in the non-depleted wells was 3-, 8- and 4-fold higher than in the mucus-depleted wells of the Calu-3, SPOC1 and UNCN3T cells respectively. The permeability of 'high mucus' cells to testosterone was significantly less in SPOC1 and UNCN3T cells (P < 0.05) but not Calu-3 cells. Mucin secretion and cytokine release were investigated as indicators of drug irritancy in the SPOC1 and UNCN3T cell lines. A number of inhaled drugs significantly increased mucin secretion at high concentrations and the release of IL-6 and IL-8 from SPOC1 or UNCN3T cells (P < 0.05). SPOC1 and UNCN3T cell lines are better able to model the effect of mucus on drug absorption than the Calu-3 cell line and are proposed for use in assessing drug-mucus interactions in inhaled drug and formulation development.

Baseline Goblet Cell Mucin Secretion in the Airways Exceeds Stimulated Secretion over Extended Time Periods, and Is Sensitive to Shear Stress and Intracellular Mucin Stores.

Airway mucin secretion studies have focused on goblet cell responses to exogenous agonists almost to the exclusion of baseline mucin secretion (BLMS). In human bronchial epithelial cell cultures (HBECCs), maximal agonist-stimulated secretion exceeds baseline by ~3-fold as measured over hour-long periods, but mucin stores are discharged completely and require 24 h for full restoration. Hence, over 24 h, total baseline exceeds agonist-induced secretion by several-fold. Studies with HBECCs and mouse tracheas showed that BLMS is highly sensitive to mechanical stresses. Harvesting three consecutive 1 h baseline luminal incubations with HBECCs yielded equal rates of BLMS; however, lengthening the middle period to 72 h decreased the respective rate significantly, suggesting a stimulation of BLMS by the gentle washes of HBECC luminal surfaces. BLMS declined exponentially after washing HBECCs (t1/2 = 2.75 h), to rates approaching zero. HBECCs exposed to low perfusion rates exhibited spike-like increases in BLMS when flow was jumped 5-fold: BLMS increased >4 fold, then decreased within 5 min to a stable plateau at 1.5-2-fold over control. Higher flow jumps induced proportionally higher BLMS increases. Inducing mucous hyperplasia in HBECCs increased mucin production, BLMS and agonist-induced secretion. Mouse tracheal BLMS was ~6-fold higher during perfusion, than when flow was stopped. Munc13-2 null mouse tracheas, with their defect of accumulated cellular mucins, exhibited similar BLMS as WT, contrary to predictions of lower values. Graded mucous metaplasia induced in WT and Munc13-2 null tracheas with IL-13, caused proportional increases in BLMS, suggesting that naïve Munc13-2 mouse BLMS is elevated by increased mucin stores. We conclude that BLMS is, [i] a major component of mucin secretion in the lung, [ii] sustained by the mechanical activity of a dynamic lung, [iii] proportional to levels of mucin stores, and [iv] regulated differentially from agonist-induced mucin secretion.

A2B adenosine receptors regulate the mucus clearance component of the lung's innate defense system.

Adenosine (ADO) signaling is altered in both asthma and chronic obstructive pulmonary disease, and the A(2B) adenosine receptor (A(2B)-R) may drive pulmonary inflammation. Accordingly, it has been proposed that specific inhibition of the A(2B)-R could treat inflammatory lung diseases. However, stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by ADO may be crucial in permitting the superficial epithelium to maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Our goal was to determine which ADO receptor (ADO-R) underlies ASL volume regulation in bronchial epithelia. We used PCR techniques to determine ADO-R expression in bronchial epithelia and used nasal potential difference measurements, Ussing chambers studies, and XZ-confocal microscopy to look at Cl- secretion and ASL volume regulation. The A(2B)-R was the most highly expressed ADO-R in donor specimens of human bronchial epithelia, and inhibition of ADO-R in vivo prevented activation of CFTR. A(2B)-R was the only ADO-R detected in cultured human bronchial epithelial cells and inhibition of this receptor with specific A(2B)-R antagonists resulted in ASL height collapse and a failure to effect ASL height homeostasis. Removal of ADO with ADO deaminase and replacement with 5'N-ethylcarboxamide adenosine resulted in dose-dependent changes in ASL height, and suggested that the cell surface (ADO) may be in excess of 1 microM, which is sufficient to activate A(2B)-R. A(2B)-R are required for ASL volume homeostasis in human airways, and therapies directed at inhibiting A(2B)-R may lead to a cystic fibrosis-like phenotype with depleted ASL volume and mucus stasis.

Novel quartz crystal microbalance based biosensor for detection of oral epithelial cell-microparticle interaction in real-time.

Recent applications of quartz crystal resonant sensor technology to monitor cell adhesion and specific ligand interaction processes has triggered the development of a new category of quartz crystal microbalance (QCM) based biosensors. In this study human oral epithelial cells (H376) were cultured on quartz sensors and their response to microspheres investigated in situ using the QCM technique. The results demonstrated that this novel biosensor was able to follow cell-microsphere interactions in real-time and under conditions of flow as would occur in the oral cavity. Unique frequency profiles generated in response to the microspheres were postulated to be due to phases of mass addition and altered cellular rigidity. Supporting microscopic evidence demonstrated that the unique frequency responses obtained to these interactions were in part due to binding between the cell surface and the microspheres. Furthermore, a cellular uptake process, in response to microsphere loading was identified and this, by influencing the rigidity of the cellular cytoskeleton, was also detectable through the frequency responses obtained.

Endothelin-1 inhibits mucin secretion from ovine airway epithelial goblet cells.

Mucus hypersecretion is a feature of several respiratory diseases and frequently leads to obstruction of small airways where the principal source of mucous glycoproteins (mucins), the major macromolecular constituents of mucus, are goblet cells. Hence, inhibition of mucin secretion from these cells may be clinically beneficial. In this study, we have developed a lectin-based assay for mucin secretion from ovine airway goblet cells and used this assay to investigate the regulation of these cells by endothelin (ET)-1. ET-1 inhibited baseline mucin secretion (maximum inhibition: 60.3 +/- 4.2%, 50% inhibitory concentration: 0.8 +/- 0.17 nM). This response was abolished by the ET(A) antagonist, BQ-123 (1 muM), but not by the ET(B) antagonist, BQ-788 (1 muM). ET-1 (1 muM) did not affect mucin secretion stimulated by ATP (100 muM) but secretion in response to ATP (10 muM) was inhibited by 63.3 +/- 11.8%. This response could be eliminated by BQ-123, but not by BQ-788. Radioligand binding and immunohistochemistry indicated the expression of both ET(A)- and ET(B)-receptors on the epithelium. In summary, ET-1, acting via ET(A)-receptors, inhibits baseline and ATP-stimulated mucin secretion from ovine airway goblet cells. This represents the first report of a physiologic mechanism for inhibiting airway goblet cell mucin secretion; an understanding of this mechanism may provide opportunities for the treatment of obstructive airways disease.