Airway Gland Structure and Function.

Submucosal glands contribute to airway surface liquid (ASL), a film that protects all airway surfaces. Glandular mucus comprises electrolytes, water, the gel-forming mucin MUC5B, and hundreds of different proteins with diverse protective functions. Gland volume per unit area of mucosal surface correlates positively with impaction rate of inhaled particles. In human main bronchi, the volume of the glands is ∼ 50 times that of surface goblet cells, but the glands diminish in size and frequency distally. ASL and its trapped particles are removed from the airways by mucociliary transport. Airway glands have a tubuloacinar structure, with a single terminal duct, a nonciliated collecting duct, then branching secretory tubules lined with mucous cells and ending in serous acini. They allow for a massive increase in numbers of mucus-producing cells without replacing surface ciliated cells. Active secretion of Cl(-) and HCO3 (-) by serous cells produces most of the fluid of gland secretions. Glands are densely innervated by tonically active, mutually excitatory airway intrinsic neurons. Most gland mucus is secreted constitutively in vivo, with large, transient increases produced by emergency reflex drive from the vagus. Elevations of [cAMP]i and [Ca(2+)]i coordinate electrolyte and macromolecular secretion and probably occur together for baseline activity in vivo, with cholinergic elevation of [Ca(2+)]i being mainly responsive for transient increases in secretion. Altered submucosal gland function contributes to the pathology of all obstructive diseases, but is an early stage of pathogenesis only in cystic fibrosis.

Early studies on the surface epithelium of mammalian airways.

This article traces the beginnings of the various areas of physiological research on airway epithelium. First mentioned in 1600, it was not until 1834 that it was found to be ciliated. Goblet and basal cells were described in 1852, to be followed by ~10 other epithelial cell types (the most recent in 2018). It also contains nerve endings and resident leukocytes. Mucociliary clearance was documented in 1835, but the first studies on the ciliary beat cycle did not appear until 1890, and a definitive description was not published until 1981. It was established in 1932 that goblet cells in the cat trachea were unresponsive to cholinergic agents; but only since 1980 or so has any significant progress been made on what does cause them to degranulate. Active transfer of salts across epithelia creates local osmotic gradients that drive transepithelial water flows. Vectorial salt transport was first described for airway epithelium in 1968, and the associated volume flows were measured in 1981. Evidence that airway epithelium releases signaling molecules first appeared in 1981. Since then, scores of molecules have been identified. The pace of research in most areas increased dramatically after the development of confluent, polarized cultures of airway epithelium in the early 1980s.

Early studies of airway submucosal glands.

This historical article provides a comprehensive review of early research on the structure and function of airway submucosal glands. The literature before 1950 or so, is virtually unknown, but in addition to being of historical interest it contains much of relevance to current research. Airway glands were first mentioned in 1602. The first description of their general form, size, and distribution was in 1712. Gland morphology was determined in 1827 by injecting mercury into their openings. Wax was later used. Detailed comparative information for all regions of the tracheobronchial tree was provided by Frankenhauser in 1879 (Untersuchungen uber den bau der Tracheo-Bronchial-Schleimhaut). Histological studies began in 1870, and by the end of the 19th century, all the major histological features had been described. The first physiological studies on airway mucous secretion were published in 1892. Kokin, in 1896 (Archiv für die gesamte Physiologie des Menschen und der Tiere 63: 622-630), was the first to measure secretion from individual glands. It was not, however, until 1933 that gland secretion was quantified. This early literature raises important questions as to the role of the collecting duct epithelium in modifying primary secretions. It also provides perhaps the most accurate measure of basal gland secretion in vivo.

Differential effects of extracellular ATP on chloride transport in cortical collecting duct cells.

Extracellular ATP in the cortical collecting duct can inhibit epithelial sodium channels (ENaC) but also stimulate calcium-activated chloride channels (CACC). The relationship between ATP-mediated regulation of ENaC and CACC activity in cortical collecting duct cells has not been clearly defined. We used the mpkCCD(c14) cortical collecting duct cell line to determine effects of ATP on sodium (Na(+)) and chloride (Cl(-)) transport with an Ussing chamber system. ATP, at a concentration of 10(-6) M or less, did not inhibit ENaC-mediated short-circuit current (I(sc)) but instead stimulated a transient increase in I(sc). The macroscopic current-voltage relationship for ATP-inducible current demonstrated that the direction of this ATP response changes from positive to negative when transepithelial voltage (V(te)) is clamped to less than -10 mV. We hypothesized that this negative V(te) might be found under conditions of aldosterone stimulation. We next stimulated mpkCCD(c14) cells with aldosterone (10(-6) M) and then clamped the V(te) to -50 mV, the V(te) of aldosterone-stimulated cells under open-circuit conditions. ATP (10(-6) M) induced a transient increase in negative clamp current, which could be inhibited by flufenamic acid (CACC inhibitor) and BAPTA-AM (calcium chelator), suggesting that ATP stimulates Cl(-) absorption through CACC. Together, our findings suggest that the status of ENaC activity, by controlling V(te), may dictate the direction of ATP-stimulated Cl(-) transport. This interplay between aldosterone and purinergic signaling pathways may be relevant for regulating NaCl transport in cortical collecting duct cells under different states of extracellular fluid volume.

A simple device to illustrate the Einthoven triangle.

The Einthoven triangle is central to the field of electrocardiography, but the concept of cardiac vectors is often a difficult notion for students to grasp. To illustrate this principle, we constructed a device that recreates the conditions of an ECG reading using a battery to simulate the electrical vector of the heart and three voltmeters for the main electrocardiographic leads. Requiring minimal construction with low cost, this device provides hands-on practice that enables students to rediscover the principles of the Einthoven triangle, namely, that the direction of the cardiac dipole can be predicted from the deflections in any two leads and that lead I + lead III = lead II independent of the position of heart's electrical vector. We built a total of 6 devices for classes of 30 students and tested them in the first-year Human Physiology course at the University of California-Davis School of Medicine. Combined with traditional demonstrations with ECG machines, this equipment demonstrated its ability to help medical students obtain a solid foundation of the basic principles of electrocardiography.

Cystic fibrosis and the relationship between mucin and chloride secretion by cultures of human airway gland mucous cells.

We investigated how cystic fibrosis (CF) alters the relationship between Cl(-) and mucin secretion in cultures of non-CF and CF human tracheobronchial gland mucous (HTGM and CFTGM, respectively) cells. Biochemical studies showed that HTMG cells secreted typical airway mucins, and immunohistochemical studies showed that these cells expressed MUC1, MUC4, MUC5B, MUC8, MUC13, MUC16, and MUC20. Effects of cumulative doses of methacholine (MCh), phenylephrine (Phe), isoproterenol (Iso), and ATP on mucin and Cl(-) secretion were studied on HTGM and CFTGM cultures. Baseline mucin secretion was not significantly altered in CFTGM cells, and the increases in mucin secretion induced by mediators were unaltered (Iso, Phe) or slightly decreased (MCh, ATP). Across mediators, there was no correlation between the maximal increases in Cl(-) secretion and mucin secretion. In HTGM cells, the Cl(-) channel blocker, diphenylamine-2-carboxylic acid, greatly inhibited Cl(-) secretion but did not alter mucin release. In HTGM cells, mediators (10(-5) M) increased mucin secretion in the rank order ATP > Phe = Iso > MCh. They increased Cl(-) secretion in the sequence ATP > MCh ≈ Iso > Phe. The responses in Cl(-) secretion to MCh, ATP, and Phe were unaltered by CF, but the response to Iso was greatly reduced. We conclude that mucin secretion by cultures of human tracheobronchial gland cells is independent of Cl(-) secretion, at baseline, and is unaltered in CF; that the ratio of Cl(-) secretion to mucus secretion varies markedly depending on mediator; and that secretions induced by stimulation of ß-adrenergic receptors will be abnormally concentrated in CF.

Interleukin-13-induced mucous metaplasia increases susceptibility of human airway epithelium to rhinovirus infection.

Infection of airway epithelium by rhinovirus is the most common cause of asthma exacerbations. Even in mild asthma, airway epithelium exhibits mucous metaplasia, which increases with increasing severity of the disease. We previously showed that squamous cultures of human airway epithelium manifest rhinoviral infection at levels many times higher than in well-differentiated cultures of a mucociliary phenotype. Here we tested the hypothesis that mucous metaplasia is also associated with increased levels of rhinoviral infection. Mucous metaplasia was induced with IL-13, which doubled the numbers of goblet cells. In both control (mucociliary) and IL-13- treated (mucous metaplastic) cultures, goblet cells were preferentially infected by rhinovirus. IL-13 doubled the numbers of infected cells by increasing the numbers of infected goblet cells. Furthermore, IL-13 increased both the maturity of goblet cells and the probability that a goblet cell would be infected. The infection of cells other than goblet cells was unaltered by IL-13. Treatment with IL-13 did not alter the levels of rhinovirus receptor ICAM-1, nor did the proliferative effects of IL-13 enhance infection, because rhinovirus did not colocalize with dividing cells. However, the induction of mucous metaplasia caused changes in the apical membrane structure, notably a marked decrease in overall ciliation, and an increase in the overall flatness of the apical surface. We conclude that mucous metaplasia in asthma increases the susceptibility of airway epithelium to infection by rhinovirus because of changes in the overall architecture of the apical surface.

CFTR and calcium-activated chloride channels in primary cultures of human airway gland cells of serous or mucous phenotype.

Using cell culture models, we have investigated the relative importance of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCC) in Cl secretion by mucous and serous cells of human airway glands. In transepithelial recordings in Ussing chambers, the CFTR inhibitor CFTR(inh)-172 abolished 60% of baseline Cl secretion in serous cells and 70% in mucous. Flufenamic acid (FFA), an inhibitor of CaCC, reduced baseline Cl secretion by ∼20% in both cell types. Methacholine and ATP stimulated Cl secretion in both cell types, which was largely blocked by treatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and partially by mucosal FFA or CFTR(inh)-172 with the exception of methacholine responses in mucous cells, which were not blocked by FFA and partially (∼60%) by CFTR(inh)-172. The effects of ionomycin on short-circuit current (I(sc)) were less than those of ATP or methacholine. Forskolin stimulated Cl secretion only if Cl in the mucosal medium was replaced by gluconate. In whole cell patch-clamp studies of single isolated cells, cAMP-induced Cl currents were ∼3-fold greater in serous than mucous cells. Ionomycin-induced Cl currents were 13 times (serous) or 26 times (mucous) greater than those generated by cAMP and were blocked by FFA. In serous cells, mRNA for transmembrane protein 16A (TMEM16A) was ∼10 times more abundant than mRNA for CFTR. In mucous cells it was ∼100 times more abundant. We conclude: 1) serous and mucous cells both make significant contributions to gland fluid secretion; 2) baseline Cl secretion in both cell types is mediated predominantly by CFTR, but CaCC becomes increasingly important after mediator-induced elevations of intracellular Ca; and 3) the high CaCC currents seen in patch-clamp studies and the high TMEM16A expression in intact polarized cells sheets are not reflected in transepithelial current recordings.

In vitro susceptibility to rhinovirus infection is greater for bronchial than for nasal airway epithelial cells in human subjects.

BACKGROUND: Human rhinoviruses (HRVs) characteristically cause upper respiratory tract infection, but they also infect the lower airways, causing acute bronchitis and exacerbating asthma. OBJECTIVE: Our purpose was to study ex vivo the differences in the response to HRV infection of nasal and bronchial epithelial cultures from the same healthy and asthmatic individuals using conditions favoring development of fully differentiated, pseudostratified mucociliary epithelium. METHODS: Cells from the inferior turbinates and bronchial tree of 5 healthy and 6 asthmatic individuals were cultured at an air-liquid interface. Cultures were infected with HRV-16, and after 48 hours, the degree of infection was measured. RESULTS: Baseline median transepithelial resistance was lower in human bronchial epithelial (HBE) cell cultures than in human nasal epithelial (HNE) cell cultures (195 Omega.cm2 [95% CI, 164-252] vs 366 Omega.cm2 [95% CI, 234-408], respectively; P < .01). Virus replicated more easily in HBE cells than in HNE cells based on virus shedding in apical wash (log tissue culture infective dose of 50%/0.1 mL = 2.0 [95% CI, 1.0-2.5] vs 0.5 [95% CI, 0.5-1.5], P < .01) and on a 20- to 30-fold greater viral load and number of infected cells in HBE cell cultures than in HNE cell cultures. The increases in expression of RANTES and double-stranded RNA-dependent protein kinase were greater in HBE cell cultures than in HNE cell cultures, as were the concentrations of IL-8, IL-1alpha, RANTES, and IP-10 in basolateral medium. However, no significant differences between asthmatic and healthy subjects (including IFN-beta1 expression) were found. CONCLUSIONS: Differentiated nasal epithelial cells might have mechanisms of increased resistance to rhinovirus infection compared with bronchial epithelial cells. We could not confirm previous reports of increased susceptibility to HRV infection in epithelial cells from asthmatic subjects.

Why Airway Gland Secretions Are Abnormally Sticky in Cystic Fibrosis, and Why Not Much Can Be Done About It.

In this issue of Developmental Cell, Xie et al. show that in cystic fibrosis, airway gland mucus gels form under conditions of high acidity and protein concentration. This causes them to be unusually stiff. This abnormal rheology cannot be corrected by changing pH or calcium levels in the bathing medium.

A Brief History of Bronchitis in England and Wales.

Chronic bronchitis is associated with hypertrophy of airway submucosal glands and with mucus and squamous metaplasia of the surface epithelium. A historical review of research on these and other pathological changes is provided. Next, from annual reports of the Registrar-General's Office (and later the Office of National Statistics), death rates per unit population from acute and chronic bronchitis (a term that here includes chronic obstructive pulmonary disease [COPD]) are calculated for England and Wales from 1838 to the present. It is argued that a large increase in the death rate between 1838 and 1879, from all forms of bronchitis combined, was due primarily to increased levels of atmospheric coal smoke, whereas a decrease from 1879 to 1935 was due to progressively cleaner air. Between 1935 and the mid-1960s, mortality from chronic bronchitis among men increased dramatically, after which it has fallen, a pattern that parallels changes in cigarette smoking. Finally, a brief historical review of the treatments for chronic bronchitis is presented.

Expansion of cultures of human tracheal epithelium with maintenance of differentiated structure and function.

We have developed a technique for expanding primary cultures of human tracheal epithelium while minimizing loss of differentiated structure and function. Cells were seeded at 2 x 10(4) cells/cm2 into T75 flasks and trypsinized when approximately 80% confluent. The dispersed cells were then passaged at the same plating density into further T75 flasks or seeded at 5 x 10(5) cells/cm2 on porous-bottomed inserts and maintained with an air-interface. Differentiation of cells on inserts was assessed from transepithelial electrical resistance (an index of tight junction formation), short-circuit current (an index of transepithelial salt transport), cell numbers, total cell protein, and histology. Unpassaged cells (P0) and cells passaged once (P1) took about a week to become 80% confluent on T75 flasks, with 10-fold and 5-fold increases in cell numbers, respectively. Confluence was achieved in approximately 3 days following plating to inserts. Functionally and structurally, P1 and P2 cells (cells passaged twice) were little different from P0 cells. Thus, within a little over 2 weeks, the numbers of confluent cell sheets can be increased 50-fold with minimal change in function. However, there was a marked decline in differentiation by cells passaged three times (P3), and not all cell preparations could be taken to P4 (cells passaged four times).

Endotoxin induces leukocyte transmigration and changes in permeability of the airway epithelium via protein-kinase C and extracellular regulated kinase activation.

Lipopolysaccharide (LPS) endotoxin of Gram-negative bacteria compromises the integrity of the airway epithelial barrier and initiates migration of leukocytes across the epithelium. The goal of the present study was to identify the role of extracellular regulated kinase (ERK1/2) transduction pathways in these processes. The first aim was to determine whether LPS induces ERK1/2 activation and changes in epithelial permeability in epithelial cells alone or only in the presence of immune cells. The second aim was to determine whether the changes in the epithelial permeability were diminished by ERK1/2 blockade. The third aim was to investigate the role of protein kinase C (PKC) activation as an upstream event in activation of ERK1/2. In vitro 20 microg/ml LPS challenge reduced epithelial barrier function, and induced ERK1/2 phosphorylation in primary cultures of bovine tracheal epithelium and in the transformed human airway epithelial cell line, Calu-3. LPS initiated migration of neutrophil-like and monocyte-like transformed HL-60 cell across sheets of Calu-3 cells. The migration rate and the associated changes in the electrical resistance, permeability to albumin, and ERK1/2 phosphorylation were all blocked by calphostin C, the specific blocker of PKC and by PD98059 (2'-amino-3'-methoxyflavone), a selective cell-permeable inhibitor of MAP kinase kinase. In rats, in vivo perfusion of the lumen of an isolated segment of trachea with LPS (0.1 mg/ml) initiated migration of neutrophils and increased the permeability to albumin. Again, these effects were markedly inhibited by PD98059 and calphostin C (by > 50%). We conclude that epithelial ERK1/2 is activated by endotoxin via PKC and is an important pathway in regulation of epithelial permeability.

Measurement of nasal potential difference in mild asthmatics.

STUDY OBJECTIVE: To determine whether ion transport or barrier function across the nasal epithelium are altered in asthmatics. DESIGN: In this pilot study, the nasal potential difference (PD) was measured using the technique established by Knowles and colleagues. A flowing agar bridge made electrical contact with the surface of the nasal epithelium along the floor of the nose. This bridge was referenced to a cutaneous electrode to determine the PD across the nasal epithelium. Changes in nasal PD in response to amiloride, chloride-free medium, and chloride-free medium containing isoproterenol were measured, and responses of asthmatics and healthy control subjects were compared. PARTICIPANTS: Measurements were made in eight adult nonasthmatic subjects and 6 adult asthmatic subjects. All asthmatics had mild intermittent asthma. MEASUREMENTS AND RESULTS: Continuous measurements of nasal PD were obtained while the nasal surface was perfused consecutively with saline solution (NaCl-containing solution), saline solution plus 100 micro mol/L amiloride, chloride-free solution plus amiloride, and chloride-free solution with amiloride plus 10 micro mol/L isoproterenol (a beta-adrenergic agonist). No significant differences in baseline PD or change in PD in response to changes in perfusate were found between the two groups. CONCLUSIONS: Our results suggest that ion transport and barrier function of patients with mild asthma are normal. Therefore, by contrast to cystic fibrosis, changes in salt and water transport across airway epithelium may not contribute to accumulation of mucous secretions in asthma.

Polarized cultures of human airway epithelium from nasal scrapings and bronchial brushings.

Airway epithelial cultures are generally derived from tracheas postmortem or from surgical specimens of nasal polyps or turbinates. Scrapings of the mucosal surface have been little used as starting material for cultures because of their low yield of epithelial cells and their contamination with mucous secretions, blood, and underlying connective tissue. For the first time, we report that human airway epithelial cells obtained from nasal scrapings or bronchial brushings can be grown in culture to produce polarized cell sheets suitable for studies of vectorial transport.

A novel extract SB-300 from the stem bark latex of Croton lechleri inhibits CFTR-mediated chloride secretion in human colonic epithelial cells.

An oligomeric proanthocyanidin (SP-303) extracted from the bark latex of the tree Croton lechleri (family Euphorbiaceae) is a potent inhibitor of cholera toxin-induced fluid accumulation and chloride secretion. The manufacturing process for SP-303 was optimized and simplified to produce an increased yield of the herbal extract. The novel extract (named SB-300) contained on average 70.6+/-7.2% SP-303 by weight (mean +/- S.D.; n=56 lots). Here, we describe the effectiveness of SB-300 on cAMP-regulated chloride secretion, which is mediated by the cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) in human colonic T84 cells. Exposure of the apical surface to SB-300 blocked forskolin-stimulated Cl- secretion by 92.2+/-3.0% with a half-maximal inhibition constant (KB) of 4.8+/-0.8 microM. For SP-303, stimulated Cl- currents were decreased by 98.0+/-7.2 % and KB averaged 4.1+/-1.3 microM. There was no significant difference between the blocking kinetics of SP-303 and SB-300. Forskolin-stimulated whole cell Cl- currents were effectively blocked by extracellular addition of SB-300 (63+/-8.5%; n=3) and to a similar extent by SP-303 (83 +/- 0.6%; n=2; at 50 microM each). Both extracts inhibited a time- and voltage-independent Cl- conductance, which indicated the involvement of CFTR Cl- channels. We conclude that both SP-303 (used in Provir) and SB-300 (used in NSF Normal Stool Formula) are novel natural products that target the CFTR Cl- channel. SB-300 is a low cost herbal extract and may present a complementary and alternative medicine approach for the treatment of fluid loss in watery diarrhea.

Distribution and size of mucous glands in the ferret tracheobronchial tree.

A transgenic ferret model of cystic fibrosis has recently been generated. It is probable that malfunction of airway mucous glands contributes significantly to the airway pathology of this disease. The usefulness of the ferret model may therefore depend in part on how closely the airway glands of ferrets resemble those of humans. Here, we show that in the ferret trachea glands are commonest in its most ventral aspect and disappear about half way up the lateral walls; they are virtually absent from the dorsal membranous portion. Further, the aggregate volume of glands per unit mucosal surface declines progressively by about 60% between the larynx and the carina. The average frequency of glands openings for the ferret trachea as a whole is only about one-fifth that in humans (where gland openings are found at approximately the same frequency throughout the trachea). Glands in the ferret trachea are on average about one-third the size of those in the human. Therefore, the aggregate volume of tracheal glands (per unit mucosal surface area) in the ferret is only about 6% that in humans. As in other mammalian species, airway glands in the ferret disappear at an airway internal diameter of ∼1 mm, corresponding approximately in this species to airway generation 6.

Quantitative real-time PCR for rhinovirus, and its use in determining the relationship between TCID50 and the number of viral particles.

The development of a quantitative real-time PCR (qPCR) assay for human rhinovirus serotype 16 (HRV16) is described using the plasmid pR16.11, which contains the full-length genome of HRV16. A standard curve was generated by plotting the critical threshold (C(t)) against numbers of plasmid. The limit of sensitivity was less than10 cDNA copies, and the curve showed a high degree of linearity over a range of 10(1) to 10(6) cDNA copies with r(2)≥0.9989. Amplification efficiency of the qPCR was greater than 97.6 percent. The standard curve was highly reproducible with low intra- and inter-assay coefficients of variation. Standard curves were also generated from cDNA derived from two viral suspensions of known TCID(50), and were exactly parallel to those generated from the plasmid. Comparison of the curves generated from the plasmid or viral cDNA showed that for the two suspensions, TCID(50) corresponded to either 142 or 2088 viral particles. This new qPCR will permit quantitative assessments of interactions between virus and epithelium such as determinations of the affinity and number of viral binding sites or of the number of virus produced per infected cell.

Transgenic animals may help resolve a sticky situation in cystic fibrosis.

Cystic fibrosis (CF) is caused by defects in the CFTR, a cAMP-activated Cl- channel of epithelia. The resulting reduction in epithelial fluid transport creates abnormally viscous secretions from airway mucous glands that may be a major factor in CF pathology. Mouse airways have few mucous glands, and the mouse model of CF exhibits no significant airway disease. Pigs and ferrets, however, have approximately the same number of airway mucous glands as humans. In this issue of the JCI, three independent research groups conclude that changes in airway mucous gland function in CFTR-deficient animals of these species resemble the changes seen in human CF. It is expected, therefore, that these animals will develop lung disease similar to human CF and prove to be valuable models on which to test potential therapies.

Cultures of human tracheal gland cells of mucous or serous phenotype.

There are two main epithelial cell types in the secretory tubules of mammalian glands: serous and mucous. The former is believed to secrete predominantly water and antimicrobials, the latter mucins. Primary cultures of human airway gland epithelium have been available for almost 20 yr, but they are poorly differentiated and lack clear features of either serous or mucous cells. In this study, by varying growth supports and media, we have produced cultures from human airway glands that in terms of their ultrastructure and secretory products resemble either mucous or serous cells. Of four types of porous-bottomed insert tested, polycarbonate filters (Transwells) most strongly promoted the mucous phenotype. Coupled with the addition of epidermal growth factor (EGF), this growth support produced "mucous" cells that contained the large electron-lucent granules characteristic of native mucous cells, but lacked the small electron-dense granules characteristic of serous cells. Furthermore, they showed high levels of mucin secretion and low levels of release of lactoferrin and lysozyme (markers of native serous cells). By contrast, growth on polyethylene terephthalate filters (Cyclopore) in medium lacking EGF produced "serous" cells in which small electron-dense granules replaced the electron-lucent ones, and the cells had high levels of lactoferrin and lysozyme but low levels of mucins. Measurements of transepithelial resistance and short-circuit current showed that both "serous" and "mucous" cell cultures possessed tight junctions, had become polarized, and were actively secreting Cl.