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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.

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.

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 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. Anat Rec, 296:1768–1774, 2013. © 2013 Wiley Periodicals, Inc.

—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 (10−5M) 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 β-adrenergic receptors will be abnormally concentrated in CF.

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 (Ct) 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 101 to 106 cDNA copies with r2 ≥ 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 TCID50, 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, TCID50 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.

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.

Horses commonly suffer from respiratory diseases associated with excess secretions in the airway lumen, some of which are presumably derived from airway mucous glands. However, these structures have been little investigated in the horse. Accordingly, we describe here the number, distribution and size of equine tracheal mucous glands, and compare the data with similar information for other mammalian species. Two types of gland acini were present. In the thick connective tissue, up to 400 microm beneath the epithelium, gland acini were grouped in thin sheets that, in cross-section, averaged 20 microm thick and were up to 4.0 mm in length. However, it is probable that most sheets had maximal diameters much less than 4.0 mm. Between 400 to 900 microm below the epithelium, the connective tissue was much more diffuse, and glands were larger and more globular. Gland volume in the ventral portion was approximately 1.7 microl/cm2 of mucosal surface, and approximately 1.1 microl/cm2 in the dorsal portion. Glands were somewhat more abundant between, rather than over, the cartilaginous rings, but the difference between the 2 locations was not marked. Mucous gland openings were small (20 microm diameter) and very unevenly distributed, generally occurring about 100 microm apart in longitudinal rows of about 5. Average frequency of openings in the ventral portions of 3 tracheas was approximately 1.0/mm2 of mucosal surface. The volume of individual glands was therefore approximately 17 nl. Although the frequency of gland openings in the horse trachea is similar to that for the tracheas of other large mammalian species, horse tracheal gland volume was only about 15% that of the other species. Therefore, the excess 'mucous' secretions seen in equine recurrent airway obstruction and other respiratory diseases are unlikely to be caused by comparatively high levels of airway mucous gland secretion. Instead, they may be caused mainly by hyperplasia of the mucus-producing cells of the surface epithelium or by vascular transudation.

Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance ( GCl) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral GCl was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl2 = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl2 > tamoxifen > 4,4′-dinitro-2,2′-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of GCl were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive GCl; 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive GCl; and 3) a forskolin-activated, linear GCl, which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl− secretion or support transcellular Cl− absorption.

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]iand [Ca2+]icoordinate electrolyte and macromolecular secretion and probably occur together for baseline activity in vivo, with cholinergic elevation of [Ca2+]ibeing 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.

We measured innate immune responses by primary human tracheal epithelial (HTE) cells grown as confluent, pseudostratified layers during exposure to inflammatory activators on apical vs. basolateral surfaces. Apical Pseudomonas aeruginosa strain PAK (but not flagellin mutant PAK.fliC), flagellin, and flagellin + PAK.fliC activated NF-kappaB and IL-8 expression and secretion. In contrast, HTE cells were insensitive to LPS compared to flagellin. Flagellin activated NF-kappaB in columnar but not basal cells. IL-1beta + TNF-alpha elicited responses similar to those of flagellin. Basolateral flagellin or IL-1beta + TNF-alpha caused 1.5- to 4-fold larger responses, consistent with the fact that NF-kappaB activation occurred in both columnar and basal cells. MyD88 (toll receptor-associated adapter), IL-1 receptor (IL1R)1, and TNF-alpha receptor (TNFR)1 were expressed in columnar and basal cells. ZO-1 was localized to tight junctions of columnar cells but not to basal cells. We infer the following. 1) Flagellin is necessary and sufficient to trigger inflammatory responses in columnar cells during accumulation of P. aeruginosa in the airway surface liquid (ASL); columnar cells express toll-like receptor 5 and MyD88, often associated with flagellin-activated cell signaling. 2) IL-1beta + TNF-alpha in the ASL also activate columnar cells, and these cells also express IL1R1 and TNFR1. 3) Apical flagellin, IL-1beta, and TNF-alpha do not activate basal cells because tight junctions between columnar cells prevent access from the apical surface to the basal cells. 4) Exposure of basolateral surfaces to inflammatory activators elicits larger responses because both columnar and basal cells are activated, likely because both cell types express receptors for flagellin, IL-1beta, and TNF-alpha.

Oxidative atmospheric pollutants represent a significant stress and cause injury to both vertebrate and invertebrate species. In both, the biosurfaces of their respiratory apparatus are directly exposed to oxidizing pollutant-induced stresses. Respiratory-tract surfaces contain integrated antioxidant systems that appear to provide a primary defense against environmental insults caused by inhaled atmospheric reactive oxygen species (ROS) and reactive nitrogen species (RNS), whether gaseous or particulate. When the biosurface antioxidant defenses are overwhelmed, oxidative and nitrosative stress to the acellular and cellular components of the exposed biosurfaces can ensue via direct chemical reactions that lead to the induction of inflammatory, adaptive, injurious, and reparative processes. The study of model invertebrates (e.g., Drosophila) has a long history of yielding valuable insights into both fundamental biology and pathobiology. Mutants and/or transgenic insects, with specific alterations in key components of innate and/or adaptive antioxidant defense systems and immune genes, offer opportunities to dissect the complex systems that maintain respiratory tract surface defenses against environmental oxidants and the ensuing host responses. In this article, we use a comparative absfont approach to consider interactions of atmospheric oxidant pollutants with selected biosystems. We focused primarily on ozone (O(3)) as the pollutant, vertebrate and invertebrate respiratory tracts as the exposed biosystems, and nonenzymatic micronutrient antioxidants as significant contributors to overall antioxidant defense strategies. We present parallels among these diverse organisms with regard to their protective strategies against environmental atmospheric oxidants, with particular focus given to using the invertebrate Drosophila as a potentially useful model for vertebrate respiratory-tract responses to inhaled oxidants specifically and pollutants in general. We conclude that the insect respiratory system has considerable promise toward understanding novel aspects of vertebrate respiratory tract responses to inhaled oxidative environmental challenges.

Lactoferrin and lysozyme are important antimicrobial compounds of airway surface liquid, derived predominantly from serous cells of submucosal glands but also from surface epithelium. Here we compared release of these compounds from the following human cell cultures: primary cultures of tracheal epithelium (HTE), Calu-3 cells (a lung adenocarcinoma cell line frequently used as a model of serous gland cells), 16HBE14o- cells (an SV40 transformed line from airway surface epithelium), T84 cells (a colon carcinoma cell line), and human foreskin fibroblasts (HFF). For lysozyme, baseline secretory rates were in the order Calu-3 > 16HBE14o- > HTE ≈ T84 > HFF = 0; for lactoferrin, the only cell type showing measurable release was HTE; for mucus, HTE > Calu-3 > 16HBE14o- ≈ T84 > HFF = 0. A wide variety of neurohumoral agents and inflammatory stimuli was without effect on lactoferrin and lysozyme release from HTE or Calu-3 cells, although forskolin did stimulate secretion of water and lysozyme from Calu-3 cells. However, the concentration of lysozyme in the forskolin-induced secretions was much less than in airway gland secretions. Thus our data cast doubt on the utility of Calu-3 cells as a model of airway serous gland cells but do suggest that HTE could prove highly suitable for studies of mucin synthesis and release.

The goal of this study was to determine whether the extracellular regulated kinases (ERK1/2) are involved in leukocyte transmigration across airway epithelium and the associated changes in epithelial permeability. In vitro, we used formyl-methionyl-leucyl-phenylalanine (fMLP) to induce migration of HL-60 cells (a human leukocyte cell line) across sheets of polarized Calu-3 airway epithelial cells and also to induce migration of human neutrophils across primary cultures of cow tracheal epithelial cells. In both systems, leukocyte migration decreased transepithelial electrical resistance (R(te)), increased epithelial permeability to albumin (P(alb)), and increased ERK1/2 phosphorylation in epithelial cells. Leukocyte migration and the associated changes in R(te), P(alb), and ERK1/2 phosphorylation were inhibited by calphostin C, a blocker of protein kinase C (PKC), and by PD98059 (a blocker of ERK1/2). Leukocyte transmigration in rat tracheas in vivo was induced with fMLP, and was associated with increased P(alb) and phosphorylation of epithelial ERK1/2. Again, migration and the associated changes were inhibited by luminal PD98059 or calphostin C though neither agent affected rat leukocyte migration in Boyden chambers in vitro. We conclude that PKC and ERK1/2 pathways are activated in airway epithelial cells during migration of leukocytes and are important regulators of airway epithelial permeability.

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 μmol/L amiloride, chloride-free solution plus amiloride, and chloride-free solution with amiloride plus 10 μmol/L isoproterenol (a β-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.

Inflammatory diseases of the upper respiratory tract are characterized by flow of plasma filtrate across the epithelium into the airway lumen (“transudation”). Elsewhere, we have proposed that extravasation from microvessels causes edema, and this is associated with elevated subepithelial hydrostatic pressure that drives transudation. To test this hypothesis, we have attempted to block transudation by elevating luminal hydrostatic pressure. We measured the appearance of plasma markers into the lumen of an isolated perfused segment of rat trachea in vivo and found that stimulation of one vagal nerve caused a rapid (half-time ∼5 min) and nonselective increase in the flow of markers from blood to airway lumen. Leukocyte migration also caused transudation that developed much more slowly (half-time = 2–3 h). In both cases, transudation was blocked by application of luminal hydrostatic pressures. The critical luminal pressure needed to block vagally induced transudation was ∼4.5 cmH2O, and, to block epithelial transudation induced by leukocyte traffic, it was 3 cmH2O, and we conclude that these are the subepithelial pressures that drive inflammatory transudation into the airway lumen.

Transport of lung liquid is essential for both normal pulmonary physiologic processes and for resolution of pathologic processes. The large internal surface area of the lung is lined by alveolar epithelial type I (TI) and type II (TII) cells; TI cells line >95% of this surface, TII cells + and K + (Rb + ) transport in TI cells isolated from adult rat lungs and compared the results to those of concomitant experiments with isolated TII cells. TI cells take up Na + in an amiloride-inhibitable fashion, suggesting the presence of Na + channels; TI cell Na + uptake, per microgram of protein, is ≈2.5 times that of TII cells. Rb + uptake in TI cells was ≈3 times that in TII cells and was inhibited by 10 −4 M ouabain, the latter observation suggesting that TI cells exhibit Na + -, K + -ATPase activity. By immunocytochemical methods, TI cells contain all three subunits (α, β, and γ) of the epithelial sodium channel ENaC and two subunits of Na + -, K + -ATPase. By Western blot analysis, TI cells contain ≈3 times the amount of αENaC/μg protein of TII cells. Taken together, these studies demonstrate that TI cells not only contain molecular machinery necessary for active ion transport, but also transport ions. These results modify some basic concepts about lung liquid transport, suggesting that TI cells may contribute significantly in maintaining alveolar fluid balance and in resolving airspace edema.

Continuous secretion of liquid into the fetal mammalian lung is important for its normal development. The main driving force for this secretion is local osmotic gradients created across alveolar and airway epithelia by active secretion of Cl – . At the time of birth, the primary type of active solute transport across pulmonary epithelia switches, within minutes, from active secretion of Cl – to active absorption of Na + , a change that promotes absorption of liquid. Recent research, however, suggests that transepithelial hydrostatic pressure gradients generated during inspiration may be of more importance than solute transport in reabsorption of fetal lung liquid. Nevertheless, active Na + absorption across pulmonary epithelia persists in the air-filled adult lung as a guard against alveolar flooding in pneumonia and other inflammatory pulmonary diseases. This chapter reviews the solute transport processes of airway epithelium, their developmental changes, the cell types responsible, and the mechanisms by which lung liquid is absorbed at birth.

We have compared the distribution, numbers and volume of mucous glands in the tracheas of 11 mammalian species. No glands were present in the rabbit. The mouse only contained glands at the border between the trachea and larynx. In the rat, glands were commonest in the cephalad third of the trachea, but on average were much scarcer than in the larger species. Between species, there was a significant correlation between airway diameter and gland volume per unit surface area, suggesting that the rate of deposition of inhaled particles may increase in large airways. In the ventral portion of the trachea of about half the species, the glands were concentrated between the cartilaginous rings; in others they were evenly distributed over and between the rings. In most species in which the trachealis muscle attached to the internal surface of the cartilaginous rings, the glands were external to the muscle. In all species in which the muscle attached to the external surface of the cartilaginous rings, the glands were internal to the muscle. In the ox, goat, dog and sheep, the volume of glands per unit tracheal surface area was markedly greater in the ventral than the dorsal aspect of the trachea. The reverse was true of the pig. In humans, gland density in the 2 regions was similar. The frequency of gland openings was determined in the ox, goat, pig, dog and sheep tracheas, and ranged from 0.3 per mm2 in the dorsal portion of the sheep trachea to 1.5 per mm2 in the ventral portion of the ox trachea. For these 5 species, the volume of gland acini per unit luminal surface area varied linearly with the numbers of gland openings, with the volume of individual glands being constant at approximately 120 nl.

In human airways, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is predominantly expressed in serous cells of the tracheobronchial glands. Despite considerable evidence that submucosal glands are important contributors to the pathophysiology of CF lung disease, most attempts at CFTR gene transfer have primarily targeted airway surface epithelial cells. In this study, we systematically evaluated CFTR gene transfer into cultures of immortalized CF human tracheobronchial submucosal gland (6CFSMEO) cells using adenovirus and cationic lipid vectors. We found that the efficiency of adenovirus-mediated gene transfer was comparable in 6CFSMEO and CFT1 cells (a surface airway epithelial cell line isolated from a subject with CF). So was the ranking order of adenovirus vectors containing different enhancers/promoters (CMVE1a approximately phosphoglycerokinase), as determined by both X-Gal staining and quantitative measurement of beta-galactosidase activity. Further, we provide the first demonstration that cationic lipids mediate efficient gene transfer into 6CFSMEO cells in vitro. The transfection efficiency at optimal conditions was higher in 6CFSMEO than in CFT1 cells. Finally, either infection with adenoviral vectors or transfection with cationic lipid:plasmid DNA complexes encoding CFTR significantly increased chloride (Cl-) permeability, as assessed using the 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) fluorescence assay, indicating restoration of functional CFTR Cl- channel activity. These data show that although the mechanisms of transfection may be different between the two cell types, 6CFSMEO cells are as susceptible as CFT1 cells to transfection by adenoviral and cationic-lipid gene transfer vectors.

Elevated levels of Na and Cl in airway surface liquid may play a major role in the airway pathology of cystic fibrosis (CF) (J. J. Smith, S. M. Travis, E. P. Greenberg, and M. J. Welsh. Cell85: 229–236, 1996) and could be caused by block of transcellular Cl absorption due to lack of a functional CF transmembrane conductance regulator (CFTR). To test for transcellular absorption of Cl across non-CF epithelium, we studied how fluid absorption was affected by the opening and closing of Cl channels. Forskolin (an activator of CFTR) tripled fluid absorption across primary cultures of bovine tracheal epithelium but had no effect on human cells. However, in both species, fluid absorption was markedly inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, a blocker of CFTR. Microelectrode studies suggested that the magnitude of the absorptive response to forskolin in bovine cells depended on the size of an inwardly directed electrochemical driving force for Cl movement across the apical membrane. Patch-clamp measurements of bovine cells revealed CFTR in the apical membrane and a cAMP-activated, inwardly rectifying Cl channel in the basolateral membrane. We conclude that a significant fraction of absorbed Cl passes transcellularly in bovine tracheal epithelial cultures, with CFTR as the path of entry in the apical membrane and a novel cAMP-activated Cl channel as the exit route in the basolateral membrane. Our data further indicate that a similar pathway may exist in non-CF human tracheal epithelium.

Fluid transport across cultures of bovine tracheal epithelium was measured with a capacitance probe technique. Baseline fluid absorption ( Jv) across bovine cells of 3.2 μl ⋅ cm−2⋅ h−1was inhibited by ∼78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with a concomitant decrease in transepithelial potential (TEP) and increase in transepithelial resistance ( Rt). Effects of P. aeruginosa were blocked by amiloride, which decreased Jvby 112% from baseline of 2.35 ± 1.25 μl ⋅ cm−2⋅ h−1, increased Rtby 101% from baseline of 610 ± 257 Ω ⋅ cm2, and decreased TEP by 91% from baseline of −55 ± 18.5 mV. Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption were due in part to a block of basolateral membrane K channels. In the presence of Cl transport inhibitors [5-nitro-2-(3-phenylpropylamino)-benzoic acid, H2-DIDS, and bumetanide], P. aeruginosa induced a fluid secretion of ∼2.5 ± 0.4 μl ⋅ cm−2⋅ h−1and decreased Rtwithout changing TEP. However, these changes were abolished when the transport inhibitors were used in a medium in which Cl was replaced by an impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect on Jv, TEP, or Rt. Mutants lacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encoding a ς factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial salt and water transport induced by P. aeruginosa may alter viscosity and ionic composition of airway secretions so as to foster further bacterial colonization.

Cystic fibrosis (CF) is caused by the loss of functional CFTR Cl- channels. However, it is not understood how this defect disrupts salt and liquid movement in the airway or whether it alters the NaCl concentration in the thin liquid film covering the airway surface. Using a new approach, we found that CF airway surface liquid had a higher NaCl concentration than normal. Both CF and non-CF epithelia absorbed salt and liquid; however, expression of CFTR Cl- channels was required for maximal absorption. Thus, loss of CFTR elevates the salt concentration in CF airway surface liquid and in sweat by related mechanisms; the elevated NaCl concentration is due to a block in transcellular Cl- movement. The high NaCl may predispose CF airways to bacterial infections by inhibiting endogenous antibacterial defenses.

The luminal surface of airways is lined by a thin film of airway surface liquid (ASL). Physiological regulation of the depth of ASL has not been reported previously. In this paper, we have used low-temperature scanning electron microscopy of rapidly frozen specimens of bovine tracheal epithelium to demonstrate alterations in the depth of ASL in response to the cholinergic agonist methacholine. We first established that methacholine selectively stimulated airway glands, with maximal secretion at approximately 2 min and a return to baseline within approximately 5 min. A 2-min exposure to methacholine increased the depth of ASL from 23 to 78 microns. Thereafter, depth decreased linearly with time, reaching 32 microns at 30 min. The initial increase in depth was blocked by bumetanide, an inhibitor of active chloride secretion, whereas the slow decline back to baseline was inhibited by amiloride, a blocker of active sodium absorption. We conclude that the methacholine-induced changes in ASL depth reflect transient gland secretion followed by liquid absorption across the surface epithelium.

The depth and composition of human airway surface liquid (ASL) may depend on secretion from airway glands, ion transport across the surface epithelium, goblet cell discharge, transepithelial gradients in hydrostatic pressure, and surface tension. Published values for the frequency of airway glands and for the secretory rates of individual glands suggest that total gland secretion in human trachea can amount to approximately 60 microL x cm(-2) x h(-1). Volume absorption directly measured across cultures of surface epithelium from human trachea is approximately 5 microL x cm(-2) x h(-1). These flows should alter the depth of ASL at +10 and -1 microm x min(-1). We have looked for changes in ASL depth of this magnitude using low-temperature scanning electron microscopy (LT-SEM) of rapidly frozen specimens of bovine trachea. Stimulation of gland secretion with methacholine led to an initial increase in depth of approximately 25 microm x min(-1) followed by a decline at approximately 1.5 microm x min(-1). Whereas the initial increase in depth was probably due to transient gland secretion, the later decline reflected active absorption of liquid across the surface epithelium. Finally, we present preliminary data showing that LT-SEM can be combined with X-ray microanalysis to determine the elemental composition of ASL.

1. There is evidence that defective submucosal gland secretion contributes to the airway pathology of cystic fibrosis (CF). Using a capacitance probe technique, we have compared fluid transport across submucosal gland cultures from individuals with and without CF. 2. Under baseline conditions, approximately 60% of non-CF cultures secreted fluid; the rest absorbed. In secreting tissues, amiloride increased secretion, whereas in absorbing tissues it reduced or reversed absorption. 5-Nitro-2(3-phenylpropylamino)-benzoate (NPPB) a blocker of the CF transmembrane conductance regulator (CFTR), converted secretion to absorption. Thus, the direction and magnitude of baseline fluid movement depended on a balance between active absorption of Na+ and cAMP-dependent secretion of Cl-. 3. 8-(4-Chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP), methacholine and luminal uridine 5'-triphosphate (UTP) all induced or increased fluid secretion across non-CF cultures. Results with NPPB and with 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS), a blocker of Ca(2+)-activated Cl- channels, suggested that fluid secretion induced by CPT-cAMP was mediated primarily by CFTR; UTP acted entirely via Ca(2+)-activated Cl- channels, and methacholine activated both pathways. 4. All CF cultures showed baseline fluid absorption, which was abolished by amiloride. 5. CF cultures showed a normal secretory response to UTP, a reduced response to methacholine, and no response to CPT-cAMP. 6. Thus, the absorptive processes of airway glands are retained in CF, but the cAMP-dependent secretory process is lost. This would markedly reduce the water content of gland secretions. The resulting change in viscosity would contribute to the accumulation of airway mucus which is characteristic of this disease.

We describe a simple hyposmotic shock procedure whereby the apical membrane of airway epithelium can be made transiently leaky to proteins and other macromolecules. Bovine or human tracheal epithelial cells were grown as confluent polarized cell sheets on porous inserts. While physiological saline was maintained on the basolateral surface, the mucosal surface was exposed to water. This led to marked increases in the uptake of [14C]mannitol across both apical and basolateral membranes. On restoring saline to the mucosal surface, the [14C]mannitol permeability returned to preexposure levels with a half-life of approximately 5 min. Mucosal water also increased efflux of lactate dehydrogenase and the uptakes of fluorescent albumin and dextran (2,000 kDa). Water-induced increases in mannitol permeability were similar at 4 and 37 degrees C, suggesting that pinocytosis was not the mechanism. Detailed time courses of the uptake of dextran and the loss of lactate dehydrogenase and 36Cl showed that the bulk of the permeability increase occurred during the first 2- to 4-min exposure to water. Transepithelial resistance was reversibly decreased by exposure to water, but short-circuit current responses to transport blockers and secretagogues remained qualitatively normal. The hyposmotic shock procedure also successfully permeabilized apical membranes of primary cultures of nasal epithelial cells from a patient with cystic fibrosis (CF) and of JME/CF 15 cells, a cell line derived from CF bronchial epithelium. This simple and efficient procedure may prove useful in studies on the cell and molecular biology of airway and other epithelia.

Effective gene delivery to specific organs is a major goal for human gene therapy. The lung's structure allows instillation of agents into the airspaces, directly adjacent to the lung epithelium. We hypothesized that the airspace instillation of hypotonic solutions would increase the permeability of the lung epithelium and increase DNA uptake. This hypothesis was tested by instilling plasmid DNA (p4241) encoding the luciferase gene in isotonic and hypotonic solutions. The highest luciferase expression in the lung was achieved after the instillation of this plasmid DNA in distilled water. Aerosolization of water just before the instillation of the plasmid DNA also enhanced the expression level of luciferase in the lung. In addition, an intralobar instillation of the plasmid DNA in water significantly increased the luciferase expression, suggesting that the instillation of the plasmid over a smaller surface area increased expression. Levels of expression could be measured for 3 days. Water increases the permeability of lung epithelial cells transiently and/or enhances gene expression and can be used to achieve gene expression in the lung airspaces for short intervals without toxicity.

We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.

Genetic defects in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, cause cystic fibrosis. Most defective forms of CFTR show improper intracellular trafficking. Because isoprenylated, small GTP-binding proteins are involved in the vesicular trafficking of other integral membrane proteins, we have investigated the role of isoprenylation in the trafficking of CFTR to the apical membranes of primary cultures of human airway epithelium and of Calu-3 cells, a human lung carcinoma cell line. CFTR function was measured as short circuit current, 125I efflux, and conductance of cell sheets with permeabilized basolateral membranes. Lovastatin, an inhibitor of isoprenyl lipid biosynthesis, markedly inhibited all measures of CFTR function. The lovastatin-induced declines in CFTR function were corrected by the simultaneous addition of mevalonate or the isoprenyl lipids geranylgeranyl and farnesyl but not cholesterol. Lovastatin reduced total cellular CFTR as assessed by immunoprecipitation. Mevalonate or isoprenyl lipids protected CFTR levels from the actions of lovastatin. Together, these results suggest a role for isoprenyl lipids, presumably through the actions of small GTP-binding proteins, in the trafficking of CFTR to the apical membrane of human airway epithelium.

Genistein, a protein tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) in transfected NIH-3T3 fibroblasts that express the CFTR (3T3-CFTR). CFTR activity was assayed by 125I efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated 125I efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate 125I efflux in mock-transfected NIH-3T3 cells. Genistein, unlike forskolin and 3-isobutyl-1-methylxanthine, did not increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) above control levels. This demonstrates that genistein-dependent activation does not involve inhibition of phosphodiesterase activity and suggests that stimulation does not involve a direct activation of protein kinase A. Genistein stimulated 125I efflux to approximately 50% of the maximal rate with forskolin. Genistein did not increase 125I efflux at saturating forskolin but decreased the concentration of forskolin required for half-maximal stimulation. Orthovanadate (VO4), a phosphotyrosine phosphatase inhibitor, inhibited genistein-induced channel activation with an inhibition constant of approximately 20 microM. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway.

This paper describes a method for measuring the increase in halide permeability of isolated airway epithelial cells induced by adenosine 3′,5′-cyclic monophosphate (cAMP). Suspensions of isolated cells, known to contain the cystic fibrosis transmembrane conductance regulator (CFTR), were placed in the upper part of a Swinnex filter holder containing a filter with pores of 0.65 micron diameter. Medium was perfused over the cells at room temperature and collected at minute intervals following its passage through the filter. Experiments were performed on Calu-3 and T84 cells (human lung and colonic epithelial cell lines), primary cultures of dog and human tracheal epithelium, and Swiss 3T3 fibroblasts stably transfected with CFTR. In all cell types, addition of agents that elevate cAMP led to increases in the rates of loss of 36Cl and 125I. However, in human tracheal epithelial cells, warming the medium from room temperature to 37 degrees C was a more effective way of stimulating tracer efflux. Increases in efflux in response to either temperature or cAMP-elevating agents were inhibited by diphenylamine-2-carboxylate, a blocker of CFTR. Reproducible increases in tracer efflux were seen with as few as 10(6) cells. Cells that had been trypsinized off their culture dishes responded better than cells that had been scraped off, although treatment of scraped cells with trypsin enhanced their responsiveness to cAMP-elevating agents. Cystic fibrosis is characterized by the lack of a cAMP-activated Cl conductance in the apical membrane of airway epithlia.(ABSTRACT TRUNCATED AT 250 WORDS)

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 mpkCCDc14cortical 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 10−6M or less, did not inhibit ENaC-mediated short-circuit current ( Isc) but instead stimulated a transient increase in Isc. 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 ( Vte) is clamped to less than −10 mV. We hypothesized that this negative Vtemight be found under conditions of aldosterone stimulation. We next stimulated mpkCCDc14cells with aldosterone (10−6M) and then clamped the Vteto −50 mV, the Vteof aldosterone-stimulated cells under open-circuit conditions. ATP (10−6M) 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 Vte, 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.

Cells from the acini of human tracheal glands were grown in culture to produce confluent cell sheets of mucous or mixed seromucous phenotype. Levels of mediator-induced Cl secretion in mucous cells were 2-18% those of seromucous cells. Levels of the cystic fibrosis transmembrane conductance regulator (an apical membrane Cl channel) were also much less in mucous than in seromucous cells. These results suggest that serous cells are more important than mucous cells in providing the fluid component of gland secretions.

Of 12 cell lines derived from human lung cancers, only Calu-3 cells showed high transepithelial resistance (Rte) and increases in short-circuit current (Isc) in response to mediators. Calu-3 cells formed polarized monolayers with tight junctions and Rte of approximately 100 omega.cm2. Baseline Isc was approximately 35 microA/cm2 and was increased by approximately 75 microA/cm2 on elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by isoproterenol. Flux studies showed that the increase in Isc was due to Cl- secretion. Forskolin and permeant analogues of cAMP also increased Isc. Consistent with the presence of cAMP-dependent Cl- secretion, immunoprecipitation demonstrated the presence of the cystic fibrosis transmembrane conductance regulator (CFTR). Bradykinin, methacholine, trypsin, and histamine all transiently (15–30 s) elevated Isc, probably by increasing intracellular Ca concentration. Experiments in which the basolateral membrane was permeabilized with nystatin indicated that CFTR was substantially activated under baseline conditions and that Ca-activated Cl- channels were absent from the apical membrane. We anticipate that Calu-3 cells will prove useful in the study of Cl- secretion and other functions of human airway epithelial cells.

A major limitation in the study of vectorial ion transport, secretion, and differentiated function in the human airway epithelium has been the lack of suitable cell culture systems. Progress in this direction has been made through the transformation of primary cultured epithelial cells. However, these transformants tend to lose differentiated properties with increasing serial passage, particularly following crisis. The suc­ cessful establishment of a postcrisis SV40 large T-antigen transformed epithelial cell line derived from human bronchial epithelium is described. This cell line, 16HBEI40-, retains differentiated epithelial mor­ phology and functions. Cell cultures show the presence of tight junctions and cilia, and monolayers gener­ ate transepithelial resistance, as measured in Ussing chambers, and retain iJ-adrenergic stimulation of cAMP-dependent chloride ion transport, measured either by ,6CI- efflux or as short-circuit current in Ussing chambers. The cells also increase chloride transport in response to bradykinin or calcium iono­ phore. In addition, 16HBE140-cells express levels of both the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein readily detectable by Northern and Western hybridization analysis, respectively. These cells provide a valuable resource for studying the modulation of CFTR and its role in regulation of chloride ion transport in human airway epithelium as well as other aspects of human airway cell biology. The human airway epithelium is pseudostratified, consisting of highly organized layers of polar cells with specific dif­ ferentiated functions. It includes ciliated columnar cells, basal cells, and secretory goblet cells that are linked by tight junctions. The tight junctions provide a barrier between the airway lumen and the underlying tissues and divide the epi­ thelial cells into apical and basolateral domains. Both of these plasma membrane compartments contain different populations of proteins that allow for directional flux of ions

In cystic fibrosis (CF), absence or dysfunction of a phosphorylation-regulated chloride channel [CF transmembrane conductance regulator (CFTR)] leads to the loss or reduction of chloride secretion into the airways. Active sodium absorption is also increased in CF, and both of these ion transport changes could alter fluid transport across the airways. Under baseline conditions, cultured human airway epithelia from normal individuals absorbed fluid, and this absorption was increased in epithelia from patients with CF. In normal and CF epithelial cultures fluid absorption was inhibited by amiloride. Adenosine 3',5'-monophosphate stimulated fluid secretion in normal epithelial cultures but not in cultures from individuals with CF. In contrast, fluid secretion induced by nucleotide triphosphates (uridine triphosphate or adenosine triphosphate) was unaltered in cultures of epithelia from patients with CF, suggesting an approach to the treatment of CF.

In T84 cells, we investigated how stimulation of protein kinase C leads to an inhibition of cAMP-dependent chloride secretion. Specifically, we tested the hypothesis that the inhibition was caused by loss of the cystic fibrosis transmembrane regulator (CFTR), an apical membrane chloride channel. As described by others (Trapnell, B. C., Zeitlin, P. L., Chu, C.-S., Yoshimura, K., Nakamura, H., Guggino, W. B., Bargon, J., Banks, T. C., Dalemans, W., Pavirani, A., Lecocq, J.-P., and Crystal, R. G. (1991) J. Biol. Chem. 266, 10319-10323), we found that treatment with the phorbol ester, phorbol myristate acetate (PMA), reduced CFTR mRNA levels by approximately 80% with a t 1/2 of approximately 2 h. Chloride secretion, measured as forskolin-induced short circuit current, was also abolished by PMA with a t 1/2 of approximately 2 h. Levels of mature glycosylated CFTR measured by Western blotting also declined to 50 +/- 8% (n = 7) of control after a 12-h PMA treatment. However, a 12-h exposure to PMA did not affect the forskolin-stimulated efflux of 125I into high potassium medium, a measure of apical membrane CFTR activity. We conclude that increased turnover of apical membrane CFTR in PMA-treated cells compensates for the decline in anion channel numbers. By contrast to its lack of effect on 125I effluxes, PMA reduced the cAMP-induced increase in 86Rb efflux, suggesting that it inhibits chloride secretion mainly by an action on basolateral potassium channels.

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 CFTRinh-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 CFTRinh-172 with the exception of methacholine responses in mucous cells, which were not blocked by FFA and partially (∼60%) by CFTRinh-172. The effects of ionomycin on short-circuit current ( Isc) 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.

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.

Here we describe the conditions which allow cultured human tracheal epithelial cells to retain the ion transport properties and ultrastructure of the original tissue. The order of potency of growth supports and media additives in elevating baseline short-circuit current (Isc) and responses to mediators were vitrogen gel (VIT) greater than extracellular matrix from bovine corneal endothelial cells (ECM) greater than human placental collagen (HPC), and 2% Ultroser G serum substitute (USG) greater than 5% fetal calf serum (FCS) greater than defined growth factors (GF). For all combinations of medium and growth supports, an air interface (AIR) gave better electrical properties than immersion feeding (IMM). As opposed to our earlier conditions (HPC/FCS/IMM), the best new combination (VIT/USG/AIR) produced higher baseline Isc (58.0 +/- 10.6 vs. 5.1 +/- 1.0 microA/cm2) and increased Isc responses to isoproterenol (6.1 +/- 1.5 vs. 0.8 +/- 0.3 microA/cm2) and bradykinin (9.6 +/- 2.0 vs. 1.0 +/- 0.2 microA/cm2), while retaining high transepithelial resistance (227 +/- 5 omega.cm2). VIT/USG/AIR led to the appearance of cilia, an increase in the depth of the cell sheets (50 vs. 10 microns), longer and more frequent apical microvilli, and increased interdigitations of the basolateral membrane. Protein and DNA content were also significantly increased. Secretory granules were present which stained with antibody to goblet cells, but not to serous or mucous gland cells. CF cells grown in VIT/USG/AIR showed high baseline Isc (69 +/- 18 microA/cm2) and a proportionately larger inhibition of Isc by amiloride (70 +/- 10 vs. 34 +/- 3%). Isc did not respond to isoproterenol, and the response to bradykinin was 22% normal.

The role of adenosine 3',5'-cyclic monophosphate (cAMP) and protein phosphorylation during beta-adrenergic receptor stimulation of bovine tracheal gland serous cells was investigated in vitro. Isoproterenol, a beta-adrenergic agonist, increased the secretion of 35S-labeled molecules. Intracellular cAMP levels were increased within 1 min after stimulation of bovine tracheal gland serous cells with isoproterenol. The dose-response relationship for isoproterenol-stimulated generation of cAMP correlated with the dose-response relationship for isoproterenol-stimulated secretion of 35S-labeled molecules. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated both isoproterenol-evoked secretion of 35S-labeled molecules and the production of intracellular cAMP, and the beta-adrenergic receptor antagonist propranolol completely blocked both effects. The secretory response of the cells to isoproterenol could be mimicked by the cAMP analogues 8-bromoadenosine 3',5'-cyclic monophosphate and dibutyryl adenosine 3',5'-cyclic monophosphate. Activity of cAMP-dependent kinase was measured in soluble and particulate cell extracts. cAMP effected the state of phosphorylation of proteins associated with the soluble but not the particulate fraction. These studies are consistent with the hypothesis that beta-adrenergic stimulation of secretion from bovine tracheal gland serous cells occurs via a cAMP-mediated pathway and that one of the molecular events in this pathway is cAMP-dependent protein phosphorylation.

In primary cultures of dog tracheal epithelium, isoproterenol produced a transient increase in short-circuit current (Isc) (duration 30 s; maximal increase, 32 +/- 5 microA/cm2). This was followed by a more slowly developing sustained increase (9 +/- 3 microA/cm2), which mimicked the response to N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP). The transient and sustained responses had dissociation constants for isoproterenol of 2 x 10(-8) and 2 x 10(-9) M, respectively. Bradykinin (in the presence of indomethacin), substance P, histamine, and thrombin produced only transient increases in Isc. The time courses of these transients closely paralleled changes in concentration of intracellular Ca ([Ca2+]i) as measured with fura 2. For different mediators, there was a significant correlation between the maximal transient increase in Isc and the maximal increase in [Ca2+]i. The transients in Isc were not associated with elevation of adenosine 3',5'-cyclic monophosphate (cAMP) and were unaffected by pretreatment with DBcAMP, which abolishes the steady-state increase in response to isoproterenol. Both the transient increases in Isc and [Ca2+]i were inhibited by pretreatment with the Ca chelator 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate abolished the transient increases in [Ca2+]i and Isc in response to isoproterenol but not to bradykinin. These results provide evidence that 1) isoproterenol and bradykinin elevate [Ca2+]i by different mechanisms, and 2) Ca elevation is associated with a transient increase in Isc, whereas increased cAMP is associated with a smaller sustained increase.

We studied the effects of arachidonic acid (AA) on Cl secretion across primary cultures of dog tracheal epithelium. Cell sheets showed mean values for baseline short-circuit current (I sc ) and transepithelial resistance of 33.8 μ A / cm 2 and 360 Ω. cm 2 ( n =44). AA (5 × 10 −5 M ) added to both sides increased I sc by 27.8 ±5.2 μ A/cm 2 (mean±SE, n=8), and elevated intracellular cAMP levels. In the presence of 5 × 10 −6 M of both indomethacin (INDO) and nordihydroguaiaretic acid (NDGA) (inhibitors of cyclooxygenase and lipoxygenase, respectively), AA reduced Isc by 4.4 ± 0.6 μ A/cm 2 (n=10) without changing cAMP. Both INDO and NDGA were necessary to abolish the Isc increase in response to AA. The effects of AA on Isc were unaffected by amiloride. In the presence of INDO and NDGA, isoproterenol (ISO) raised cAMP and increased Isc by 27.6 ± 4.3 (transient) and 12.8 ± 3.2 μ A/cm 2 (sustained) (n=9). With AA present as well as INDO and NDGA, the transient and sustained responses to ISO were significantly reduced to 13.2 ± 2.4 and 3.9 ± 0.8 μ A/cm 2 (n=10), respectively; the increase in cAMP was unaltered. AA approximately halved baseline efflux of 125 I from confluent cell sheets in high K medium and reduced the isoproterenol-induced increase in efflux to 20% of control. These data are consistent with the reported inhibitory effect of AA on apical membrane chloride channels.

Acini of tracheal glands from 12 humans without cystic fibrosis (CF) and from two CF patients were isolated by enzymatic digestion. They were plated on flasks coated with human placental collagen (HPC) in media containing Ultroser G serum substitute and a variety of growth factors. Confluent cell sheets formed after 20 days. Cells were then isolated by trypsinization and replated at 10(6) cells/cm2 onto porous-bottomed inserts coated with HPC. Confluent sheets formed on day 1 after replating and were studied on day 10 in Ussing chambers. Transepithelial resistance (Rte) and baseline short-circuit current (Isc) of CF cultures (171 +/- 67 omega.cm2, 3.8 +/- 0.8 microA/cm2; n = 5) were significantly less than non-CF (541 +/- 116 omega.cm2, 9.9 +/- 2.3 microA/cm2; n = 14). Responses in Isc to mediators were also significantly reduced in CF: isoproterenol (10(-5)M) = 0.04 +/- 0.04 vs. 1.9 +/- 0.6; methacholine (10(-5)M) = 0.2 +/- 0.1 vs. 7.1 +/- 1.7; bradykinin (10(-6)M) = 0.4 +/- 0.1 vs. 5.0 +/- 1.0 microA/cm2; n = 5 for CF, n = 14 for non-CF. When CF and non-CF cells were matched for baseline Isc and Rte, the responses of CF cells to mediators still remained statistically lower than normal. The reduced responses of CF cells to bradykinin and methacholine, in addition to isoproterenol, suggest that both Ca-dependent and adenosine 3',5'-cyclic monophosphate-dependent regulation of Cl secretion are defective in CF tracheobronchial glands. The resulting reduction in fluid secretion by glands may contribute to the accumulation of airway mucus in CF.