Search

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Cultures of dog tracheal epithelium have proved very useful in studies of ion transport. Their short-circuit current (Isc), however, is usually much less than the original tissue. We have tested a variety of conditions in an attempt to produce large numbers of cells with electrical properties comparable with the original tissue. Of several growth supports, human placental collagen (HPC) gave the best results. When plated at 2.5 x 10(5) cells/cm2 onto HPC, cells grown in serum-free, growth factor-supplemented medium (GF medium) showed increases in cells per unit area, thickness of cell sheet, numbers of domes, numbers of apical microvilli, and degree of basolateral membrane interdigitation compared with cells grown in medium containing 5% fetal calf serum (FCS medium). Transepithelial resistance (Rte) and the increases in Isc and intracellular Ca in response to isoproterenol were also increased. However, baseline Isc and adenosine 3',5'-cyclic monophosphate levels were not changed. The improved electrical properties were maintained for up to 4 mo. GF medium combined with an air interface produced further increases in Rte, Isc, and changes in Isc in response to amiloride and isoproterenol. Ultrastructural features such as the presence of cilia, greater thickness of the cell sheet, and increased amplification of apical and basolateral membranes also indicated improved differentiation. Our results show that GF medium and an air interface can be combined with a simple growth support and a relatively low-plating density to allow the easy production of greater than 500 cm2 of cultured cells from a single trachea, with a level of differentiation similar to that of the original tissue.

Apical membranes from cow tracheal epithelium were prepared in a two-step process. First, most of the unwanted membranes in the crude homogenate were aggregated with Mg and removed by a low-speed spin. The membranes remaining in the supernatant were pelleted by a high-speed spin, resuspended, and exposed to ouabain-affinity chromatography. This step removed approximately 50% of the protein, all the Na-K-adenosinetriphosphatase, but had no effect on total levels of alkaline phosphatase (a marker for apical membranes). The specific activity of the apical membrane marker, alkaline phosphatase, was 21 +/- 7-fold (mean +/- SD) greater in the apical membranes than in the homogenate. Markers for nuclei, mitochondria, and basolateral membranes were excluded compared with the homogenate. Similar results were obtained with primary cultures of cow tracheal epithelium. The vesicular nature of the membranes was demonstrated in isotope uptake studies that revealed an osmotically active space.

The secretion of fluid into the airway lumen is due in part to active chloride secretion across the epithelium. Changes in Cl secretion require coordinated changes in the turnover of several transport proteins in order to avoid lethal changes in cell volume. Several different Ca- and cAMP-dependent pathways effect this coordination. Many mediators raise cyclic AMP which activates protein kinase A which in turn opens the apical membrane Cl channel. Cyclic AMP also raises Cai (without change in levels of inositol triphosphate, IP3), and this Ca is probably responsible for activation of basolateral K channels and NaK2Cl cotransporters. Other mediators raise Cai by elevating IP3, and seem to stimulate chloride secretion entirely by Ca-dependent pathways with no change in cyclic AMP levels. Protein kinase C and arachidonic acid inhibit Cl secretion.

Because an increase in the HCO3− concentration of oviductal liquid at midcycle is believed to markedly enhance fertility, we have studied active secretion of HCO3− across highly differentiated cultures of monkey oviductal epithelium. Cultured cell sheets were mounted in Ussing chambers and bathed in medium containing 25 mM HCO3−. Purinergic agents potently stimulated short-circuit current ( Isc) with an initial transient response declining within ∼2 min to a sustained response. The potency sequence of ATP ≈ UTP > ADP >> AMP suggested that the Isc response was mediated mainly by P2Y2 receptors. Acetazolamide, an inhibitor of carbonic anhydrase, had little or no effect on baseline Isc or the transient response to ATP but abolished the sustained response to ATP. Similar results were obtained on sheets of native epithelium. In pH-stat experiments, the abluminal medium of cell cultures was bathed in HCO3−-CO2 medium, and the pH of the unbuffered luminal medium was maintained at ∼7.4 by addition of strong acid or base. ATP stimulated base secretion, and this was inhibited by acetazolamide. Furthermore, these changes in secretion of base were in good quantitative agreement with the Isc responses. When phenol red (an estrogen) was removed from the culture medium, ATP-dependent HCO3− secretion was markedly reduced but could be restored by treatment with estradiol. Estrogens also markedly increased ciliation of the cultures. These results suggest that the midcycle increase in the HCO3− concentration of oviductal liquid may be mediated by the effects of estradiol on purinergic pathways or on ATP secretion.

In 1984, primary cultures of dog tracheal epithelium were shown to retain the ion transport processes of the original tissue. Since then, ion transport in both nasal and tracheal cell cultures has been studied with Ussing chambers, conventional and ion-sensitive microelectrodes, radiotracers, Cl-sensing fluorescent dyes, and patch clamping. Much of the resulting information could not have been obtained using the native tissues and has been especially useful in determining the ion transport defect(s) in cystic fibrosis. Recent improvements in growth conditions should make highly differentiated cell cultures routinely available, further increasing the usefulness of cultured cells for the study of ion transport by airway epithelium.

The generation of a pig model of cystic fibrosis (CF) is a multistep process. Initial steps in this process involved the design and cloning of a small DNA fragment (SDF) or large oligodeoxynucleotide (LODN) that contains the F508del mutation and a silent restriction fragment length polymorphism causing mutation. This SDF/LODN was transfected into wild-type (WT) pig fetal fibroblast with the intention of modifying the pig genomic DNA by small fragment homologous replacement (SFHR). The targeted deletion (F508del) was detected in a subpopulation of transfected cells by allele-specific polymerase chain reaction (AS-PCR).

One of the main functions of the airway epithelium is to inactivate and remove infectious particles from inhaled air and thereby prevent infection of the distal lung. This function is achieved by mucociliary and cough clearance and by antimicrobial factors present in the airway surface liquid (ASL). There are indications that airway defenses are affected by the pH of the ASL and historically, acidification of the airway surfaces has been suggested as a measure of airway disease. However, even in health, the ASL is slightly acidic, and this acidity might be part of normal airway defense. Only recently research has focused on the mechanisms responsible for acid and base secretion into the ASL. Advances resulted from research into the airway disease associated with cystic fibrosis (CF) after it was found that the CFTR Cl(-) channel conducts HCO (3) (-) and, therefore, may contribute to ASL pH. However, the acidity of the ASL indicated parallel mechanisms for H(+) secretion. Recent investigations identified several H(+) transporters in the apical membrane of the airway epithelium. These include H(+) channels and ATP-driven H(+) pumps, including a non-gastric isoform of the H(+)-K(+) ATPase and a vacuolar-type H(+) ATPase. Current knowledge of acid and base transporters and their potential roles in airway mucosal pH regulation is reviewed here.

Amiloride-sensitive, epithelial Na+ channel (ENaC)-mediated, active absorption of Na+ is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, d- myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na+ and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and 22Na+ flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives.

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 permeabilityin 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 investigatethe role of protein kinase C (PKC) activation as an upstream event in activation of ERK1/2. In vitro 20 μg/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.

The airways are lined with a film of liquid about 10 microm deep that is in two layers. Around the cilia is the watery periciliary sol. Over this is a mucous blanket that traps inhaled particles. The low viscosity of the periciliary sol allows the cilia to beat and propel the mucous blanket to the mouth. In large airways, mucus comes predominantly from the mucous glands but also from goblet cells in the surface epithelium. Water is added to the airway surface by gland secretion that is driven by active Cl secretion by serous cells. During inflammation elevation of the subepithelial hydrostatic pressure may also add significant volumes of water to the airway lumen. Water is removed by active Na transport across the surface epithelium. In airway diseases, the balance is shifted from water secretion to mucus secretion. In bronchitis and asthma this is due mainly to conversion of gland serous to mucous cells. In cystic fibrosis, gland serous cells cannot secrete water because they lack functioning CFTR in their apical membranes (CFTR is the cystic fibrosis transmembrane conductance regulator, a Cl channel that is abundant in serous cells). In all three diseases, the result is secretion of excessively concentrated gland secretions that are poorly moved by the cilia and accumulate. Altered salt and water transport by the surface epithelium may also contribute to the pathology of cystic fibrosis.

We used scanning electron microscopy to count the number of mucous gland openings in the tracheae and lower portion of the larynges of the rat, guinea pig, hamster, mouse and rabbit. Cells of the airway surface epithelium were removed by protease digestion better to visualise the gland openings. The distribution of glands was further studied by conventional histology and by PAS/Alcian blue staining of whole mounts. In all rodent species, gland openings in the larynx occurred with a frequency of 1-2 per mm2. Mice had no gland openings in their tracheae, and hamsters, only a handful. Rat tracheae contained 126+/-42 gland openings (+/-S.D.; n = 6) at a frequency of approximately 0.6 per mm2 at the top of the trachea and approximately 0.15 per mm2 at the bottom. Guinea pig tracheae contained 153+/-90 gland openings (+/-S.D.; n = 5), with 54% being in the top 40% of the trachea. In both rat and guinea pig, tracheal glands were found in the ventral aspect between the cartilaginous rings, and were absent from the dorsal membranous portion. Gland openings in most species were simple circles of approximately 50 microm diameter. However, glands in the rat trachea generally opened obliquely into shallow (approximately 20 microm deep) oval troughs (approximately 150 x 75 microm), which had their long axes oriented from head to tail. In the rabbit, there was no evidence of tracheal or laryngeal glands histologically. However, the tracheal and laryngeal surfaces contained numerous pits (approximately 30 microm diameter) distributed evenly over and between cartilages at a frequency of approximately 4 per mm2. These may correspond to the 'nests' of goblet cells described by others.

In 1983, Paul Quinton showed that the chloride permeability of sweat duct epithelium was essentially abolished in cystic fibrosis (CF) (1). Shortly after, cyclic adenosine monophosphate (cAMP)–dependent transport of chloride across airway epithelia was also shown to be greatly reduced in CF, and the defect was localized to the apical membrane (2, 3). Therefore, it was to be anticipated that, within a couple of years of cloning the CF gene, its protein product (the cystic fibrosis transmembrane conductance regulator [CFTR]) was shown to be a cAMP-activated chloride channel (4). The situation is now more complex. First, CFTR itself has been proposed to transport water, urea, formate, adenosine triphosphate (ATP), glutathione, and organic anions in addition to halide ions (5–9). Second, CFTR has been shown to influence a large number of cell functions in addition to transepithelial transport of chloride ( see Table 1). Not all of the effects listed in Table 1 are generally accepted, and in some cases the initial report has not been confirmed by subsequent studies. For instance, the defective acidification hypothesis was highly appealing in that it could explain the pleiotropic effects of CF (10). According to the hypothesis, failure of CFTR in intracellular vesicles led to decreased acidification of their contents. This would clearly affect the posttranslational processing and trafficking of many proteins. But several later studies failed to show differences in intravesicular pH in CF (11). The key questions to the many proposed actions of CFTR are ( 1 ) whether the methods are adequate and ( 2 ) do the results have any relevance to the function of affected human epithelia in vivo. Specific methodologic problems include random variation among cell lines, use of nonpolarized cells to draw conclusions about epithelial function, overexpression of CFTR, inadequacies in the transgenic mouse model of CF, and use of nonspecific pharmacologic agents. Many studies have compared CF and non-CF cell lines, most of which are poor models of native epithelium because they do not form tight junctions and polarize. Realizing this, many workers supplement their results on cell lines with data from primary cell cultures. These can very closely resemble the native epithelium in both their structure and function (12), and CF and non-CF cultures can be closely matched for cell number, structure, transepithelial resistance, and other parameters. However, the primary cultures used are generally very poorly characterized. At minimum, investigators should check whether their primary cultures are polarized, but even this simple assay (for transepithelial resistance) is rarely performed. Transfection of cell lines and expression of exogenous CFTR is also commonly used. The most sophisticated application of this approach involves the “recovery” of CF cell lines by expression of wild-type CFTR. The problem, however, with expression experiments is that the expressed CFTR is usually present at levels far greater than found in native epithelia. Excess of any ion channel in the membrane might be expected to have multiple effects on function. It is also possible that CFTR trafficking pathways may become saturated and then CFTR trafficked to unusual locations. In retinal pigment epithelium, for instance, CFTR is normally trafficked to the apical membrane, but exogenously expressed CFTR appears in the basolateral (S. Miller, personal communication). Similarly, in rat Fischer thyroid cells, the relative amounts of exogenous CFTR passing to the apical and basolateral membranes depend on the level of expression (13). Furthermore, if trafficking pathways are overwhelmed with CFTR, then trafficking of proteins other than CFTR may be affected. To control for the effects of overexpression, it is usual to transfect control cells with an irrelevant reporter gene such as lacZ. Yet this is not an entirely adequate control because the reporter gene is usually not an integral membrane protein and uses trafficking pathways different from CFTR. The CF mouse is a powerful investigational tool (14), but it must be remembered that mouse airways and their epithelia differ greatly from those of humans. Mouse airway epithelium has comparatively large numbers of Caactivated chloride channels (15) and lacks mucous glands and goblet cells (16). In fact, ion transport by the tracheal epithelium of CF transgenic mice is indistinguishable from ( Received in original form October 5, 1999 )

Abnormal folding of mutant cystic fibrosis transmembrane conductance regulator (CFTR) and subsequent degradation in the endoplasmic reticulum is the basis for most cases of cystic fibrosis. Structural differences between wild-type (WT) and mutant proteins, however, remain unknown. Here we examine the intracellular trafficking, degradation, and transmembrane topology of two mutant CFTR proteins, G85E and G91R, each of which contains an additional charged residue within the first putative transmembrane helix (TM1). In microinjected Xenopus laevis oocytes, these mutations markedly disrupted CFTR plasma membrane chloride channel activity. G85E and G91R mutants (but not a conservative mutant, G91A) failed to acquire complex N-linked carbohydrates, and were rapidly degraded before reaching the Golgi complex thus exhibiting a trafficking phenotype similar to DeltaF508 CFTR. Topologic analysis revealed that neither G85E nor G91R mutations disrupted CFTR NH2 terminus transmembrane topology. Instead, WT as well as mutant TM1 spanned the membrane in the predicted C-trans (type II) orientation, and residues 85E and 91R were localized within or adjacent to the plane of the lipid bilayer. To understand how these charged residues might provide structural cues for ER degradation, we examined the stability of WT, G85E, and G91R CFTR proteins truncated at codons 188, 393, 589, or 836 (after TM2, TM6, the first nucleotide binding domain, or the R domain, respectively). These results indicated that G85E and G91R mutations affected CFTR folding, not by gross disruption of transmembrane assembly, but rather through insertion of a charged residue within the plane of the bilayer, which in turn influenced higher order tertiary structure.

The presence of blood proteins and excess liquid in the airway lumen during airway inflammation may be secondary to extravasation and elevation of subepithelial hydrostatic pressure. This study examines how hydrostatic pressures of 5-20 cm H2O affect hydraulic conductivity and macromolecular permeability of primary cultures of bovine tracheal epithelium. Hydraulic conductivity was not altered by transepithelial pressure gradients of up to 20 cm H2O directed from the mucosal to serosal side of the tissue (m-s). By contrast, a 20-cm H2O s-m pressure resulted in a marked increase in hydraulic conductivity with the critical pressure lying between 10 and 20 cm H2O. Electrical conductance (i.e., permeability to ions) was not altered by m-s pressure gradients, or by a 5-cm H2O s-m gradient, but was increased by s-m pressures > or = 10 cm. Fluxes (s-m and m-s) of fluorescein and fluorescent dextrans (70 and 2000 kDa) were not altered by pressures of up to 20 cm H2O m-s. By contrast s-m pressure gradients of 20 cm H2O dramatically increased the s-m fluxes of these probes. The increases in flux were completely reversible. The results indicate that s-m pressure gradients greatly increase the hydraulic conductivity of airway epithelium by creating pores with an effective diameter greater than 54 nm.

A layer of liquid lines the airways in the lung. Previous microscopic studies have suggested that it is in two phases, with a mucous gel lying above a periciliary sol. However, shrinkage artifacts due to chemical fixation, dehydration, and drying have prevented reliable estimates of the depth of these layers. To avoid such problems, we have studied the surface liquid of bovine trachea by low-temperature scanning electron microscopy (LTSEM). A polished copper probe cooled to liquid nitrogen temperature was applied to the mucosal surface of sheets of excised tracheal epithelium to effect rapid freezing of surface liquid. Tissue sheets were then mounted in an LTSEM (AMRay 1000A with Biochamber) which maintains samples at -180 degrees C with a Joule-Thompson refrigerator built into the stage. Tissues were fractured at right angles to the epithelial surface, coated with gold, and viewed, all at 10(-5) to 10(-6) torr without transfer through air. The sample was stable under the electron beam at accelerating voltages up to 20 kV. Epithelial features (nuclei, cilia, microvilli, mucous granules) were well preserved. The mucosal surface of the cells was covered with material on the order of 8 microns in depth. The mucous gel and periciliary sol could be seen as distinct layers and could be distinguished by the size and pattern of ice crystal voids generated by radiant-etching of the fractured surface of the sample.

The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. Recent advances in culturing primary epithelial cells and the development of transformed airway epithelial cell lines have been particularly important in enhancing our understanding of the pathology associated with cystic fibrosis and lung cancer. The establishment of conditions that enhance the proliferative capacity of airway epithelial cells in primary culture was the first technical hurdle overcome in the development of in vitro culture systems. Research is now being geared toward the development of cell culture conditions that facilitate the expression in culture of the differentiated characteristics found in the native epithelium. Aside from the advances that have been made in defining the growth media and extracellular matrixes that enhance the expression of differentiated features, the use of an air-liquid interface has been a significant advance in the culture of airway epithelial cells. The implementation of the in vitro cell culture systems that have now been established and the research into optimizing the conditions for the growth of airway epithelial cells have been and will continue to be essential in the development of therapies for airway disease.

Human airways are lined with a film of liquid from 5-100 microns in depth, consisting of a periciliary sol around and a mucous gel above the cilia. Microscopical studies have shown the sol to be invariably the same depth as the length of the cilia, and we discuss possible reasons for this. The composition and sources of the airway surface liquid are also described. In addition the forces regulating its volume are analyzed. Several airway diseases are characterised by dramatic changes in the volume and composition of airway liquid. We review recent research suggesting that the accumulation of airway mucous secretions in cystic fibrosis is caused by alterations in active transport of ions and water across both the surface and gland epithelia.

We measured fluxes of radiotracers in Xenopus oocytes expressing the cystic fibrosis transmembrane conductance regulator (CFTR). Addition of adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents [forskolin and 3-isobutyl-1-methylxanthine (I/F)] led to large increases in uptake of 36Cl, 125I, and 82Br into oocytes expressing wild-type CFTR or delta F508 CFTR but not sham-injected oocytes. I/F also stimulated halide efflux from CFTR and delta F508 oocytes in the sequence Cl > Br > I. cAMP-induced increases in 36Cl efflux from delta F508 oocytes were approximately 20% of those in CFTR oocytes. Increases in halide efflux were blocked by diphenylamine-2-carboxylic acid but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The phorbol ester, phorbol 12-myristate 13-acetate, also stimulated 36Cl efflux from CFTR oocytes. ATP uptakes into CFTR and sham oocytes were similar, and both were reduced by I/F. However, ATP uptake into I/F-treated CFTR oocytes was slightly greater (approximately 40%) than into I/F-treated sham oocytes. Urea uptake into CFTR and sham oocytes was similar and in both cases was increased by I/F. However, the I/F-induced increase in urea uptake into CFTR oocytes was significantly greater than for sham oocytes. I/F stimulated formate uptake into CFTR oocytes but not into sham oocytes. Fluxes of 22Na, 86Rb, 35SO4, 32PO4, and mannitol were unaltered by expression and activation of CFTR.

Tracheal epithelial cells were grown on Nuclepore filters coated with human placental collagen. When grown immersed in medium containing fetal bovine serum, cells displayed an undifferentiated ultrastructure (no cilia and a cell height of approximately 10 microns). Short-circuit current (Isc) was approximately 1/10 that of the native epithelium. By contrast, when grown in hormonally defined, serum-free medium with an air interface, cells showed Isc equal to or greater than the original tissue, possessed cilia, and had a cell height of approximately 50 microns. Responses in Isc to mediators were similar to those of the original tissue, but differed from those of dog or human tracheal epithelium. Given the ready availability and low cost of the native tissues, bovine tracheal cultures grown in serum-free medium with an air interface should prove useful in studies of airway epithelial physiology.

We tested the hypothesis that, in asthma, the airway epithelial damage and leakage of blood proteins into the lumen are the result of edema and raised submucosal hydrostatic pressure. Sheets of dog tracheal epithelium were mounted in Ussing chambers, and the effects of transepithelial hydrostatic pressure differences (delta P) on conductance (G), [3H]mannitol flux (Jman), and fluorescein isothiocyanate-albumin flux (Jalb) were determined. delta P values of 20 cmH2O directed from the mucosal to submucosal side of the tissue (m----s) had no significant effects on G, Jman, Jalb, or tissue ultrastructure. delta Ps----;m caused increases in conductance (G) with a maximal effect at approximately 20 cmH2O. delta Ps----m of 20 cmH2O significantly (P less than 0.05) increased G (4.3 +/- 0.6 to 10.6 +/- 1.6 mS/cm2), Jman s—m (18 +/- 5 to 411 +/- 54 nmol.cm-2.h-1), J(alb)s----m (0.3 +/- 0.1 to 6.0 +/- 2.0 micrograms.cm-2.h-1), and J(alb)m—s (0.7 +/- 0.3 to 1.8 +/- 0.4 micrograms.cm-2.h-1). Jman m----s was not affected. On removal of delta P, G and Jman s----m returned to preexposure values, though J(alb)s----m remained slightly elevated at 1.1 +/- 0.3 micrograms.cm-2.h-1. Morphologically, delta Ps----m caused dilation of lateral intercellular spaces, disruption of tight junctions, and submucosal edema. The large increases in s----m fluxes of albumin and mannitol are consistent with bulk flow of fluid toward the lumen via the areas of epithelial damage.

1. Radioisotopes and intracellular microelectrodes were used to characterize the permeability of Xenopus oocytes to chloride and other halides. 2. Uptake of 36Cl had a half-time for equilibration of approximately 3 h, with an initial rate of Cl- entry corresponding to a permeability coefficient of 3.9 x 10(-7) cm/s, and an equilibrium uptake of 36Cl of 33 mM. 3. Replacement of bathing Na+ by K+ depolarized the oocytes from -46 to -7 mV and stimulated influx approximately 3-fold. 4. Influx was linearly dependent on bathing [Cl-] and was temperature dependent with an activation energy of 46 kJ/mol. Influx of 125I of 36Cl was not affected by the presence of equal concentrations of other halides or thiocyanate. These results are consistent with a channel-mediated entry mechanism. 5. Diphenylamine-2-carboxylate (DPAC) and 9-anthracene carboxylate (9-AC), blockers of Cl- channels in other cells, inhibited Cl- entry with dissociation constants (Kds) of approximately 5 x 10(-4) and approximately 10(-3) M, respectively. Inhibitors of Cl(-)-HCO3- exchange or Na(+)-K(+)-2Cl- co-transport did not affect Cl- influx. 6. Attempts to lower or raise intracellular Ca2+ with BAPTA or A23187, respectively, were also without effect on Cl- influx. 7. The halide selectivity sequence determined with isotopes was I- (3.2) greater than Br- (1.3) greater than Cl- (1.0). However, DPAC inhibited almost all of the 36Cl influx but only a small fraction of 125I influx. 8. Replacement of bathing Cl- by I- or Br-resulted in hyperpolarizations, from which the same selectivity sequence was determined. 9. Replacement of bathing Cl- by gluconate caused a marked depolarization, which was inhibited by DPAC and, less potently, by 9-AC.

To study C1 conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was greater than 100 omega.cm2, and short circuit current (Isc = 2-20 microA/cm2), representing active secretion of Cl, increased greater than threefold with addition of 10 microM isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the C1-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5 mM, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360 +/- 5 nm, emission greater than 410 nm). SPQ leakage from the cells was less than 10% in 60 min at 37 degrees C. Intracellular calibration of SPQ fluorescence vs. [C1] (0-90 nM) was carried out using high-K buffers containing the ionophores nigericin (5 microM) and tributyltin (10 microM); SPQ fluorescence was quenched with a Stern-Volmer constant of 13 M-1. Intracellular Cl activity was 43 +/- 4 mM. Cl flux was measured in response to addition and removal of 114 mM Cl from the bathing solution. Addition of 10 microM isoproterenol increased Cl efflux from 0.10 to 0.27 mM/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1 mM). In the absence of isoproterenol, removal of external Na or addition of 0.5 mM furosemide, reduced Cl influx by greater than fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5 mM barium diminished Cl influx by greater than twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the real-time measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism.

We have established well-differentiated, polarized cultures of monkey oviductal epithelium. Oviductal epithelial cells were isolated by protease digestion and plated on collagen-coated, porous cell culture inserts. About 5 d after plating, cells developed detectable transepithelial electrical resistance of up to 2000 Omega.cm(2) (an index of tight junction formation) and transepithelial voltages of up to 20 mV (an index of vectorial transepithelial ion transport). Measurements of short-circuit current in Ussing chambers indicated that active secretion of Cl was the major transepithelial active ion transport process, and that this was stimulated by elevation of either cAMP or Ca(i). Furthermore, estimates of the volume of mucosal liquid were consistent with Cl secretion mediating fluid secretion. Various microscopical methods showed that the cultures were densely ciliated and contained mature secretory cells. Transport across the oviductal epithelium determines the composition of the oviductal fluid, and the study of the relevant transport processes will be greatly enhanced by well-differentiated cultures of oviductal epithelium of the kind established here.

During fluid absorption induced by amphotericin B, the lateral intercellular spaces (LIS) of the dog tracheal epithelium were widely dilated as compared to untreated time controls. When fluid absorption was inhibited by ouabain, or by replacement of luminal Na by choline, amphotericin B failed to cause dilation of the LIS. These data suggest that, as in other epithelia, a significant amount of transepithelial fluid flow passes down the LIS, and that these spaces may provide the local osmotic compartment which is responsible for linking transepithelial fluid movement to active ion transport.

Under short-circuit conditions, vasoactive intestinal peptide (VIP) did not alter net Na+ movement but selectively stimulated net Cl- secretion across dog tracheal epithelium with a high affinity (Km congruent to 10(-8) M). The increase in Cl- secretion was not different from the rise in short-circuit current (Isc). However, stimulation of Cl- secretion was not maximal, because the addition of isoproterenol (10(-6) M) to VIP-treated tissues further increased the Isc by 54%. The effect of exogenous VIP was not blocked by a combination of atropine, phentolamine, propranolol (10(-5) or 10(-6) M), or tetrodotoxin (10(-6) M). Under open-circuit conditions, VIP caused an increase in the net secretion of Cl- and Na+, but the changes did not reach statistical significance. We conclude that VIP acts directly on receptors on the surface of epithelial cells to stimulate active Cl- secretion. The abundance of VIP nerves in the submucosa suggests that VIP may be important in regulation of fluid movement across the epithelium.

Airway epithelia possess transepithelial ion transport processes which may help to regulate the fluid content of airway secretions. Chloride secretion promotes fluid movement from blood to airway lumen. Active absorption of Na favours fluid movement in the opposite direction. The balance between these two processes can be altered by a number of agents which stimulate Cl secretion. The importance of ion transport for normal mucociliary clearance is suggested by the finding that airway epithelia in patients with cystic fibrosis are unable to secrete chloride. This defect may cause the characteristically sticky and tenacious mucous secretions which are the major cause of death in this disease.

1. Tissues with raised intracellular Na levels, produced by incubation in K-free media, were used throughout. The uptake of 42K by these Na-loaded tissues was followed for 10 min in the presence and absence of 1-37 X 10(-4) M ouabain, this being sufficient to inhibit Na pumping maximally. Subtraction of the uptake seen in the presence from that seen in the absence of ouabain gave estimates of the pumped ouabain-sensitive K uptake. 2. In Na-free (MgCl2) medium this depended on the [K]0 in a sigmoidal fashion with a half maximal [K]0 for activation of some 4mM. The maximal uptake of K was 3 m-mole/kg.min corresponding to a transmembrane flux of some 12-5 p-mole. cm-2.sec-1. 3. In the presence of Na the K activation curve became more obviously sigmoid and higher concentrations of K were needed to achieve a given active K influx. The results were well fitted by assuming that Na and K competed for two identical, non-interacting sites on the external pump face. 4. Addition of K during the efflux of 24Na into a Na-free (MgCl2) medium led to an increased rate of tracer loss. The magnitude of this increase depended on the [K] used in a hyperbolic fashion and it was abolished by addition of ouabain. The [K] causing half-maximal activation of ouabain-sensitive Na efflux was in the order of 1-2 mM. 5. When the [K] in the uptake media was 1-5 mM; Na, Li, Rb and Cs all inhibited ouabain-sensitive K uptake, the order of effectiveness being Rb greater than Cs greater than Na greater than Li. With a E1TKA10 OF 0-15 MM low concentrations of Cs and Rb were shown to stimulate K uptake. Such an effect is predicted by assuming two ion binding sites on the pump's outer face, and that the pump can translocate mixtures of K and either Rb or Cs...