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The Calu-3 cell line is being investigated as a model for human submucosal gland serous cells. In a previous investigation of basal short-circuit current ( I sc) in Calu-3 cells, high levels of bumetanide-insensitive basal I sc (∼60 μA/cm2) were measured in cells grown at an air interface. Basal I sc was reduced only 7% by bumetanide, and the largest component of basal I sc required both Cl− and[Formula: see text] in the bathing solutions. Because I sc could be partially inhibited by basolateral 4,4′-dinitrostilbene-2,2′-disulfonic acid and because the only known apical exit pathway for anions is the cystic fibrosis transmembrane conductance regulator, which has a relatively poor conductance for [Formula: see text], it was concluded that most basal I sc is[Formula: see text]-dependent Cl− secretion [M. Singh, M. Krouse, S. Moon, and J. J. Wine. Am. J. Physiol. 272 ( Lung Cell. Mol. Physiol. 16): L690–L698, 1997]. We have now measured isotopic fluxes of36Cl−and22Na+across short-circuited Calu-3 cells and found that virtually none of the basal I sc is Cl− secretion or Na+ absorption. Thus, in contrast to the earlier report, we conclude that the major component of basal I sc is[Formula: see text] secretion. Stimulation recruits primarily Cl− secretion, as previously proposed.

Hepatocyte growth factor (HGF) can influence epithelial cell growth and differentiation. We examined the actions of recombinant human HGF (rhHGF) on the differentiation of human primary tracheal epithelial (HTE) cells cultured in vitro for up to 96 h. Basolateral, but not apical, treatment of confluent HTE cell sheets for 48 h with rhHGF led to increases in cell height, cell volume, cilia, and total protein content. Basolateral rhHGF treatment produced a decrease in HGF receptor (c-met) expression but had no effect on c-met mRNA levels. HTE cell sheets treated with rhHGF for 48 h showed a significant increase in mediator-induced Cl- secretion and a decrease in amiloride-sensitive sodium absorption. No effect on transepithelial resistance was observed with rhHGF treatment. The enhancement of short-circuit responses by basolateral rhHGF was dose dependent. Our results demonstrate that rhHGF has hypertrophic actions on, and can influence the differentiation of, human airway epithelia in vitro, presumably through the activation of c-met at the basolateral surface of these cells.

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.

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.

Calu-3, a cell line derived from a lung adenocarcinoma, forms tight junctions, expresses cystic fibrosis transmembrane conductance regulator (CFTR), and secretes Cl- in response to adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents. Anion conductance of Calu-3 cells was assessed with isotopic flux and patch-clamp methods at 22 degrees C. Iodide efflux was increased by cAMP-elevating agents and brief trypsin treatment. A 7.1 +/- 0.4-pS voltage-independent Cl- channel with linear current-voltage relation was the most common channel observed in cell-attached recordings and was identified as CFTR on the basis of shared features with recombinant CFTR. In unstimulated cells, the mean minimum number of active CFTR channels per patch was 1 +/- 1 (n = 12), increasing to 6 +/- 8 (n = 40) after stimulation with cAMP-elevating agents or after brief trypsin treatment. Channel closure after excision was biexponential with tau 1 approximately 4 s and tau 2 approximately 79 s; typically channels were open continuously until closing permanently. In 11 of 12 excised patches, channels were reactivated by exposure to cAMP-dependent protein kinase (PKA) plus ATP. Efficacy of reactivation was inversely related to the duration from excision to addition of PKA. Channels were blocked by 20–40 microM 5-nitro-2-(3-phenylpropylamino)benzoate on cytosolic but not external side. Active CFTR channels were recorded in 83% of total patches. Other types of Cl- channels were observed in 5 of 52 (10%) cell-attached patches and in 17 of 34 (50%) excised patches, including an outwardly rectifying channel in 2 patches. CFTR channels are the predominant pathway for cAMP-stimulated Cl- conductance in Calu-3 cells; the long open times in the absence of ATP are not explained by present models of CFTR activation.

Surface epithelium and gland cells from human trachea were cultured on porous-bottom inserts and loaded with fura 2 to permit measurement of the intracellular calcium concentration ([Ca2+]i). Short-circuit current (Isc), an index of transepithelial active ion transport, was measured on cells from the same cultures. Surface epithelial [Ca2+]i of 82 +/- 15 nM was increased transiently by isoproterenol, histamine, and bradykinin with maximal increases of 88 +/- 17, 480 +/- 149, and 978 +/- 214 nM (n = 15), respectively. Baseline [Ca2+]i in cultured gland cells of 68 +/- 11 nM was increased transiently by isoproterenol, histamine, methacholine, and bradykinin with maximal increases of 105 +/- 19, 233 +/- 47, 327 +/- 121, and 634 +/- 151 nM (n = 17-21), respectively. In both cell types, mediators that increased [Ca2+]i also increased Isc with a time course identical to the increase in [Ca2+]i. Pretreatment with the calcium chelator, 1,2-bis-(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, acetoxymethyl ester (BAPTA-AM), had no effect on basal Isc or transepithelial resistance but markedly inhibited both the Isc and [Ca2+]i responses to agonists. Forskolin (10(-5) M), 3-isobutyl-1-methylxanthine (10(-3) M), dibutyryl adenosine 3',5'-cyclic monophosphate (10(-3) M), and 8-(4-chlorophenylthio)-cAMP (10(-3) M) had no or only trivial effects on Isc and [Ca2+]i. We suggest that mediators increase Isc across human airway epithelium by activating Ca-dependent basolateral K channels, resulting in hyperpolarization and an increased driving force for Cl exit through apical membrane Cl channels.

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.

Acini of human tracheobronchial submucosal glands were isolated by enzymatic disaggregation, and, when plated on flasks coated with human placental collagen (HPC) in media containing Ultroser G serum substitute (USG) and a variety of growth factors (GF), they became confluent after 14–20 days. The cells were then isolated by trypsinization and replated in media containing USG and GF at 10(6) cells/cm2 on porous-bottomed inserts coated with HPC. Confluent monolayers formed on day 1 after replating and were studied on day 10. Transepithelial resistance and short-circuit current (Isc) were 578 +/- 89 omega.cm2 and 12.9 +/- 1.9 microA/cm2 (means +/- SE, n = 23 cell sheets). The potency sequence for stimulation of Isc by mediators was methacholine greater than bradykinin greater than isoproterenol approximately or equal to phenylephrine. Amiloride decreased baseline Isc by 42 +/- 9% (n = 6 cell sheets) but had little effect on the Isc response to mediators. Diphenylamine-2-carboxylic acid, however, had no effect on baseline Isc but markedly inhibited the Isc response to all mediators. These results show that submucosal gland cells from human trachea can be grown in culture to produce epithelial sheets of high resistance, which secrete Cl in response to bradykinin and alpha- and beta-adrenergic and cholinergic agents.

The pathways of ion movement across canine tracheal epithelium, a Cl-secreting tissue, were examined by three techniques. First, the measurement of simultaneous, unidirectional fluxes of Na or Cl and mannitol, a large hydrophilic molecule that serves as a marker of the paracellular pathway, indicated that a significant fraction of both the Na flux from submucosa to mucosa (J Na sm) and the flux of Cl from mucosa to submucosa (J Cl ms) traverse the cellular pathway. The ratio of the Na-to-Cl diffusion coefficients through the paracellular pathway was 0.23, in contrast to the free solution ratio of 0.63. Second, in voltage-clamp experiments we examined the effect of transepithelial voltage differences on the unidirectional fluxes of Na and Cl. The results agree with the previous findings, suggesting that there are voltage-independent, or transcellular, backfluxes of Na and Cl, and that the relative permeability of Na to Cl through the voltage-dependent (presumably paracellular) pathway was 0.28. Third, measurement of transepithelial diffusion potentials gave a Na-to-Cl permeability ratio of 0.31 +/- 0.02 (mean +/- SE). These results suggest that there are significant transcellular backfluxes of Na and Cl and that the paracellular pathway in the canine trachea is anion selective. An anion-selective pathway would tend to shunt the secreted Cl back through the paracellular pathway, thus minimizing the net ion and fluid movement across the tissue in the open-circuit condition.

The "loop" diuretics MK-196, bumetanide, piretanide, and furosemide are all potent inhibitors of Cl transport by the dog's tracheal epithelium. In short-circuited tissues, the drugs caused significant decreases in both unidirectional Cl fluxes and in the net flux of Cl toward the lumen; the change in net Cl flux was not significantly different from the change in short-circuit current. The drugs had no effect on active Na absorption. All drugs caused a significant fall in tissue conductance. All drugs, except MK-196, were more potent from the serosal bath; MK-196 was equipotent from either side of the tissue. In experiments with isolated cells, the diuretics caused no significant changes in intracellular Na and K concentrations, a fall in intracellular Cl concentration, and approximately equal falls in Na and Cl influxes. These results suggest that the site of action of these drugs is on a basolateral linked Na-Cl entry process. Additional evidence for such a linked entry process was provided by experiments in which removal of Cl reduced Na influx and removal of Na reduced Cl influx.

We have developed a preparation of isolated cells from dog tracheal mucosa. The three major cell types of the intact epithelium (ciliated, secretory, and basal) are present in approximately the same proportions as in the intact tissue. These cells show high viability as judged by exclusion of vital dyes, high O2 consumption, and incorporation of amino acids into protein. The intracellular ion contents (in mmol/l cell H2O) are [K]i, 150; [Na]i, 20; and [Cl]i, 50. Ouabain (10(-4) M) causes a rise in [Na]i and a reciprocal loss of intracellular K.

The effects of stimulating protein kinase C on Cl- secretion across dog tracheal epithelium were investigated. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the synthetic diacylglycerol, 1-oleolyl-2-acetylglycerol (OAG), which stimulate protein kinase C (PKC), both stimulated short-circuit current (Isc) with Kd of 10 nM and 1 microM, respectively. In Cl(-)-free solution, the increases in Isc were virtually abolished, suggesting that these compounds stimulate Cl- secretion, a hypothesis confirmed for TPA by measurement of 36Cl- fluxes. The stimulations of Cl- secretion were not sensitive to indomethacin, nor were cAMP levels elevated during stimulation. In addition to its transient stimulatory effect, TPA at high doses caused the eventual lowering of the base-line Isc and a block of subsequent stimulation by cAMP-mediated agonists. This was probably not the result of toxicity or an effect on adenylate cyclase or on cAMP-dependent protein kinase. Cell extracts from both cultured and native dog tracheal epithelial cells showed strong PKC activities. These results suggest that PKC may play a role in regulating Cl- secretion across dog tracheal epithelium.

Confluent monolayers of polygonal epithelioid cells were obtained from enzymatically and mechanically dispersed bovine anterior pituitaries (AP) and pars tuberali (PT). The ultrastructure of the cells composing the monolayer was consistent with the follicular or folliculostellate cells (FC) of the pituitary, i.e., lack of secretory granules; formation of follicles in culture; interdigitations with neighboring cells with numerous tight junctions; presence of extensive microfilaments; and sparse rough endoplasmic reticulum and Golgi apparatus. Culture media from monolayers of first passage cultures contained little if any of the AP hormones' luteinizing hormone, prolactin, and ACTH. Shortly after reaching confluency, regions of the monolayer bulge away from the surface of the culture dish to form domes. Dome formation has been described only with cultures of cells that function as transport epithelia in vivo. FC cultured on polycarbonate filters were placed in Ussing chambers. A transepithelial potential difference of approximately 1.1 mV and a resistance greater than 300 omega cm2 were detectable 4–5 days after plating. The short-circuit current (Isc) was decreased 70% by amiloride applied to the mucosal surface and further decreased by the addition of ouabain at the serosal surface. The beta-adrenergic agonist isoproterenol increased the Isc and this action was prevented by a beta-antagonist. These observations indicate that pituitary FC in culture behave as a transport epithelium. Considering the organization of FC in the AP and PT, they suggest a regulatory role for FC in the maintenance of the ionic composition of the interstitial fluid of the pituitary gland.

Release of cyclooxygenase products from primary cultures of dog or human tracheal epithelium was measured by radioimmunoassay. In both species, bradykinin, platelet-activating factor (PAF), and A23187 (a calcium ionophore) caused increases in the rate of release of prostaglandin (PG) E2 and smaller increases in PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane B2 output. Isoproterenol, vasoactive intestinal peptide (VIP), methacholine, and leukotrienes C4 and D4 had no effect on release of these cyclooxygenase products. Gas chromatography-mass spectrometry showed that the ratio of PGE2 to PGD2 released from dog cells by A23187 was 30:1. Short-circuit current across dog cells was stimulated by bradykinin, A23187, PAF, VIP, methacholine, and isoproterenol. Only the responses to bradykinin and A23187 were reduced by pretreatment with indomethacin.

Bradykinin increased mean short-circuit current (Isc) when added either to the mucosal (KD = 1.1 nM; delta Imaxsc = 29.8 +/- 4.4 microA/cm2) or submucosal bath (KD = 108 nM; delta Imaxsc = 27.1 +/- 4.9 microA/cm2). Bumetanide or replacement of Cl reduced the maximal change in Isc. In paired tissues, the increase in net 36Cl flux toward the mucosa equaled the change in Isc. Net 22Na flux toward the submucosa was unchanged. Involvement of intramural nerves was ruled out because bradykinin-induced increases in Isc were not inhibited by phentolamine, propranolol, atropine, or tetrodotoxin. A direct action of bradykinin on the epithelium was also made probable by the autoradiographic demonstration of specific bradykinin binding sites. The antagonists of arachidonic acid metabolism, indomethacin and eicosa-5,8,11,14-tetraynoic acid, inhibited the increase in Isc induced by bradykinin. Finally, prostaglandin E2 release was significantly increased by submucosal addition of bradykinin, and this effect was abolished by pretreatment with indomethacin. We conclude that bradykinin stimulates Cl secretion in canine tracheal epithelium by increasing prostaglandin release.