Human Tracheal Gland Research Articles

Neutrophil Elastase Promotes Rapid Exocytosis in Human Airway Gland Cells by Producing Cytosolic Ca2 +Oscillations

The molecular and ionic mechanisms responsible for the regulation of mucus exocytosis in human airway gland cells remain poorly defined. To determine whether dynamic changes of intracellular free Ca2+ concentration [Ca2+]i can promote different exocytotic responses, we monitored dynamic changes in [Ca2+]i and secretory granule (SG) exocytosis in individual human tracheal submucosal serous gland (HTG) cells. These changes were in response to exposure of the cells to three different secretagogues associated with airway inflammation and disease: human neutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca2+]i from single cells were determined with Indo-1/AM using quantitative UV laser microspectrofluorometry. The rate of SG exocytosis was measured in single cells by fluorescence videomicroscopy of SG degranulation and by the ELISA method. Exposure of HTG cells to a low concentration of HNE (1.0 microM) caused a high rate of SG exocytosis (52% decrease in the initial quinacrine fluorescence) during the first 8-min stimulation period compared with that observed following exposure of the cells to 100 microM histamine (10% decrease) or 100 microM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca2+]i induced by histamine (1.0-100 microM) and ATP (10-100 microM), HNE (0.01-1 microM) generated asynchronous oscillations in [Ca2+]i over the first 8-min period. Depletion of internal Ca2+ stores with thapsigargin (500 nM) induced a significant reduction (P < 0.01) in the observed increases in [Ca2+]i upon addition of each of the secretagogues, but did not greatly affect the SG exocytotic responses. Interestingly, the removal of extracellular Ca2+ (+5 mM EGTA) significantly reduced (P < 0.01) both the [Ca2+]i increases and the rate of SG exocytosis following exposure to the secretagogues. We also demonstrate that the influx of extracellular Ca2+ and [Ca2+]i oscillations rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of exocytosis process in human airway gland cells.

Altered cytokine production by cystic fibrosis tracheal gland serous cells.

Human submucosal tracheal glands are now believed to play a major role in the physiopathology of cystic fibrosis (CF). We successfully developed techniques for culturing human tracheal gland serous cells from normal individuals (HTGS cells) and from CF patients (CF-HTGS cells) and have shown that the cultured cells have retained most of their in vivo epithelial and secretory characteristics. In order to determine to what extent the serous cells may participate in the lung defense against infection, we examined the effects of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa on HTGS and CF-HTGS cells, with special reference to tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-8 secretion. HTGS cells showed a daily basal secretion of IL-6 (1.68 +/- 0.14 ng/10(6) cells) and IL-8 (9.6 +/- 1.3 ng/10(6) cells) and no constitutive secretion of TNF-alpha. Treatment with P. aeruginosa LPS resulted in a significant increase in the basal production of IL-6 (increase of 200% +/- 12%) and IL-8 (525% +/- 40%) as well as a rapid production of TNF-alpha (250 +/- 38 pg/10(6) cells). The LPS-induced secretion of IL-6 and IL-8, but not that of TNF-alpha, was inhibited by glucocorticoids. CF-HTGS cells showed a much higher basal secretion of IL-6 (13.2 +/- 0.5 ng/10(6) cells) and IL-8 (45.6 +/- 7.2 ng/10(6) cells) than normal cells. Treatment with the LPS of P. aeruginosa induced increased production of IL-6 (increase of 100% +/- 8%) and IL-8 (55% +/- 18%) but did not induce the secretion of TNF-alpha. Neither intracellular TNF-alpha nor TNF-alpha transcripts were found in CF-HTGS cells, whereas they were found in normal HTGS cells. In addition, dexamethasone was found to stimulate IL-6 and IL-8 secretion (in the presence or absence of LPS) but did not induce any secretion of TNF-alpha. All these data indicate that HTGS cells are responsive to P. aeruginosa LPS, which results in an increased secretion of IL-6, IL-8, and TNF-alpha, the secretion of which appeared to be impaired in CF-HTGS cells.

Pseudomonas aeruginosaLipopolysaccharide Induces CF-like Alteration of Protein Secretion by Human Tracheal Gland Cells

Human tracheal gland (HTG) serous cells are now believed to play a major role in the physiopathology of cystic fibrosis. Because of the persistent inflammation and the specific infection byPseudomonas aeruginosain the lung, we looked for the action of the lipopolysaccharide (LPS) of this bacteria on human tracheal gland cells in culture by studying the secretion of the secretory leukocyte proteinase inhibitor (SLPI) which is a specific serous secretory marker of these cells. Treatment withPseudomonas aeruginosaLPS resulted in a significant dose-dependent increase in the basal production of SLPI (+250 ± 25%) whilst the SLPI transcript mRNA levels remained unchanged. This LPS-induced increase in secretion was inhibited by glucocorticoides. Furthermore, LPS treatment of HTG cells induces a loss of responsiveness to carbachol and isoproterenol but not to adenosine triphosphate. These findings indicate that HTG cells treated byPseudomonas aeruginosaLPS have the same behavior as those previously observed with CF-HTG cells. Exploration by using reverse transcriptase polymerase chain reaction amplification showed that LPS downregulated cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in HTG cells indicative of a link between CFTR function and consequent CF-like alteration in protein secretory process.

Mucins secreted by a transformed cell line derived from human tracheal gland cells.

High-molecular-mass glycoconjugates are secreted by the continuous cell line MM-39, which has been obtained from cultured human tracheal gland cells transformed by simian virus 40. They were purified on Sepharose(R) CL-4B and then by two steps of density-gradient centrifugation. High-molecular-mass glycoproteins resistant to digestion by hyaluronidase, chondroitin ABC lyase and heparitinase were obtained, in addition to hyaluronic acid and proteoglycans. They were susceptible to beta-elimination. They contained polylactosaminoglycan chains as well as carbohydrate chains with a terminal sialic acid in the NeuAc alpha2-3 sequence. Most of them have a buoyant density of 1.45 g/ml in CsCl-density-gradient centrifugation, except for MUC1. The MM-39 cells were also characterized by a high expression of MUC1 and MUC4 genes, but they did not express MUC2, MUC3, MUC5B and MUC5AC. Therefore the MM-39 cells synthesized mucin-like glycoproteins as well as lysozyme and mucous proteinase inhibitor [Merten, Kammouni, Renaud, Birg, Mattéi and Figarella (1996) Am. J. Respir. Cell. Mol. Biol. 15, 520-528]; they should be considered as having a mixed, both serous and mucous, phenotype.

Fluid transport across cultures of human tracheal glands is altered in cystic fibrosis.

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.

A transformed human tracheal gland cell line, MM-39, that retains serous secretory functions.

Infection with the wild type SV40 virus was used to transform primary cultures of human tracheal gland serous (HTGS) cells. Over 80 different cell lines were obtained, but the majority had lost some of their epithelial and secretory features. However, one of these cell lines, MM-39, was shown to have conserved the physiologic characteristics of the genuine HTGS cells-i.e., the presence of cytokeratin, expression of cystic fibrosis transmembrane conductance regulator mRNA, a level of secretory leukocyte proteinase inhibitor secretion comparable to that of the native cells (25 +/- 3 ng/10(6) cells/h), and the responsiveness to pharmacological agonists: carbachol (+260 +/- 40%), isoproterenol (+260 +/- 40%), and adenosine 5'-triphosphate (+280 +/- 30%). These characteristics describe a transformed cell line of human tracheal gland cells which has retained the features of the native serous cells. As a result, this cell line appears to be a useful tool for large-scale physiologic and pharmacologic studies of bronchial secretion at the cellular level.

Spatial and Temporal Mg2+Signaling in Single Human Tracheal Gland Cells

The combined use of Mag-indo-1 probe and laser confocal UV-microspectrofluorometry allowed us to investigate the spatial and temporal dynamic changes of the Mg2+variations in human tracheal gland (HTG) cells at the single cell level. Stimulation of HTG cells with either bradykinin, ouabain or extracellular high Mg2+concentrations (up to 10 mM) induced increases in intracellular Mg2+concentration [Mg2+]i. From a cytosolic basal concentration of 0.8 ± 0.3 mM in a medium free of Mg2+, an increase in extracellular Mg2+concentration from 1 to 10 mM, increased cytosolic [Mg2+]ifrom 1.4 ± 0.6 to 1.8 ± 0.8 mM after 10 min (p<0.05). We also demonstrated using line-scanned spectral images within single cells, that the [Mg2+]iis distributed uniformally in the nucleoplasm, but in contrast, showed marked local differences among different cytoplasmic regions, thus suggesting a functional heterogeneity in the intracellular Mg2+stores involved. The influx pathway for Mg2+in HTG cells was not inhibited by verapamil and appeared to be independent of [Ca2+]i.

P2u-purinoceptor regulation of chloride secretion in cultured human tracheal submucosal glands

We examined the purinergic regulation of chloride secretion and intracellular calcium ion concentration ([Ca2+]i) in the cultured epithelial cells passaged from human tracheal submucosal glands. Adenosine, AMP, ADP, ATP, and UTP induced transient short-circuit current (Isc) responses in a concentration-dependent fashion. The rank order of peak Isc responses at 10 microM concentrations was UTP = ATP > ADP > AMP = adenosine. Diphenylamine-2-carboxylic acid (1 mM) and 4,4'- diisothiocyano-2,2'-stilbene disulfonate (0.5 mM), and quinidine (1 mM) inhibited Isc responses induced by extracellular nucleotides. However, amiloride (10 microM) was without effect. Theophylline (1 mM) did not alter ATP (10 microM)-induced Isc response. Increases in Isc induced by ATP and UTP were significantly larger than those by alpha, beta-methylene ATP, beta, gamma-methylene ATP, or 2-methylthio ATP. Extracellular nucleotides caused increases in [Ca2+]i, and the rank order of [Ca2+]i responses was same as that of Isc responses. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester (50 microM) inhibited increases in Isc and [Ca2+]i induced by extracellular nucleotides. These results suggest that extracellular nucleotides induce chloride secretion across cultured human tracheal glands via a P2u-purinoceptor, and this chloride secretion appears to be dependent on increases in [Ca2+]i and activation of Ca-dependent K channels.

Intracellular free Ca 2+ dynamic changes to histamine are reduced in cystic fibrosis human tracheal gland cells

This study documents a difference between cystic fibrosis human (CF-HTG) and normal human (HTG) tracheal gland cells: the ability of histamine to induce an increase of intracellular free calcium concentration [Ca 2+] i was abnormally reduced in CF-HTG cells. The magnitude of the [Ca 2+] i peak rise in response to histamine is smaller in CF-HTG cells than in HTG cells, and the percentage of CF-HTG cells that increase [Ca 2+] i is decreased compared with HTG cells. In contrast to histamine, the human neutrophil elastase (HNE) stimulation of both CF-HTG and HTG cells generated [Ca 2+] i asynchronous oscillations and the magnitude of the peak [Ca 2+] i response as well as the percentage of responding cells were similar for both groups. By videomicroscopy observations, the secretory response (exocytosis of secretion granules) of CF-HTG cells occurred with HNE, but not with histamine, thus suggesting that [Ca 2+] i asynchronous oscillations may be linked to the exocytosis process in human tracheal gland cells.

Evidence for, and characterization of, a lipopolysaccharide-inducible adenosine A2 receptor in human tracheal gland serous cells

Human tracheal glands are considered as the principle secretory structures in the bronchotracheal tree. In earlier studies, we successfully performed primary cultures of human tracheal gland (HTG) serous cells and noted that these cells were responsive to many secretagogues including purinergic agonists but not to the inflammatory mediator adenosine. In this study, we demonstrate that adenosine was capable of inducing stimulation of protein secretion by HTG serous cells which had previously been cultured in pro-inflammatory conditions (induced by lipopolysaccharide (LPS)). This stimulation was inhibited by 8-phenyltheophylline but not by dipyridamole, which is indicative of a P1 purinoceptor. This inducible receptor is of the adenosine A2 subtype [rank potency order: 5′-( N-ethyl)-carboxamidoadenosine (NECA) > adenosine > N 6-(phenylisopropyl)-adenosine (PIA); and stimulation of adenylyl cyclase]. The adenosine-induced protein secretion was concentration-dependent, however, increased intracellular cyclic adenosine monophosphate (cAMP) was not dependent on the concentration of adenosine. The adenosine-induced secretion and the ATP-induced secretion were shown to be additive. This study concludes that there is evidence of a LPS-inducible adenosine A2 receptor in human tracheal gland serous cells.

Asynchronous Dynamic Changes of Intracellular Free Ca2+ and Possible Exocytosis in Human Tracheal Gland Cells Induced by Neutrophil Elastase

Measurements of the intracellular free calcium concentration [Ca2+]i in single cells of the human tracheal gland cell line MM 39 demonstrate dynamic changes in [Ca2+]i after their exposure to human neutrophil elastase (HNE). A heterogeneity in [Ca2+]i responses measured cell to cell in monolayer culture is evident : cells generate an initial [Ca2+]i peak rise with or without a delayed time (up to 180 sec) followed either by a rapid return to baseline, asynchronous oscillations or a sustained plateau phase. From basal concentration of 85± 15 nM, HNE (1 μM) produces a [Ca2+]i increase of 91 ± 66 nM in about 50 % of responding cells. At lower concentrations of HNE (0.1 μM, 0.01 μM), the [Ca2+]i rise remains similar, but only 30-40 % of the cells are responding. Pretreatment of cells with the recombinant elafin protein, a specific elastase inhibitor, reduces both the [Ca2+]i response to HNE and the number of responding cells. Electron microscopy observations reveal an increased number of secretory granules located beneath the cell plasma membrane after HNE treatment. These results suggest that intracellular [Ca2+]i changes may be associated to the HNE-induced exocytosis in human tracheal gland cells. These findings could have implications with regard to the pathogenesis of increased mucus secretion in human airway diseases.

Glandular-like morphogenesis and secretory activity of human tracheal gland cells in a three-dimensional collagen gel matrix.

The extracellular matrix has been demonstrated to affect the differentiation of epithelial cells. We present evidence that in a three-dimensional (3-D) type I collagen gel matrix, isolated human adult tracheal gland (HTG) cells are capable of reconstructing new functional gland-like tubules in vitro. During the first two weeks in culture, HTG cells developed globular epithelial cell aggregates in which lumina is absent. By the third week in culture, the tubulogenesis and the formation of branching structures became evident with a polarized morphology, which in many aspects resembles the in vivo morphology. A central lumen was lined by polarized secretory epithelial cells exhibiting well-developed microvilli and apical secretory granules. Furthermore, we showed that the capacity of in vitro tracheal gland differentiation was associated with the basal deposition of laminin and type IV collagen around the gland-like tubules. A cell-associated 72 kDa type IV collagenase was expressed in developing tubule cells, as shown by immunocytochemistry. The secretion of the antileucoprotease (ALP), a protein marker of tracheal gland serous cells, was bidirectional in gland-like tubules, since up to 65% of released ALP was in the basolateral direction. Taken together, these observations indicate that isolated HTG cells in a 3-D collagen matrix form functional tracheal gland-like tubules and suggest that similar new tracheobronchial gland formations may occur during the human normal gland development and remodeling.

Chloride secretion and function of serous and mucous cells of human airway glands.

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.

Increased Coexpression of CFTR and S-100 Calcium Binding Proteins MRP8 and MRP14 mRNAs in Cystic Fibrosis Human Tracheal Gland Cells

We have demonstrated for the first time, by Northern analysis, the presence of the S-100 calcium binding proteins MRP8 (also called "cystic fibrosis protein") and MRP14 mRNAs in cultured human tracheal gland cells, obtained from normal and cystic fibrosis (CF) patients. A significant increase of these mRNAs in cells of CF origin (as well as that of CFTR mRNA) is shown. These results allow us to assume a potential pretranslational regulation of MRP8 and MRP14 gene expression related to the presence of a mutated CFTR gene.

Neuropeptide Y and norepinephrine cooperatively inhibit human tracheal gland cell secretion.

The effects of neuropeptide Y (NPY) and norepinephrine (NE) on the secretion of human tracheal gland (HTG) cells in culture were examined using the bronchial inhibitor (BrI) as a secretory marker. NPY by itself provoked no change in secretion, neither in intracellular Ca2+ nor in adenosine 3',5'-cyclic monophosphate (cAMP) levels. NE induced transient intracellular Ca2+ mobilization and an increase of intracellular cAMP and also, as previously described, a short-time concentration-dependent decrease of BrI secreted by HTG cells. When applied together on HTG cells, NPY and NE induced an increase of intracellular cAMP, a discrete sustained increase of basal intracellular Ca2+, and a long-lasting inhibition of secretion. Our data suggest that NPY acts for human tracheal gland cells as a NE neuromodulator rather than as a neurotransmitter. Thus culture of HTG cells appears to be an interesting pharmacological support for studying inhibition of bronchial secretion and could be applied in the study of pathologies where hypersecretion is observed, such as cystic fibrosis.

ATP and UTP increase secretion of bronchial inhibitor by human tracheal gland cells in culture.

The effects of ATP and UTP on intracellular Ca2+ levels and on the secretion of the bronchial inhibitor and high-molecular-weight glycoproteins were studied in cultures of human bronchotracheal gland cells. ATP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP increased intracellular Ca2+ levels in a manner that was partially dependent on the presence of extracellular Ca2+. Other nucleotides (ADP, alpha,beta-methylene ATP, beta,gamma-methylene ATP, and 2-methylthio ATP) and adenosine were ineffective, thus suggesting the presence of a "nucleotide" receptor specific for ATP and UTP. At concentrations similar to those that raised intracellular Ca2+ concentration, ATP, UTP, and ATP gamma S stimulate the secretion of the bronchial inhibitor. ATP and UTP also increase the production of sulfated high-molecular-weight glycoproteins. These results indicate the presence in human tracheal gland cells of a nucleotide receptor that mediates intracellular Ca2+ mobilization and controls the secretion of macromolecules.

Expression and immunohistochemical localization of laminin and type IV collagen in developing human fetal tracheal glands.

The expression and distribution of laminin and type IV collagen, two major components of the basement membrane, were investigated at the epithelio-mesenchymal interface of the human developing tracheal glands from 10 to 37 weeks of gestation. The localization of these molecules was assessed by indirect immunoperoxidase and indirect immunofluorescence staining and correlated to morphogenesis and epithelial cell differentiation. Laminin and type IV collagen were detected as early as 10 weeks of gestation in a continuous, linear pattern in the basement membrane surrounding the epithelial tracheal tube. By 12 weeks of gestation the basement membrane developed large openings at the tips of the budding glands beneath poorly differentiated cells, concomitant with the onset of morphogenetic movements. The remodeling of the basement membrane led to branching epithelial morphogenesis. The maturation and the functional differentiation of the secretory cells appeared later in the epithelium, when the basement membrane was strongly labeled with both anti-laminin and anti-type IV collagen antibodies, after 24 weeks of gestation. At this time the basement membrane became regular and thick and the maturation of serous cells increased progressively. These results suggest that the remodeling of the basement membrane takes place very early during gestation and that the morphogenesis and the maturation of the tracheal glands are rapidly achieved in humans.

Calcium-dependent chloride secretion across cultures of human tracheal surface epithelium and glands.

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.

Epinephrine Promotes Growth and Differentiation of Human Tracheal Gland Cells in Culture

Submucosal gland cells isolated from human tracheas by enzymatic digestion and cultured in the absence or presence of epinephrine (E) were used to investigate the possible action of this catecholamine on the physiology of the gland secretory cells issued from the human trachea. A 3 x 10(-6) M concentration of E shortens the doubling time of growth and increases the cells' confluency rate. On the other hand, E appears to induce cell polarity in terms of differential secretion apically versus basolaterally. Furthermore, when human tracheal gland cells are cultured in the presence of E, a maximal cell stimulability by different agonists occurs from 8 days after confluency and then remains identical for 10 days, allowing us to compare the action of different adrenergic and cholinergic agonists on the proteinase bronchial inhibitor and the radiolabeled glycoconjugate secretion. As previously described, secretions of bronchial inhibitor and high molecular weight glycoconjugates were stimulated both by alpha- and beta-adrenergic and by cholinergic agonists but at a much higher rate when cells were cultured in the presence of E. These results indicate that E improves cultured human tracheal glandular cell growth and differentiation in that it increases their polarity and their ability to respond to adrenergic and cholinergic agonists.

Characterization of cAMP dependent CFTR-chloride channels in human trache gland cells

Human tracheal gland cells are believed to be a major site at the origin of cystic fibrosis. Since this disease is due to mutations in a protein called CFTR, we looked for the activity of CFTR in human tracheal gland cells in culture. We have identified CFTR-like chloride-selective channels as having a linear current-voltage relationship and unitary conductance of 7 pS in these cells. In cell-attached patches, theophylline (1 mM), IBMX (1 mM), or a cocktail of dibutyryl cAMP (1 mM) and IBMX (0.1 mM) promoted the opening of channels. The unitary current had a reversal potential close to the cell resting potential. Replacement of choline by K + or Na + in the pipette solution was without effect on the current-voltage relationship, the reversal potential or the unitary conductance, which is consistent with the chloride selectivity of the channel. Channels were always found clustered and their opening probability was not noticeably dependent on membrane potential. This work therefore represents the first observation of a CFTR-like channel activity in submucosal gland cells.