CFTR is involved in membrane endocytosis but not in fluid-phase and receptor-mediated endocytosis in human respiratory epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) protein has been reported to be a cAMP-regulator of plasma membrane recycling in epithelial cells overexpressing CFTR. To assess its role in the different endocytic processes in human respiratory epithelial cells, the rates of internalization of membrane, fluid-phase and receptor-mediator tracers were compared, under control conditions and after treatment with the cAMP agonist forskolin in normal and cystic fibrosis (CF) cells. In both control and treated-cells, CFTR was only present in the plasma membrane of normal but not in CF cells. Similarly, activation of Cl- efflux only occurred in normal and not in CF-treated cells. The rate of membrane endocytosis was significantly decreased by 35% in normal treated-cells, whereas it was not significantly decreased (12%) in CF-treated cells. Upon forskolin treatment, the decrease of the rate of both fluid-phase and receptor-mediated endocytosis was not significantly different between normal and CF cells. These results demonstrate that CFTR is involved in membrane endocytosis but not in fluid-phase and receptor-mediated endocytosis in human respiratory epithelial cells.

Intracellular free Ca2+ 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 [Ca2+]i was abnormally reduced in CF-HTG cells. The magnitude of the [Ca2+]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 [Ca2+]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 [Ca2+]i asynchronous oscillations and the magnitude of the peak [Ca2+]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 [Ca2+]i asynchronous oscillations may be linked to the exocytosis process in human tracheal gland cells.

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.

Vectorial delivery of newly-synthesized secretory proteins by human tracheal gland cells in culture.

The polarized secretion of newly-synthesized proteins of human tracheal submucosal gland cells was studied. Human tracheal gland cells were cultured on permeable filter supports allowing a separate biochemical analysis of apical and basal secretion. By transmission electron microscopy, confluent filter-grown cells were seen to form a continuous sheet of both multilayer and monolayer epithelial cells. Junctional complexes between adjacent cells were observed. On immunofluorescence microscopy, human tracheal gland cells in cultured exhibited characteristics of epithelial and secretory cells, including cytoplasmic staining for cytokeratin and for two secretory protein markers specific to the glandular serous type cell: lysozyme and antileucoprotease. [35S]methionine metabolic labelling experiments demonstrated that at least 90% of newly-synthesized secretory proteins were recovered in the apical medium. Moreover, lysozyme secretion was strongly polarized since 85% was released into the apical medium. Conversely, antileucoprotease secretion was more bidirectional since nearly 40% of released antileucoprotease was present in the basal medium. The fact that these two secretory proteins are released with differing relative polarity emphasizes that human tracheal gland cells exhibit at least two different exocytotic routing operations.

Localization of the cystic fibrosis transmembrane conductance regulator in airway secretory glands.

Cystic fibrosis (CF) is caused by mutations in the gene coding for the CF transmembrane conductance regulator (CFTR). From human normal tracheal submucosal gland cells in culture, we identified endogenous CFTR as a 170 kDa protein, consistent with that of fully glycosylated, mature CFTR molecule. This observation led to the hypothesis that airway secretory glands could be an important site for the CFTR expression. Using anti-human CFTR polyclonal and monoclonal antibodies, we examined the cellular and subcellular localization of the CFTR protein in airway submucosal glands from human and bovine tracheal tissues as well as in tracheal gland cell cultures. In human tracheal tissue, CFTR immunolabelling was present along both the apical and basolateral plasma membranes of glandular mucous cells. In contrast, CFTR was associated with the secretory granules of glandular serous cells. Using immunogold electron microscopy, we demonstrated that CFTR protein was more specifically associated with the membrane of serous cell secretory granules. In bovine tracheal tissue CFTR labelling was also identified in the secretory granules of glandular serous cells. In contrast, when bovine and human tracheal gland cells were cultured, no mature secretory granules were present, but a predominantly intracytoplasmic distribution of CFTR was observed. Our data thus suggest that in airway tissues, CFTR could be involved in intracellular processes of the mucus exocytosis in submucosal secretory glands.

[Structure and secretory functions of the respiratory epithelium]. / Structure et fonctions sécrétoires de l'épithélium respiratoire.

Airway secretions actively participate in respiratory epithelium protection. Apart from its main participation in transport of inhaled microorganisms and particles by mucociliary clearance, respiratory mucus also contributes to other protective purposes such as the control of airway humidification. Biochemical components found in secretions, such as mucins, lipids, antibacterial agents (secretory IgA, lysozyme, lactoferrin), antioxidant and antiprotease components, contribute significantly to the airway epithelium defense.

Molecular analysis of the Bg-rbg transposable element system of Zea mays L.

The two components of the Bg-rbg transposable element system of maize have been cloned. The Bg element, isolated from the mutable allele wx-m32:: Bg is inserted in the intron of the Waxy (Wx) gene between exons 12 and 13. The length of the element is of 4869 bp. Bg has 5 bp terminal inverted repeats, and generates upon insertion an 8 bp direct duplication of the target sequence. Both ends of the Bg element contain a 76 bp direct repeat adjacent to the terminal inverted repeats. The hexamer motif TATCGGC is here repeated several times in direct or inverse orientation. The rbg element was isolated from the mutable allele o2m(r) where it is located in the promoter region of the Opaque-2 (O2) gene. rbg is approximately 4.5 kb in length, has terminal inverted repeats identical to those of the Bg element, and is also flanked by an 8 bp direct duplication at the target site. Like Bg, rbg carries the 76 bp direct repeats. Restriction enzyme analysis reveals that, compared to Bg, the receptor element is distinguishable by small deletion and insertion events. Sequence data indicate that not more than 75% homology exists at the DNA level between the rbg element and the autonomous Bg element.

Modulation of albumin-like protein and lysozyme production by bovine tracheal gland serous cells. Dependence on culture conditions.

Bovine tracheal submucosal gland serous cells were cultured in medium supplemented with either 10% fetal calf serum or 2% Ultroser G, a commercial serum substitute for cell culture. The proteins synthesized and secreted into the culture medium during [35S]methionine pulse, chase and isoproterenol-stimulated periods were analyzed. Marked differences in the patterns of secretory radiolabeled proteins with Mr values ranging from 15,000 to 95,000 were observed between pulse and chase media of cells cultured in fetal calf serum and Ultroser G. In the presence of Ultroser G, albumin-like protein production was inhibited 95% as compared to cultures incubated with fetal calf serum. A bovine lysozyme-type enzymatic activity was detected only in medium from stimulated cells cultured in Ultroser G. The results suggest that bovine tracheal serous cells synthesize different proteins according to the composition of culture medium and release certain proteins when adrenergically stimulated.