Cultures Of Hamster Tracheal Epithelial Cells Research Articles

Immunological characterization of a mucin-associated protein from hamster tracheal epithelial cell culture.

Airway mucins are high molecular mass (>10(6) dalton) glycoproteins with various types of associated molecules including glycoproteins, lipoproteins, and lipids. The study of mucin-associated proteins is limited largely due to the lack of specific probes. In this study, we produced a monoclonal antibody, MAbHT10, against a 190-kDa mucin associated-protein by immunizing mice with hamster airway mucin purified in nondissociative condition. Using HT10, the 190-kDa mucin-associated protein was characterized immunologically. The 190-kDa mucin-associated protein is glycoprotein and HT10 recognized carbohydrate containing portion of the protein. The association of 190-kDa protein with mucin is strong enough that heat and detergent treatment is required to dissociate it from mucin as evidenced by gel filtration chromatography, Western blot, enzyme-linked immunoadsorbent assay (ELISA), and co-immunoprecipitation. The expression of the 190-kDa protein is increased with the development of hamster tracheal epithelial cells in culture, but showed differences with the pattern of the regulation of mucin expression. Adenosine triphosphate (ATP), a known strong mucin secretagogue, dose-dependently increased mucin release but caused only marginal increase in the release of the 190-kDa protein. The MAb should be useful in the structural and functional analysis of the 190-kDa mucin-associated proteins in physiological and pathological situations such as chronic airway diseases.

Muscarinic-induced mucin secretion and intracellular signaling by hamster tracheal goblet cells.

Hamster tracheal epithelial cell cultures were used to investigate muscarinic regulation of high-molecular-weight glycoconjugate (HMWG) secretion by airway goblet cells. HMWG were radiolabeled with N-acetyl-D-[1-(3)H]glucosamine, precipitated with trichloroacetic acid and phosphotungstic acid, and counted by liquid scintillation. Carbachol (100 microM) increased HMWG secretion (166.6 +/- 18.7%, P < 0.001, n = 20), and this response was blocked by the muscarinic receptor antagonist atropine. Ca2+ may not be essential for carbachol response since 1) carbachol-activated secretion was not inhibited by chelating extracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by reducing both extracellular and intracellular Ca2+ with BAPTA-acetoxymethyl ester in low-Ca2+ medium; 2) the carbachol response was only partially blocked in low-Ca2+ medium; and 3) calcium ionophore did not stimulate HMWG secretion. However, carbachol-stimulated secretion was abolished by pertussis toxin (PTX), indicating the involvement of a PTX-sensitive guanine nucleotide-binding regulatory protein (G protein), and by the protein kinase C (PKC) inhibitor chelerythrine chloride. Furthermore, carbachol-stimulated secretion was not inhibited by overnight incubation with phorbol 12-myristate 13-acetate. In conclusion, carbachol-stimulated secretion of HMWG appears to be coupled to a PTX-sensitive G protein and requires the activation of a phorbol ester-insensitive PKC isoform.

Type IV collagen localization in hamster tracheal epithelial cell cultures

Hamster tracheal epithelial (HTE) cell cultures when grown on collagen coated Millicell-HA filters for 28 d in vitro have an incomplete basal lamina-like structure at the basal plasmalemma/collagen interface. EM evaluation of 42 d HTE cell cultures also revealed the presence of hemidesmosome-like structures on the basal plasmalemma. In order to better characterize this discontinuous basal lamina-like material, HTE cultures at 7, 14, 21, and 28 d were evaluated for the presence of type IV collagen, a basal lamina component. Immunocytochemical treatment of HTE cultures at these time points resulted in the presence of reaction product at the base of the cell layer. When immunocytochemical procedures for the localization of type IV collagen were carried out with normal mouse trachea, the results were also positive. Immunoblotting evaluation of HTE cell supernatants and conditioned media also indicated the presence of type IV collagen. Taken altogether, the presence of what appears to be basal lamina, hemidesmosome-like structures and immunocytochemical and immunoblot data showing the presence of type IV collagen in HTE cultures suggest that HTE cells may be producing basal lamina components but cannot organize them into a complete basal lamina.

O-65 - Inward and outward currents in differentiated hamster tracheal epithelial cell culture.

O-65 - Inward and outward currents in differentiated hamster tracheal epithelial cell culture.

Immunocytochemical localization of laminin in hamster tracheal epithelial cell cultures

EM examination of 28 day cultures of enzymatically dissociated hamster tracheal epithelial (HTE) cells grown on collagen coated millipore filters reveals that fragments of basal lamina may be present at the basal plasmalemma. Since the basal lamina consists of several major components including type IV collagen, heparan sulfate proteoglycans, entactin/nidogen, and laminin, questions naturally arise concerning the presence of such a structure in this cell culture system. When immunocytochemical procedures utilizing anti-laminin antibody and PAP techniques are carried out with paraffin sections of HTE culture at 1, 2,3, and 4 weeks in vitro, LM analysis reveals that a thin, dense line of reaction product is present between the basal surface of the HTE cells and the underlying collagen substrate. Immunoblotting evaluation carried out with supernatants of 7d HTE cell homogenates and HTE cell conditioned media also indicate that laminin is being produced by the tracheal cells. Thus, the presence of basal lamina-like fragments, the immunocytochemical localization of laminin, and immunoblot identification of laminin in hamster tracheal epithelial cell cultures, suggest that, although basal lamina components may be produced by HTE cells, at the time points tested, they are not yet being organized into a complete basal lamina.

The Effect of Retinoic Acid on Growth and Proto-oncogene Expression in Hamster Tracheal Epithelial Cells

Retinoic acid (RA) has been shown to be required for the maintenance of epithelial differentiation. Vitamin A deficiency in hamsters induces the tracheal epithelial cells to undergo squamous metaplasia. Reversing the vitamin deficiency restores the tracheal epithelial cells to their normal morphology and function. Using a hamster tracheal epithelial (HTE) cell culture system which undergoes differentiation to predominantly secretory cells in vitro, we found that RA can convert flat, squamous-like cells to compact, cuboidal-like cells, and that it stimulated cell proliferation. The mitogenic response to RA was maximal at 10(-7) M and required at least 48 h of treatment to observe the effect. RNA levels of growth-related genes during the growth and differentiation phases of primary HTE cultures were examined by Northern analysis. RA maintained a high level of c-myc RNA expression in preconfluent cultures, whereas untreated cells had low amounts of c-myc RNA. Expression of RNA for the replication-dependent histone 3.2 followed a similar pattern, i.e., its level was high in retinoid-treated versus control preconfluent cultures. In confluent (fully differentiated) HTE cell cultures, both retinoid-treated and control cells had low RNA levels of c-myc and histone 3.2. c-fos RNA levels were undetectable in either control or treated cells at any stage during primary culture. The RNA level of c-Ha-ras was very low in both control and treated cultures and did not vary with the state of growth or differentiation, except that when RA-treated cultures reached confluence, no c-Ha-ras RNA was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of ciliated and mucus cells from basal cells in hamster tracheal epithelial cell cultures

Hamster tracheal epithelia consist of three cell types: ciliated, mucus and basal cells. Autoradiographic data from several studies suggest that either basal or non-ciliated columnar cells may serve as stem cells for regeneration of lost or damaged ciliated and mucus cells. The objective of the present study was to examine the role of basal cells in the formation of ciliated and mucus cells in hamster tracheal epithelial (HTE) cell cultures via tritialed thymidine ([ 3H]-TdR) autoradiography. When 3 day cultures were pulsed with [ 3H]-TdR for 6hr and incubated for 2 additional days in non-radioactive media (5 day total) label was present in the nuclei of basal and columnar epithelial cells suggesting that the labeled columnar cells may be derived from basal cells. However, the morphological reorganization occurring during this 2 day interval may create difficulties in this interpretation. Since these morphological changes are minimal during the 6 day to 8 day in vitro period, 6 day HTE cultures were pulsed with [ 3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (8 day total), and examined to further study the fate of labeled basal cells during this period. Analysis of these 8 day cultures revealed that labeled nuclei were present in both basal cells and adjacent ciliated and mucus cells. These results do not exclude the possibility of non-basal cell origin of ciliated and mucus cells in other systems but suggest that, at least in HTE cultures, undifferentiated basal cells have the ability to develop into ciliated and mucus cells.