Hamster Tracheal Surface Epithelial Cells Research Articles

Effects of betaine, coumarin and flavonoids on mucin release from cultured hamster tracheal surface epithelial cells.

Betaine, coumarin, hesperidin and kaempferol are the components derived from Lycium chinense, Angelicae decursiva, Poncirus trifoliata and Polygonatum odoratum, respectively. These plants have been used for the treatment of respiratory diseases in oriental medicine and their respective components were reported to have various biological effects. In this study, we investigated whether these natural products affect mucin release from cultured hamster tracheal surface epithelial cells and compared the possible activities of these agents with the inhibitory action on mucin release by poly-L-lysine and the stimulatory action by adenosine triphosphate. Confluent primary hamster tracheal surface epithelial cells were metabolically radiolabeled using (3)H-glucosamine for 24 h and treated for 30 min in the presence of varying concentrations of each agent to assess the effects on (3)H-mucin release. The results were as follows: (i) Coumarin and kaempferol did not affect mucin release significantly; (ii) Betaine and hesperidin increased mucin release at the highest concentration; (iii) Poly-L-lysine inhibited and adenosine triphosphate increased mucin release. We conclude that betaine and hesperidin can increase mucin release by direct acting on airway mucin-secreting cells and suggest these agents be further studied for the possible use as mild expectorants during the treatment of chronic airway diseases.

Effects of polycationic peptides on mucin release from airway goblet cells: relationship between polymer size and activity.

Various sizes of poly-L-lysine (PLL) and poly-L-arginine (PLA) were tested for their possible effects on airway goblet cell mucin release using primary hamster tracheal surface epithelial (HTSE) cells in an attempt to identify the smallest size of the polycationic peptide to suppress mucin release without cytotoxicity. HTSE cells were metabolically labeled using 3H-glucosamine and chased in the presence of varying concentrations of various sizes of the polycationic peptides. The amount of 3H-mucin in the spent media was measured by Sepharose CL-4B gel-filtration column chromatography. Possible cytotoxicity of the peptides was assessed by measuring the release of lactic dehydrogenase (LDH) during the treatment period. (1) PLL (MW 78,000) inhibited whereas PLA (MW 92,000) stimulated mucin release. However, these peptides were cytotoxic at the effective concentrations; (2) Both PLL (MW 9,600) and PLA (MW 8,900) could inhibit mucin release in a dose dependent manner without cytotoxicity; (3) Both PLL and PLA were effective in suppressing mucin release in 20-mer but not in either 10-mer or 5-mer; (4) 14-mers of both PLL and PLA also inhibited mucin release without cytotoxicity; (5) PLL and poly-D-lysine (PDL) of 14-mer were equipotent in its ability to suppress mucin release. Both PLL and PLA are cytotoxic at 'high' molecular weights, but have an ability to suppress mucin release without cytotoxicity at 'low' molecular weights. 14-mer seems to be the small, effective size, if not the smallest, for both PLL and PLA to suppress mucin release without cytotoxicity. The inhibitory effect of these polycationic peptides seems to be determined by the presence and the absolute number of positive charges and also to be independent of optical isomerism.

ATP-induced mucin release from cultured airway goblet cell involves, in part, activation of phospholipase A2.

Adenosine triphosphate (ATP) has been shown to stimulate mucin release by activation of protein kinase C (PKC) following activation of phospholipase C (PLC) coupled to the P2 receptor via G-proteins. The aim of the present study was to investigate pathways downstream to the PKC activation in ATP-induced mucin release from primary hamster tracheal surface epithelial (HTSE) cells. The release of mucin was determined by chromatographic procedure after metabolic labeling of mucin with [3H]-glucosamine. The results were: i) ATP induced the release of arachidonic acid, which caused the release of mucin. Pretreatment with mepacrine (0.3 mM), a phospholipase A2 (PLA2) inhibitor, inhibited the ATP-induced arachidonic acid and mucin release. Oleoyloxyethylphosphocholine, another PLA2 inhibitor, gave similar results. ii) An activator of PKC, 4 beta-phorbol-12 alpha-myristate-13-acetate (PMA, 1 microM) induced mucin release, which was inhibited by mepacrine pretreatment. iii) Downregulation of PKC by prolonged (16 h) PMA treatment caused inhibition of ATP-induced mucin release. Treatment of PKC downregulated HTSE cells with mepacrine did not further decrease the ATP-induced mucin release. These results suggest that PLA2 is involved in ATP-induced mucin release and its activation is sequential to the PLC-PKC pathway.

Squamous Differentiation Downregulates Muc1 Mucin in Hamster Tracheal Surface Epithelial Cell

To investigate whether the squamous differentiation of primary hamster tracheal epithelial cell, which is induced by retinoic acid deficiency or chronic PMA treatment, regulates Muc1 expression, we first produced and characterized a monoclonal antibody against hamster tracheal Muc1 mucin using pGEX-Muc1 fusion protein as an antigen and the changes of Muc1 mucin expression was determined by Western blot. Squamous differentiation downregulated the expression of Muc1 mucin from HTSE cells. The decrease in the immunoreactivity of Muc1 mucin was parallel to the decrease in the immunoreactivity of high molecular weight mucin, which is secreted from HTSE cells. The data from the present study implicate a possible role of Muc1 mucin in squamous differentiation of HTSE cells.

Inhibition of mucin release from airway goblet cells by polycationic peptides.

In the present study, we investigated whether polycationic peptides affect mucin release from cultured airway goblet cells. Confluent primary hamster tracheal surface epithelial cells were metabolically radiolabeled with [(3)H]glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of either poly-L-arginine (PLA) or poly-L-lysine (PLL) to assess the effects on [(3)H]mucin release. Possible cytotoxicity by the polycations was assessed by measuring lactate dehydrogenase release, (51)Cr release, and cell exfoliation. The results were as follows: 1) both PLA and PLL inhibited mucin release in a dose-dependent fashion; 2) there was no significant difference in either lactate dehydrogenase release, (51)Cr release, or the number of floating cells between control and treatment groups; 3) the effects of both PLA and PLL on mucin release were completely blocked by neutralizing the positive charges either by pretreatment with heparin or by N-acetylation of the polycations; and 4) both PLA and PLL completely masked the stimulatory effect of ATP on mucin release. We conclude that these polycationic peptides can inhibit mucin release from airway goblet cells without any apparent cytotoxicity, and the inhibitory effect seems to be attributable to their positive charges. These are the first nonsteroidal agents, to the best of our knowledge, that have been shown to inhibit mucin release from airway goblet cells.

Two eosinophil granule proteins, eosinophil peroxidase and major basic protein, inhibit mucin release from hamster tracheal surface epithelial cells in primary culture.

Effects of eosinophil proteins on airway goblet cell mucin release were investigated using primary hamster tracheal surface epithelial (HTSE) cell cultures. HTSE cells were metabolically labeled using 3H-glucosamine and chased in the presence of varying concentrations of eosinophil proteins. The amount of 3H-mucin in the spent media was measured by Sepharose CL-4B gel-filtration column chromatography. Possible cytotoxicity by the eosinophil proteins was assessed by measurements of both lactic dehydrogenase and mucin release during as well as after the treatment period. (1) Neither eosinophil cationic protein nor eosinophil-derived neurotoxin affected mucin release at concentrations up to 10(-6) M; (2) both major basic protein (MBP) and eosinophil peroxidase (EPO) inhibited mucin release in a dose-dependent fashion, and the inhibitory effect by these proteins appeared to be reversible; (3) neither MBP nor EPO caused any apparent cytotoxicity at concentrations up to 10(-6) M. Eosinophil proteins such as MBP and EPO inhibit mucin release from primary HTSE cells without causing any apparent cytotoxicity.

Identification and characterization of high molecular-mass mucin-like glycoproteins in the plasma membrane of airway epithelial cells.

A previous lectin binding study demonstrated the presence of high molecular-mass mucin-like glycoproteins (HMGP) on the surface of hamster tracheal surface epithelial (HTSE) secretory cells (Proc. Natl. Acad. Sci. USA 1987;84:9304). In the present study, we intended to isolate and characterize these HMGP from the plasma membrane of the primary HTSE cells and then to determine whether or not these membrane HMGP are Muc-1 mucins, a type of mucins originally discovered on the surface of some carcinomas. A subcellular fraction enriched with the plasma membrane was obtained using a sucrose density gradient centrifugation. This fraction contained high molecular-mass glycoconjugates which were excluded from Sepharose CL-4B gel. Biochemical characterization of these glycoconjugates revealed the following characteristics: (1) susceptibility to both pronase and mild alkaline treatments, but totally resistant to proteoglycan-digesting enzymes; (2) partitioning in the detergent phase of Triton X-114 and resistance to digestion by phosphatidylinositol phospholipase C or D; (3) a buoyant density of 1.5 g/ml based on CsCl density gradient centrifugation; (4) polydispersity in terms of both size and charge density; and (5) lack of immunoreactivity with an anti-Muc-1 mucin antibody. We conclude that the plasma membrane of HTSE cells at confluence contains HMGP, which seem to be the integral membrane proteins but different from Muc-1 mucins, and that these membrane HMGP appear to share some similarities with secreted mucins in terms of size and charge.

Expression of MUC1 mucin gene by hamster tracheal surface epithelial cells in primary culture.

Primary hamster tracheal surface epithelial (HTSE) cells carry mucin-like glycoproteins on the apical surface which are releasable by neutrophil elastase. In some cancer cells, mucins are localized on the cell surface and have been shown to be encoded by the MUC1 mucin gene. The objectives of the present experiments were: (I) to determine if HTSE cells express MUC1 mucin gene; (2) if they do, to isolate and characterize the hamster MUC1 complementary DNA (cDNA); and (3) to examine the pattern of MUC1 mRNA expression at different stages of culture. Reverse transcriptase-polymerase chain reaction amplification of HTSE cell RNAs using degenerate primers based on homologous sequences between the human and mouse MUC1 genes revealed the presence of a cDNA (0.5 kb) which has an 88% similarity in sequence with the mouse MUC1 cDNA. Using this 0.5 kb cDNA as a probe, an HTSE cell cDNA library was screened to isolate a hamster MUC1 cDNA clone. Sequence analysis of the cDNA revealed that it encodes an integral membrane protein of 676 amino acids which consists of (1) an N-terminal signal sequence, (2) the tandem repeat domain encoding 12 repeats of 20 amino acids, and (3) the C-terminal region consisting of degenerate tandem repeats and a unique sequence containing both the transmembrane and cytoplasmic domains. The presence of seven tyrosine residues in the cytoplasmic domain suggests a potential role as a receptor. Finally, expression of MUC1 mucin gene in HTSE cells appears to be associated with differentiation of secretory cells.

Nucleotide-induced mucin release from primary hamster tracheal surface epithelial cells involves the P2u purinoceptor

Mucin release by airway surface epithelial cells is regulated by extracellular adenosine triphosphate (ATP) via a P2 purinoceptor-mediated mechanism. The objective of the present experiment was to examine the possible involvement of uridine triphosphate (UTP) in this purinergic signal transduction pathway. Using primary hamster tracheal surface epithelial cells, ATP and UTP were compared in their abilities: 1) to displace ATP gamma S35-binding to intact cells; 2) to accumulate inositol phosphates; and 3) to stimulate mucin release. Finally, the presence of a P2u receptor message was examined. Our results showed that: 1) UTP was much less effective than ATP in displacing ATP gamma S35-binding (median inhibitory concentrations (IC50S) 240 vs 2.9 microM); 2) UTP was more potent than ATP in accumulating inositol phosphates (100 vs 43% increase at 2mM); 3) UTP was equipotent with ATP in stimulating mucin release; 4) Northern blot analysis of messenger ribonucleic acids (mRNAs) with a mouse P2u receptor complementary deoxyribonucleic acid (cDNA) probe revealed a single specific band (2.8 kb), partial sequencing of which showed a great homology with those of human or mouse P2u receptors. We conclude that, although both ATP and UTP are equipotent in stimulating mucin release, their binding kinetics to the cell surface are quite different, suggesting the presence of a common binding domain which may be responsible for the mucin release by these nucleotides. We suggest that the P2u purinoceptor is likely to be responsible for mucin release by these nucleotides, probably via activation of phospholipase C.

Binding kinetics of ATP gamma S35 on cultured primary tracheal surface epithelial cells.

Extracellular ATP can stimulate mucin release from primary hamster tracheal surface epithelial (HTSE) cells via a P2 purinoceptor-mediated mechanism, based on agonist potency studies of mucin release (Br. J. Pharmacol. 1991; 103:1053-1056). In the present study, we examined the kinetics of ATP binding to the surface of intact HTSE cells at 4 degrees C using ATP gamma S35 as a radioligand. We found that ATP gamma S35 bound to HTSE cells in a saturable, reversible manner, reaching an equilibrium at about 30 min. Scatchard analysis of equilibrium binding suggested the presence of two binding sites with Kd values of 0.47 and 9.4 microM. Competitive binding experiments, based on the ability of nucleotides and ATP analogs to block ATP gamma S35 revealed a rank order of ATP > ADP > alpha,beta-methylene ATP > 2-methylthio ATP > or = beta, gamma-methylene ATP. Neither AMP nor adenosine could inhibit the ATP gamma S35 binding. A comparison between the ability of nucleotides to compete with ATP gamma S35 binding and their ability to induce mucin release revealed a rather poor correlation (r2 = 0.67) with all of the above nucleotides but a good correlation (r2 = 0.96) without 2-methylthio ATP, indicating the presence of heterogenous ATP binding sites on the HTSE cell surface. UTP, a pyrimidine nucleotide, which is almost equipotent with ATP in its ability to stimulate mucin release, was much less potent than ATP in its ability to displace the ATP gamma S35 binding in these HTSE cells.

Effect of floating a gel matrix on mucin release in cultured airway epithelial cells.

Confluent cultures of primary hamster tracheal surface epithelial (HTSE) cells grown on a thick collagen gel are highly enriched with secretory cells and constitutively release mucins. In the present experiment, we examined the possible effect of mechanical strain of cultured HTSE cells on the release of mucin. The mechanical strain of cells was accomplished by several methods: 1) by floating the gel from the culture dish by rimming; 2) by treatment with EGTA which interrupts intercellular tight junctions; 3) by treatment with collagenase which disrupts the cell-matrix adhesion; and 4) by mechanically flexing the collagen gel matrix. All these conditions caused increases of mucin release without damage on the plasma membrane. We conclude that a number of mechanical strains which might alter cell shape can stimulate mucin release from cultured HTSE cells. Such a mechanism might be operative in the physiological regulation of airway goblet cell mucin secretion where mechanical strains may be induced on epithelial cells by underlying smooth muscles.

Mucin-like Glycoproteins Secreted from Cultured Hamster Tracheal Surface Epithelial Cells: Their Hydrophobic Nature and Amino Acid Composition

Confluent cultures of primary hamster tracheal surface epithelial cells synthesize and secrete high molecular weight mucin-like glycoproteins (HMW MLGP). Secreted HMW MLGP are highly associated with various lipids, indicating that they are extremely hydrophobic. These HMW MLGP are also associated with varying amounts of "small" glycoproteins via hydrophobic interactions and they can be dissociated by heat and detergent treatments. HMW MLGP free of these "small" glycoconjugates have a buoyant density of 1.5 g/ml and can enter 4% but not 7.5% gels during SDS-polyacrylamide gel electrophoresis. Amino acid composition analysis of HMW MLGP free of these "small" glycoproteins shows that they are relatively rich in serine, threonine, and proline, but the total content of these amino acids seems somewhat lower than that of in vivo airway mucins.

Hydrophobicity of Mucin-Like Glycoproteins Secreted by Cultured Tracheal Epithelial Cells: Association with Lipids

Confluent cultures of primary hamster tracheal surface epithelial cells are highly enriched with secretory cells resembling airway goblet cells. The cultures secrete lipids, most of which appear to be associated with high-molecular-weight mucinlike glycoproteins (HMW MLGP). In the present communication, we examined the nature of the association and analyzed the lipids associated with the secreted HMW MLGP. The HMW MLGP purified in the presence of 4 M guanidinium HCl were highly associated with lipids. When these HMW MLGP were gel filtered using a Sepharose CL-4B column in the presence of detergents, more than 97% of the associated lipids were dissociated from HMW MLGP and eluted in fractions with Kd = 0.70. On the other hand, when [3H]palmitic acid-labeled spent medium was gel filtered, column elution profiles varied among elution buffers, a major change being an appearance of a 3H peak at Kd = 0.70 in the presence of 50 mM sodium acetate, phosphate-buffered saline (PBS), or PBS/sodium dodecyl sulfate, but not with 4 M guanidinium HCl. Lipids associated with MMW MLGP under a "physiological" condition consisted of neutral, phospho-, and glycolipids. We conclude that (1) HMW MLGP secreted from cultured airway epithelial cells are extremely hydrophobic and associated with a variety of lipids, mostly via noncovalent binding, and (2) the degree of the lipid association seems to depend on the ionic environment of the solution.

Mechanisms of airway goblet cell mucin release: studies with cultured tracheal surface epithelial cells.

Confluent hamster tracheal surface epithelial (HTSE) cells in primary culture are enriched with secretory cells that synthesize and release mucins. Using this cell culture system, we investigated possible mechanisms of goblet cell mucin release by altering the media bathing the apical surface of HTSE cells: medium hyperosmolarity decreased mucin release, whereas hypo-osmolarity increased release without causing a cytoplasmic leak due to plasma membrane damage. A Ca2+ ionophore, A23187, did not influence mucin release. Both acidic (pH less than 4) and basic (pH greater than 9) media caused significant increases in mucin release secondary to cell membrane damage. Physiologic concentrations of chemical mediators such as prostaglandins (PGE2 and PGF2 alpha) and leukotrienes (LTC4 and LTD4) did not influence mucin release. Both elastase and cathepsin G derived from human neutrophils caused marked increases in release, whereas trypsin from the porcine pancreas produced a small increase only at a high concentration. We conclude that mucin release by cultured airway goblet cells can be enhanced by: (1) irritant gases, (2) luminal fluid osmolarity, (3) pharmacologic concentrations of LTC4 and LTD4, and (4) cationic proteases, each presumably acting by different mechanisms. Each of these mechanisms may play a role in epithelial mucin secretion associated with airway inflammation.

Secretions from primary hamster tracheal surface epithelial cells in culture: mucin-like glycoproteins, proteoglycans, and lipids.

Surface epithelial cells dissociated from hamster tracheas and grown on a thick collagen gel in the presence of 5% fetal bovine serum become highly enriched with secretory cells at confluence. In the present communication, we have analyzed secretory products from this primary hamster tracheal surface epithelial (HTSE) cell culture. The secreted glycoconjugates included high-molecular-weight mucin-like glycoproteins (HMW MLGP) and proteoglycans that comprised 22% and 5% of the total [3H]glycoconjugates secreted when [3H]glucosamine was added as a metabolic precursor. Among the proteoglycans were hyaluronic acids (53%), heparan sulfate proteoglycans (29%), and chrondroitin sulfate proteoglycans (18%). Chondroitin sulfates were mostly 4-sulfated. On the other hand, the secreted lipids included cholesterol, phospholipids, and glycolipids, and most of them were associated with HMW MLGP.