Secretory activity of hamster tracheal explants and isolated tracheal epithelial cells and the effects of cystic fibrosis serum

Abstract

Serum from cystic fibrosis patients has been shown by scanning electron microscopy to cause release of large quantities of mucus from the cultured tracheal rings of 3-4-month-old male Golden Syrian hamsters. In order to study this phenomenon on single cells, an epithelial (HTE) cell culture has been established from the hamster tracheal rings using the cell rescue method of Goldman and Baseman (1980a, In Vitro, 16:313). The cells were demonstrated to be epithelial by histochemical staining and immunofluorescent detection of laminin. Proteins secreted by HTE cells were partially characterized and shown to consist, at least in part, of acidic glycoproteins. The proteins were precipitated by addition of buffered alcian blue (AB) to the cell-free medium under conditions in which all of a polyanionic protein [3H]-labeled mucin, was precipitated without carrier. [14C] galactosamine-labeled AB precipitate was beta-eliminated and, after neutralization and centrifugation, the material in the supernatant was sized by chromatography on a calibrated Bio-Gel P2 column. The label eluted with a molecular weight close to a disaccharide. HTE cells pulse-labeled for 1.0 hr with [3H] leucine or [14C] galactosamine secreted increasing amounts of labeled glycoprotein during the chase. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography of labeled AB precipitates revealed three major bands, two with molecular weights greater than 100 kd. Secretion was stimulated by retinoate (50% increase), but not by retinol. Exposure of HTE cells to whole sera from cystic fibrosis patients resulted in heightened secretion rates as compared to results obtained with normal sera. Heterozygote sera produced secretion rates intermediate between the two extremes.

Effects of cystic fibrosis serum on calcium influx and secretion using isolated tracheal epithelial cells

“…Thus, after 6 hr there was a 2.2-fold difference between NHS and CFS, a 1.5-fold difference between NHS and HHS, and a 1.4-fold difference between HHS and CFS. These data confirm earlier work which demonstrated that the observed differences were statistically significant at P < 0.01 (Rudick et al, 1984). 45Ca2+ uptake When 45Ca2f influx was measured, there was very low uptake in the presence of NHS compared to that seen with CFS (Fig.…”

“…The cells were routinely grown in 100-mm plastic culture dishes containing Ham's F12 medium supplemented with M retinoate and 10% fetal calf serum (FCS). Culture conditions have been previously reported (Rudick et al, 1984).…”

“…This assay was carried out as previously described (Rudick et al, 1984). HTE cells were grown to near confluency (less than 100,000 cells/dish) in 35-mm diameter plastic dishes.…”

“…Furthermore, McPherson et al (1983) have demonstrated that CF serum stimulated release of mucins from rat submandibular acini to a significantly qreater degree than did control serum as measured by ( 4C)-glucosamine incorporation. Corroborating the findings of Boat et al (1982) and McPherson et al (1983), we have recently used a homogeneous cell culture method to detect and assay for the putative CF serum factor (Rudick et al, 1984). Epithelial cells isolated from the hamster trachea and placed in tissue culture respond to some component of CF serum by an increased rate of secretion.…”

Growth Requirements of Ferret Tracheal Epithelial Cells in Primary Culture

“…Much interest in the culture of normal airway epithelial cells has stemmed from the potential usefulness of such systems in the study of carcinogenesis (1 8, 19), pulmonary disease (20), and microbial pathogenicity (2 1). In the present study the isolation and growth in culture of airway epithelial cells from the ferret are described.…”