Search

We have investigated the effect of both adenosine triphosphate (ATP) and uridine triphosphate (UTP) on the fluid transport, transepithelial electric potential difference (PD), and unidirectional chloride flux when applied apically to cultured human surface respiratory epithelial (HSRE) cells in a double-compartment chamber. The effects of ATP and UTP (both 100 microM) were examined in cells either untreated or pretreated with 100 microM amiloride in lactated Ringer's solution. ATP or UTP was added to the apical solution in a 100 microliters final volume. After a 2-h incubation period, the change in fluid transport was measured by weighing the apical fluid. Compared with control, amiloride blocked the fluid absorption by HSRE cells. The addition of ATP or UTP, either alone or after pretreatment with amiloride, induced a similar and significant increase in the apical fluid and chloride flux (P0.001 and P0.005, respectively). The changes in both fluid transport and chloride flux were accompanied by changes in PD. A blocker for chloride transport, 4,4'-diisothiocyano-2,2'-stilbene disulfonate, at 500 microM significantly blocked the ATP-stimulated fluid transport (P0.05) and chloride flux (P0.01). These results support the hypothesis that extracellular ATP and UTP increase the fluid transport by respiratory epithelial cells and may be useful in the hydration of mucus and respiratory mucosa.

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number. The maintenance of the epithelial serous phenotype of BTG cells during the first passages (1 to 4) suggests that the study of the regulation of these gland cells should be limited to these early passages.

Human airway lysozyme (HAL) was separated into fractions of distinct molecular forms using a Mono S cation-exchange column on a fast-protein liquid chromatography system. This new and rapid (30 min) purification procedure of human lysozyme enabled the preparation of fractions, highly enriched in different isoenzymes of HAL. Purified HAL from pathological purulent airway secretions, nonpurulent airway secretions, and normal tracheobronchial tissue culture medium was characterized by four, three, and only one enzymatically active molecular forms, respectively. All charge forms (separated or combined) recovered from either purulent or nonpurulent airway secretions or tracheobronchial culture medium exhibited the same apparent molecular weight of 15,000.

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 15000 to 95000 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.

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15 +/- 4 (X 10(6] cells per g of tracheal submucosa with 72 +/- 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1:1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelioid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS-staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post-confluent culture of BTG cells on EHS matrix, we observed numerous dome-like structures formed by differentiated cells which were joined together around luminal spaces.

Summary Bovine tracheal submucosal gland serous cells were incubated with ( 35 S) methionine. The proteins synthesized and secreted into the culture medium during pulse and chase periods were analyzed. Three major protein bands with apparent molecular weight values of 67 kD, 47 kD and 32 kD were detected by fluorography following SDS-polyacrylamide gel electrophoresis. Based on the molecular weight, immuno-reactive cross reactivity and surface molecular charge characteristics, the labeled Mr 67 kD protein was identified as an albumin-like protein. Our results suggest that albumin found in airway secretions can originate, at least in part, from tracheal gland serous cells.