| Abstrakt: |
Principal cells of the pancreatic ductal epithelium have been reported to secrete high molecular weight (HMW) glycoconjugates such as mucins into the ductal lumen. We used a human pancreatic carcinoma cell line of ductal origin (PANC-1) which has retained some of the morphological and biochemical characteristics of normal ductal principal cells as a source for isolation of HMW secretory products. The present study was designed to isolate these HMW secretory products, partially characterize them through biochemical and immunological approaches, and determine the effects of secretin on their synthesis and discharge from PANC-1 cells. Our results indicated that when PANC-1 cells are grown on collagen-coated beads in defined serum-free medium, at least three HMW secretory products could be isolated from the medium. These secretory products had a mass of approximately 200, 280, and 370 kDa based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The 200–kDa species made up proportionally less of the three in the gel-staining pattern. Polyclonal antibodies raised to the 370–kDa secretory product cross-reacted with the 280–kDa species. The 370–kDa secretory product was sulfated and wheat germ agglutinin (WGA) binding indicated that the 370–kDa species was a glycoconjugate. The 280–kDa and 200–kDa species were sulfated to a much lesser degree than the 370–kDa species and WGA binding could not be clearly demonstrated with the 280 kDa or 200 kDa species. Glycosidase and selective degradation studies, however, indicated that all three species contained glycosaminoglycan moieties. Antibodies raised to the 370–kDa secretory product localized to the epithelium of human pancreatic carcinomas but not to other cell types in this neoplastic tissue. The antibody also cross-reacted with the ductal epithelium of normal human pancreas and could be localized to centroacinar cells, the epithelium of intralobular ducts, and the epithelium of interlobular ducts. The antibody did not cross-react with goblet cells of the human small or large intestine, indicating no generalized reactivity to gastrointestinal mucins. ELISA and pulse-chase immunoprecipitation studies indicated an increase in the cellular content and synthesis of these HMW secretory products after stimulation of PANC-1 cells with 10−8- to 10−11M secretin. We correlated secretin stimulation with the appearance of numerous membrane bound vesicles throughout the cytoplasm as monitored at the ultrastructural level. Optimal secretin concentrations were in the range of 10−10M.The content of these vesicles was immunoreactive to the antibody raised against the 370–kDa secretory product as determined by ultrastructural immunocytochemical techniques. These results indicate that principal cells of the pancreatic ductal epithelium produce HMW secretory products that (a) may be unique to the pancreas and (b) are responsive to secretin stimulation in terms of their synthesis and discharge. |
