Dog tracheal epithelial cells in culture synthesize sulfated macromolecular glycoconjugates and release them from the cell surface upon exposure to extracellular proteinases.
To determine whether glycoconjugates can be released into airways by surface epithelial cells that do not contain secretory granules and, if so, whether extracellular proteinases can affect this release, we studied dog tracheal epithelial cells after 8-10 days in culture. Ultrastructurally, these cells showed an extensive cell surface coat and no secretory granules. Cells were pulse labeled with radioactive sulfate (Na2 35SO4, 50 microCi/ml/24 h) and washed free of the unbound label. Release of sulfated products was then measured at 20-min intervals under basal conditions and again after 20 min of incubation with various extracellular proteinase. We found that these cells synthesized sulfated products and released them spontaneously and continuously into the medium. In addition, trypsin, Pseudomonas aeruginosa elastase, thermolysin, Staphylococcus aureus proteinase, mast cell chymase, plasmin, and kallikrein (each at 10(-7) M except plasmin, at 5 X 10(-6) M) increased the release of sulfated products to 77-667% over baseline release (p less than 0.01, n = 5 dogs for each); preliminary results showed that human neutrophil elastase was also very potent. The sulfated products released by trypsin had an apparent molecular weight of greater than or equal to 10(6) da as determined by gel filtration on Sepharose Cl-4B. Over 50% of these 35S-labeled products were digested to low-molecular-weight products (500-2000 da) upon incubation with endo-beta-galactosidase or with keratanase, suggesting that they are glycoconjugates containing poly(N-acetyllactosamine)-type carbohydrate chains. Decrease in cell staining by lectins specific for poly(N-acetyllactosamine), which accompanied the release of glycoconjugates, indicates that these sulfated glycoconjugates were released by proteinases from the apical cell surface. We conclude that cultured tracheal epithelial cells synthesize and transport sulfated macromolecular glycoconjugates to apical cell surfaces. These glycoconjugates are released from cell surfaces when exposed to extracellular proteinases. We therefore suggest that macromolecular glycoconjugates in airway secretions can originate not only from secretory granules but also from epithelial cell surfaces during airway inflammation.
Varsano
S
S
Cardiovascular Research Institute, University of California, San Francisco 94143.
Basbaum
C B
CB
Forsberg
L S
LS
Borson
D B
DB
Caughey
G
G
Nadel
J A
JA
D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004285:Dogs; D010450:Endopeptidases / Q000494:pharmacology*; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D006001:Glycoconjugates / Q000096:biosynthesis*; D006026:Glycoside Hydrolases / Q000494:pharmacology*; D037102:Lectins / Q000494:pharmacology; D046911:Macromolecular Substances / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D011975:Receptors, Mitogen / Q000032:analysis; D013431:Sulfates / Q000378:metabolism*; D014132:Trachea / Q000187:drug effects / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D006001:Glycoconjugates; D037102:Lectins; D046911:Macromolecular Substances; D011975:Receptors, Mitogen; D013431:Sulfates; D006026:Glycoside Hydrolases; D010450:Endopeptidases; D014357:Trypsin