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Free keywords:
cyclic AMP; epithelial polarity; MDCK cellmultilayer
Abstract:
The effect of hormones on the morphology and cell surface polarity of the epithelial cell line MDCK was examined. When MDCK cells were seeded in high densities in media containing FCS a regular monolayer was formed. However, in serum-free medium supplemented with insulin, transferrin, prostaglandin E1, hydrocortisone and triiodothyronine, the development of a multilayer with intercellular lumina was observed. In hormone-depletion studies we identified PGE1 as the inducer of these multilayers. Since dibutyryl cyclic AMP and the phosphodiesterase inhibitor isobutyl methylxanthine could substitute for PGE1, we conclude that an elevated intracellular cAMP level resulted in formation of the multilayer. Further analysis by electron microscopy and immunocytochemistry revealed a polarized organization of the multilayered cells. Junctional complexes, enclosing microvilli-rich membrane domains, were found at the apices of adjacent cells facing the medium and those surrounding the intercellular lumina. Surprisingly, cells participating in the formation of both the free surface and the surface of the intercellular lumen, exhibited two distinct membranes with microvilli, each separated by junctional complexes. Immunolocalization of membrane marker proteins demonstrated that an apical 114 kDa membrane protein was localized to the free cell surfaces, the same membrane domains where extensive microvilli were also observed. The distribution of a basolateral 58 kDa membrane protein was restricted to sites of cell contact. These results provided evidence that nontransformed epithelial MDCK cells form multilayers in response to elevated cAMP levels; however, they retain the potential of developing cell surface polarity.
