Adsorptive and fluid phase endocytosis by cultured rabbit corneal endothelium

In addition to maintaining corneal transparency via a pump-leak mechanism, the corneal endothelium plays an active role in the transport of certain proteins to supply nutrients for the stroma and to remove metabolites. In order to investigate transcellular transport mechanisms across the endothelium, we exposed cultured rabbit corneal endothelial cells to tracers that are commonly used to describe various forms of endocytosis. The cells were incubated on their apical surfaces in solutions containing different HRP conjugated lectins (concanavalin A and lens culinaris = alpha-D-mannose, alpha-D-glucose; peanut agglutinin = alpha-D-galactose), cationized ferritin (CF), native ferritin (NF), and HRP alone for 5-60 min and processed for EM cytochemistry. At early times, the lectins and CF were seen bound to the apical plasma membrane, thus indicating adsorptive endocytosis. The NF and the lectins in the presence of their competing sugar as well as HRP showed little or no surface binding, thus being markers for fluid phase endocytosis. At later times, large amounts of lectins and CF were located in round, tubular, or U-shaped vesicles of various sizes, large vacuoles, multivesicular bodies, and in other cytoplasmic compartments. Very little or no uptake was observed with NF, or when the lectins were used in presence of their competing sugars. HRP was seen in moderate amounts only in round or oval shaped vesicles. This study suggests that adsorptive endocytic pathways play a major role in transcellular transport through the corneal endothelium, whereas the transport of macro-molecules via fluid phase endocytosis is more limited. In addition, our observations suggest that adsorptive endocytic tracers undergo various intracellular fates and also appear to be transported through the cells at different rates.