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J.R. Sabah, B.D. Schultz, Z.W. Brown, M. Burton, L.J. Takemoto; Mode of Alexa–BSA Internalization into the Bovine Lens . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1869.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Recently our laboratory showed that rat albumin was internalized into rat lenses upon in vivo injection of the protein into the aqueous or the vitreous humor. The objectives of this study were: 1) to assess if albumin passage through the lens occurs via an intracellular route 2) to characterize the possible involvement of clathrin and/or caveolin in this passage. Methods: Epithelial cells were isolated from bovine lenses and cultured in 75–cm2 flasks containing DMEM supplemented with 10% FBS, antibiotics and fungicides. Confluent monolayers were treated with fluorescently labeled bovine albumin (Alexa–BSA) in serum free DMEM (DMEM SF). Following incubation, cells were washed in DMEM SF pH 2.0, fixed in 2% paraformaldehyde, and stained with the nuclear stain To–Pro 3 prior to visualization using confocal microscopy. To assess the mode of internalization of albumin into lens cells, capsule and lens epithelial cells layers were excised from fresh bovine lenses and mounted in modified Ussing flux chambers symmetrically containing DMEM SF in the absence (control) or presence of filipin (1.25 µg/ml), or dansylcadaverine (6.5 µg/ml). After the tissues were allowed to equilibrate in their respective medium for 30 min, Alexa–BSA (25 mg/ml) was added to either the hemichamber bathing the capsule/basolateral side of the epithelial cells or the hemichamber bathing the apical aspect of the lens epithelium. Samples of medium were removed from the opposite hemichamber after 1, 5, 15, and 30 min and analyzed for fluorescence to quantitate unidirectional albumin flux. The transepithelial electrical resistance, a sensitive indicator of epithelial barrier integrity, was determined at 100 second intervals throughout the experiment. Results: Confocal microscopy showed that Alexa–BSA was internalized into cultured lens epithelial cells. Results from Ussing flux chamber experiments with freshly isolated bovine lens demonstrated that Alexa–BSA passes through the capsule/epithelium in a time dependent manner. Preincubation with filipin or dansylcadaverine reduces transepithelial Alexa–BSA flux. Conclusions: These results suggest that albumin is internalized into the lens through an intracellular pathway that involves clathrin and caveolin endocytic mechanisms.
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