Transcytotic passage of albumin through lens epithelial cells.

PURPOSE: To characterize the transcytotic passage of albumin through lens epithelial cells. METHODS: N/N 1003A rabbit lens epithelial cells were grown to a confluent monolayer on porous filter supports (Transwell Corning, Inc., Corning, NY). Monolayers were exposed apically to Alexa 488-labeled albumin (Alexa 488-BSA) in the absence and presence of endocytic inhibitors (filipin; dansylcadaverine [DCV]). Transcytotic passage of albumin was monitored for 4 hours by quantitating fluorescence in the basolateral compartment. The mechanism of albumin passage was studied by labeling cell monolayers and cryosections of whole rat lenses for clathrin or caveolin. RESULTS: The monolayer of cells formed a barrier to the passage of albumin, as shown by the 44% reduction in albumin passage in comparison to nonseeded membranes. Treatment with filipin or DCV reduced the passage of Alexa 488-BSA through lens epithelial cells by 73% and 66%, respectively. Confocal microscopy showed that albumin passage was predominantly transcellular and demonstrated colocalization of albumin with caveolin-1 and clathrin in lens epithelial and fiber cells. CONCLUSIONS: The Transwell apparatus is an excellent system to monitor transport systems across cell monolayers. In this study, rabbit lens epithelial cells formed a confluent monolayer that acted as a barrier to the passive diffusion of albumin. The kinetics of albumin movement across the monolayer and the inhibitor pharmacology suggests that lens cells actively transport albumin from the apical to the basolateral compartment. The inhibitory profile suggests the involvement of caveolae and clathrin-coated vesicles in the transcytotic process.

In vivo passage of albumin from the aqueous humor into the lens.

PURPOSE: To determine if albumin, the major protein component of the aqueous humor, passes into the lens in vivo. METHODS: Rat albumin was covalently-labeled with Alexa 488 fluorophore, purified by gel permeation chromatography, then injected into the aqueous chamber of living rats. At 5 min postinjection, lenses were removed and analyzed by HPLC gel permeation chromatography, confocal microscopy, and immunogold electron microscopy. RESULTS: At 5 min postinjection, HPLC analysis detected measurable amounts of Alexa-labeled albumin in the lens. The results were confirmed by confocal microscopy, which showed passage into epithelial and cortical fiber cells. Immunogold electron microscopy using antibody to the Alexa fluorophore demonstrated intracellular location of the Alexa-albumin complex. CONCLUSIONS: In vivo, significant amounts of albumin pass from the aqueous chamber into cells of the lens, consistent with a possible physiological role for this process involving passage of metabolites into the lens.