Transcytotic passage of albumin through lens epithelial cells.
OBJECTIVE
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.
Sabah
Judith R
JR
Division of Biology, Kansas State University, Manhattan, Kansas, USA.
Schultz
Bruce D
BD
Brown
Zach W
ZW
Nguyen
Annelise T
AT
Reddan
John
J
Takemoto
Larry J
LJ
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D015153:Blotting, Western; D002103:Cadaverine / Q000031:analogs & derivatives / Q000494:pharmacology; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D017097:Electric Impedance; D004705:Endocytosis / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism*; D005372:Filipin / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D006834:Hydrazines / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology*; D008297:Male; D018613:Microscopy, Confocal; D011817:Rabbits; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D019108:Tight Junctions / Q000378:metabolism*
C417561:Alexa 488 hydrazide; D051242:Caveolin 1; D002966:Clathrin; D005456:Fluorescent Dyes; D006834:Hydrazines; D012710:Serum Albumin, Bovine; D005372:Filipin; C008542:monodansylcadaverine; D002103:Cadaverine