Kinetic analysis of transcytosis of epidermal growth factor in Madin-Darby canine kidney epithelial cells.
OBJECTIVE
The aim of this study is to clarify the intracellular fate and a rate limiting step in transcytosis of epidermal growth factor (EGF) in Madin-Darby Canine Kidney (MDCK) epithelial cells.


METHODS
The kinetics of transcytosis of 125I-EGF was investigated. To examine the fate of EGF molecules bound to its receptor on the cell surface. 125I-EGF was prebound to the basal surface at 4 degrees C, followed by extensive washing and subsequent incubation at 37 degrees C in EGF-free medium.


RESULTS
Saturable transport of 125I-EGF through the cell monolayer could only be observed from the basal to apical side. Most (approximately 90%) of the EGF molecules bound to the surface receptor are internalized with a half-life of 1-3 min, followed by intracellular degradation with a half-life of 20-50 min. The exocytosis of internalized EGF into the apical medium is much slower with a half-life of 130-250 min. Even when 125I-EGF was incubated with MDCK cells at 37 degrees C and washed with acid to remove cell-surface 125I-EGF, intact 125I-EGF appeared in the basal medium with a half life of 160-170 min.


CONCLUSIONS
The exocytosis of internalized EGF into the apical medium is a rate limiting step in EGF transcytosis. At least a small amount of internalized EGF is recycled.
Kozu
A
A
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Kato
Y
Y
Shitara
Y
Y
Hanano
M
M
Sugiyama
Y
Y
D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D004815:Epidermal Growth Factor / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D011869:Radioligand Assay; D051381:Rats
D004815:Epidermal Growth Factor