AJP - Cell Physiology, Vol 257, Issue 2 C190-C196, Copyright © 1989 by American Physiological Society
Retroendocytosis of insulin in a cultured kidney epithelial cell line
D. C. Dahl, T. Tsao, W. C. Duckworth, M. J. Mahoney and R. Rabkin
Department of Medicine, Stanford University, California 94305.
It has been generally accepted that in renal tubular epithelium endocytosed
proteohormones are transported to lysosomes where they undergo complete
hydrolysis. En route, as endosomal pH falls, the proteohormone uncouples
from the endocytosed membrane binding site, which recycles to the cell
surface. However, studies in other tissues have uncovered alternate
intracellular pathways for proteins. One such pathway is retroendocytosis
(endocytosis then exocytosis). To determine whether a retroendocytotic
pathway exists for insulin in renal epithelium, a study was carried out
with confluent monolayers of a proximal-like opossum kidney cell line that
exhibits receptor-mediated endocytosis of insulin. Cells were preloaded
with 125I-labeled insulin (4 X 10(-10) M) for 30 min, surface-bound insulin
was then removed by acid washing, and over the next 60 min the release of
intracellular radioactivity into the medium was monitored. At 37 degrees C,
control cells released on average 7-15% of the intracellular radioactivity
as intact insulin [trichloroacetic acid (TCA)-precipitable radioactivity]
and approximately 62% as TCA-soluble degradation products. In the presence
of 0.1 mM chloroquine (an acidotropic agent) the release of intact insulin
increased approximately twofold while degradation fell by nearly one-half.
With Sephadex G-50 chromatography we found that the released radioactivity
included insulin-size material that increased in the presence of
chloroquine. High-performance liquid chromatography revealed that 53
(controls) and 81% (chloroquine treatment) of this latter material
consisted of intact insulin. We conclude that, in addition to a major
degradative pathway, cultured kidney epithelial cells exhibit a
retroendocytotic pathway for insulin. Chloroquine inhibits degradation and
appears to divert insulin from the degradative into the retroendocytotic
pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
