Retroendocytosis of insulin in a cultured kidney epithelial cell line.
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)
Dahl
D C
DC
Department of Medicine, Stanford University, California 94305.
Tsao
T
T
Duckworth
W C
WC
Mahoney
M J
MJ
Rabkin
R
R
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D004705:Endocytosis; D004848:Epithelium / Q000502:physiology; D007328:Insulin / Q000378:metabolism*; D007668:Kidney / Q000502:physiology*; D007700:Kinetics; D009893:Opossums
D007328:Insulin; D002738:Chloroquine