Basolateral and apical binding, internalization, and degradation of insulin by cultured kidney epithelial cells.
In vivo, filtered insulin is absorbed and degraded in proximal tubules after binding to the apical membrane. Peritubular removal also occurs and involves basolateral receptor binding and degradation. Whether basolateral degradation proceeds within the cell or on the cell surface is unknown. Because of the difficulties in addressing this question in vivo, this study was carried out with a cultured opossum kidney epithelium cell line with proximal-like features and insulin receptors. Cells were grown in partitioned wells on polycarbonate filters and, when confluent, the monolayer effectively separated the culture well into apical and basolateral compartments. Apical and basolateral binding, internalization, and degradation were studied separately by incubating monolayers with 125I-insulin added to either the apical or basal compartment. At 37 degrees C insulin associated with either pole in a time-dependent manner. This interaction was specific, for it was competitively inhibited by cold insulin but not by unrelated peptides. Separation of surface-bound from internalized insulin was achieved by lowering extracellular pH. At 4 degrees C, 92% of the radioactivity added to either side of the monolayer was surface-bound, whereas at 37 degrees C and after 1 h, 57% was surface-bound and 43% internalized. Affinity of apical and basolateral receptors were similar (1-2 nM), but basolateral receptor number was greater, for at high insulin concentrations (5 x 10(-8) M) basolateral membrane binding exceeded apical by fivefold (250 +/- 81 vs. 56 +/- 11 fm/10(6) cells). Degradation followed exposure to either pole of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)
Rabkin
R
R
Department of Medicine, Stanford University 94305.
Yagil
C
C
Frank
B
B
D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002116:Calcitonin / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure; D005934:Glucagon / Q000494:pharmacology; D013006:Growth Hormone / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008854:Microscopy, Electron; D009893:Opossums; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism*
D007328:Insulin; D000324:Adrenocorticotropic Hormone; D013006:Growth Hormone; D002116:Calcitonin; D005934:Glucagon; D011972:Receptor, Insulin