AJP - Endocrinology and Metabolism, Vol 257, Issue 6 E895-E902, Copyright © 1989 by American Physiological Society
Basolateral and apical binding, internalization, and degradation of insulin by cultured kidney epithelial cells
R. Rabkin, C. Yagil and B. Frank
Department of Medicine, Stanford University 94305.
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)
