Kinetic study of internalization and degradation of 131I-labeled follicle-stimulating hormone in mouse Sertoli cells and its relevance to other systems.
The behavior of 131I-labeled follicle-stimulating hormone (FSH) after binding to cell-surface receptors in cultured Sertoli cells of C57BL/6NCrj mice was investigated. Sertoli cells cultured in F12/DME were pulse-labeled with 131I-FSH for 10 min at 4 degrees C, followed by cold chase for various periods of time. After the cold chase Sertoli cells were treated with 0.2 M acetate (pH 2.5) to dissociate membrane-bound 131I-FSH (surface radioactivity). The medium containing radioactivity after cold chase was mixed with 20% trichloroacetic acid, centrifuged, and the radioactivity of the supernatant was measured (degraded hormone). The radiolabeled materials associated with each process (surface binding, internalization, and degradation) were concentrated with ultrafiltration and characterized with gel filtration and/or thin layer chromatography. The effects of lysosomotropic agents, NH4Cl and chloroquine, were studied. The cold chase study at 32 degrees C showed that the surface radioactivity was the largest among the three kinds of radioactivities associated with each process immediately after pulse labeling, but the surface radioactivity rapidly decreased, while the internalized radioactivity increased. The cold chase study at 4 degrees C did not show such time-related changes in radioactivities, and a high level of surface radioactivity constantly persisted. The surface and internalized radioactivities were due to 131I-FSH, and the degraded radioactivity was mainly due to [131I]monoiodotyrosine. When Sertoli cells were cultured with lysosomotropic agents, the internalized radioactivity increased, while the degraded radioactivity decreased. Based on these observations, a kinetic model was proposed and the relationships among the surface, internalized, and degraded radioactivities and cold chase time were calculated algebraically. The rate constants of dissociation, internalization, and degradation were calculated to be 8.28 x 10(-4), 4.30 x 10(-2), and 6.46 x 10(-3) min-1, respectively. The simulated theoretical values of surface, internalized, and degraded radioactivities were almost the same as our experimental data. The present results indicate that 131I-FSH bound to the cell-surface receptors internalizes into Sertoli cells and that the degraded products are instantaneously released from the cells as [131I]monoiodotyrosine. The intracellular processes may be subdivided into 1) translocation, 2) net degradation of internalized hormone in the lysosomes, and 3) release of degraded hormone mainly as [131I]monoiodotyrosine. The rate-limiting step of these serial reactions was in the degradation of 131I-FSH, which consisted of a few substeps.
Shimizu
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A
Hayashibara Biochemical Laboratory, Okayama, Japan.
Kawashima
S
S
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D005640:Follicle Stimulating Hormone / Q000378:metabolism*; D006728:Hormones / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008433:Mathematics; D051379:Mice; D008810:Mice, Inbred C57BL; D008962:Models, Theoretical; D011865:Radioisotope Dilution Technique; D011962:Receptors, FSH / Q000378:metabolism*; D012708:Sertoli Cells / Q000378:metabolism*
D006728:Hormones; D007457:Iodine Radioisotopes; D011962:Receptors, FSH; D000643:Ammonium Chloride; D002738:Chloroquine; D005640:Follicle Stimulating Hormone