Secretion of insulin-like growth factor I and its binding proteins by collecting duct cells.
Insulin-like growth factor I (IGF-I) has been found in the kidney, particularly in the collecting duct in the rat. Since cultured rabbit collecting duct cells constitute a convenient system for in vitro studies, we have examined whether these cells secrete IGF-I. Culture medium conditioned by collecting duct cells was concentrated by reverse phase chromatography and applied to a Sephadex G100 column equilibrated in a denaturing buffer. Two major species with apparent molecular weights of 7.5 and greater than 25 kilodaltons (kD) were identified by IGF-I RIA. A smaller amount of 10 kD species was also observed. Further characterization of 7.5 kD IGF-I immunoreactive species by reverse phase HPLC showed that it eluted in a single peak. To determine whether the higher molecular weight species possessed IGF-I binding activity, appropriate fractions were desalted, incubated with [125I]IGF-I (thr59) for two hours at 30 degrees C and applied to a Sephadex G100 column equilibrated in a non-dissociating buffer. The major peak of radioactivity was confined to a high molecular weight region; there was no radioactivity in the fractions corresponding to 7.5 kD. Western ligand analysis of unreduced conditioned medium identified two IGF-I binding species of 25 and 30 kD, similar in size to species observed in normal rabbit serum. 125I-IGF-I binding as assessed in a charcoal adsorption assay could be displaced by IGF-I and IGF-II but not by insulin. Further characterization of the 10 kD peak of IGF-I immunoreactivity indicated that it did not possess IGF-I binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Aron
D C
DC
Department of Medicine, University Hospitals, Cleveland, Ohio.
Saadi
H F
HF
Nye
C N
CN
Douglas
J G
JG
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism* / Q000502:physiology; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D008297:Male; D011817:Rabbits; D011863:Radioimmunoassay
D002352:Carrier Proteins; D003470:Culture Media; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I