Iron-loading of cultured adult rat hepatocytes reversibly enhances lactoferrin binding and endocytosis.
Isolated rat hepatocytes bind and internalize bovine lactoferrin (Lf) protein and Lf-bound Fe3+ via Ca2+-dependent recycling Lf binding sites (McAbee, 1995, Biochem. J., 311:603-609). In this study, we determined if iron loading of primary cultures of adult rat hepatocytes altered their ability to bind and internalize Lf. Rat hepatocytes were cultured 16-24 h with or without ferric ammonium citrate (FAC) and then assayed for Ca2+-dependent 125I-Lf binding at 4 degrees C or 125I-Lf endocytosis at 37 degrees C. Cells pretreated with FAC (5 microg/mL) internalized two- to sixfold more 125I-Lf than did control cells. The FAC-induced increase in 125I-Lf endocytosis required 4-8 h of culture at 37 degrees C and was fully reversible if cells were incubated an additional 24 h without FAC either in the presence or absence of the Fe3+ chelator desferrioxamine. Maximal endocytic rates for untreated and FAC-treated cells were 370 and 2,300 molecules 125I-Lf cell(-1) sec(-1), respectively. Both 125I-Lf binding at 4 degrees C and endocytosis at 37 degrees C increased up to sixfold between 0.3 10 microg/mL FAC, indicating that iron-induced enhancement of 125I-Lf uptake was due to an increase in the number of Lf receptors present on the cells. 125I-Lf bound to untreated and FAC-treated cells at 4 degrees C with similar affinities (K(d) approximately 1.5 microM). Cycloheximide but not actinomycin D blocked the FAC-induced increase in 125I-Lf binding, indicating that the increase in the number of Lf binding sites required translation but not transcription. Notably, iron loading blocked endocytosis of asialoorosomucoid by hepatocytes by up to 80%, reducing the number of active intracellular asialoglycoprotein receptors >65% without altering the number of active cell surface receptors. We conclude from these studies that Lf receptor activity on hepatocytes is regulated posttranscriptionally by the iron status of the cells.
McAbee
D D
DD
Department of Biological Sciences, University of Notre Dame, Indiana 46556, USA.
Ling
Y Y
YY
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004705:Endocytosis; D007501:Iron / Q000378:metabolism*; D007781:Lactoferrin / Q000378:metabolism / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley
D007501:Iron; D007781:Lactoferrin