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 site s (McAbee, 1995, Biochem. J., 311:603-609). In this study, we determin ed if iron loading of primary cultures of adult rat hepatocytes altere d their ability to bind and internalize Lf. Rat hepatocytes were cultu red 16-24 h with or without ferric ammonium citrate (FAG) and then ass ayed for Ca2+-dependent I-125-Lf binding at 4 degrees C or I-125-Lf en docytosis at 37 degrees C. Cells pretreated with FAC (5 mu g/mL) inter nalized two- to sixfold more I-125-Lf than did control cells. The FAC- induced increase in I-125-Lf endocytosis required 4-8 h of culture at 37 degrees C and was fully reversible if cells were incubated an addit ional 24 h without FAC either in the presence or absence of the Fe3+ c helator desferrioxamine. Maximal endocytic rates for untreated and FAG -treated cells were 370 and 2,300 molecules I-125-Lf cell(-1) sec(-1), respectively. Both I-125-Lf binding at 4 degrees C and endocytosis at 37 degrees C increased up to sixfold between 0.3-10 mu g/mL FAC, indi cating that iron-induced enhancement of I-125-Lf uptake was due to an increase in the number of Lf receptors present on the cells. I-125-Lf bound to untreated and FAC-treated cells at 4 degrees C with similar a ffinities (K-d similar to 1.5 mu M). Cycloheximide but not actinomycin D blocked the FAC-induced increase in I-125-Lf binding, indicating th at the increase in the number of Lf binding sites required translation but not transcription. Notably, iron loading blocked endocytosis of a sialoorosomucoid by hepatocytes by up to 80%, reducing the number of a ctive intracellular asialoglycoprotein receptors >65% without altering the number of active cell surface receptors. We conclude from these s tudies that Lf receptor activity on hepatocytes is regulated posttrans criptionally by the iron status of the cells. (C) 1997 Wiley-Liss, Inc .