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 Fe 3+ via Ca 2+ ‐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 Ca 2+ ‐dependent 125 I‐Lf binding at 4°C or 125 I‐Lf endocytosis at 37°C. Cells pretreated with FAC (5 μg/mL) internalized two‐ to sixfold more 125 I‐Lf than did control cells. The FAC‐induced increase in 125 I‐Lf endocytosis required 4–8 h of culture at 37°C and was fully reversible if cells were incubated an additional 24 h without FAC either in the presence or absence of the Fe 3+ chelator desferrioxamine. Maximal endocytic rates for untreated and FAC‐treated cells were 370 and 2,300 molecules 125 I‐Lf cell ‐1 sec ‐1 , respectively. Both 125 I‐Lf binding at 4°C and endocytosis at 37°C increased up to sixfold between 0.3–10 μg/mL FAC, indicating that iron‐induced enhancement of 125 I‐Lf uptake was due to an increase in the number of Lf receptors present on the cells. 125 I‐Lf bound to untreated and FAC‐treated cells at 4°C with similar affinities (K d ∼ 1.5 μM). Cycloheximide but not actinomycin D blocked the FAC‐induced increase in 125 I‐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. J. Cell. Physiol. 171:75–86, 1997. © 1997 Wiley‐Liss, Inc.