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  • Title: Iron-loading of cultured adult rat hepatocytes reversibly enhances lactoferrin binding and endocytosis.
    Author: McAbee DD, Ling YY.
    Journal: J Cell Physiol; 1997 Apr; 171(1):75-86. PubMed ID: 9119894.
    Abstract:
    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.
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