ENDOCYTOSIS AND DEGRADATION OF BOVINE APO-LACTOFERRIN AND HOLO-LACTOFERRIN BY ISOLATED RAT HEPATOCYTES ARE MEDIATED BY RECYCLING CALCIUM-DEPENDENT BINDING-SITES

We characterized endocytosis of iron-saturated(holo) and iron-depleted (apo) I-125-labeled bovine lactoferrin (Lf) by isolated rat hepatocyt es. Hepatocytes ingested both Lf forms-determined by EGTA/dextran sulf ate removal of surface-bound Lf-at maximal endocytic rates of 1.85 and 1.52 fmol cell-1 min-1 for I-125-apo-Lf and I-125-holo-Lf, respective ly. First-order endocytic rate constants (37-degrees-C) for I-125-apo- Lf and I-125-holo-Lf were 0.276 and 0.292 min-1, respectively. Regardl ess of Lf's iron content, hyperosmotic media (approximately 500 mmol/k g) inhibited Lf uptake by approximately 90%, indicating endocytosis of both Lf forms was primarily clathrin-dependent. Endocytosis of both L f forms was not altered significantly in the presence of excess iron c helator desferrioxamine or rat holo-transferrin, or by cycloheximide t reatment. Fluorescein isothiocyanate- and cyclohexanedione-modified Lf competed fully with native Lf for binding and endocytosis, indicating that, unlike human Lf, modification of lysine or arginine residues do es not block the interaction of bovine Lf with cells. After binding Lf at 4-degrees-C, cells at 37-degrees-C internalized approximately 90% of Lf bound to Ca2+-dependent sites but not Lf bound to Ca2+-independe nt sites. Following uptake, hepatocytes released acid-soluble (degrade d) products of I-125-Lf biphasically at 37-degrees-C, an initial rapid phase within the first 20 min-more pronounced with I-125-holo-Lf-foll owed by a sustained linear release of 298 and 355 molecule equiv cell- 1 min-1 for I-125-apo-Lf and I-125-holo-Lf, respectively. At 4-degrees -C, both digitonin-permeabilized and intact cells bound approximately 1.1 x 10(6) I-125-Lf molecules to Ca2+-dependent sites per cell, indic ating that hepatocytes do not contain a sizeable intracellular pool of these sites. Moreover, cells retained >70% of Ca2+-dependent sites on the surface during sustained Lf endocytosis. Thus, these Lf binding s ites recycle during endocytosis at an estimated 4-5 min/circuit.