Receptor-mediated uptake of ferritin-bound iron by human intestinal Caco-2 cells

Ferritin (Ft) is a large iron (Fe)-binding protein ( approximately 450 kDa) that is found in plant and animal cells and can sequester up to 4500 Fe atoms per Ft molecule. Our previous studies on intestinal Caco-2 cells have shown that dietary factors affect the uptake of Fe from Ft in a manner different from that of Fe from FeSO4, suggesting a different mechanism for cellular uptake. The objective of this study was to determine the mechanism for Ft-Fe uptake using Caco-2 cells. Binding of (59)Fe-labeled Ft at 4 degrees C showed saturable kinetics, and Scatchard analysis resulted in a K(d) of 1.6 muM, strongly indicating a receptor-mediated process. Competitive binding studies with excess unlabelled Ft significantly reduced binding, and uptake studies at 37 degrees C showed saturation after 4 h. Enhancing and blocking endocytosis using Mas-7 (a G-protein activator) and hypertonic medium (0.5 M sucrose), respectively, demonstrated that Ft-Fe uptake by Mas-7-treated cells was 140% of control cells, whereas sucrose treatment resulted in a statistically significant reduction in Ft-Fe uptake by 70% as compared to controls. Inhibition of macropinocytosis with 5-(N,N-dimethyl)-amiloride (Na+/H+ antiport blocker) resulted in a decrease (by approximately 20%) in Ft-Fe uptake at high concentrations of Ft, suggesting that enterocytes can use more than one Ft uptake mechanism in a concentration-dependent manner. These results suggest that Ft uptake by enterocytes is carried out via endocytosis when Ft levels are within a physiological range, whereas Ft at higher concentrations may be absorbed using the additional mechanism of macropinocytosis.