Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anem ia. Transferrin (Tf)-dependent iron uptake is defective because of a mutati on in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nat ure permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe ut ilization to increase understanding of the phenotype of the b mutation. The distribution of Fe-59(2+) into intact erythroid cells and cytosolic, strom al, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe u ptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI up take. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was similar to 20% of +/b, a decre ase similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibi ted by bafilomycin A1, concanamycin, NH4Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited . NTBI uptake was unaffected after removal of Tf receptors by Pronase or de pletion of endogenous Tf. Concentration dependence revealed that NTBI uptak e into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process , as did an expression assay for DMT1. DMT1 serves in both apparent high-af finity NTBI membrane transport and the exit of iron from the endosome durin g Tf delivery of iron in rat reticulocytes; the low-affinity membrane trans porter, however, exhibits little dependence on DMT1. J. Cell. Physiol. 178: 349-358, 1999. (C) 1999 Wiley-Liss, Inc.