USE OF INHIBITORS OF ION-TRANSPORT TO DIFFERENTIATE IRON TRANSPORTERSIN ERYTHROID-CELLS

Iron uptake by rabbit reticulocytes and mature erythrocytes was invest igated using 4 incubation systems: 1. Fe-transferrin in NaCl at pH 7.4 , 2. Fe-transferrin in sucrose at pH 5.9, 3. Fe(II)-sucrose in sucrose at pH 6.5, and 4. Fe(II)-sucrose in KCl at pH 7.0. These systems were compared with respect to their magnitude and response to many membran e transport inhibitors and modifying agents. Iron uptake eia the first 3 systems had many similar features that were quite distinct from tho se of iron uptake in the fourth system. On the basis of these results, it is concluded that erythroid cells contain two iron transport mecha nisms, one with a high affinity and relatively low capacity for iron t ransport, which can be studied using incubation systems 1-3, and the o ther of low affinity but high capacity (incubation system 4). High-aff inity transport is present only in immature erythroid cells, is relati vely sensitive to inhibition by N-ethylmaleimide (NEM), N,N-1-dicycloh exylcarbodiimide (DCCD), and 7-chloro-4-nitrobenz-2-oxa-1,3 diazole (N BD), and is probably the mechanism by which iron, released from transf errin within endosomes, is transported across the endosomal membrane i nto the cytosol. DCCD and NBD are also inhibitors of the endosomal H+- ATPase, which is in keeping with the hypothesis that this ATPase funct ions as the iron transporter in endosomal membranes. However, the more specific inhibitor of this enzyme, bafilomycin A(1), inhibited iron u ptake only in incubation system 1, where its action can be attributed to inhibition of endosomal acidification. Hence, it is unlikely that t he ATPase also functions as the iron transporter. The low-affinity upt ake mechanism is sensitive to inhibition by amiloride, valinomycin, qu inidine, imipramine, quercetin, and diethylstilbestrol (to all of whic h high-affinity transport is relatively resistant), and is present in mature erythrocytes as well as reticulocytes.