Inhibition of reticulocyte iron uptake by NH 4Cl and CH 3NH 2

Abstract

The aim of this investigation was to test the hypothesis that elevation of intracellular pH would inhibit iron uptake by reticulocytes. The experiments were performed with rabbit reticulocytes and iron bound to rabbit transferrin. Incubation of the cells with NH 4Cl, (NH 4) 2CO 3, CH 3NH 2 and (CH 3) 2NH was used in an attempt to increase intracellular pH. These substances were all found to inhibit iron uptake by reticulocytes. The mechanism of action of NH 4Cl and CH 3NH 2 was investigated in detail. Similar results were found with both reagents. They inhibited iron uptake in a concentration-dependent manner, but produced a small increase in the cellular uptake of transferrin. The onset of action was rapid and the effect was reversible. There was no decrease in the number of transferrin-binding sites per cell and their apparent affinity for transferrin increased slightly, while the efficiency of iron removal from transferrin per binding site diminished greatly. The rate of transferrin release from reticulocytes was unaffected. NH 4Cl did not affect the rate of iron release from transferrin in a cell-free system. Incubation of reticulocytes with 10 mM NH 4Cl or CH 3NH 2 was found to produce an increase in intracellular pH of 0.05—0.15 pH units. The intracellular pH determined by used of the weak acid 5,5-dimethyl-oxazolidine-2,4-dione was significantly higher than that obtained with the weak base (CH 3) 2NH. By transmission electron microscopy it was shown that reticulocytes treated with NH 4Cl or CH 3NH 2 have enlarged intracellular vesicles. The results are considered to support the hypothesis that iron release from transferrin in reticulocytes occurs as a result of protonation of the transferrin within intracellular vesicles. According to this hypothesis, weak bases such as NH 3 and CH 3NH 2 inhibit iron release by neutralizing H + within the vesicles.