No abstract

The effect of different drugs which affect lysosomal function (chloroquine, methylamine) and endocytosis (colchicine) or of the iron chelators desferrioxamine B, rhodotorulic acid, bipyridyl, 2,3-dihydroxybenzoic acid, isoniazid pyridoxal hydrazone on the uptake of 59Fe-loaded 3H-labelled transferrin by cultured rat embryo fibroblasts has been studied. All these substances, except isoniazid pyridoxal hydrazone reduce the uptake of iron by fibroblasts, without any significant effect on the uptake of [3H]transferrin, nor of its specific binding to the cells. Cell fractionation experiments indicate that in the presence of methylamine and chloroquine, most of the cell associated 3H label is no longer transferred into lysosomes and remains associated with subcellular compartments which have a 5'-nucleotidase activity (a marker enzyme of plasma membrane and related structures such as endocytic vesicles).Short-term kinetic experiments with fibroblasts incubated for 1 min at 37 "C with doubly labelled transferrin show that on reincubation in fresh medium almost all of the 59Fe initially bound remains associated with the cells whereas the 3H-labelled transferrin is released with a tljz of about 3.5 min. In the presence of desferrioxamine B both "Fe and transferrin, the former in part as ferrioxamine, are released from the cells with the same kinetics as is observed for transferrin in the control experiments. Similar studies with cells preincubated for 16 h with chloroquine or methylamine indicate that the release of transferrin from the cells is slowed down considerably. Chloroquine-treated cells also release large amounts of 59Fe.These results are consistent with the model for iron release from transferrin proposed previously [I] involving receptor mediated endocytosis of transferrin followed by release of iron during a rapid passage of the transferrinreceptor complex in lysosomes and recycling of the complex to the plasma membrane where the iron-depleted transferrin is released. The inhibition of iron uptake by iron chelators such as desferrioxamine is consistent with complexation of iron by the chelator subsequent to its release from transferrin inside the cells and transfer of the iron-chelate into the extracellular medium.