The surface of rat hepatocytes can transfer iron from stable chelates to external acceptors.

The chelator diethylenetriaminepentaacetate (DTPA) forms a stable complex with iron that does not donate iron to transferrin under physiological conditions, i.e., pH above 7 and isotonic milieu. It does, however, deliver iron to hepatocytes. This uptake is initiated by a mobilization of the metal from the complex by the cell surface. When an external chelator is added simultaneously, it can bind the iron and inhibit its accumulation by the cells. This is shown here with the impermeant siderophore conjugate hydroxyethyl-starch coupled desferrioxamine, as well as with apotransferrin. We also demonstrate exchange of iron between DTPA and holo-transferrin, or at least movement from the chelator to the protein, which may have lost its iron to the cell in advance, providing new binding sites for mobilized iron. The efficient hepatocyte iron donor lactoferrin greatly stimulates iron uptake from DTPA, apparently by binding iron and transferring it into the cells by endocytosis. Ferritin is unable to do this; therefore, the mobilization of iron is not caused by a reducing activity at the cell surface, because iron is readily transferred from DTPA to ferritin by the reductant ascorbic acid. The transfer process is dependent on the temperature, the time, and the amount of cells present, and is partly inhibited by sulfhydryl reagents. We conclude that this activity represents a hitherto unidentified first step in the movement of iron through the cell membrane and may be relevant for transferrin-bound, as well as for non-transferrin-bound, iron uptake by hepatocytes.

Hepatic uptake of iron by receptor-mediated and receptor-independent mechanisms.

Hepatocytes can accumulate iron from transferrin via receptor- or non-receptor-mediated endocytosis or from non-transferrin iron complexes. The latter is several times more efficient than the transferrin-mediated uptake. Both pathways have some properties in common and mutually influence each other: Whereas on the one hand the non-permeant chelator DTPA as well as a polymer-conjugated desferrioxamine inhibit uptake of iron from transferrin, transferrin (in both forms, diferric or apo) itself inhibits uptake from the Fe(3+)-DTPA complex. At neutral pH, strong stimulation by reductant is observed, as well as inhibition by the prototropic agent chloroquine. This situation is reversed at acidic pH. Weak chelators stimulate uptake of iron from Fe-DTPA in hepatocytes. We conclude that the cell can labilize the chelate complex. The further mechanism of membrane passage is dependent on the environment. All the acquired iron can be found in ferritin.

Uptake of iron by isolated rat hepatocytes from a hydrophilic impermeant ferric chelate, Fe(III)-DTPA.

We studied uptake of iron from Fe(III)-diethylenetriamine pentaacetate (DTPA) in isolated rat hepatocytes. This uptake is specific with an affinity of 600 nM and shows an optimum pH of 6. The specificity is indicated by inhibition by ferric citrate and diferric transferrin. Iron uptake from Fe(III)-DTPA is completely inhibited by trypsinization of the cell surface, by strong impermeant ferric chelators (DTPA, apo-transferrin, polymer-conjugated desferrioxamine), both hexacyanoferrates, copper and zinc, and partly by dipyridyl, manganese, cobalt, N-ethylmaleimide, and citrate. The lysosomotropic agent chloroquin inhibits weakly; proton pump inhibitors are without effect. Ascorbate and Tiron both effectively stimulate the uptake and also mobilize iron from DTPA in vitro. Approximately 75% of the freshly acquired intracellular iron is found in ferritin even after uptake at lowered temperature (16 degrees C). We conclude that a rate-limiting mobilization of iron from the DTPA chelate by a cell-surface activity is required before iron can actually enter the cell. This can be enhanced by mediators of iron release, but does not seem to require reduction of iron. The use of DTPA as chelator offers the possibility of studying this putative activity because the Fe(III)-DTPA chelate is stable in the presence of transferrin or desferroxamine, in contrast to ferric citrate or Fe(NTA)2.

Non-transferrin iron uptake by HeLa cells cultured in serum-free media with different iron sources.

HeLa cells cultured in defined serum-free media supplied with iron wither in the form of diferric transferrin (transferrin-dependent cells), ferric citrate at 500 micromol/l (high-iron dependent cells) or ferric citrate at 5 micromol/l (low-iron dependent cells) accumulate iron from ferric citrate in different ways. The uptake rate in transferrin-dependent cells is always much lower in the other two lines. In all three, the uptake rate rises almost linearly with the concentration of iron up to 10 micromol/l. In high-iron dependent cells, the uptake of radiolabelled iron is suppressed by a 100-fold excess of the iron complex, whereas this same excess stimulates iron uptake in the other two lines. The same concentrations of pure citrate completely inhibit iron uptake by all three types of cell. Only high-iron dependent cells take up citrate at measurable and reproducible rates. These rates are independent on the presence of iron, and the uptake is inhibited by an unlabelled surplus. The pH-dependence of iron uptake in high-iron dependent cells is also different from that of the other cells. Low-iron dependent cells transferred to medium containing 500 micromol/l iron show increased uptake rates within 3 to 7 h, and after overnight maintenance in this medium they acquire the uptake characteristics of high-iron dependent cells. The special characteristics of iron uptake by high-iron dependent cells are paralleled by low binding activity of iron-regulatory protein to iron-responsive elements of RNA. We conclude that low-iron dependent cells maintain their iron supply from the culture medium by unspecific uptake of oligomeric complexes, while cells in media with a high content of low-molecular weight iron induce a specific uptake system which might have a protective function.

Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor and transport to endosomes.

LDL receptor related protein (LRP) is a ubiquitously expressed cell surface receptor that binds, at least in vitro, a plethora of ligands among them alpha 2-macroglobulin and lactoferrin (Lf). The function of LRP in internalisation and distribution of ligands within cellular metabolism is still unclear. We here investigated by combined ligand- and immunoblotting the participation of LRP/alpha 2MR and its associated protein (RAP) in receptor mediated endocytosis of Lf into rat liver. We found LRP highly enriched in sucrose density gradient fractions around density 1.10 g/ml, previously characterised as endosomal fractions. RAP was concentrated in distinct fractions around density 1.14 g/ml. This separation of RAP from LRP/alpha 2MR is physiologically meaningful as RAP avidly binds to LRP/alpha 2MR and by that shuts off all ligand binding function. In endosomal fractions we found one single binding protein for 125I-labelled Lf. With a specific anti LRP/alpha 2MR antibody and ligand blotting with 125I-labelled RAP this endosomal Lf binding site was verified to be LRP/alpha 2MR. Endosomes did not bind labelled Lf when prepared from rats that received an intravenous injection of Lf (20 mg per animal) 20 min prior to preparation. Surprisingly we immunodetected Lf in these endosomes at a position around 600 kDa, comigrating with LRP/alpha 2MR. We determined Lf binding to be optimal at pH 5.8, what led us to suggest the existence of a very stable LF-LRP/alpha 2MR complex in endosomes. These data support the idea of effective binding of Lf at pH as found in inflamed tissue environment where Lf is reported to be involved in leukocyte mediated inflammation regulation.

Biochemical aspects of iron metabolism, transport and regulation.

Iron metabolism in man is controlled by homeostatic mechanisms mainly based on intracellular regulation of iron uptake, utilisation and storage. Despite impressive accumulation of knowledge in recent years, the question how iron actually passes cellular membranes and how this transport process is regulated remains largely unanswered. Various models and hypotheses are discussed in context with other well established features of iron homeostasis.

NAD(P)H:ferric iron reductase in endosomal membranes from rat liver.

Endosomes isolated from rat liver, characterized by high enrichment of endocytosed ligand after liver perfusion, displayed ferric reductase activity with higher affinity for NADH (1.7 microM) than for NADPH (7.1 microM). The ferric-NTA complex was reduced by NADH with a molar stoichiometry of 2:1 for the iron complex to pyridine nucleotide ratio under near anaerobic conditions. Superoxide radicals were not apparently involved in the reduction of ferric iron under these conditions, despite measurable generation of superoxide under aerobic atmosphere. The reaction was inhibited by sulfhydryl reagents, was heat labile, and may account for reduction of ferric to ferrous iron during hepatic iron uptake from transferrin or from other iron sources.