Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine.

Rat liver ferritin is an effective donor of iron to rat hepatocytes. Uptake of iron from ferritin by the cells is partially inhibited by including apotransferrin in the culture medium, but not by inclusion of diferric transferrin. This inhibition is dependent on the concentration of apotransferrin, with a 30% depression in iron incorporation in the cells detected at apotransferrin concentrations above 40 micrograms/ml. However, apotransferrin does not interfere with uptake of 125I-labeled ferritin, suggesting that apotransferrin decreases retention of iron taken up from ferritin by hepatocytes by sequestering a portion of released iron before it has entered the metabolic pathway of the cells. The iron chelators desferrioxamine (100 microM), citrate (10 mM) and diethylenetriaminepentaacetate (100 microM) reduce iron uptake by the cells by 35, 25 and 8%, respectively. In contrast, 1 mM ascorbate increases iron accumulation by 20%. At a subtoxic concentration of 100 microM, chloroquine depresses ferritin and iron uptake by hepatocytes by more than 50% after 3 h incubation. Chloroquine presumably acts by retarding lysosomal degradation of ferritin and recycling of ferritin receptors.

Transferrin protein and iron uptake by cultured hepatocytes.

The binding and uptake of 59Fe-loaded 3H-labelled rat transferrin by cultured rat hepatocytes was investigated. At 4 degrees C, there is no evidence for a specific binding of transferrin which could be related to the association of neo-synthesized transferrin with plasma membrane receptors. At 37 degrees C, iron uptake is much more important than transferrin uptake; it proceeds linearly over the time of incubation, is largely proportional to the extracellular transferrin concentration, and is compatible with uptake by fluid phase endocytosis. The difference observed between iron and transferrin uptake implies the existence of a mechanism allowing the reutilization of transferrin after iron delivery.

Interactions between isolated hepatocytes and Kupffer cells in iron metabolism: a possible role for ferritin as an iron carrier protein.

Like the rat peritoneal macrophage, the isolated Kupffer cell is capable of processing and releasing iron acquired by phagocytosis of immunosensitized homologous red blood cells. When erythrophagocytosis is restrained to levels which do not affect cell viability, about one red cell per macrophage, close to 50% of iron acquired from red cells is released within 24 hr in the form of ferritin. Immunoradiometric assay of the extracellular medium indicates that 160 ng ferritin are released by 10(6) Kupffer cells after 24-hr incubation at 37 degrees C. Iron release is temperature-dependent, the rate at 37 degrees C being nearly 5-fold greater than at 4 degrees C. As estimated by sucrose-gradient ultracentrifugation, ferritin released by the erythrophagocytosing Kupffer cell averages 2,400 iron atoms per molecule. When reincubated with isolated hepatocytes, this released ferritin is rapidly taken up by the cells. Via this process, hepatocytes may accumulate more than 160,000 iron atoms per cell per min. Such accumulation is not impeded by the presence of iron-loaded transferrin in the culture medium, but is markedly depressed by rat liver ferritin. In contrast to the conservation of transferrin during its interaction with hepatocytes, the protein shell of the ferritin molecule is rapidly degraded into trichloroacetic acid-soluble fragments. Ferritin-mediated transfer of iron from Kupffer cells to hepatocytes may help explain the resistance of the liver to iron deficiency as well as the liver's susceptibility to iron overload.

Hydroxyl radical formation and iron-binding proteins. Stimulation by the purple acid phosphatases.

The effect of the purple acid phosphatases with binuclear iron centers (uteroferrin and bovine spleen phosphatase) on hydroxyl radical formation by iron-catalyzed Haber-Weiss-Fenton chemistry has been compared to that of lactoferrin and transferrin. Using 5,5-dimethyl-1-pyrroline-1-oxide to detect superoxide and hydroxyl radicals and the xanthine-xanthine oxidase system to generate superoxide and hydrogen peroxide, we have observed by ESR spectroscopy that both phosphatases were able to promote hydroxyl radical formation. Lactoferrin and transferrin were found incapable of giving rise to these reactive species. This can be explained by the fact that lactoferrin and transferrin carry two Fe(III) atoms per molecule, neither of which are readily reduced by biological reductants. In contrast, the phosphatases possess a binuclear iron center in which one of the iron atoms is stabilized in the ferric state, but the other freely undergoes one-electron redox reactions. The redox-active iron may act as a catalyst of the Haber-Weiss-Fenton sequence, thus enabling the reactions generating hydroxyl radical to proceed. The iron complex of diethylenetriamine penta-acetic acid, also redox active, was investigated and found as well to promote Haber-Weiss-Fenton chemistry.

Subcellular localization of ferritin and iron taken up by rat hepatocytes.

The subcellular localization of ferritin and its iron taken up by rat hepatocytes was investigated by sucrose-density-gradient ultracentrifugation of cell homogenates. After incubation of hepatocytes with 125I-labelled [59Fe]ferritin, cells incorporate most of the labels into structures equilibrating at densities where acid phosphatase and cytochrome c oxidase are found, suggesting association of ferritin and its iron with lysosomes or mitochondria. Specific solubilization of lysosomes by digitonin treatment indicates that, after 8 h incubation, most of the 125I is recovered in lysosomes, whereas 59Fe is found in mitochondria as well as in lysosomes. As evidenced by gel chromatography of supernatant fractions, 59Fe accumulates with time in cytosolic ferritin. To account for these results a model is proposed in which ferritin, after being endocytosed by hepatocytes, is degraded in lysosomes, and its iron is released and re-incorporated into cytosolic ferritin and, to a lesser extent, into mitochondria.

Subcellular localization of transferrin protein and iron in the perfused rat liver. Effect of Triton WR 1339, digitonin and temperature.

The subcellular localization of 3H-labelled 59Fe-loaded transferrin accumulated by the liver has been studied by means of cell fractionation techniques. More than 96% of the 59Fe present in the liver of rats perfused with 59Fe-labelled transferrin is recovered in the parenchymal cells. Rat livers were perfused with 10 micrograms/ml 3H-labelled 59Fe-saturated transferrin, homogenized separated in nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and supernatant (S) fractions; M, L and P fractions were further analysed by isopycnic centrifugation in sucrose gradients. 3H label distributes essentially around densities of 1.13-1.14 g/ml overlapping to a large extent with the distribution of galactosyltransferase, the marker enzyme of the Golgi complex. However, after treatment with low concentrations of digitonin the 3H label dissociates from galactosyltransferase and is shifted to higher densities, suggesting an association of transferrin with cholesterol-rich endocytic vesicles which could derive from the plasma membrane. 59Fe is mostly found in the supernatant fraction largely in the form of ferritin, as indicated by its reaction with antiferritin antibodies. In the mitochondrial fraction the density distribution of 59Fe suggests an association with lysosomes and/or mitochondria. In contrast to the lysosomal enzyme cathepsin B, the density distribution of 59Fe was only slightly affected by pretreatment of the rats with Triton WR 1339, suggesting its association with the mitochondria. At 15 degrees C, 59Fe and 3H labels are recovered together in low-density endocytic vesicles. On the basis of our results we suggest that, at low extracellular transferrin concentration, iron uptake by the liver involves endocytosis of the transferrin protein. The complex is interiorized in low-density acidic vesicles where iron is released. The iron passes into the cytosol, where it is incorporated into ferritin and into the mitochondria. The iron-depleted transferrin molecule would then be returned to the extracellular medium during the recycling of the plasma membrane.

Microbial glycolipids: possible virulence factors that scavenge oxygen radicals.

Two important pathogens of developing countries, Mycobacterium leprae, the etiologic agent of leprosy, and Leishmania donovani, the protozoal parasite that causes kalaazar, persist in the human host primarily in mononuclear phagocytes. The mechanisms by which they survive in these otherwise highly cytocidal cells are presently unknown. Since the best understood cytocidal mechanism of these cells is the oxygen-dependent system that provides lethal oxidants including the superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH), and singlet oxygen (1O2), we sought specific microbial products of these organisms that might enable them to elude oxidative cytocidal mechanisms. Phenolic glycolipid I of M. leprae and lipophosphoglycan of L. donovani are unique cell-wall-associated glycolipids produced in large amounts by the organisms. In this study, phenolic glycolipid I derivatives and lipophosphoglycan were examined for their ability to scavenge potentially cytocidal oxygen metabolites in vitro. Electron spin resonance and spin-trapping indicate that phenolic glycolipid I derivatives and lipophosphoglycan are highly effective in scavenging hydroxyl radicals and superoxide anions. The results suggest that complex glycolipids and carbohydrates of intracellular pathogens that can scavenge oxygen radicals may contribute to their pathogenicity and virulence.