Chelator-mediated iron efflux from reticulocytes

The mechanism by which bipyridine and phenanthroline types of iron chelator inhibit iron uptake from transferrin and iron efflux mediated by pyridoxal isonicotinoyl hydrazone was investigated using rabbit reticulocytes with the aim of providing more information on the normal process of iron uptake by developing erythroid cells. It was shown that the chelators block cellular uptake by chelating the iron immediately after release from transferrin while it is still in the membrane fraction of the cells. The iron-chelator is then released from the cells by a process which is very similar to that of transferrin release with respect to kinetics and sensitivity to incubation temperature and the effects of metabolic inhibitors and other chemical reagents. These results are compatible with the conclusion that both transferrin and the iron-chelators in the cells are mainly present in endocytotic vesicles and are released from the cells by exocytosis. The chelators were also shown to block the pyridoxal isonicotinoyl hydrazone-mediated efflux of iron from cells which had taken up iron in the presence of isoniazid, an inhibitor of haem synthesis, by chelating the iron in the cytosol and the mitochondria. In this case, the iron-chelator complexes were not released from the cells. Measurement of the diethyl ether/water partition coefficients of bipyridine and 1,10-phenanthroline and their iron complexes gave much higher values for the free chelators, supporting the concept that the chelators trap the iron intracellularly because of differences in the lipid solubility and, hence, membrane permeability to the free chelators and their iron complexes.     

Membrane transport of non-transferrin-bound iron by reticulocytes

The transport of non-transferrin-bound iron into rabbit reticulocytes was investigated by incubating the cells in 0.27 M sucrose with iron labelled with 59Fe. In most experiments the iron was maintained in the reduced state, Fe(II), with mercaptoethanol. The iron was taken up by cytosolic, haem and stromal fractions of the cells in greater amounts than transferrin-iron. The uptake was saturable, with a Km value of approx. 0.2 microM and was competitively inhibited by Co2+, Mn2+, Ni2+ and Zn2+. It ceased when the reticulocytes matured into erythrocytes. The uptake was pH and temperature sensitive, the pH optimum being 6.5 and the activation energy for iron transport into the cytosol being approx. 80 kJ/mol. Ferric iron and Fe(II) prepared in the absence of reducing agents could also be transported into the cytosol. Sodium chloride inhibited Fe(II) uptake in a non-competitive manner. Similar degrees of inhibition was found with other salts, suggesting that this effect was due to the ionic strength of the solution. Iron chelators inhibited Fe(II) uptake by the reticulocytes, but varied in their ability to release 59Fe from the cells after it had been taken up. Several lines of evidence showed that the uptake of Fe(II) was not being mediated by transferrin. It is concluded that the reticulocyte can transport non-transferrin-bound iron into the cytosol by a carrier-mediated process and the question is raised whether the same carrier is utilized by transferrin-iron after its release from the protein.     

Failure of rabbit reticulocytes to incorporate conalbumin or lactoferrin iron.

Despite the remarkable molecular similarity of human lactoferrin and human transferrin, the results of this investigation indicate that human lactoferrin was unable to furnish rabbit reticulocytes with iron for heme synthesis. Although conalbumin closely resembles transferrin in many of its properties, conalbumin iron-binding differs from human transferrin iron-binding. There are conflicting reports in the literature regarding conalbumin's ability to furnish iron to reticulocytes. In this study, small amounts of lactoferrin or conalbumin were adsorbed to mature and immature cell surfaces but neither of these iron-binding proteins surrendered iron intracellularly to reticulocytes for heme synthesis.     

Transferrin and iron uptake by rat reticulocytes

The uptake of transferrin labeled with 3H and 59Fe by rat reticulocytes was studied to clarify the characteristics of the uptake process and intracellular transport. Rat reticulocytes took up transferrin in a saturable, time- and temperature-dependent manner. Scatchard analysis of the binding parameters indicated that transferrin molecules were bound to cell-surface receptors with high affinity. Monodansyl- cadaverine, a potent inhibitor of transglutaminase, reduced the amount of internalized transferrin but has no effect on the total amount of cell-associated transferrin, suggesting that transferrin is taken up by rat reticulocytes via receptor-mediated endocytosis. About 50% of the internalized 3H label was released from the cells after reincubation for 1 h in fresh medium. In contrast, no release of 59Fe label was observed. By immunoprecipitation and subsequent SDS-PAGE the released 3H-labeled product was identified as apotransferrin. Lysosomotropic reagents and a proton ionophore reduced the uptake of 59Fe. These results indicated that iron was removed from transferrin at an intracellular site in an acidic environment. The released iron was found not to associate with any intermediate ligands before it was utilized for heme synthesis in mitochondria.     

Removal of iron from diferric rabbit serum transferrin by rabbit reticulocytes.

When radioiron-labelled transferrin with 55Fe located predominantly in the N-terminal iron-binding site and 59Fe predominantly in the C-terminal iron-binding site was incubated with rabbit reticulocytes, both radioisotopes of iron were removed at similar rates. Electrophoresis of transferrin samples taken during the course of an incubation, in polyacrylamide gels containing 6 M-urea, showed that iron was removed in a pairwise fashion, giving rise to iron-free transferrin.     

Binding sites of iron transferrin on rat reticulocytes. Inhibition by specific antibodies

Rat ventral prostate contains an acidic protein which can bind spermine selectively. The relative binding affinities of various aliphatic amines for the protein are, in decreasing order, spermine greater than thermine greater than greater than putrecine greater than 1,10-diaminodecane, cadaverine and 1,12-diaminododecane. The binding protein has an isoelectric point at pH 4.3 and a sedimentation coefficient of 3 S. Its molecular weight is approx. 30 000. Histones and nuclear chromatin preparations of the prostate can interact with the binding protein. The spermine-binding activity of the purified prostate protein can be inactivated by treatment with intestinal alkaline phosphatases. The phosphatase treated preparation can then be reactivated by beef heart protein kinase in the presence of cyclic AMP and ATP. The spermine-binding activity of the prostate cytosol protein fraction decreases after castration, but increases very rapidly after the castrated rats are injected with 5alpha-dihydrotestosterone. This finding raises the possibility that, in the postate, certain androgen actions may be dependent on the androgen-induced increase in the acidic protein binding of polyamines and their translocation to a functional cellular site such as nuclear chromatin. In the prostate cytosol, spermine also binds to 4-S tRNAs and to a unique RNA which has a sedimentation coefficient of 1.5 S.     

Amines as inhibitors of iron transport in rabbit reticulocytes

The effect of the known inhibitors of iron uptake, n-butylamine and NH4Cl, was examined at the molecular level to more precisely define the mechanisms by which these lysosomotropic agents block iron uptake by rabbit reticulocytes. Utilizing a rapid pulse-chase technique to follow the handling of a cohort of 59Fe, 125I-transferrin bound to rabbit reticulocytes, both amines were observed to have no effect on the cell-mediated release of 59Fe from internalized transferrin. The results indicated, however, that both agents acted to 1) retard the internalization of transferrin bound to transferrin receptors on the plasma membrane of reticulocytes, 2) retard the externalization of internalized transferrin, and 3) block the transport into the cytosol of iron released from transferrin.     

Low-Mr iron isolated from guinea pig reticulocytes as AMP-Fe and ATP-Fe complexes.

Guinea pig reticulocytes were pulse-labelled with 59Fe bound to transferrin. Haemolysates prepared from these reticulocytes were subjected to rapid (NH1)2SO1 precipitation and then chromatography on an anion-exchange resin. ATP-bound 59Fe was the dominant species in the reticulocyte cytosol; 2,3-bisphosphoglycerate and GTP iron complexes were not detected despite the fact that these were stable with (NH1)2SO1 precipitation and readily detected with anion-exchange chromatography. AMP-bound Fe was a minor component of the cytosol following rapid (NH1)2SO4 precipitation, and the major component when iron was released from transferrin by haemolysates. We speculate that ATP-Fe may be degraded in the cell to permit utilization of its iron for haem synthesis.     

Kinase cascades regulating entry into apoptosis.

All cells are constantly exposed to conflicting environment cues that signal cell survival or cell death. Survival signals are delivered by autocrine or paracrine factors that actively suppress a default death pathway. In addition to survival factor withdrawal, cell death can be triggered by environmental stresses such as heat, UV light, and hyperosmolarity or by dedicated death receptors (e.g., FAS/APO-1 and tumor necrosis factor [TNF] receptors) that are counterparts of growth factor or survival receptors at the cell surface. One of the ways that cells integrate conflicting exogenous stimuli is by phosphorylation (or dephosphorylation) of cellular constituents by interacting cascades of serine/threonine and tyrosine protein kinases (and phosphatases). Survival factors (e.g., growth factors and mitogens) activate receptor tyrosine kinases and selected mitogen-activated, cyclin-dependent, lipid-activated, nucleic acid-dependent, and cyclic AMP-dependent kinases to promote cell survival and proliferation, whereas environmental stress (or death factors such as FAS/APO-1 ligand and TNF-alpha) activates different members of these kinase families to inhibit cell growth and, under some circumstances, promote apoptotic cell death. Because individual kinase cascades can interact with one another, they are able to integrate conflicting exogenous stimuli and provide a link between cell surface receptors and the biochemical pathways leading to cell proliferation or cell death.     

Kinetics of iron passage through subcellular compartments of rabbit reticulocytes

Summary The kinetics of the separate processes of Fe₂(III)-transferrin binding to the transferrin receptor, transferrin-receptor internalization, iron dissociation from transferrin, iron passage through the membrane, and iron mobilization into the cytoplasm were studied by pulse-chase experiments using rabbit reticulocytes and⁵⁹Fe,¹²⁵I-labeled rabbit transferrin. The binding of⁵⁹Fe-transferrin to transferrin receptors was rapid with an apparent rate constant of 2×10⁵ m ^–1 sec^–1. The rate of internalization of⁵⁹Fe-transferrin was directly measured at 520±100 molecules of Fe₂(III)-transferrin internalized/sec/cell with 250±43 sec needed to internalize the entire complement of reticulocyte transferrin receptors. Subsequent to Fe₂(III)-transferrin internalization the flux of⁵⁹Fe was followed through three compartments: internalized transferrin, membrane, and cytosol.A process preceding iron dissociation from transferrin and a reaction involving membrane-associated iron required 17±2 sec and 34±5 sec, respectively. Apparent rate constants of 0.0075±0.002 sec^–1 and 0.0343±0.0118 sec^–1 were obtained for iron dissociation from transferrin and iron mobilization into the cytosol, respectively. Iron dissociation from transferrin is the rate-limiting step. An apparent rate constant of 0.0112±0.0025 sec^–1 was obtained for processes involving iron transport through the membrane although at least two reactions are likely to be involved. Based on mechanistic considerations, iron transport through the membrane may be attributed to an iron reduction step followed by a translocation step. These data indicate that the uptake of iron in reticulocytes is a sequential process, with steps after the internalization of Fe₂(III)-transferrin that are distinct from the handling of transferrin.     

Failure of a cell-free system from rabbit reticulocytes to remove iron from transferrin.

1. Cell-free solutions prepared from rabbit reticulocytes were not able to release iron from rabbit transferrin. 2. The results, which differ from those obtained with Rana catesbeiana immature erythrocytes, indicate that cellular integrity is a requirement for this process in the rabbit reticulocyte system.     

Preferential utilization in vitro of iron bound to diferric transferrin by rabbit reticulocytes.

59Fe uptake by rabbit reticulocytes from human transferrin-bound iron was studied by using transferrin solutions (35, 50, 65, 80 and 100% saturated with iron) whose only common characteristic was their content of diferric transferrin. During the early incubation period, 59Fe uptake from each preparation by reticulocytes was identical despite wide variations in amounts of total transferrin, total iron, monoferric transferrin and apotransferrin in solution. During the later phase of incubation, rate of uptake declined and was proportional to each solution's monoferric transferrin content. Uptake was also studied in a comparative experiment which used two identical, partially saturated transferrin preparations, one uniformly 59Fe-labelled and the other tracer-labelled with [59Fe]diferric transferrin. In both experiments, iron uptake by reticulocytes corresponded to utilization of a ferric ion from diferric transferrin before utilization of iron from monoferric transferrin.     

Effect of metabolic inhibitors on uptake of non-transferrin-bound iron by reticulocytes

The relationship between transferrin-free iron uptake and cellular metabolism was investigated using rabbit reticulocytes in which energy metabolism was altered by incubation with metabolic inhibitors (antimycin A, 2,4-dinitrophenol, NaCN, NaN3 and rotenone) or substrates. Measurements were made of cellular ATP concentration and the rate of uptake of Fe(II) from a sucrose solution buffered at pH 6.5. There was a highly significant correlation between the rate of iron uptake into cytosolic and stromal fractions of the cells and ATP levels. Iron transport into the cytosol showed saturation kinetics. The metabolic inhibitors all reduced the Vmax but had no effect on the Km values for this process. It is concluded that the uptake of transferrin-free iron by reticulocytes is dependent on the cellular concentration of ATP and that it crosses the cell membrane by an active, carrier-mediated transport process. Additional studies were performed using transferrin-bound iron. The metabolic inhibitors also reduced the uptake of this form of iron but the inhibition could be accounted for entirely by reduction in the rate of transferrin endocytosis.     

Bidirectional entry of poliovirus into polarized epithelial cells.

The interactions of viruses with polarized epithelial cells are of some significance to the pathogenesis of disease because these cell types comprise the primary barrier to many virus infections and also serve as the sites for virus release from the host. Poliovirus-epithelial cell interactions are of particular interest since this virus is an important enteric pathogen and the host cell receptor has been identified. In this study, poliovirus was observed to adsorb to both the apical and basolateral surfaces of polarized monkey kidney (Vero C1008) and human intestinal (Caco-2) epithelial cells but exhibited preferential binding to the basolateral surfaces of both cell types. Localization of the poliovirus receptor by a receptor-specific monoclonal antibody (D171) revealed a similar distribution predominantly on basolateral membranes, and treatment of cells with antibody D171 inhibited virus adsorption to both membrane surfaces. Poliovirus was able to initiate infection with similar efficiency following adsorption to either surface, and infection was blocked at both surfaces by D171, indicating that functional receptor molecules are expressed on both surfaces at sufficient density to mediate efficient infection at the apical and basolateral plasma membranes. Poliovirus infection resulted in a decrease in transepithelial resistance which was inhibited by prior treatment with monoclonal antibody D171 and occurred prior to other visible cytopathic effects. These results have interesting implications for viral pathogenesis in the human gut.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

Studies on the mechanism of transferrin iron uptake by rat reticulocytes

Mechanism of transferrin iron uptake by rat reticulocytes was studied using ⁵⁹Fe- and ¹²⁵I-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound ⁵⁹Fe was located in the cytoplasmic fraction, only 25–30% of ¹²⁵I-labelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10–15% of the cytoplasmic ¹²⁵I-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound ¹²⁵I-labelled transferrin to be inside the reticulocytes. Both the electron microscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharose-linked transferrin was found to be capable of delivering ⁵⁹Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.