Effects of Iron Chelators,Iron Salts,and Iron Oxide Nanoparticles on the Proliferation and the Iron Content of Oligodendroglial OLN-93 Cells

The oligodendroglial cell line OLN-93 was used as model system to investigate the consequences of iron deprivation or iron excess on cell proliferation. Presence of ferric or ferrous iron chelators inhibited the proliferation of OLN-93 cells in a time and concentration dependent manner, while the application of a molar excess of ferric ammonium citrate (FAC) prevented the inhibition of proliferation by the chelator deferoxamine. Proliferation of OLN-93 cells was not affected by incubation with 300 μM iron that was applied in the form of FAC, FeCl₂, ferrous ammonium sulfate or iron oxide nanoparticles, although the cells efficiently accumulated iron during exposure to each of these iron sources. The highest specific iron content was observed for cells that were exposed to the nanoparticles. These data demonstrate that the proliferation of OLN-93 cells depends strongly on the availability of iron and that these cells efficiently accumulate iron from various extracellular iron sources.     

Formaldehyde induces rapid glutathione export from viable oligodendroglial OLN-93 cells

Formaldehyde is a neurotoxic environmental pollutant that can also be produced in the body by certain enzymatic reactions. To test for the potential consequences of an exposure of oligodendrocytes to formaldehyde, we used OLN-93 cells as a model system. Treatment with formaldehyde altered the cellular glutathione (GSH) content of these cells by inducing a rapid time- and concentration-dependent export of GSH. Half-maximal effects were observed for a formaldehyde concentration of about 0.2 mM. While the basal GSH efflux from OLN-93 cells was negligible even when the cellular GSH content was doubled by pre-incubation of the cells with cadmium chloride, the formaldehyde-stimulated export increased almost proportionally to the cellular GSH content. In addition, the stimulated GSH export required the presence of formaldehyde and was almost completely abolished after removal of the aldehyde. Analysis of kinetic parameters of the formaldehyde-induced GSH export revealed similar K_m and V_max values of around 100 nmol/mg and 40 nmol/(h mg), respectively, for both OLN-93 cells and cultured astrocytes. The transporter responsible for the formaldehyde-induced GSH export from OLN-93 cells is most likely the multidrug resistance protein 1 (Mrp1), since this transporter is expressed in these cells and since the inhibitor MK571 completely prevented the formaldehyde-induced GSH export. The rapid export of GSH from formaldehyde-treated viable oligodendroglial cells is likely to compromise the cellular antioxidative and detoxification potential which may contribute to the known neurotoxicity of formaldehyde.     

Kynurenic Acid Induces Impairment of Oligodendrocyte Viability: On the Role of Glutamatergic Mechanisms

Kynurenic acid (KYNA) is an end stage product of tryptophan metabolism with a variety of functions in the human body, both in the central nervous system (CNS) and in other organs. Although its activity in the human brain has been widely studied and effects on neural cells were emphasized, the effect of KYNA on oligodendroglial cells remains unknown. Present study aims at describing the activity of high concentration of KYNA in OLN-93 cells. The inhibition of OLN-93 oligodendrocytes viability by KYNA in a medium with reduced serum concentration has been demonstrated. Although decreased metabolic activity of KYNA treated OLN-93 cells was shown, the cells proliferation was not altered. KYNA treatment did not alter morphology as well as expression level of cell cycle and proliferation regulating proteins. Furthermore, glutamate receptor antagonists and agonists did not alter the inhibitory effect of KYNA on viability of OLN-93 oligodendrocytes. This study contributes to the elucidation of effects of KYNA on oligodendrocytes in vitro, yet further analyses are necessary to explain the mechanisms behind the damage and loss of myelin sheaths.     

Formaldehyde induces rapid glutathione export from viable oligodendroglial OLN-93 cells

Formaldehyde is a neurotoxic environmental pollutant that can also be produced in the body by certain enzymatic reactions. To test for the potential consequences of an exposure of oligodendrocytes to formaldehyde, we used OLN-93 cells as a model system. Treatment with formaldehyde altered the cellular glutathione (GSH) content of these cells by inducing a rapid time- and concentration-dependent export of GSH. Half-maximal effects were observed for a formaldehyde concentration of about 0.2 mM. While the basal GSH efflux from OLN-93 cells was negligible even when the cellular GSH content was doubled by pre-incubation of the cells with cadmium chloride, the formaldehyde-stimulated export increased almost proportionally to the cellular GSH content. In addition, the stimulated GSH export required the presence of formaldehyde and was almost completely abolished after removal of the aldehyde. Analysis of kinetic parameters of the formaldehyde-induced GSH export revealed similar K_m and V_max values of around 100 nmol/mg and 40 nmol/(h mg), respectively, for both OLN-93 cells and cultured astrocytes. The transporter responsible for the formaldehyde-induced GSH export from OLN-93 cells is most likely the multidrug resistance protein 1 (Mrp1), since this transporter is expressed in these cells and since the inhibitor MK571 completely prevented the formaldehyde-induced GSH export. The rapid export of GSH from formaldehyde-treated viable oligodendroglial cells is likely to compromise the cellular antioxidative and detoxification potential which may contribute to the known neurotoxicity of formaldehyde.     

Monitoring of the Cytoskeleton-Dependent Intracellular Trafficking of Fluorescent Iron Oxide Nanoparticles by Nanoparticle Pulse-Chase Experiments in C6 Glioma Cells

Iron oxide nanoparticles (IONPs) are used for various biomedical and therapeutic approaches. To investigate the uptake and the intracellular trafficking of IONPs in neural cells we have performed nanoparticle pulse-chase experiments to visualize the internalization and the fate of fluorescent IONPs in C6 glioma cells and astrocyte cultures. Already a short exposure to IONPs for 10 min at 4 °C (nanoparticle pulse) allowed binding of substantial amounts of nanoparticles to the cells, while internalization of IONPs into the cell was prevented. The uptake of bound IONPs and the intracellular trafficking was started by increasing the temperature to 37 °C (chase period). While hardly any cellular fluorescence nor any iron staining was detectable directly after the nanoparticle pulse, dotted cellular fluorescence and iron patterns appeared already within a few minutes after start of the chase incubation and became intensified in the perinuclear region during further incubation for up to 90 min. Longer chase incubations resulted in separation of the fluorescent coat from the core of the internalized IONPs. Disruption of actin filaments in C6 cells strongly impaired the internalization of IONPs, whereas destabilization of microtubules traped IONP-containing vesicles to the plasma membrane. In conclusion, nanoparticle pulse-chase experiments allowed to synchronize the cellular uptake of fluorescent IONPs and to identify for C6 cells an actin-dependent early and a microtubule-dependent later process in the intracellular trafficking of fluorescent IONPs.     

Collapsin response mediator protein-2 phosphorylation promotes the reversible retraction of oligodendrocyte processes in response to non-lethal oxidative stress

The extension of processes of oligodendrocyte (OLG) and their precursor cells are crucial for migration, axonal contact and myelination. Here we show that a non-lethal oxidative stress induced by 3-nitropropionic acid (3-NP) elicited a rapid shortening of processes (~24%) in primary OLGs and in oligodendroglial cell line (OLN-93) cells (~36%) as compared with vehicle-exposed cells. This was reversible and prevented by antioxidants. Proteomics of OLG lysates with and without 3-NP treatment yielded collapsin response mediator protein 2 (CRMP-2) as a candidate effector molecule. Inhibition of rho kinase was sufficient to prevent process retraction in both OLGs and OLN-93 cells. Oxidative stress increased phosphorylation of CRMP-2 at T555 that was completely prevented by Y27632. Moreover, transfection of OLN-93 cells with the mutant CRMP-2 T555A which cannot be phosphorylated by rho kinase, prevented process shortening induced by 3-NP as compared with wild-type CRMP-2. Our results suggest a role for endogenous reactive oxygen species in a pathway that regulates OLG process extension. The vulnerability of late myelinated neurons in the adult brain and the presence of white matter pathology in human dementias warrant the study of this oligodendroglial pathway in the early stages of neurodegenerative conditions characterized by oxidative stress.     

Demonstration of Kynurenine Aminotransferases I and II and Characterization of Kynurenic Acid Synthesis in Oligodendrocyte Cell Line (OLN-93)

In the present study we demonstrate for the first time that both kynurenine aminotransferase (KAT) isoforms I and II are present in the permanent immature rat oligodendrocytes cell line (OLN-93). Moreover, we provide evidence that OLN-93 cells are able to synthesize kynurenic acid (KYNA) from exogenously added l-kynurenine and we characterize its regulation by extrinsic factors. KYNA production in OLN-93 cells was depressed in the presence of aminotransferase inhibitor, aminooxyacetic acid and was not affected by depolarizing agents such as 50 mM K⁺ and 4-aminopyridine. Glutamate agonists, l-glutamate and d,l-homocysteine significantly decreased KYNA production. Selective agonist of ionotropic glutamate receptors Amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropionic acid (AMPA) lowered KYNA production in OLN-93 cell line, whereas N-methyl-d-aspartate (NMDA) had no influence on KYNA production. Furthermore, KYNA synthesis in OLN-93 cells was decreased in a concentration-dependent manner by amino acids transported by l-system, l-leucine, l-cysteine and l-tryptophan. The role of KYNA synthesis in oligodendrocytes needs further investigation.     

Comparison of Primary and Secondary Rat Astrocyte Cultures Regarding Glucose and Glutathione Metabolism and the Accumulation of Iron Oxide Nanoparticles

Astrocyte-rich primary cultures (APCs) are frequently used as a model system for the investigation of properties of brain astrocytes. However, as APCs contain a substantial number of microglial and oligodendroglial cells, biochemical parameters determined for such cultures may at least in part reflect also the presence of the contaminating cell types. To lower the potential contributions of microglial and oligodendroglial cells on properties of the astrocytes in APCs we prepared rat astrocyte-rich secondary cultures (ASCs) by subculturing of APCs and compared these ASCs with APCs regarding basal metabolic parameters, specific enzyme activities and the accumulation of iron oxide nanoparticles. Immunocytochemical characterization revealed that ASCs contained only minute amounts of microglial and oligodendroglial cells. ASCs and APCs did not significantly differ in their specific glucose consumption and lactate production rates, in their specific iron and glutathione contents, in their specific activities of various enzymes involved in glucose and glutathione metabolism nor in their accumulation of iron oxide nanoparticles. Thus, the absence or presence of some contaminating microglial and oligodendroglial cells appears not to substantially modulate the investigated metabolic parameters of astrocyte cultures.     

Effect of γFe2O3 nanoparticles on photosynthetic characteristic of soybean (Glycine max (L.) Merr.): foliar spray versus soil amendment

There is an urgent need to address comprehensive biosafety issues associated with the use of Fe₂O₃ nanoparticles (IONPs). The present study was performed to investigate the effect of 6-nm IONPs and citrate-coated IONPs (IONPs-Cit) on photosynthetic characteristics and root elongation during germination of Glycine max (L) Merr. Plant physiological performance was assessed after foliar and soil IONPs fertilization. No adverse impacts at any growth stage of the soybeans were observed after application of IONPs. The Fe₂O₃ nanoparticles produced a significant positive effect on root elongation, particularly when compared to the bulk counterpart (IOBKs) suspensions of concentrations greater than 500 mg L^?1. Furthermore, IONPs-Cit significantly enhanced photosynthetic parameters when sprayed foliarly at the eight-trifoliate leaf stage (P < 0.05). The increases in photosynthetic rates following spraying were attributed to increases in stomatal opening rather than increased CO₂ uptake activity at the chloroplast level. We observed more pronounced positive effects of IONPs via foliar application than by soil treatment. This study concluded that IONPs coated with citric acid at IONPs to citrate molar ratio of 1:3 can markedly improve the effectiveness of insoluble iron oxide for Fe foliar fertilization.     

Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms

The only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are targeted to distinct subcellular locations, which is governed by the presence or absence of exon-II, although their functional expression is often less clear. Here, we investigated the role of exon-II-containing MBP isoforms and their link with cell proliferation. Live-cell imaging and FRAP analysis revealed a dynamic nucleocytoplasmic translocation of the exon-II-containing postnatal 21.5-kDa MBP isoform upon mitogenic modulation. Its nuclear export was blocked upon treatment with leptomycin B, an inhibitor of nuclear protein export. Next to the postnatal MBP isoforms, embryonic exon-II-containing MBP (e-MBP) is expressed in primary (immature) oligodendrocytes. The e-MBP isoform is exclusively present in OLN-93 cells, a rat-derived oligodendrocyte progenitor cell line, and interestingly, also in several non-CNS cell lines. As seen for postnatal MBPs, a similar nucleocytoplasmic translocation upon mitogenic modulation was observed for e-MBP. Thus, upon serum deprivation, e-MBP was excluded from the nucleus, whereas re-addition of serum re-established its nuclear localization, with a concomitant increase in proliferation. Knockdown of MBP by shRNA confirmed a role for e-MBP in OLN-93 proliferation, whereas the absence of e-MBP similarly reduced the proliferative capacity of non-CNS cell lines. Thus, exon-II-containing MBP isoforms may regulate cell proliferation via a mechanism that relies on their dynamic nuclear import and export, which is not restricted to the oligodendrocyte lineage.     

The participation of nitric oxide in cell free- and its restriction of macrophage-mediated oxidation of low-density lipoprotein

The potential role of nitric oxide radical (NO ·) in macrophage-mediated oxidation and conversion of human low density lipoprotein (LDL) to a high-uptake form was examined by exposing LDL to aerobic solutions to either NO · or 3-morpholinosydnonimine-hydrochloride (SIN-1, a compound that spontaneously forms NO · and superoxide anion radical) or to mouse peritoneal macrophages in the presence and absence of modulators of cellular NO · synthesis. Incubation with NO · alone caused oxidation of LDL's ubiquinol-10 and accumulation of small amounts of lipid hydroperoxidases, but failed to form any high-uptake ligand for endocytosis by macrophages and did not alter the LDL particle charge or the integrity of apoB. Exposure of LDL to SIN-1 resulted in complete consumption of all antioxidants and substantial formation of lipid hydroperoxidases, but again had little effect on the lipoprotein particle charge or generation of high-uptake form. Preincubation of macrophages with interferon-γ increased the cells ability to generate reactive nitrogen metabolites. The extent of cell-mediated oxidation of LDL and the generation of high-uptake LDL was substantial in resident cells in which NO · synthesis was barely detectable, depressed in cells active in NO · synthesis and restored when NO · synthesis was suppressed by the arginine analogue, NMMA. These results suggest that, while longer with superoxide anion radical, NO · can oxidize LDL, its synthesis is not required for macrophage-mediated oxidation of LDL in vitro; rather it exerts a protective role in preventing oxidative LDL modification by macrophages.     

Defective calmodulin‐dependent rapid apical endocytosis in zebrafish sensory hair cell mutants

Vertebrate mechanosensory hair cells contain a narrow “pericuticular” zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1‐43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 424–434, 1999     

The effects of an antibody to the rat transferrin receptor and of rat serum albumin on the uptake of diferric transferrin by rat hepatocytes

The role of high-affinity specific transferrin receptors and low-affinity, non-saturable processes in the uptake of transferrin and iron by hepatocytes was investigated using fetal and adult rat hepatocytes in primary monolayer culture, rat transferrin, rat serum albumin and a rabbit anti-rat transferrin receptor antibody. The intracellular uptake of transferrin and iron occurred by saturable and non-saturable mechanisms. Treatment of the cells with the antibody almost completely eliminated the saturable uptake of iron but had little effect on the non-saturable process. Addition of albumin to the incubation medium reduced the endocytosis of transferrin by the cells but had no significant effect on the intracellular accumulation of iron. The maximum effect of rat serum albumin was observed at concentrations of 3 mg/ml and above. At a low incubation concentration of transferrin (0.5 microM), the presence of both rat albumin and the antibody decreased the rate of iron uptake by the cells to about 15% of the value found in their absence, but to only 40% when the diferric transferrin concentration was 5 microM. These results confirm that the uptake of transferrin-bound iron by both fetal and adult rat hepatocytes in culture occurs by a specific, receptor-mediated process and a low-affinity, non-saturable process. The low-affinity process increases in relative importance as the iron-transferrin concentration is raised.     

Uptake and Toxicity of Copper Oxide Nanoparticles in C6 Glioma Cells

Copper oxide nanoparticles (CuO-NPs) are frequently used for many technical applications, but are also known for their cell toxic potential. In order to investigate a potential use of CuO-NPs as a therapeutic drug for glioma treatment, we have investigated the consequences of an application of CuO-NPs on the cellular copper content and cell viability of C6 glioma cells. CuO-NPs were synthesized by a wet-chemical method and were coated with dimercaptosuccinic acid and bovine serum albumin to improve colloidal stability in physiological media. Application of these protein-coated nanoparticles (pCuO-NPs) to C6 cells caused a strong time-, concentration- and temperature-dependent copper accumulation and severe cell death. The observed loss in cellular MTT-reduction capacity, the loss in cellular LDH activity and the increase in the number of propidium iodide-positive cells correlated well with the specific cellular copper content. C6 glioma cells were less vulnerable to pCuO-NPs compared to primary astrocytes and toxicity of pCuO-NPs to C6 cells was only observed for incubation conditions that increased specific cellular copper contents above 20 nmol copper per mg protein. Both cellular copper accumulation as well as the pCuO-NP-induced toxicity in C6 cells were prevented by application of copper chelators, but not by endocytosis inhibitors, suggesting that liberation of copper ions from the pCuO-NPs is the first step leading to the observed toxicity of pCuO-NP-treated glioma cells.     

Phenanthrolines Protect Astrocytes from Hemin Without Chelating Iron

Hemin, the degradation product of hemoglobin, contributes to the neurodegeneration that occurs in the weeks following a hemorrhagic stroke. The breakdown of hemin in cells releases redox-active iron that can facilitate the production of toxic hydroxyl radicals. The present study used 3-week old primary cultures of mouse astrocytes to compare the toxicity of 33 μM hemin in the presence of the iron chelator 1,10-phenanthroline or its non-chelating analogue, 4,7-phenanthroline. This concentration of hemin killed approximately 75 % of astrocytes within 24 h. Both isoforms of phenanthroline significantly decreased the toxicity of hemin, with the non-chelating analogue providing complete protection at concentrations of 33 μM and above. The decrease in toxicity was associated with less cellular accumulation of hemin. Approximately 90 % of the hemin accumulated was not degraded, irrespective of treatment condition. These observations indicate that chelatable iron is not the cause of hemin toxicity. Cell-free experiments demonstrated that hemin can inactivate a molar excess of hydrogen peroxide (H₂O₂), and that the rate of inactivation is halved in the presence of either isoform of phenanthroline. We conclude that phenanthrolines may protect astrocytes by limiting hemin uptake and by impairing the capacity of intact hemin to interact with endogenous H₂O₂.     

Lectin-stimulated cellular iron uptake and toxin generation in the freshwater cyanobacterium Microcystis aeruginosa

The lectin family is composed of mono- and oligosaccharide binding proteins that could activate specific cellular activities, such as cell-cell attachment and toxin production. In the present study, the effect of the external addition of lectins to culture media containing the freshwater cyanobacterium Microcystis aeruginosa on its metabolic activities, such as iron uptake and toxin production was investigated. Among the three lectins examined in this study (concanavalin A [Con A], wheat germ agglutinin [WGA] and peanut agglutinin [PNA]), PNA substantially increased the accumulated intracellular and extracellular iron content. The binding of PNA and Con A to M. aeruginosa cells was visualized via fluorescence microscopy using a lectin adjunct with fluorescein isothiocyanate, and resulted in carbohydrate and protein accumulation in the cellular capsule. Given that the highest carbohydrate accumulation was seen in the Con A system (where iron accumulation was relatively lower), carbohydrate quality is likely important factor that influences cellular iron accumulation. Since PNA specifically binds to sugars such as galactose and N-acetylgalactosamine, these saccharide species could be important candidates for intracellular and extracellular iron accumulation and transport. Microcystin biosynthesis was stimulated in the presence of PNA and WGA, whereas cellular iron uptake increased only in the presence of PNA. Thus, the iron uptake was not necessarily congruent with the upregulation of microcystin synthesis, which suggested that the positive effect of lectin on iron uptake is probably attributable to the PNA-assisted iron accumulation around the cell surface. Overall, the present study provides insights into the interactions of lectin that influence cellular metabolic activities such as iron uptake, extracellular polymeric substance accumulation, and toxin production.     

Uptake and Metabolism of Iron Oxide Nanoparticles in Brain Cells

Magnetic iron oxide nanoparticles (IONPs) are used for various applications in biomedicine, for example as contrast agents in magnetic resonance imaging, for cell tracking and for anti-tumor treatment. However, IONPs are also known for their toxic effects on cells and tissues which are at least in part caused by iron-mediated radical formation and oxidative stress. The potential toxicity of IONPs is especially important concerning the use of IONPs for neurobiological applications as alterations in brain iron homeostasis are strongly connected with human neurodegenerative diseases. Since IONPs are able to enter the brain, potential adverse consequences of an exposure of brain cells to IONPs have to be considered. This article describes the pathways that allow IONPs to enter the brain and summarizes the current knowledge on the uptake, the metabolism and the toxicity of IONPs for the different types of brain cells in vitro and in vivo.     

The mechanism of iron uptake by the rat placenta

The mechanism of iron uptake from transferrin by the rat placenta in culture has been studied. Transferrin endocytosis preceded iron accumulation by the cells. Both transferrin internalisation and iron uptake were inhibited by low temperature. Transferrin endocytosis was less susceptible to the effects of metabolic inhibitors such as sodium fluoroacetate, potassium cyanide, 2,4, dinitrophenol or carbonylcyanide M-chlorophenyl hydrazone (CCCP) than was iron uptake. Iron accumulation was decreased if the cells were incubated in the presence of weak bases such as chloroquine or ammonium chloride. These results suggest that, following internalisation, the vesicles containing the transferrin and iron became acidified, and that this acidification was a necessary prerequisite for the accumulation of iron by the cell. Further, the results indicate that the intravesicular pH was maintained at the expense of metabolic energy, suggesting that a pump may be involved. The importance of the permeability properties of the vesicle membrane in the iron uptake process was investigated by incubating the cells with labelled transferrin and iron in the presence of different cation and anion ionophores. Irrespective of the normal cation that the ionophores carried, all inhibited iron uptake without altering transferrin levels. In contrast, phloridzin, a Cl- transport inhibitor, did not affect either the levels of transferrin within the cells or the amount of iron accumulated.     

Modulation of transferrin-receptor activity and recycling after induced differentiation of BeWo choriocarcinoma cells.

BeWo human choriocarcinoma cells normally grow as cytotrophoblast cells. However, in the presence of 100 microM-forskolin or 5 mM-theophylline, these cells form syncytia similar to morphologically well differentiated syncytiotrophoblasts. We have examined the effect of syncytia formation on transferrin-receptor activity and recycling. Although cellular proliferation stops upon growth in the presence of forskolin or theophylline, the number of cell-surface transferrin-receptors unexpectedly increased 2-fold, whereas the total cellular number increased at most 15%. The rate of biosynthesis of the transferrin receptor as well as class I MHC glycoprotein did not change measurably during syncytium formation. The biosynthesis of human chorionic gonadotropin increased 35-fold after 30 h of growth in the presence of theophylline. The redistribution of the transferrin receptor in syncytia is maintained by a decreased rate constant of endocytosis (0.141 min-1 compared with 0.231 min-1 for control cells) and an increased rate constant of externalization (0.122 min-1 compared with 0.060 min-1 for control cells). These altered rates of endocytosis and externalization resulted in an increased rate of iron accumulation in the syncytia. Furthermore, the recycling time of the transferrin receptor decreased in cells grown in the presence of theophylline (14.6 min compared with 21.2 min in control cells).