The significance of the mutated divalent metal transporter (DMT1) on iron transport into the Belgrade rat brain

Brain iron transport and distributional pattern of divalent metal transporter I (DMT1) were studied in homozygous Belgrade rats (b/b) which suffer from a mutation in the DMT1 gene. In adult rats, brain uptake of transferrin-bound iron injected intravenously (i.v.) was significantly lower compared with that in heterozygous Belgrade (+/b) and Wistar rats, whereas transferrin uptake was identical. The difference in iron uptake was not apparent until 30 min after injection. The brain iron concentration was lower, and neuronal transferrin receptor-immunoreactivity higher, in adult b/b rats, thus confirming their iron-deficient stage. Antibodies targeting different sites on the DMT1 molecule consistently detected DMT1 in neurones and choroid plexus at the same level irrespective of strain, but failed to detect DMT1 in brain capillary endothelial cells (BCECs), or macro- or microglial cells. The absence of DMT1 in BCECs was confirmed in immunoblots of purified BCECs. DMT1 was virtually undetectable in neurones of rats aged 18 post-natal days irrespective of strain. Neuronal expression of transferrin receptors and DMT1 in adult rats implies that neurones at this age acquire iron by receptor-mediated endocytosis of transferrin followed by iron transport out of endosomes mediated by DMT1. The existence of the mutated DMT1 molecule in neurones suggests that the low cerebral iron uptake in b/b rats derives from a reduced neuronal uptake rather than an impaired iron transport through the blood-brain barrier.     

Olfactory ferric and ferrous iron absorption in iron-deficient rats

The absorption of metals from the nasal cavity to the blood and the brain initiates an important route of occupational exposures leading to health risks. Divalent metal transporter-1 (DMT1) plays a significant role in the absorption of intranasally instilled manganese, but whether iron uptake would be mediated by the same pathway is unknown. In iron-deficient rats, blood (59)Fe levels after intranasal administration of the radioisotope in the ferrous form were significantly higher than those observed for iron-sufficient control rats. Similar results were obtained when ferric iron was instilled intranasally, and blood levels of (59)Fe were even greater in the iron-deficient rats compared with the amount of ferrous iron absorbed. Experiments with Belgrade (b/b) rats showed that DMT1 deficiency limited ferric iron uptake from the nasal cavity to the blood compared with +/b controls matched for iron deficiency. These results indicate that olfactory uptake of ferric iron by iron-deficient rats involves DMT1. Western blot experiments confirmed that DMT1 levels are significantly higher in iron-deficient rats compared with iron-sufficient controls in olfactory tissue. Thus the molecular mechanism of olfactory iron absorption is regulated by body iron status and involves DMT1.     

Efflux of iron from the cerebrospinal fluid to the blood at the blood-CSF barrier: effect of manganese exposure

The blood-cerebrospinal fluid (CSF) barrier (BCB) resides within the choroid plexus, with the apical side facing the CSF and the basolateral side towards the blood. Previous studies demonstrate that manganese (Mn) exposure in rats disrupts iron (Fe) homeostasis in the blood and CSF. The present study used a primary culture of rat choroidal epithelial cells grown in the two-chamber Transwell system to investigate the transepithelial transport of Fe across the BCB. Free, unbound Fe as [(59)Fe] was added to the donor chamber and the radioactivity in the acceptor chamber was quantified to determine the direction of Fe fluxes. Under the normal condition, the [(59)Fe] efflux (from the CSF to the blood) was 128% higher than that of the influx (P < 0.01). Mn exposure significantly increased the efflux rate of [(59)Fe] (P < 0.01) and the effect was inhibited when the cells were pre-incubated with the antibody against divalent metal transport 1 (DMT1). Moreover, when the siRNA knocked down the cellular DMT1 expression, the elevated Fe uptake caused by Mn exposure in the choroidal epithelial Z310 cells was completely abolished, indicating that Mn may facilitate Fe efflux via a DMT1-mediated transport mechanism. In vivo subchronic exposure to Mn in rats reduced Fe clearance from the CSF, as demonstrated by the ventriculo-cisternal brain perfusion, along with up-regulated mRNAs encoding DMT1 and transferrin receptor (TfR) in the same animals. Taken together, these data suggest that free Fe appears to be favorably transported from the CSF toward the blood by DMT1 and this process can be facilitated by Mn exposure. Enhanced TfR-mediated influx of Fe from the blood and ferroportin-mediated expelling Fe toward the CSF may compromise DMT1-mediated efflux, leading to an increased Fe concentration in the CSF as seen in Mn-exposed animals.     

Effects of chelators on iron uptake and release by the brain in the rat

The iron chelators desferrioxamine (DFO), pyridoxal isonicotinoyl hydrazone (PIH), 2,2-bipyridine, diethylenetriamine penta-acetic acid (DTPA) and 1,2 dimethyl-3-hydroxy pyrid-4-one (CP20) were analysed for their ability to change⁵⁹Fe uptake and release from the brain of 15- and 63-day rats either during or after intravenous injection of⁵⁹Fe-¹²⁵I-transferrin. DTPA was the only chelator unable to significantly reduce iron uptake into the brain of 15-day rats. This indicates that iron is not released from transferrin at the luminal surface of brain capillary endothelial cells. CP20 was able to reduce iron uptake in the brain by 85% compared to 28% with DFO. Only CP20 was able to significantly reduce brain iron uptake in 63 day rats. Once⁵⁹Fe had entered the brain no chelator used was able to mediate its release. All of the chelators except CP20 had similar effects on femur iron uptake as they did on brain uptake, suggesting similar iron uptake mechanisms. It is concluded that during the passage of transferrin-bound iron into the brain the iron is released from transferrin within endothelial cells after endocytosis of transferrin.     

Restricted transport of anti-transferrin receptor antibody (OX26) through the blood-brain barrier in the rat

Anti-transferrin receptor IgG2a (OX26) transport into the brain was studied in rats. Uptake of OX26 in brain capillary endothelial cells (BCECs) was > 10-fold higher than isotypic, non-immune IgG2a (Ni-IgG2a) when expressed as % ID/g. Accumulation of OX26 in the brain was higher in 15 postnatal (P)-day-old rats than in P0 and adult (P70) rats. Iron-deficiency did not increase OX26 uptake in P15 rats. Three attempts were made to investigate transport from BCECs further into the brain. (i) Using a brain capillary depletion technique, 6-9% of OX26 was identified in the post-capillary compartment consisting of brain parenchyma minus BCECs. (ii) In cisternal CSF, the volume of distribution of OX26 was higher than for Ni-IgG2a when corrected for plasma concentration. (iii) Immunohistochemical mapping revealed the presence of OX26 almost exclusively in BCECs; extravascular staining was observed only in neurons situated periventricularly. The data support the hypothesis of facilitated uptake of OX26 due to the presence of transferrin receptors at the blood-brain barrier (BBB). However, OX26 accumulation in the post-capillary compartment was too small to justify a conclusion of receptor-mediated transcytosis of OX26 occurring in BCECs. Accumulation of OX26 in the post-capillary component may result from a diphasic transport that involves high-affinity accumulation of OX26 by the BCECs, clearly exceeding that of Ni-IgG2a, followed by a second transport mechanism that releases OX26 non-specifically further into the brain. The periventricular localization suggests that OX26 probably also derives from transport across the blood-CSF barrier.     

Gene expression of divalent metal transporter 1 and transferrin receptor in duodenum of Belgrade rats

Regulation of iron absorption is thought to be mediated by the amount of iron taken up by duodenal crypt cells via the transferrin receptor (TfR)-transferrin cycle and the activity of the divalent metal transporter (DMT1), although DMT1 cannot be detected morphologically in crypt cells. We investigated the uptake of transferrin-bound iron by duodenal enterocytes in Wistar rats fed different levels of iron and Belgrade (b/b) rats in which iron uptake by the transferrin cycle is defective because of a mutation in DMT1. We showed that DMT1 in our colony of b/b rats contains the G185R mutation, which in enterocytes results in reduced cellular iron content and increased DMT1 gene expression similar to levels in iron deficiency of normal rats. In all groups the uptake of transferrin-bound iron by crypt cells was directly proportional to plasma iron concentration, being highest in iron-loaded Wistar rats and b/b rats. We conclude that the uptake of transferrin-bound iron by developing enterocytes is largely independent of DMT1.     

Brain capillary endothelium and choroid plexus epithelium regulate transport of transferrin-bound and free iron into the rat brain

Iron transport into the CNS is still not completely understood. Using a brain perfusion technique in rats, we have shown a significant brain capillary uptake of circulating transferrin (Tf)-bound and free 59Fe (1 nm) at rates of 136 +/- 26 and 182 +/- 23 microL/g/min, respectively, while their respective transport rates into brain parenchyma were 1.68 +/- 0.56 and 1.52 +/- 0.48 microL/g/min. Regional Tf receptor density (Bmax) in brain endothelium determined with 125I-holo-Tf correlated well with 59Fe-Tf regional brain uptake rates reflecting significant vascular association of iron. Tf-bound and free circulating 59Fe were sequestered by the choroid plexus and transported into the CSF at low rates of 0.17 +/- 0.01 and 0.09 +/- 0.02 microL/min/g, respectively, consistent with a 10-fold brain-CSF concentration gradient for 59Fe, Tf-bound or free. We conclude that transport of circulating Tf-bound and free iron could be equally important for its delivery to the CNS. Moreover, data suggest that entry of Tf-bound and free iron into the CNS is determined by (i) its initial sequestration by brain capillaries and choroid plexus, and (ii) subsequent controlled and slow release from vascular structures into brain interstitial fluid and CSF.     

Manganese metabolism is impaired in the Belgrade laboratory rat

Homozygous Belgrade rats have a hypochromic anaemia due to impaired iron transport across the cell membrane of immature erythroid cells. This study aimed at investigating whether there are also abnormalities of Mn metabolism in erythroid and other types of cells. The experiments were performed with homozygous (b/b) and heterozygous (+/b) Belgrade rats and Wistar rats and included measurements of Mn uptake by reticulocytes in vitro, Mn absorption from in situ closed loops of the duodenum, and plasma clearance and uptake by several organs after intravenous injection of radioactive Mn bound to transferrin (Tf ) or mixed with serum. Similar measurements were made with ⁵⁹Fe-labelled Fe in several of the experiments. Mn uptake by reticulocytes and absorption from the duodenum was impaired in b/b rats compared with +/b or Wistar rats. The plasma clearance of Mn-Tf was much slower than Mn-serum, but both were faster than the clearance of Fe-Tf. Uptake of ⁵⁴Mn by the kidneys, brain and femurs was less in b/b than Wistar or +/b rats, but uptake by the liver was greater in b/b rats. Similar differences were found for ⁵⁹Fe uptake by kidneys, brain and femurs but ⁵⁹Fe uptake by the liver was also impaired in the liver. It is concluded that the genetic abnormality present in b/b rats affects Mn metabolism as well as Fe metabolism and that Mn and Fe share similar transport mechanisms in the cells of erythroid tissue, duodenal mucosa, kidney and blood-brain barrier. Accepted: 20 February 1997     

Iron absorption by Belgrade rat pups during lactation

Divalent metal transporter-1 (DMT1) mediates dietary nonheme iron absorption. Belgrade (b) rats have defective iron metabolism due to a mutation in the DMT1 gene. To examine the role of DMT1 in neonatal iron assimilation, b/b and b/+ pups were cross-fostered to F344 Fischer dams injected with (59)FeCl(3) twice weekly during lactation. Tissue distribution of the radioisotope in the pups was determined at weaning (day 21). The b/b pups had blood (59)Fe levels significantly lower than b/+ controls but significantly higher (59)Fe tissue levels in heart, bone marrow, skeletal muscle, kidney, liver, spleen, stomach, and intestines. To study the pharmacokinetics of nonheme iron absorption at the time of weaning, (59)FeCl(3) was administered to 21-day-old b/b and b/+ rats by intragastric gavage. Blood (59)Fe levels measured 5 min to 4 h postgavage were significantly lower in b/b rats, consistent with impaired DMT1 function in intestinal iron absorption. Tissue (59)Fe levels were also lower in b/b rats postgavage. Combined, these data suggest that DMT1 function is not essential for iron assimilation from milk during early development in the rat.     

Molecular mechanisms of non‐transferrin‐bound and transferring‐bound iron uptake in primary hippocampal neurons

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell‐derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co‐localize, indicating these two proteins may function in Fe³⁺ reduction prior to Fe²⁺ permeation. Zip8, DMT1, and Steap2 co‐localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin‐mediated iron assimilation. In brain interstitial fluid, transferring‐bound iron (TBI) and non‐transferrin‐bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin‐⁵⁹Fe³⁺) and NTBI, whether presented as ⁵⁹Fe²⁺‐citrate or ⁵⁹Fe³⁺‐citrate; reductase‐independent ⁵⁹Fe²⁺ uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn²⁺ inhibition of Fe²⁺ uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of ⁵⁹Fe from TBI relies at least in part on an endocytosis‐independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons.

     

Dietary iron induces rapid changes in rat intestinal divalent metal transporter expression

The divalent metal transporter (DMT1, also known as NRAMP2 or DCT1) is the likely target for regulation of intestinal iron absorption by iron stores. We investigated changes in intestinal DMT1 expression after a bolus of dietary iron in iron-deficient Belgrade rats homozygous for the DMT1 G185R mutation (b/b) and phenotypically normal heterozygous littermates (+/b). Immunofluorescent staining with anti-DMT1 antisera showed that DMT1 was located in the brush-border membrane. Duodenal DMT1 mRNA and protein levels were six- and twofold higher, respectively, in b/b rats than in +/b rats. At 1.5 h after dietary iron intake in +/b and b/b rats, DMT1 was internalized into cytoplasmic vesicles. At 1.5 and 3 h after iron intake in +/b and b/b rats, there was a rapid decrease of DMT1 mRNA and a transient increase of DMT1 protein. The decrease of DMT1 mRNA was specific, because ferritin mRNA was unchanged. After iron intake, an increase in ferritin protein and decrease in iron-regulatory protein binding activity occurred, reflecting elevated intracellular iron pools. Thus intestinal DMT1 rapidly responds to dietary iron in both +/b and b/b rats. The internalization of DMT1 may be an acute regulatory mechanism to limit iron uptake. In addition, the results suggest that in the Belgrade rat DMT1 with the G185R mutation is not an absolute block to iron.     

Iron trafficking inside the brain

Iron, an essential element for all cells of the body, including those of the brain, is transported bound to transferrin in the blood and the general extracellular fluid of the body. The demonstration of transferrin receptors on brain capillary endothelial cells (BCECs) more than 20 years ago provided the evidence for the now accepted view that the first step in blood to brain transport of iron is receptor-mediated endocytosis of transferrin. Subsequent steps are less clear. However, recent investigations which form the basis of this review have shed some light on them and also indicate possible fruitful avenues for future research. They provide new evidence on how iron is released from transferrin on the abluminal surface of BCECs, including the role of astrocytes in this process, how iron is transported in brain extracellular fluid, and how iron is taken up by neurons and glial cells. We propose that the divalent metal transporter 1 is not involved in iron transport through the BCECs. Instead, iron is probably released from transferrin on the abluminal surface of these cells by the action of citrate and ATP that are released by astrocytes, which form a very close relationship with BCECs. Complexes of iron with citrate and ATP can then circulate in brain extracellular fluid and may be taken up in these low-molecular weight forms by all types of brain cells or be bound by transferrin and taken up by cells which express transferrin receptors. Some iron most likely also circulates bound to transferrin, as neurons contain both transferrin receptors and divalent metal transporter 1 and can take up transferrin-bound iron. The most likely source for transferrin in the brain interstitium derives from diffusion from the ventricles. Neurons express the iron exporting carrier, ferroportin, which probably allows them to excrete unneeded iron. Astrocytes lack transferrin receptors. Their source of iron is probably that released from transferrin on the abluminal surface of BCECs. They probably to export iron by a mechanism involving a membrane-bound form of the ferroxidase, ceruloplasmin. Oligodendrocytes also lack transferrin receptors. They probably take up non-transferrin bound iron that gets incorporated in newly synthesized transferrin, which may play an important role for intracellular iron transport.     

The mechanism of hepatic iron uptake from native and denatured transferrin and its subcellular metabolism in the liver cell.

Hepatic iron uptake and metabolism were studied by subcellular fractionation of rat liver homogenates after injection of rats with a purified preparation of either native or denatured rat transferrin labelled with 125I and 59Fe. (1) With native transferrin, hepatic 125I content was maximal 5 min after injection and then fell. Hepatic 59Fe content reached maximum by 16 h after injection and remained constant for 14 days. Neither label appeared in the mitochondrial or lysosomal fractions. 59Fe appeared first in the supernatant and, with time, was detectable as ferritin in fractions sedimented with increasingly lower g forces. (2) With denatured transferrin, hepatic content of both 125I and 59Fe reached maximum by 30 min. Both appeared initially in the lysosomal fraction. With time, they passed into the supernatant and 59Fe became incorporated into ferritin. The study suggests that hepatic iron uptake from native transferrin does not involve endocytosis. However, endocytosis of denatured transferrin does occur. After the uptake process, iron is gradually incorporated into ferritin molecules, which subsequently polymerize; there is no incorporation into other structures over 14 days.     

Role of transferrin in iron uptake by the brain: a comparative study

Summary The role of specific transferrin (Tf) and Tf receptor interaction on brain capillary endothelial cells in iron transport from the plasma to the brain was investigated by using Tf from several species of animals labeled with ⁵⁹Fe and ¹²⁵I, and 15-day and adult rats. The rate of iron transfer was much greater in the 15-day rats. It was greatest with Tf from the mammals, rat, rabbit and human, but much lower with chicken ovotransferrin and quokka (a marsupial), toad, lizard, crocodile, and fish Tf. The uptake of Tf by the brain showed a similar pattern, except for a very high uptake of ovotransferrin (ovo Tf). Iron uptake by the femurs (a source of bone marrow) was also high with Tf from the mammalian species and low with the other types of Tf, but showed little change with aging of the animals. It is concluded that iron transport into the brain is dependent on the function of Tf receptors, probably on capillary endothelial cells, and that these receptors show the same type of species specificity as the receptors on immature erythroid cells. Also, the decrease in iron uptake by the brain as rats age from 15 days to adulthood is specific for the brain and is not a general effect of the aging process.Abbreviations Tf transferrin - ovo Tf ovotransferrin     

The transferrin homologue, melanotransferrin (p97), is rapidly catabolized by the liver of the rat and does not effectively donate iron to the brain

Melanotransferrin (MTf) or melanoma tumor antigen p97 is a membrane-bound transferrin (Tf) homologue that binds iron (Fe). This protein is also found as a soluble form in the plasma (sMTf) and was suggested to be an Alzheimer's disease marker. In addition, sMTf has been recently suggested to cross the blood-brain barrier (BBB) and accumulate in the brain of the mouse following intravenous infusion. Considering the importance of this observation to the physiology and pathophysiology of the BBB and the function of sMTf in vivo, we investigated the uptake and distribution of 59Fe-125I-sMTf and compared it to 59Fe-125I-Tf that were injected intravenously in rats. Studies were also performed to measure 59Fe and 125I-protein uptake by reticulocytes using these radiolabelled proteins. The results showed that sMTf was rapidly catabolized, mainly in the liver and to a lesser extent by the kidneys. The 59Fe was largely retained by these organs but the 125I was released into the plasma. Only a small amount of 125I-sMTf or its bound 59Fe was taken up by the brain, less than that from 59Fe-125I-Tf. There was much less 59Fe uptake by erythropoietic organs (spleen and femurs) from 59Fe-sMTf than from 59Fe-Tf, and no evidence of receptor-mediated uptake of sMTf was obtained using reticulocytes. It is concluded that compared to Tf, sMTf plays little or no role in Fe supply to the brain and erythropoietic tissue. However, a small amount of sMTf was taken up from the plasma by the brain and a far greater amount by the liver.     

ATP-driven copper transport across the intestinal brush border membrane

The divalent metal ion transporter DMT1 is localized in the brush border membrane (BBM) of the upper small intestine and has been shown to be able to transport Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. Belgrade rats have a glycine-to-arginine (G185R) mutation in DMT1, which affects its function. We investigated copper transport with BBM vesicles of Belgrade rats loaded with calcein, which exhibits fluorescence quenching by various metal ions. Transport of copper was disrupted in unenergized BBM vesicle of b/b Belgrade rats, as had been described for iron transport, while +/b vesicles exhibited normal transport by DMT1. When either b/b or +/b vesicles were loaded with ATP and magnesium, similar high-affinity accumulation of copper was observed in both types of vesicles. Thus, brush border membranes possess an ATP-driven, high-affinity copper transport system which could serve as the primary route for copper uptake by the intestine.     

Manganese and iron transport across pulmonary epithelium

Pathways mediating pulmonary metal uptake remain unknown. Because absorption of iron and manganese could involve similar mechanisms, transferrin (Tf) and transferrin receptor (TfR) expression in rat lungs was examined. Tf mRNA was detected in bronchial epithelium, type II alveolar cells, macrophages, and bronchus-associated lymphoid tissue (BALT). Tf protein levels in lung and bronchoalveolar lavage fluid did not change in iron deficiency despite increased plasma levels, suggesting that lung Tf concentrations are regulated by local synthesis in a manner independent of body iron status. Iron oxide exposure upregulated Tf mRNA in bronchial and alveolar epithelium, macrophages, and BALT, but protein was not significantly increased. In contrast, TfR mRNA and protein were both upregulated by iron deficiency. To examine potential interactions with lung Tf, rats were intratracheally instilled with (54)Mn or (59)Fe. Unlike (59)Fe, interactions between (54)Mn and Tf in lung fluid were not detected. Absorption of intratracheally instilled (54)Mn from the lungs to the blood was unimpaired in Belgrade rats homozygous for the functionally defective G185R allele of divalent metal transporter-1, indicating that this transporter is also not involved in pulmonary manganese absorption. Pharmacological studies of (54)Mn uptake by A549 cells suggest that metal uptake by type II alveolar epithelial cells is associated with activities of both L-type Ca(2+) channels and TRPM7, a member of the transient receptor potential melastatin subfamily. These results demonstrate that iron and manganese are absorbed by the pulmonary epithelium through different pathways and reveal the potential role for nonselective calcium channels in lung metal clearance.     

Interactions between tissue uptake of lead and iron in normal and iron-deficient rats during development

Environmental lead intoxication, which frequently causes neurological disturbances, and iron deficiency are clinical problems commonly found in children. Also, iron deficiency has been shown to augment lead absorption from the intestine. Hence, there is evidence for an interaction between lead and iron metabolism which could produce changes in lead and iron uptake by the brain and other tissues. These possibilities were investigated using 15-, 21-, and 63-old rats with varying nutritional iron and lead status. Dams were fed diets containing 0 or 3% lead-acetate and 0.2% lead-acetate in the drinking water. After weaning, 0.2% lead-acetate in the drinking water became the sole source of dietary lead. Measurements were made of tissue lead and nonheme iron levels and the uptake of⁵⁹Fe after intravenous injection of transferrin-bound⁵⁹Fe. Iron deficiency was associated with increased intestinal absorption of lead as indicated by blood and kidney lead levels in rats exposed to dietary lead. However, iron deficiency did not increase lead deposition in the brain, and in all rats brain lead levels were relatively low (<0.1 μg/g). Lead concentrations in the liver were below 2 μg/g, whereas kidneys had almost 20 times this concentration. Animals with iron deficiency had lower liver iron levels and had increased brain⁵⁹Fe uptake in comparison to control rats. However, iron levels in brain and kidneys were unaffected by lead intoxication regardless of the animal's iron status.⁵⁹Fe uptake rates were also unaffected by lead, but increased rates of uptake were apparent in iron-deficient rats. Lead did increase liver iron levels in all iron-adequate rats, but iron deficiency had little effect. It is concluded that, compared with other tissues, the blood-brain barrier largely restricts lead uptake by the brain and that the uptake that does occur is unrelated to the iron status of the animal. Also, the level of lead intoxication produced in this investigation did not influence iron uptake by the brain and kidneys, but liver iron stores could be incresed if iron levels were already adequate.     

Effects of iron deficiency and iron overload on manganese uptake and deposition in the brain and other organs of the rat

Managanese (Mn) is an essential trace element at low concentrations, but at higher concentrations is neurotoxic. It has several chemical and biochemical properties similar to iron (Fe), and there is evidence of metabolic interaction between the two metals, particularly at the level of absorption from the intestine. The aim of this investigation was to determine whether Mn and Fe interact during the processes involved in uptake from the plasma by the brain and other organs of the rat. Dams were fed control (70 mg Fe/kg), Fe-deficient (5–10 mg Fe/kg), or Fe-loaded (20 g carbonyl Fe/kg) diets, with or without Mn-loaded drinking water (2 g Mn/L), from day 18–19 of pregnancy, and, after weaning the young rats, were continued on the same dietary regimens. Measurements of brain, liver, and kidney Mn and nonheme Fe levels, and the uptake of⁵⁴Mn and⁵⁹Fe from the plasma by these organs and the femurs, were made when the rats were aged 15 and 63 d. Organ nonheme Fe levels were much higher than Mn levels, and in the liver and kidney increased much more with Fe loading than did Mn levels with Mn loading. However, in the brain the increases were greater for Mn. Both Fe depletion and loading led to increased brain Mn concentrations in the 15-d/rats, while Fe loading also had this effect at 63 d. Mn loading did not have significant effects on the nonheme Fe concentrations.⁵⁴Mn, injected as MnCl₂ mixed with serum, was cleared more rapidly from the circulation than was⁵⁹Fe, injected in the form of diferric transferrin. In the 15-d-rats, the uptake of⁵⁴Mn by brain, liver, kidneys, and femurs was increased by Fe loading, but this was not seen in the 63-d rats. Mn supplementation led to increased⁵⁹Fe uptake by the brain, liver, and kidneys of the rats fed the control and Fe-deficient diets, but not in the Fe-loaded rats. It is concluded that Mn and Fe interact during transfer from the plasma to the brain and other organs and that this interaction is synergistic rather than competitive in nature. Hence, excessive intake of Fe plus Mn may accentuate the risk of tissue damage caused by one metal alone, particularly in the brain.     

Kinetics of transferrin endocytosis and iron uptake by intact isolated rat seminiferous tubules and Sertoli cells in culture

The receptor-mediated endocytotic cycle of rat and human transferrin has been studied in intact, isolated rat seminiferous tubules and Sertoli cells in culture. Double-labeled [( 59Fe125I]) transferrin has been used to study the fate of transferrin and iron. Diferric transferrin binds to the tubules and the cultured Sertoli cells and is internalized. The iron remains inside, while the transferrin recycles and is released into the medium. Although, as reported before (Wauben-Penris et al., 1986), "extra" binding sites for human transferrin exist as compared to rat transferrin, this does not result in extra uptake of transferrin or iron. Both rat and human transferrin transport iron into the cells and recycle back to the surface, and do so with identical kinetics. A striking difference has been found between the mean efficient recycling times of the transferrin receptors in intact tubules (90 min) and in Sertoli cells in culture (21 min). Possible explanations of this difference are discussed. Light-microscopic autoradiography of [125 I]-labeled transferrin has revealed that the transferrin protein is excluded from the adluminal compartment, even after 21 h of incubation. This indicates that externally added transferrin itself does not deliver iron to the postmeiotic germ cells in intact, isolated rat seminiferous tubules.