Transferrin and Transferrin Receptor Function in Brain Barrier Systems

1. Iron (Fe) is an essential component of virtually all types of cells and organisms. In plasma and interstitial fluids, Fe is carried by transferrin. Iron-containing transferrin has a high affinity for the transferrin receptor, which is present on all cells with a requirement for Fe. The degree of expression of transferrin receptors on most types of cells is determined by the level of Fe supply and their rate of proliferation.2. The brain, like other organs, requires Fe for metabolic processes and suffers from disturbed function when a Fe deficiency or excess occurs. Hence, the transport of Fe across brain barrier systems must be regulated. The interaction between transferrin and transferrin receptor appears to serve this function in the blood–brain, blood–CSF, and cellular–plasmalemma barriers. Transferrin is present in blood plasma and brain extracellular fluids, and the transferrin receptor is present on brain capillary endothelial cells, choroid plexus epithelial cells, neurons, and probably also glial cells.3. The rate of Fe transport from plasma to brain is developmentally regulated, peaking in the first few weeks of postnatal life in the rat, after which it decreases rapidly to low values. Two mechanisms for Fe transport across the blood–brain barrier have been proposed. One is that the Fe–transferrin complex is transported intact across the capillary wall by receptor-mediated transcytosis. In the second, Fe transport is the result of receptor-mediated endocytosis of Fe–transferrin by capillary endothelial cells, followed by release of Fe from transferrin within the cell, recycling of transferrin to the blood, and transport of Fe into the brain. Current evidence indicates that although some transcytosis of transferrin does occur, the amount is quantitatively insufficient to account for the rate of Fe transport, and the majority of Fe transport probably occurs by the second of the above mechanisms.4. An additional route of Fe and transferrin transport from the blood to the brain is via the blood–CSF barrier and from the CSF into the brain. Iron-containing transferrin is transported through the blood–CSF barrier by a mechanism that appears to be regulated by developmental stage and iron status. The transfer of transferrin from blood to CSF is higher than that of albumin, which may be due to the presence of transferrin receptors on choroid plexus epithelial cells so that transferrin can be transported across the cells by a receptor-mediated process as well as by nonselective mechanisms.5. Transferrin receptors have been detected in neurons in vivo and in cultured glial cells. Transferrin is present in the brain interstitial fluid, and it is generally assumed that Fe which transverses the blood–brain barrier is rapidly bound by brain transferrin and can then be taken up by receptor-mediated endocytosis in brain cells. The uptake of transferrin-bound Fe by neurons and glial cells is probably regulated by the number of transferrin receptors present on cells, which changes during development and in conditions with an altered iron status.6. This review focuses on the information available on the functions of transferrin and transferrin receptor with respect to Fe transport across the blood–brain and blood–CSF barriers and the cell membranes of neurons and glial cells.     

Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

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.     

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.     

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 interaction of transferin with isolated hepatocytes

Freshly isolated rat heptocytes display about 36 700 high-affinity sites to which deferric transferrin may bind with an apparent association constant of 1.62·10⁷ 1·mol^?1.Uptake of iron from diferric transferrin by hepatocytes is linear with time and is accelerated at increased differric transferrin concentrations.Apotransferrin is able to decrease net iron uptake by hepatocytes from diferric transferrin by a process not dependent on the apotransferrin concentrations used or on the rate at which the cells take up iron. Immunoprecipitation of the apotransferrin during these incubations indicates that iron is being released from the cells to apotransferrin at the same time as iron is being taken up from diferric transferrin. The simultaneous uptake and release of iron, and the insensitivity to apotransferrin concentration, suggest that the processes of iron uptake and release occur via separate mechanisms. The effect of apotransferrin on net retention of iron may be one way in which the in vivo distribution of iron between sites of storage and utilization is controlled.     

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.     

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.     

Iron Uptake in Blood-Brain Barrier Endothelial Cells Cultured in Iron-Depleted and Iron-Enriched Media

Abstract: Iron is essential in the cellular metabolism of all mammalian tissues, including the brain. Intracerebral iron concentrations vary with age and in several (neurological) diseases. Although it is evident that endothelial cells lining the capillaries in the brain are of importance, factors governing the regulation of intracerebral iron concentration are unknown. To investigate the role of blood-brain barrier endothelial cells in cerebral iron regulation, primary cultures of porcine blood-brain barrier endothelial cells were grown in either iron-enriched or iron-depleted medium. Iron-enriched cells showed a reduction in surface-bound and total transferrin receptor numbers compared with iron-depleted cells. Transferrin receptor kinetics showed that the transferrin receptor internalization rate in iron-enriched cultures was higher, whereas the transferrin receptor externalization rate in iron-enriched cultures was lower than the rate in iron-depleted cultures. Moreover, blood-brain barrier endothelial cells cultured in iron-enriched medium were able to accumulate more iron intracellularly, which underlines our kinetic data on transferrin receptors. Our results agree with histopathological studies on brain tissue of patients with hemochromatosis, suggesting that at high peripheral iron concentrations, the rate of iron transport across the blood-brain barrier endothelial cells is to some extent proportional to the peripheral iron concentration.     

Uptake and Distribution of Iron and Transferrin in the Adult Rat Brain

Brain uptake of iron-59 and iodine-125-labelled transferrin from blood in the adult rat has been investigated using graphical analysis to determine the blood-brain barrier permeability to these tracers in experiments that lasted between 5 min and 8 days. The blood-brain barrier permeability (K(in)) to 59Fe was 89 x 10(-5) ml/min/g compared to the value of 7 x 10(-5) ml/min/g for 125I-transferrin, which is similar to that of albumin, a plasma marker. The autoradiographic distribution of these tracers in brain was also studied to determine any regional variation in brain uptake after the tracers had been administered either systemically or applied in vitro. No regional uptake was seen for 125I-transferrin even after 24 h of circulation. In contrast, 59Fe showed selective regional uptake by the choroid plexus and extra-blood-brain barrier structures 4 h after administration. After 24 h of circulation, 59Fe distribution in brain was similar to the transferrin receptor distribution, as determined in vitro, but was unlike the distribution of nonhaem iron determined histochemically. The data suggest that brain iron uptake does not involve any significant transcytotic pathway of transferrin-bound iron into brain. It is proposed that the uptake of iron into brain involves the entry of iron-loaded transferrin to the cerebral capillaries, deposition of iron within the endothelial cells, followed by recycling of apotransferrin to the circulation. The deposited iron is then delivered to brain-derived transferrin for extracellular transport within the brain, and subsequently taken up via transferrin receptors on neurones and glia for usage or storage.     

Studies on the role of transferrin and endocytosis in the uptake of Fe3+ from Fe-nitrilotriacetate by mouse duodenum

Addition of iron-binding proteins (human serum transferrin, mouse serum transferrin, human lactoferrin) to the luminal fluid in tied-off segments of mouse intestine in vivo led to reduced 59Fe3+ absorption from 59Fe3+-nitrilotriacetate when compared to 59Fe3+-nitrilotriacetate alone. Assay of transferrin in luminal fluid from tied segments revealed only trace amounts of immunoreactivity. The levels of luminal transferrin are unaltered in chronic hypoxia where iron absorption is significantly enhanced. Studies in vitro revealed that NH4Cl, dansylcadavarine, para-chloromercuribenzoate and trinitrobenzenesulphonate have no effect on initial 59Fe3+ uptake rates from 59Fe3+-nitrilotriacetate, while N-ethylmaleimide (1 mM) caused a 40% inhibition. In vivo 59Fe3+ uptake was unaffected by preincubation of tied-off segments with colchicine (5 mM) for up to 2 h. These results suggest that receptor-mediated endocytosis of transferrin is not a significant mechanism in the uptake of luminal Fe3+ by mouse duodenum.     

Gallium uptake by transferrin and interaction with receptor 1

The kinetics and thermodynamics of Ga(III) exchange between gallium mononitrilotriacetate and human serum transferrin as well as those of the interaction between gallium-loaded transferrin and the transferrin receptor 1 were investigated in neutral media. Gallium is exchanged between the chelate and the C-site of human serum apotransferrin in interaction with bicarbonate in about 50 s to yield an intermediate complex with an equilibrium constant K ₁ = (3.9 ± 1.2) × 10⁻², a direct second-order rate constant k ₁ = 425 ± 50 M⁻¹ s⁻¹ and a reverse second-order rate constant k ₋₁ = (1.1 ± 3) × 10⁴ M⁻¹ s⁻¹. The intermediate complex loses a single proton with proton dissociation constant K _1a = 80 ± 40 nM to yield a first kinetic product. This product then undergoes a modification in its conformation which lasts about 500 s to produce a second kinetic intermediate, which in turn undergoes a final extremely slow (several hours) modification in its conformation to yield the gallium-saturated transferrin in its final state. The mechanism of gallium uptake differs from that of iron and does not involve the same transitions in conformation reported during iron uptake. The interaction of gallium-loaded transferrin with the transferrin receptor occurs in a single very fast kinetic step with a dissociation constant K _d = 1.10 ± 0.12 μM and a second-order rate constant k _d = (1.15 ± 0.3) × 10¹⁰ M⁻¹ s⁻¹. This mechanism is different from that observed with the ferric holotransferrin and suggests that the interaction between the receptor and gallium-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of gallium incorporation by the transferrin receptor-mediated iron-acquisition pathway is discussed.     

Kinetics of Iron Import into Developing Mouse Organs Determined by a Pup-swapping Method

The kinetics of dietary iron import into various organs of mice were evaluated using a novel pup-swapping approach. Newborn pups whose bodies primarily contained ⁵⁶Fe or ⁵⁷Fe were swapped at birth such that each nursed on milk containing the opposite isotope. A pup from each litter was euthanized weekly over a 7-week period. Blood plasma was obtained, and organs were isolated typically after flushing with Ringer''s buffer. ⁵⁶Fe and ⁵⁷Fe concentrations were determined for organs and plasma; organ volumes were also determined. Mössbauer spectra of equivalent ⁵⁷Fe-enriched samples were used to quantify residual blood in organs; this fraction was excluded from later analysis. Rates of import into brain, spleen, heart, and kidneys were highest during the first 2 weeks of life. In contrast, half of iron in the newborn liver exited during that time, and influx peaked later. Two mathematical models were developed to analyze the import kinetics. The only model that simulated the data adequately assumed that an iron-containing species enters the plasma and converts into a second species and that both are independently imported into organs. Consistent with this, liquid chromatography with an on-line ICP-MS detector revealed numerous iron species in plasma besides transferrin. Model fitting required that the first species, assigned to non-transferrin-bound iron, imports faster into organs than the second, assigned to transferrin-bound-iron. Non-transferrin-bound iron rather than transferrin-bound-iron appears to play the dominant role in importing iron into organs during early development of healthy mice.     

Iron release from transferrin induced by mixed ligand complexes of copper(II)

Summary Copper(II) complexes CuL₁L₂ with the ligand pairs 3-phosphoglycerate (PG)/ethylenediamine (en), phosphoserine (PS)/ethylenediamine, phosphoserine/malonate (mal) are shown to be effective in inducing the release of both iron atoms from di-ferric transferrin (Fe2Tf; human serum transferrin) at pH 7.3 in 1 M NaCl at 25°C. Half-times of the reaction with Cu(PG)(en)^– were less than 1 min at 0.02 M concentration. The iron(III) products are polynuclear hydroxo complexes. There is weaker interaction with Cu(PS) ₂ ^4– and virtually none with Cu(serine)(en) nor Cu(PS)(2,2-bipyridyl)^–, revealing crucial effects of the combined ligand sphere including the phosphomonoester group. The results suggest that the release of iron from Fe2Tf, or from either monoferric transferrins, occurred due to the breakdown of the stability of iron binding in conjunction with the expulsion of the synergistic anion carbonate (or oxalate). The active copper(II) complexes are postulated to be models of membrane components that could liberate iron from transferrin succeeding its uptake at the receptor sites of cells.Abbreviations PG phosphoglycerate - PS phosphoserine - en ethylenediamine - Fe2Tf diferric transferrin - Fe_cTf and Fe_NTf transferrin with iron bound to the lobe containing the C- or N-terminus, respectively - apoTf apotransferrin - K-3 all-cis-1,3,5-tris(trimethylammonio)-2,4,6-cyclo-hexanetriol - NTA nitrilotriacetic acid; bipy, 2,2-bipyridine; mal, malonate     

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 metabolism of established human hematopoietic cell lines in vitro

Three malignant hematopoietic cell lines were used in studies on cellular iron metabolism. Our results show that iron-carrying transferrin became bound to specific dimeric cell surface receptors. Iron accumulated within the cell with time, whereas intact transferrin was released back to the medium. Chloroquine and NH₄Cl, known as pH-raising agents in vesicles of the lysosomal system, inhibited iron accumulation and transferrin binding in a dose-dependent manner. This suggests that the acid pH in endosomes leads to the cleavage of the iron-transferrin bonds. Transferrin degradation was not found, which leads us to suggest a process of ‘acid flushing’ for the dissociation of iron from transferrin without the involvement of endosome-lysosome fusion. Taken together, the data agree with the concept of receptor-mediated endocytosis, as described for many macromolecules. Iron was stored in ferritin in the cell types tested. Only a minor part (less than 15%) of the iron was bound in hemoglobin in the K-562 cell line. The relationship between iron stores and exogenously added iron in heme synthesis was investigated using a double labelling (⁵⁵Fe/⁵⁹Fe) technique. The results showed that exogenous iron was preferentially used before the iron stored in ferritin. The results are discussed in relation to various hypotheses on cellular iron uptake and transport.     

Iron exchange between transferrin molecules mediated by phosphate compounds and other cell metabolites

The ability of a large number of cellular metabolites to release iron from transferrin was investigated by measuring the rate at which they could mediate iron exchange between two types of transferrin. Rabbit transferrin labelled with ⁵⁹Fe was incubated with human apotransferrin in the presence of the metabolites. After varying periods of incubation the human transferrin was separated from the rabbit transferrin by immunoprecipitation.GTP, 2,3-diphosphoglycerate, ATP, ADP and citrate produced the most rapid exchange of iron between the two types of transferrin, but many other compounds showed some degree of activity. Iron exchange mediated by the organic phosphates had the characteristics of a single first-order reaction and was sensitive to changes of incubation temperature and pH. The activation energy for the exchange reaction was approx. 13 kcal/mol. The rate of iron exchange from the oxalate · iron · transferrin complex was much lower than from bicarbonate · iron · transferrin.It is concluded that several organic phosphates have the capacity of releasing iron from transferrin. These compounds may represent the means by which the iron is released during the process of cellular uptake.     

Large cooperativity in the removal of iron from transferrin at physiological temperature and chloride ion concentration

Iron removal from serum transferrin by various chelators has been studied by gel electrophoresis, which allows direct quantitation of all four forms of transferrin (diferric, C-monoferric, N-monoferric, and apotransferrin). Large cooperativity between the two lobes of serum transferrin is found for iron removal by several different chelators near physiological conditions (pH 7.4, 37 °C, 150 mM NaCl, 20 mM NaHCO₃). This cooperativity is manifested in a dramatic decrease in the rate of iron removal from the N-monoferric transferrin as compared with iron removal from the other forms of ferric transferrin. Cooperativity is diminished as the pH is decreased; it is also very sensitive to changes in chloride ion concentration, with a maximum cooperativity at 150 mM NaCl. A mechanism is proposed that requires closure of the C-lobe before iron removal from the N-lobe can be effected; the open conformation of the C-lobe blocks a kinetically significant anion-binding site of the N-lobe, preventing its opening. Physiological implications of this cooperativity are discussed.     

Calcium-dependent ATPase unlike ecto-ATPase is located primarily on the luminal surface of brain endothelial cells

Numerous cytochemical studies have reported that calcium-activated adenosine triphosphatase (Ca²⁺-ATPase) is localized on the abluminal plasma membrane of mature brain endothelial cells. Since the effects of fixation and co-localization of ecto-ATPase have never been properly addressed, we investigated the influence of these parameters on Ca²⁺-ATPase localization in rat cerebral microvessel endothelium. Formaldehyde at 2% resulted in only abluminal staining while both luminal and abluminal surfaces were equally stained following 4% formaldehyde. Fixation with 2% formaldehyde plus 0.25% glutaraldehyde revealed more abluminal staining than luminal while 2% formaldehyde plus 0.5% glutaraldehyde produced vessels with staining similar to 4% and 2% formaldehyde plus 0.25% glutaraldehyde. The abluminal reaction appeared unaltered when ATP was replaced by GTP, CTP, UTP, ADP or when Ca²⁺ was replaced by Mg²⁺ or Mn²⁺ or p-chloromercuribenzoate included as inhibitor. But the luminal reaction was diminished. Contrary to previous reports, our results showed that Ca²⁺-specific ATPase is located more on the luminal surface while the abluminal reaction is primarily due to ecto-ATPase. The strong Ca²⁺-specific-ATPase luminal localization explains the stable Ca²⁺ gradient between blood and brain, and is not necessarily indicative of immature or pathological vessels as interpreted in the past.     

The Pivotal Role of Astrocytes in the Metabolism of Iron in the Brain

Iron is essential for the normal functioning of cells but since it is also capable of generating toxic reactive oxygen species, the metabolism of iron is tightly regulated. The present article advances the view that astrocytes are largely responsible for distributing iron in the brain. Capillary endothelial cells are separated from the neuropil by the endfeet of astrocytes, so astrocytes are ideally positioned to regulate the transport of iron to other brain cells and to protect them if iron breaches the blood-brain barrier. Astrocytes do not appear to have a high metabolic requirement for iron yet they possess transporters for transferrin, haemin and non-transferrin-bound iron. They store iron efficiently in ferritin and can export iron by a mechanism that involves ferroportin and ceruloplasmin. Since astrocytes are a common site of abnormal iron accumulation in ageing and neurodegenerative disorders, they may represent a new therapeutic target for the treatment of iron-mediated oxidative stress.