Expression of the neonatal Fc receptor (FcRn) at the blood-brain barrier

The blood-brain barrier (BBB) restricts transport of immunoglobulin G (IgG) in the blood to brain direction. However, IgG undergoes rapid efflux in the brain to blood direction via reverse transcytosis across the BBB after direct intracerebral injection. This BBB IgG transport system has the characteristics of an Fc receptor (FcR), but there is no molecular information on the putative BBB FcR. The present study uses confocal microscopy and an antibody to the rat neonatal FcR (FcRn), and demonstrates the expression of the FcRn at the brain microvasculature and choroid plexus epithelium. Co-localization with the Glut1 glucose transporter indicates the brain microvascular FcRn is expressed in the capillary endothelium. The capillary endothelial FcRn may mediate the 'reverse transcytosis' of IgG in the brain to blood direction.     

Brain to blood efflux transport of adenosine: blood-brain barrier studies in the rat

The blood-brain barrier (BBB) efflux transport of [(14)C] adenosine was studied using the brain efflux index (BEI) technique. BEI increased linearly over the first 2 min after injection, with deviation from linearity thereafter; 90.12 +/- 1.5% of the injected [(14)C] radioactivity remained within the brain after 20 min. The remaining tracer appears to be mainly intracellular, trapped by phosphorylation, as an almost linear increase of BEI over 20 min was observed after intracerebral injection of [(14)C] adenosine together with 5-iodo tubercidin. The BBB efflux clearance of [(14)C] radioactivity was estimated to be 27.62 +/- 5.2 micro L/min/g, almost threefold higher than the BBB influx clearance estimated by the brain uptake index technique. High-performance liquid chromatography (HPLC) analysis of blood plasma collected from the jugular vein after the intracerebral injection revealed metabolic breakdown of [(14)C] adenosine into nucleobases. The BBB efflux transport was saturable with apparent K(m) = 13.22 +/- 1.75 micro m and V(max) = 621.07 +/- 71.22 pmole/min/g, which indicated that BBB efflux in vivo is 6.2-12p mole/min/g, negligible when compared to the reported rate of adenosine uptake into neurones/glia. However, these kinetic parameters also suggest that under conditions of elevated ISF adenosine in hypoxia/ischaemia, BBB efflux transport could increase up to 25% of the uptake into neurones/glia and become an important mechanism to oppose the rise in ISF concentration. HPLC-fluorometry detected 93.6 +/- 5.25 nm of adenosine in rat plasma, which is 17- to 220-fold lower than the reported K(m) of adenosine BBB influx in rat. Together with the observed rapid degradation inside endothelial cells, this indicated negligible BBB influx of intact adenosine under resting conditions. Cross-inhibition studies showed that unlabelled inosine, adenine and hypoxanthine caused a decrease in BBB efflux of [(14)C] radioactivity in a concentration-dependent manner, with K(i) of 16.7 +/- 4.88, 65.1 +/- 14.1 and 71.1 +/- 16.9 micro m, respectively. This could be due to either competition of unlabelled molecules with [(14)C] adenosine or competition with its metabolites hypoxanthine and adenine for the same transport sites.     

The l-isomer-selective transport of aspartic acid is mediated by ASCT2 at the blood-brain barrier

Aspartic acid (Asp) undergoes l-isomer-selective efflux transport across the blood-brain barrier (BBB). This transport system appears to play an important role in regulating l- and d-Asp levels in the brain. The purpose of this study was to identify the responsible transporters and elucidate the mechanism for l-isomer-selective Asp transport at the BBB. The l-isomer-selective uptake of Asp by conditionally immortalized mouse brain capillary endothelial cells used as an in vitro model of the BBB took place in an Na+- and pH-dependent manner. This process was inhibited by system ASC substrates such as l-alanine and l-serine, suggesting that system ASC transporters, ASCT1 and ASCT2, are involved in the l-isomer selective transport. Indeed, l-Asp uptake by oocytes injected with either ASCT1 or ASCT2 cRNA took place in a similar manner to that in cultured BBB cells, whereas no significant d-Asp uptake occurred. Although both ASCT1 and ASCT2 mRNA were expressed in the cultured BBB cells, the expression of ASCT2 mRNA was 6.7-fold greater than that of ASCT1. Moreover, immunohistochemical analysis suggests that ASCT2 is localized at the abluminal side of the mouse BBB. These results suggest that ASCT2 plays a key role in l-isomer-selective Asp efflux transport at the BBB.     

Involvement of OCTN2 and B0,+ in the transport of carnitine through an in vitro model of the blood-brain barrier

Carnitine is known to accumulate in brain, therefore transport of carnitine through the blood-brain barrier was studied in an in vitro system using bovine brain capillary endothelial cells (BBCEC) grown on filter inserts in a co-culture system with glial cells. Long-term exposure of BBCEC to carnitine resulted in a high accumulation of long-chain acyl carnitines, which decreased dramatically upon removal of carnitine. Kinetic analysis of carnitine accumulation indicated a possibility of functioning of more than one transporter. BBCEC were incubated in the presence of substrates and inhibitors of known carnitine transporters added from either apical or basolateral side. Inhibition by replacement of sodium and expression of OCTN2 (RT-PCR) were in agreement with earlier reports on the functioning of OCTN2 in apical membrane. For the first time, functioning of OCTN2 was demonstrated in the basolateral membrane, as well as functioning in both membranes of a low affinity carnitine transporter B(0,+). Expression of B(0,+) in BBCEC was confirmed by RT-PCR. These results suggest that OCTN2 and B(0,+) could be involved in carnitine transport in both the apical and basolateral membrane.     

The blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells

Antibodies against receptors that undergo transcytosis across the blood-brain barrier (BBB) have been used as vectors to target drugs or therapeutic peptides into the brain. We have recently discovered a novel single domain antibody, FC5, which transmigrates across human cerebral endothelial cells in vitro and the BBB in vivo. The purpose of this study was to characterize mechanisms of FC5 endocytosis and transcytosis across the BBB and its putative receptor on human brain endothelial cells. The transport of FC5 across human brain endothelial cells was polarized, charge independent and temperature dependent, suggesting a receptor-mediated process. FC5 taken up by human brain endothelial cells co-localized with clathrin but not with caveolin-1 by immunochemistry and was detected in clathrin-enriched subcellular fractions by western blot. The transendothelial migration of FC5 was reduced by inhibitors of clathrin-mediated endocytosis, K+ depletion and chlorpromazine, but was insensitive to caveolae inhibitors, filipin, nystatin or methyl-beta-cyclodextrin. Following internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes and remained intact after transcytosis. The transcytosis process was inhibited by agents that affect actin cytoskeleton or intracellular signaling through PI3-kinase. Pretreatment of human brain endothelial cells with wheatgerm agglutinin, sialic acid, alpha(2,3)-neuraminidase or Maackia amurensis agglutinin that recognizes alpha(2,3)-, but not with Sambucus nigra agglutinin that recognizes alpha(2,6) sialylgalactosyl residues, significantly reduced FC5 transcytosis. FC5 failed to recognize brain endothelial cells-derived lipids, suggesting that it binds luminal alpha(2,3)-sialoglycoprotein receptor which triggers clathrin-mediated endocytosis. This putative receptor may be a new target for developing brain-targeting drug delivery vectors.     

Characterization of the blood-brain barrier choline transporter using the in situ rat brain perfusion technique

Choline enters brain by saturable transport at the blood-brain barrier (BBB). In separate studies, both sodium-dependent and passive choline transport systems of differing affinity have been reported at brain capillary endothelial cells. In the present study, we re-examined brain choline uptake using the in situ rat brain perfusion technique. Saturable brain choline uptake from perfusion fluid was best described by a model with a single transporter (V:(max) = 2.4-3.1 nmol/min/g; K(m) = 39-42 microM) with an apparent affinity (1/Km)) for choline five to ten-fold greater than previously reported in vivo, but less than neuronal 'high-affinity' brain choline transport (K(m) = 1-5 microM). BBB choline uptake from a sodium-free perfusion fluid using sucrose for osmotic balance was 50% greater than in the presence of sodium suggesting that sodium is not required for transport. Hemicholinium-3 inhibited brain choline uptake with a K(i) (57 +/- 11 microM) greater than that at the neuronal choline system. In summary, BBB choline transport occurs with greater affinity than previously reported, but does not match the properties of the neuronal choline transporter. The V:(max) of this system is appreciable and may provide a mechanism for delivering cationic drugs to brain.     

Blood-to-retina transport of creatine via creatine transporter (CRT) at the rat inner blood-retinal barrier

The purpose of this study was to elucidate the mechanisms of blood-to-retina creatine transport across the blood-retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The creatine transport across the BRB in vivo and creatine uptake in an in vitro model of the inner BRB (TR-iBRB2 cells) were examined using [(14)C]creatine. Identification and localization of the creatine transporter (CRT) were carried out by RT-PCR, western blot, and immunoperoxidase electron microscopic analyses. An in vivo intravenous administration study suggested that [(14)C]creatine is transported from the blood to the retina against the creatine concentration gradient that exists between the retina and blood. [(14)C]Creatine uptake by TR-iBRB2 cells was saturable, Na(+)- and Cl(-)-dependent and inhibited by CRT inhibitors, suggesting that CRT is involved in creatine transport at the inner BRB. RT-PCR and western blot analyses demonstrated that CRT is expressed in rat retina and TR-iBRB2 cells. Moreover, using an immunoperoxidase electron microscopic analysis, CRT immunoreactivity was found at both the luminal and abluminal membranes of the rat retinal capillary endothelial cells. In conclusion, CRT is expressed at the inner BRB and plays a role in blood-to-retina creatine transport across the inner BRB.     

High transcytosis of melanotransferrin (P97) across the blood-brain barrier

The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14-fold higher than that of holo-transferrin, with no apparent intra-endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration- and conformation-dependent, suggesting that the transport of P97 is due to receptor-mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low-density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.     

Rapid transferrin efflux from brain to blood across the blood-brain barrier

The brain efflux index method is used to examine the extent to which transferrin effluxes from brain to blood across the blood-brain barrier (BBB) following intracerebral injection. Whereas high-molecular-weight dextran is nearly 100% retained in brain for up to 90 min after intracerebral injection in the Par2 region of the parietal cortex of brain, there is rapid efflux of transferrin from brain to blood across the BBB. The efflux of apotransferrin is 3.5-fold faster than the efflux of holo-transferrin. The brain to blood efflux of apotransferrin is completely saturable by unlabeled transferrin, but is not inhibited by other plasma proteins. These studies provide evidence for reverse transcytosis of transferrin from brain to blood across the BBB. As circulating transferrin is known to undergo transcytosis across the BBB in the blood-to-brain direction, these studies support the model of bidirectional transcytosis of transferrin through the BBB in vivo.     

Alcohol-induced blood-brain barrier dysfunction is mediated via inositol 1,4,5-triphosphate receptor (IP3R)-gated intracellular calcium release

The blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC), pericytes and astrocytes controls the transport of ions, peptides and leukocytes in and out of the brain. Tight junctions (TJ) composed of TJ proteins (occludin, claudins and zonula occludens) ensure the structural integrity of the BMVEC monolayer. Neuropathologic studies indicated that the BBB was impaired in alcohol abusers; however, the underlying mechanism of BBB dysfunction remains elusive. Using primary human BMVEC, we previously demonstrated that oxidative stress induced by ethanol (EtOH) metabolism in BMVEC activated myosin light chain kinase (MLCK), resulting in the enhanced phosphorylation of either cytoskeletal or TJ proteins, and in BBB impairment. We proposed that EtOH metabolites stimulated inositol 1,4,5-triphosphate receptor (IP(3)R)-operated intracellular calcium (Ca(2+)) release, thereby causing the activation of MLCK in BMVEC. Indeed, treatment of primary human BMVEC with EtOH or its metabolites resulted in the increased expression of IP(3)R protein and IP(3)R-gated intracellular Ca(2+) release. These functional changes paralleled MLCK activation, phosphorylation of cytoskeletal/TJ proteins, loss of BBB integrity, and enhanced leukocyte migration across BMVEC monolayers. Inhibition of either EtOH metabolism or IP(3)R activation prevented BBB impairment. These findings suggest that EtOH metabolites act as signaling molecules for the activation of MLCK via the stimulation of IP(3)R-gated intracellular Ca(2+) release in BMVEC. These putative events can lead to BBB dysfunction in the setting of alcoholism, and to neuro-inflammatory disorders promoting leukocyte migration across the BBB.     

Localization of organic cation/carnitine transporter (OCTN2) in cells forming the blood-brain barrier

Carnitine β-hydroxy-γ-(trimethylammonio)butyrate – a compound necessary in the peripheral tissues for a transfer of fatty acids for their oxidation within the cell, accumulates in the brain despite low β-oxidation in this organ. In order to enter the brain, carnitine has to cross the blood–brain barrier formed by capillary endothelial cells which are in close interaction with astrocytes. Previous studies, demonstrating expression of mRNA coding two carnitine transporters – organic cation/carnitine transporter 2 (OCTN2) and B^0,+ in endothelial cells, did not give any information on carnitine transporters polarity in endothelium. Therefore more detailed experiments were performed on expression and localization of a high affinity carnitine transporter OCTN2 in an in vitro model of the blood–brain barrier by real-time PCR, western blot analysis, and immunocytochemistry. The amount of mRNA was comparable in endothelial cells and kidney, when referred to house-keeping genes, it was, however, significantly lower in astrocytes. Polarity of OCTN2 localization was further studied in an in vitro model of the blood–brain barrier with use of anti-OCTN2 antibodies. Z -axis analysis of the confocal microscope pictures of endothelial cells, with anti-P-glycoprotein antibodies as the marker of apical membrane, showed OCTN2 localization at the basolateral membrane and in the cytoplasmic region in the vicinity of nuclei. Localization of OCTN2 suggest that carnitine can be also transported from the brain, playing an important role in removal of certain acyl esters.     

Influence of breast cancer resistance protein (Abcg2) and p-glycoprotein (Abcb1a) on the transport of imatinib mesylate (Gleevec) across the mouse blood-brain barrier

Imatinib, a protein tyrosine kinase inhibitor, may prevent the growth of glioblastoma cells. Unfortunately, its brain distribution is restricted by p-glycoprotein (p-gp or multidrug resistance protein Mdr1a), and probably by breast cancer resistance protein (Bcrp1), two efflux pumps expressed at the blood-brain barrier (BBB). We have used in situ brain perfusion to investigate the mechanisms of imatinib transport across the mouse BBB. The brain uptake of imatinib in wild-type mice was limited by saturable efflux processes. The inhibition of p-gp, by valspodar and zosuquidar, increased imatinib uptake (2.5-fold), as did the deficiency of p-gp in Mdr1a/1b(-/-) mice (5.5-fold). Perfusing imatinib with the p-gp/Bcrp1 inhibitor, elacridar, enhanced the brain uptake of imatinib in wild-type (4.1-fold) and Mdr1a/1b(-/-) mice (1.2-fold). However, the brain uptake of imatinib was similar in wild-type and Bcrp1(-/-) mice when it was perfused at a non-saturating concentration. The brain uptake of CGP74588, an active metabolite of imatinib, was low. It was increased by perfusion with elacridar (twofold), but not with valspodar and zosuquidar. CGP74588 uptake was 1.5 times greater in Bcrp1(-/-) mice than in wild-type mice. These data suggest that imatinib transport at the mouse BBB is limited by p-gp and probably by Bcrp1, and that CGP74588 transport is restricted by Bcrp1.     

Interaction between flavonoids and the blood-brain barrier: in vitro studies

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity.     

Brain-to-blood elimination of 24S-hydroxycholesterol from rat brain is mediated by organic anion transporting polypeptide 2 (oatp2) at the blood-brain barrier

24S-Hydroxycholesterol (24S-OH-chol), a major cerebral cholesterol metabolite, is an endogenous ligand for the liver X receptor and is a potential stimulant of cholesterol release from glial cells. The elimination mechanism of 24S-OH-chol from the brain is one of the key issues for understanding cerebral cholesterol homeostasis. The purpose of the present study was to clarify the molecular mechanism of the elimination process of 24S-OH-chol across the blood–brain barrier (BBB). After an intracerebral injection in rats, [³H]24S-OH-chol was eliminated from the brain and the radioactivity derived from [³H]24S-OH-chol was detected in the plasma, while [³H]cholesterol was not significantly eliminated from the brain. Co-administration of unlabeled 24S-OH-chol significantly inhibited the [³H]24S-OH-chol elimination, while no inhibitory effect was seen at the same concentration of cholesterol. The [³H]24S-OH-chol elimination was inhibited by co-administration of probenecid, but not by benzylpenicillin. Pre-administration of digoxin completely inhibited the elimination. Xenopus laevis oocytes expressing rat oatp2 exhibited significant transport of [³H]24S-OH-chol, and this was inhibited by unlabeled 24S-OH-chol and digoxin, indicating that rat oatp2 transports 24S-OH-chol. These results are the first direct demonstration that 24S-OH-chol undergoes elimination from the brain to blood across the BBB via a carrier-mediated process, which involves oatp2 expressed at the BBB in rats.     

Evidence for an active transport of morphine-6-beta-d-glucuronide but not P-glycoprotein-mediated at the blood-brain barrier

Morphine-6-beta-d-glucuronide (M6G) is an active metabolite of morphine with high analgesic potency despite a low blood-brain barrier (BBB) permeability. The aim of the study was to elucidate its transport mechanism across the BBB. We first checked if M6G was effluxed by the P-glycoprotein (P-gp), as previously reported by others. Second, we investigated the role of anionic transporters like the multidrug resistance-associated protein mrp1 and the glucose transporter GLUT-1. The brain uptake of [14C]M6G was measured by the in situ brain perfusion technique in wild-type and deficient mice [mdr1a(-/-) and mrp1(-/-)], with and without probenecid, digoxin, PSC833 or d-glucose. No difference was found between P-gp and mrp1 competent and deficient mice. The brain uptake of [14C]M6G co-perfused with probenecid in wild-type mice was not significantly different from that found in group perfused with [14C]M6G alone. The co-perfusion of [14C]M6G with digoxin or PSC833 was responsible of a threefold decrease of its uptake in mdr1a competent and deficient mice, suggesting that another transporter than P-gp and sensitive to digoxin and PSC833, may be involved. The co-perfusion of [14C]M6G with d-glucose revealed a threefold decrease in M6G uptake. In conclusion, P-gp and mrp1 are not involved in the transport of M6G at the BBB level in contrast to GLUT-1 and a digoxin-sensitive transporter (probably oatp2), which can actively transport M6G but with a weak capacity.     

Microsphere embolism-induced endothelial nitric oxide synthase expression mediates disruption of the blood-brain barrier in rat brain

Microsphere embolism (ME)-induced up-regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells of brain microvessels was observed 2-48 h after ischemia. eNOS induction preceded disruption of the blood-brain barrier (BBB) observed 6-72 h after ischemia. In vascular endothelial cells, ME-induced eNOS expression was closely associated with protein tyrosine nitration, which is a marker of generation of peroxynitrite. Leakage of rabbit IgG from microvessels was also evident around protein tyrosine nitration-immunoreactive microvessels. To determine whether eNOS expression and protein tyrosine nitration in vascular endothelial cells mediates BBB disruption in the ME brain, we tested the effect of a novel calmodulin-dependent NOS inhibitor, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), which inhibits eNOS activity and, in turn, protein tyrosine nitration. Concomitant with inhibition of protein tyrosine nitration in vascular endothelial cells, DY-9760e significantly inhibited BBB disruption as assessed by Evans blue (EB) excretion. DY-9760e also inhibited cleavage of poly (ADP-ribose) polymerase as a marker of the apoptotic pathway in vascular endothelial cells. Taken together with previous evidence in which DY-9760e inhibited brain edema, ME-induced eNOS expression in vascular endothelial cells likely mediates BBB disruption and, in turn, brain edema.     

Selective gene silencing of rat ATP-binding cassette G2 transporter in an in vitro blood-brain barrier model by short interfering RNA

The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.     

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.