Glutamine transport at the blood-brain and blood-cerebrospinal fluid barriers

Glutamine has multiple physiological and pathophysiological roles in the brain. Because of their position at the interface between blood and brain, the cerebral capillaries and the choroid plexuses that form the blood-brain barriers (BBB) and blood-cerebrospinal fluid (CSF) barriers, have the potential to influence brain glutamine concentrations. Despite this, there has been a paucity of data on the mechanisms and polarity of glutamine transport at these barrier tissues. In situ brain perfusion in the rat, indicates that blood to brain L-[14C]glutamine transport at the blood-brain barrier is primarily mediated by a pH-dependent, Na(+)-dependent, System N transporter, but that blood to choroid plexus transport is primarily via a pH-independent System N transporter and a Na(+)-independent carrier that is not System L. Transport studies in isolated rat choroid plexuses and primary cultures of choroid plexus epithelial cells indicate that epithelial L-[14C]glutamine transport is polarized (apical uptake>basolateral) and that uptake at the apical membrane is mediated by pH dependent System N transporters (identified as SN1 and SN2 by polymerase chain reaction) and the Na(+)-independent System L. Blood-brain barrier System N transport is markedly effected by cerebral ischemia and may be a good marker of endothelial cell dysfunction. The multiple glutamine transporters at the blood-brain and blood-CSF barriers may have role in meeting the metabolic needs of the brain and the barrier tissues themselves. However, it is likely that the main role of these transporters is removing glutamine, and thus nitrogen, from the brain.     

Specific AHNAK expression in brain endothelial cells with barrier properties

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. Because disruption of the BBB may contribute to many brain disorders, they are of considerable interests in the identification of the molecular mechanisms of BBB development and integrity. We here report that the giant protein AHNAK is expressed at the plasma membrane of endothelial cells (ECs) forming specific blood-tissue barriers, but is absent from the endothelium of capillaries characterized by extensive molecular exchanges between blood and extracellular fluid. In the brain, AHNAK is widely distributed in ECs with BBB properties, where it co-localizes with the tight junction protein ZO-1. AHNAK is absent from the permeable brain ECs of the choroid plexus and is down-regulated in permeable angiogenic ECs of brain tumors. In the choroid plexus, AHNAK accumulates at the tight junctions of the choroid epithelial cells that form the blood-cerebrospinal fluid (CSF) barrier. In EC cultures, the regulation of AHNAK expression and its localization corresponds to general criteria of a protein involved in barrier organization. AHNAK is up-regulated by angiopoietin-1 (Ang-1), a morphogenic factor that regulates brain EC permeability. In bovine cerebral ECs co-cultured with glial cells, AHNAK relocates from the cytosol to the plasma membrane when endothelial cells acquire BBB properties. Our results identify AHNAK as a protein marker of endothelial cells with barrier properties.     

Functional characterization of rat brain-specific organic anion transporter (Oatp14) at the blood-brain barrier: high affinity transporter for thyroxine

Oatp14/blood-brain barrier-specific anion transporter 1 (Slc21a14) is a novel member of the organic anion transporting polypeptide (Oatp/OATP) family. Northern blot analysis revealed predominant expression of Oatp14 in the brain, and Western blot analysis revealed its expression in the brain capillary and choroid plexus. Immunohistochemical staining indicated that Oatp14 is expressed in the border of the brain capillary endothelial cells. When expressed in human embryonic kidney 293 cells, Oatp14 transports thyroxine (T4; prothyroid hormone) (Km = 0.18 mum), as well as amphipathic organic anions such as 17beta estradiol-d-17beta-glucuronide (Km = 10 mum), cerivastatin (Km = 1.3 mum), and troglitazone sulfate (Km = 0.76 mum). The uptake of triiodothyronine (T3), an active form produced from T4, was significantly greater in Oatp14-expressed cells than in vector-transfected cells, but the transport activity for T3 was approximately 6-fold lower that for T4. The efflux of T4, preloaded into the cells, from Oatp14-expressed cells was more rapid than that from vector-transfected cells (0.032 versus 0.006 min-1). Therefore, Oatp14 can mediate a bidirectional transport of T4. Sulfobromophthalein, taurocholate, and estrone sulfate were potent inhibitors for Oatp14, whereas digoxin, p-aminohippurate, or leukotriene C4, or organic cations such as tetraetheylammonium or cimetidine had no effect. The expression levels of Oatp14 mRNA and protein were up- and down-regulated under hypo- and hyperthyroid conditions, respectively. Therefore, it may be speculated that Oatp14 plays a role in maintaining the concentration of T4 and, ultimately, T3 in the brain by transporting T4 from the circulating blood to the brain.     

Characterization of two splice variants of human organic anion transporting polypeptide 3A1 isolated from human brain

In the present study we isolated two splice variants of organic anion transporting polypeptide 3A1 (OATP3A1_v1 and OATP3A1_v2) from human brain. OATP3A1_v2 lacks 18 amino acids (aa) at the COOH-terminal end (692 aa) but is otherwise similar in sequence to OATP3A1_v1 (710 aa). OATP3A1_v1 exhibits a wide tissue distribution, with expression in testis, various brain regions, heart, lung, spleen, peripheral blood leukocytes, and thyroid gland, whereas OATP3A1_v2 is predominantly expressed in testis and brain. On the cellular and subcellular levels OATP3A1_v1 could be immunolocalized in testicular germ cells, the basolateral plasma membrane of choroid plexus epithelial cells, and neuroglial cells of the gray matter of human frontal cortex. Immunolocalization of OATP3A1_v2 included Sertoli cells in testis, apical and/or subapical membranes in choroid plexus epithelial cells, and neurons (cell bodies and axons) of the gray and white matter of human frontal cortex. The rodent ortholog Oatp3a1 was also widely distributed in rat brain, and its localization included somatoneurons as well as astroglial cells. Transport studies in cRNA-injected Xenopus laevis oocytes and in stably transfected Chinese hamster ovary FlpIn cells revealed a similar broad substrate specificity for both splice variants. Transported substrates include prostaglandin (PG)E₁ and PGE₂, thyroxine, and the cyclic oligopeptides BQ-123 (endothelin receptor antagonist) and vasopressin. These studies provide further evidence for the involvement of OATPs in oligopeptide transport. They specifically suggest that OATP3A1 variants might be involved in the regulation of extracellular vasopressin concentration in human brain and thus might influence the neuromodulation of neurotransmission by cerebral neuropeptides such as vasopressin. peptide; transport; neuron     

Glutaric aciduria type I and methylmalonic aciduria: Simulation of cerebral import and export of accumulating neurotoxic dicarboxylic acids in in vitro models of the blood–brain barrier and the choroid plexus

Intracerebral accumulation of neurotoxic dicarboxylic acids (DCAs) plays an important pathophysiological role in glutaric aciduria type I and methylmalonic aciduria. Therefore, we investigated the transport characteristics of accumulating DCAs – glutaric (GA), 3-hydroxyglutaric (3-OH-GA) and methylmalonic acid (MMA) – across porcine brain capillary endothelial cells (pBCEC) and human choroid plexus epithelial cells (hCPEC) representing in vitro models of the blood–brain barrier (BBB) and the choroid plexus respectively. We identified expression of organic acid transporters 1 (OAT1) and 3 (OAT3) in pBCEC on mRNA and protein level. For DCAs tested, transport from the basolateral to the apical site (i.e. efflux) was higher than influx. Efflux transport of GA, 3-OH-GA, and MMA across pBCEC was Na⁺-dependent, ATP-independent, and was inhibited by the OAT substrates para-aminohippuric acid (PAH), estrone sulfate, and taurocholate, and the OAT inhibitor probenecid. Members of the ATP-binding cassette transporter family or the organic anion transporting polypeptide family, namely MRP2, P-gp, BCRP, and OATP1B3, did not mediate transport of GA, 3-OH-GA or MMA confirming the specificity of efflux transport via OATs. In hCPEC, cellular import of GA was dependent on Na⁺-gradient, inhibited by NaCN, and unaffected by probenecid suggesting a Na⁺-dependent DCA transporter. Specific transport of GA across hCPEC, however, was not found. In conclusion, our results indicate a low but specific efflux transport for GA, 3-OH-GA, and MMA across pBCEC, an in vitro model of the BBB, via OAT1 and OAT3 but not across hCPEC, an in vitro model of the choroid plexus.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na(+)/K(+)-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na⁺/K⁺-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Immunoreactivity for GABA,GAD65, GAD67 and Bestrophin-1 in the Meninges and the Choroid Plexus: Implications for Non-Neuronal Sources for GABA in the Developing Mouse Brain

Neural progenitors in the developing neocortex, neuroepithelial cells and radial glial cells, have a bipolar shape with a basal process contacting the basal membrane of the meninge and an apical plasma membrane facing the lateral ventricle, which the cerebrospinal fluid is filled with. Recent studies revealed that the meninges and the cerebrospinal fluid have certain roles to regulate brain development. γ-aminobutyric acid (GABA) is a neurotransmitter which appears first during development and works as a diffusible factor to regulate the properties of neural progenitors. In this study, we examined whether GABA can be released from the meninges and the choroid plexus in the developing mouse brain. Immunohistochemical analyses showed that glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67), both of which are GABA-synthesizing enzymes, are expressed in the meninges. The epithelial cells in the choroid plexus express GAD65. GABA immunoreactivity could be observed beneath the basal membrane of the meninge and in the epithelial cells of the choroid plexus. Expression analyses on Bestrophin-1, which is known as a GABA-permeable channel in differentiated glial cells, suggested that the cells in the meninges and the epithelial cells in the choroid plexus have the channels able to permeate non-synaptic GABA into the extracellular space. Further studies showed that GAD65/67-expressing meningeal cells appear in a manner with rostral to caudal and lateral to dorsal gradient to cover the entire neocortex by E14.5 during development, while the cells in the choroid plexus in the lateral ventricle start to express GAD65 on E11–E12, the time when the choroid plexus starts to develop in the developing brain. These results totally suggest that the meninges and the choroid plexus can work as non-neuronal sources for ambient GABA which can modulate the properties of neural progenitors during neocortical development.     

The choroid plexus removes beta-amyloid from brain cerebrospinal fluid

beta-Amyloid (Abeta) concentration in the cerebrospinal fluid (CSF) of the brain may be regulated by the choroid plexus, which forms a barrier between blood and brain CSF. Abeta uptake from CSF was determined as its volume of distribution (V(D)) into isolated rat choroid plexus tissue. The V(D) of [125I]Abeta1-40 was corrected by subtraction of the V(D) of [14C]sucrose, a marker for extracellular space and diffusion. Abeta uptake into choroid plexus was time and temperature dependent. Uptake of [125I]Abeta was saturable. Abeta uptake was not affected by addition of transthyretin or apolipoprotein E3. In studies with primary culture monolayers of choroidal epithelial cells in Transwells, Abeta permeability across cells, corrected by [(14)C]sucrose, was greater from the CSF-facing membrane than from the blood-facing membrane. Similarly, cellular accumulation of [125I]Abeta was concentrative from both directions and was greater from the CSF-facing membrane, suggesting a bias for efflux. Overall, these results suggest the choroid plexus selectively cleanses Abeta from the CSF by an undetermined mechanism(s), potentially reducing Abeta from normal brains and the brains of Alzheimer's disease patients.     

Localization of organic anion transporting polypeptide 3 (oatp3) in mouse brain parenchymal and capillary endothelial cells

Organic anion transporting polypeptide 3 (oatp3) transports various CNS-acting endogenous compounds, including thyroid hormones and prostaglandin E2, between extra- and intracellular spaces, suggesting a possible role in CNS function. The purpose of this study was to clarify the expression and localization of oatp3 in the mouse brain. RT-PCR analysis revealed that oatp3 mRNA is expressed in brain capillary-rich fraction, conditionally immortalized brain capillary endothelial cells, choroid plexus, brain and lung, but not in liver or kidney, where oatp1, 2 and 5 mRNAs were detected. Immunohistochemical analysis with anti-oatp3 antibody suggests that oatp3 protein is localized at the brush-border membrane of mouse choroid plexus epithelial cells. Furthermore, intense immunoreactivity was detected in neural cells in the border region between hypothalamus and thalamus, and in the olfactory bulb. Immunoreactivity was also detected in brain capillary endothelial cells in the cerebral cortex. These localizations in the mouse brain suggest that oatp3 plays roles in blood-brain and -cerebrospinal fluid barrier transport of organic anions and signal mediators, and in hormone uptake by neural cells.     

Asymmetric localization of Notch2 on the microvillous surface in choroid plexus epithelial cells

Notch family molecules are transmembrane receptors that play various roles in contact-dependent cell–cell interactions in a wide range of organs. In the brain, Notch2, but not the other members of Notch, is expressed in the choroid plexus at an exceptionally high level. We immunohistochemically examined the cellular and subcellular localization of Notch2 protein in the choroid plexus using confocal and electron microscopy. Unexpectedly, Notch2 was asymmetrically localized on the microvillous surface of epithelial cells in the choroid plexus of both postnatal and adult rats. This localization pattern of Notch2 suggests its novel and unknown role independent of contact with adjacent cells in the choroid plexus. In organotypic cultures of the choroid plexus, the addition of anti-Notch2 antibody resulted in deformation of microvilli in epithelial cells, which suggests a role of Notch2 in the maintenance of the microvillous structure in choroid plexus epithelial cells.     

Brain induces the expression of an early cell surface marker for blood-brain barrier-specific endothelium.

Capillaries derived from the perineural vascular plexus invade brain tissue early in embryonic development. Considerably later they differentiate into blood-brain barrier (BBB)-forming blood vessels. In the chick, the BBB as defined by impermeability for the protein horseradish peroxidase develops around embryonic day 13. We have previously found that brain endothelial cells start to express a number of proteins at around the same time, suggesting that these proteins play a role in BBB function. Here we describe a 74 kd protein defined by the monoclonal antibody HT7 that is expressed on the surface of chick embryonic blood cells and brain endothelial but on no other endothelial cells. This protein is not detectable on early embryonic brain endothelium, but is expressed by these cells on embryonic day 10. It is absent in choroid plexus endothelial cells which represent permeable fenestrated endothelial cells. The antigen is expressed on choroid plexus epithelium which is the site of the blood-cerebrospinal fluid barrier. Since it is also found in basolateral membranes of kidney tubules, it may be involved in specific carrier mechanisms. Embryonic mouse brain tissue transplanted on the chick chorio-allantoic membrane induces the expression of this antigen on endothelial cells derived from the chorio-allantois. Brain tissue can therefore induce in endothelial cells in vivo the expression of a molecule characteristic of brain endothelium.     

Expression of facilitative glucose transporter in rat liver and choroid plexus. A histochemical study in native cryostat sections.

Two isoforms of facilitative glucose transporters (GLUT), namely the erythroid/brain-type GLUT 1 and the liver-type GLUT 2, were demonstrated in native cryostat sections of normal rat liver and brain by immunofluorescence and a very sensitive immunoalkaline phosphatase reaction. Fixation with 0.1% alcoholic periodic acid resulted in an excellent localization of GLUT 2 in liver and GLUT 1 in brain. GLUT 1 in liver, however, could successfully be demonstrated after fixation with 1% alcoholic formaldehyde. GLUT 2 occurred in all hepatocytes as a basolateral membrane protein with a gradient of high expression in the periportal area and a lower one in the perivenous part. The first layer of hepatocytes adjacent to the hepatic vein coexpressed GLUT 1. In addition, GLUT 1 could be detected in the smooth muscle layer of the portal vein and in the apical and lateral plasma membrane of the bile duct epithelium. In brain, GLUT 1 showed a high expression in the microvessels, the ependyma and in the basal plasma membrane of choroid plexus epithelial cells. The blood capillaries associated with the choroidal epithelium were, however, negative for GLUT 1. The importance of the new findings in this study for the physiological role of the respective facilitative glucose transport proteins is discussed.     

Identification of the Cationic Amino Acid Transporter (System y+) of the Rat Blood-Brain Barrier

Abstract: Cationic amino acids are transported from blood into brain by a saturable carrier at the blood-brain barrier (BBB). The transport properties of this carrier were examined in the rat using an in situ brain perfusion technique. Influx into brain via this system was found to be sodium independent and followed Michaelis-Men-ten kinetics with half-saturation constants (K_m) of 50–100 μM and maximal transport rates of 22–26 nmol/min/g for L-lysine, L-arginine, and L-ornithine. The kinetic properties matched that of System y⁺, the sodium-independent cationic amino acid transporter, the cDNA for which has been cloned from the mouse. To determine if the cloned receptor is expressed at the BBB, we assayed RNA from rat cerebral microvessels and choroid plexus for the presence of the cloned transporter mRNA by RNase protection. The mRNA was present in both cerebral microvessels and choroid plexus and was enriched in microvessels 38-fold as compared with whole brain. The results indicate that System y⁺ is present at the BBB and that its mRNA is more densely expressed at cerebral microvessels than in whole brain.     

Expression of facilitative glucose transporter in rat liver and choroid plexus

Summary Two isoforms of facilitative glucose transporters (GLUT), namely the erythroid/brain-type GLUT 1 and the liver-type GLUT 2, were demonstrated in native cryostat sections of normal rat liver and brain by immunofluorescence and a very sensitive immunoalkaline phosphatase reaction. Fixation with 0.1% alcoholic periodic acid resulted in an excellent localization of GLUT 2 in liver and GLUT 1 in brain. GLUT 1 in liver, however, could successfully be demonstrated after fixation with 1% alcoholic formaldehyde. GLUT 2 occurred in all hepatocytes as a basolateral membrane protein with a gradient of high expression in the periportal area and a lower one in the perivenous part. The first layer of hepatocytes adjacent to the hepatic vein coexpressed GLUT 1. In addition, GLUT 1 could be detected in the smooth muscle layer of the portal vein and in the apical and lateral plasma membrane of the bile duct epithelium. In brain, GLUT 1 showed a high expression in the microvessels, the ependym and in the basal plasma membrane of choroid plexus epithelial cells. The blood capillaries associated with the choroidal epithelium were, however, negative for GLUT 1. The importance of the new findings in this study for the physiological role of the respective facilitative glucose transport proteins is discussed.     

Temporal expression profiles of organic anion transport proteins in placenta and fetal liver of the rat

Physiological cholestasis linked to immature hepatobiliary transport systems for organic anions occurs in rat and human neonates. In utero, the placenta facilitates vectorial transfer of certain fetal-derived solutes to the maternal circulation for elimination. We compared the ontogenesis of organic anion transporters in the placenta and the fetal liver of the rat to assess their relative abundance throughout gestation and to determine whether the placenta compensates for the late maturation of transporters in the developing liver. The mRNA of members of the organic anion transporting polypeptide (Oatp) superfamily, the multidrug resistance protein (Mrp) family, one organic anion transporter (OAT), and the bile acid carriers Na(+)-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) was quantified by real-time PCR. The most abundant placental transporters were Oatp4a1, whose mRNA increased 10-fold during gestation, and Mrp1. Mrp1 immunolocalized predominantly to epithelial cells of the endoplacental yolk sac, suggesting an excretory role that sequesters fetal-derived solutes in the yolk sac cavity, and faintly to the basal syncytiotrophoblast surface. The mRNA levels of Oatp2b1, Mrp3, and Bsep in the placenta exceeded those in the fetal liver until day 20 of gestation, suggesting that the fetus relies on placental clearance of substrates when expression in the developing liver is low. Mrp3 immunolocalized to the epithelium of the endoplacental yolk sac and less abundantly in the labyrinth zone and endothelium of the maternal arteries. The placental expression of Oatp1a1, Oatp1a4, Oatp1a5, Oatp1b2, Oat, Ntcp, Mrp2, and Mrp6 was low.     

Blood-brain barrier transport of pramipexole, a dopamine D2 agonist

The blood-brain barrier (BBB) transport of pramipexole, a potent dopamine receptor agonist with high efficacy for Parkinson's disease, was mainly characterized using immortalized rat brain capillary endothelial cells (RBEC)1 as an in vitro BBB model. [(14)C]Pramipexole uptake by RBEC1 was dependent on temperature and pH, but not sodium ion concentration or membrane potential. The uptake was inhibited by several organic cations including pyrilamine. Mutual inhibition was observed between pramipexole and pyrilamine. In addition, [(14)C]pramipexole uptake was stimulated by preloading unlabeled pramipexole. RT-PCR analysis for organic cation transporters (rOCT1-3, rOCTN1-2) in RBEC1 was performed. The mRNA level of rOCTN2 was the highest, followed by rOCTN1, while expression of rOCT1, rOCT2 and rOCT3 was negligible. The brain uptake of [(14)C]pramipexole, which was measured by the in situ rat brain perfusion technique, was significantly inhibited by unlabeled pramipexole. These results suggest that pramipexole is, at least in part, transported across the BBB by an organic cation-sensitive transporter. The pramipexole transport in RBEC1 was pH-dependent, but sodium- and membrane potential-independent.     

A critical role for pannexin-1 in activation of innate immune cells of the choroid plexus

Epiplexus cells are a population of innate immune cells in the choroid plexus of the brain ventricles. They are thought to contribute to the immune component of the blood-cerebrospinal-fluid-barrier (BCSFB). Here we have developed a novel technique for studying epiplexus cells in acutely isolated, live and intact choroid plexus. We show that epiplexus cells are potently activated by exogenous ATP, increasing their motility within the tissue. This ATP-induced chemokinesis required activation of pannexin-1 channels, which are expressed by the epithelial cells of the choroid plexus and not the epiplexus cells themselves. Furthermore, ATP acts at least in part through the P2X4 ionotropic purinergic receptor. Thus, the resident immune cells of the choroid plexus appear to be in communication with the epithelial cells through pannexin-1 channels.