Bovine Blood-brain Barrier Research Articles

KCa3.1 in the early stages of ischemic stroke (893.19)

In the early hours of ischemic stroke, a luminal Na‐K‐Cl cotransporter (NKCC) at the blood‐brain barrier (BBB) works with an abluminal Na/K pump to transport Na+ (with water following) from blood to brain. Ischemic factors (hypoxia, aglycemia and vasopressin) increase BBB NKCC activity in a manner dependent on elevation of intracellular Ca2+. Here, we identified the presence of the calcium‐activated KCa3.1 channel on cultured bovine BBB endothelial cells (CMEC). In airway and gastrointestinal epithelia, inhibition of KCa3.1 channels slows Cl‐ efflux and increases intracellular Cl‐ ([Cl‐]i). Since NKCC activity is highly sensitive to inhibition by [Cl‐]i increasing [Cl‐]i in BBB endothelial cells should reduce Na+ secretion into the brain during ischemic stroke. We therefore hypothesized that pharmacological blockade of KCa3.1 will reduce or prevent brain edema. The KCa3.1 blocker TRAM‐34 reduced Rb86 efflux in a dose‐dependent manner and inhibited vasopressin‐induced rises in CMEC [Ca2+]i MR spectroscopy and imaging studies in rat middle cerebral artery occlusion showed that pre‐treatment with TRAM‐34 (40mg/kg, IP) significantly slowed brain Na+ uptake and cytotoxic edema at a time when the BBB is still largely intact. Our findings suggest that KCa3.1 blockade constitutes an attractive approach for reducing edema in the early stages of ischemic stroke or during brain surgeries.Grant Funding Source: Supported by AHA & NIHGM

Pyroglutamate stimulates Na +-dependent glutamate transport across the blood–brain barrier

Regulation of Na +-dependent glutamate transport was studied in isolated luminal and abluminal plasma membranes derived from the bovine blood–brain barrier. Abluminal membranes have Na +-dependent glutamate transporters while luminal membranes have facilitative transporters. This organization allows glutamate to be actively removed from brain. γ-Glutamyl transpeptidase, the first enzyme of the γ-glutamyl cycle (GGC), is on the luminal membrane. Pyroglutamate (oxoproline), an intracellular product of GGC, stimulated Na +-dependent transport of glutamate by 46%, whereas facilitative glutamate uptake in luminal membranes was inhibited. This relationship between GGC and glutamate transporters may be part of a regulatory mechanism that accelerates glutamate removal from brain.

THE BLOOD-BRAIN BARRIER SODIUM-DEPENDENT MULTIVITAMIN TRANSPORTER: A MOLECULAR FUNCTIONAL IN VITRO-IN SITU CORRELATION

The molecular mechanism of biotin brain uptake was investigated using an in vitro bovine blood-brain barrier (BBB) cell model and an in situ mouse brain perfusion technique. A functional uptake/transport correlation of the in vitro and in situ characteristics of biotin uptake was investigated. Morphological and immunochemical characteristics (e.g., factor VIII expression) of the primary culture of brain microvessel endothelial cells (BMECs) were confirmed. Gene expression of the multidrug resistance (Mdr1) and sodium-dependent multivitamin (SMVT) transporters was also determined in BMECs. Biotin transport was saturable and Na(+)-dependent at the luminal side of the BBB. The estimated half-saturation concentrations (K(m)) of biotin uptake in vitro and in situ were 49.1 and 35.5 microM, respectively, supporting the presence of a carrier-mediated biotin transport system. Inhibition studies using various biotin derivatives and structural analogs demonstrated the structural requirements for biotin-SMVT interaction. Desthiobiotin and pantothenic acid significantly inhibited the uptake of biotin, whereas 2-iminobiotin and diaminobiotin were very weak inhibitors. Based on our results, there was a good correlation between the in vitro and in situ BBB models, suggesting that when a single membrane transporter is involved in substrate uptake, flexibility in choosing the experimental model can be afforded. The current results are also consistent with the suggestion that the properties of the BBB are likely to be organ-specific rather than species-specific. Further mechanistic and comparative studies are needed to validate these results. In conclusion, the in vitro transporter-based mechanism studies produced valuable molecular functional transport results that correlated well with in situ results.

Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability.

The blood-brain barrier (BBB) ensures the homeostasis of the brain microenvironment, mostly through complex tight junctions between brain endothelial cells that prevent the passage of hydrophilic molecules from blood to brain and vice versa. A recent study has shown in vivo that systemic administration of erythropoietin (Epo) protects against brain injury. Using an in vitro model of the bovine BBB, we observed that the expression of the Epo receptor is modulated by its ligand and hypoxic stimuli such as vascular endothelial growth factor (VEGF) treatment. In addition, Epo protects against the VEGF-induced permeability of the BBB, decreases the levels of endothelial nitric oxide synthase and restores junction proteins. The kinetic transport experiments revealed the capacity of Epo to cross the in vitro BBB in a saturable and specific way. Our results suggest a new mechanism for Epo-induced neuroprotection, in which circulating Epo controls and maintains the BBB through an Epo receptor signalling pathway and the re-establishment of cell junctions.

Glucose Transporter Asymmetries in the Bovine Blood-Brain Barrier

The transport of glucose across the mammalian blood-brain barrier is mediated by the GLUT1 glucose transporter, which is concentrated in the endothelial cells of the cerebral microvessels. Several studies supported an asymmetric distribution of GLUT1 protein between the luminal and abluminal membranes (1:4) with a significant proportion of intracellular transporters. In this study we investigated the activity and concentration of GLUT1 in isolated luminal and abluminal membrane fractions of bovine brain endothelial cells. Glucose transport activity and glucose transporter concentration, as determined by cytochalasin B binding, were 2-fold greater in the luminal than in the abluminal membranes. In contrast, Western blot analysis using a rabbit polyclonal antibody raised against the C-terminal 20 amino acids of GLUT1 indicated a 1:5 luminal:abluminal distribution. Western blot analysis with antibodies raised against either the intracellular loop of GLUT1 or the purified erythrocyte protein exhibited luminal:abluminal ratios of 1:1. A similar ratio was observed when the luminal and abluminal fractions were exposed to the 2-N-4[(3)H](1-azi-2,2,2,-trifluoroethyl)benzoxyl-1,3-bis-(d-mannos-4-yloxyl)-2-propylamine ([(3)H]ATB-BMPA) photoaffinity label. These observations suggest that either an additional glucose transporter isoform is present in the luminal membrane of the bovine blood-brain barrier or the C-terminal epitope of GLUT1 is "masked" in the luminal membrane but not in the abluminal membranes.

Selective expression of the large neutral amino acid transporter at the blood-brain barrier.

Amino acid supply in brain is regulated by the activity of the large neutral amino acid transporter (LAT) at the brain capillary endothelial cell, which forms the blood-brain barrier (BBB) in vivo. Bovine BBB poly(A)(+) RNA was isolated from 2.0 kg of fresh bovine brain and size fractionated on a sucrose density gradient, and a size-fractionated bovine BBB cDNA library in the pSPORT vector was prepared. The full-length cDNA encoding the bovine BBB LAT was isolated from this library, and the predicted amino acid sequence was 89-92% identical to the LAT1 isoform. The bovine BBB LAT1 mRNA produced a 10-fold enhancement in tryptophan transport into frog oocytes coinjected with bovine BBB LAT1 mRNA and the mRNA for 4F2hc, which encodes the heavy chain of the heterodimer. Tryptophan transport into the mRNA-injected oocytes was sodium independent and was specifically inhibited by other large neutral amino acids, and the K(m) of tryptophan transport was 31.5 +/- 5.5 microM. Northern blotting with the bovine BBB LAT1 cDNA showed that the LAT1 mRNA is 100-fold higher in isolated bovine brain capillaries compared with C6 rat glioma cells or rat brain, and the LAT1 mRNA was not detected in rat liver, heart, lung, or kidney. These studies show that the LAT1 transcript is selectively expressed at the BBB compared with other tissues, and the abundance of the LAT1 mRNA at the BBB is manyfold higher than that of transcripts such as the 4F2hc antigen, actin, or the Glut1 glucose transporter.

Glucose transport by isolated plasma membranes of the bovine blood-brain barrier.

Luminal and abluminal endothelial plasma membrane vesicles were isolated from bovine cerebral microvessels, the site of the blood-brain barrier. Glucose transport across each membrane was measured using a rapid-filtration technique. Glucose transport into luminal vesicles occurred by a stereospecific energy-independent transporter [Michaelis-Menten constant (K(m)) = 10.3 +/- 2.8 (SE) mM and maximal velocity (Vmax) = 8.6 +/- 2.0 nmol.mg protein(-1).min-1]. Kinetic analysis of abluminal vesicles also showed a transport system with characteristics similar to the luminal transporter (K(m) = 12.5 +/- 2.3 mM and Vmax = 10.0 +/- 1.0 nmol.mg protein-1.min-1). These functional, facilitative glucose transporters were symmetrically distributed between the luminal and abluminal membrane domains, providing a mechanism for glucose movement between blood and brain. The studies also revealed a Na-dependent transporter on the abluminal membrane with a higher affinity and lower capacity than the facilitative transporters (K(m) = 130 +/- 20 microM and Vmax = 1.59 +/- 0.44 nmol.mg protein-1.min-1. The abluminal Na-dependent glucose transporter is in a position to transport glucose from the brain extracellular fluid into the endothelial cells of the blood-brain barrier. The functional significance of its presence there remains to be determined.

Endothelin-1 as a mediator of endothelial cell-pericyte interactions in bovine brain capillaries.

Endothelial cells and pericytes are closely associated in brain capillaries. Together with astrocytic foot processes, they form the blood-brain barrier. Capillaries were isolated from bovine brain cortex. Pure populations of endothelial cells and pericytes were isolated and cultured in vitro. Polarized monolayers of endothelial cells preferentially secreted immunoreactive endothelin-1 (Et-1) at their abluminal (brain-facing) membrane. They did not express receptors for Et-1. Pericytes expressed BQ-123-sensitive ETA receptors for endothelins as evidenced by 125I-Et-1 binding experiments. These receptors were coupled to phospholipase C as demonstrated by intracellular calcium measurements using indo-1-loaded cells. Addition of Et-1 to pericytes induced marked changes in the cell morphology that were associated with a reorganization of F-actin and intermediate filaments. It is concluded that Et-1 is a paracrine mediator at the bovine blood-brain barrier and that capillary pericytes are target cells for endothelium-derived Et-1.

Role of Oxoproline in the Regulation of Neutral Amino Acid Transport across the Blood-Brain Barrier

Regulation of neutral amino acid transport was studied using isolated plasma membrane vesicles derived from the bovine blood-brain barrier. Neutral amino acids cross the blood-brain barrier by facilitative transport system L1, which may allow both desirable and undesirable amino acids to enter the brain. The sodium-dependent amino acid systems A and Bo,+ are located exclusively on abluminal membranes, in a position to pump unwanted amino acids out. gamma-Glutamyl transpeptidase, the first enzyme of the gamma-glutamyl cycle, is an integral protein of the luminal membrane of the blood-brain barrier. We demonstrate that oxoproline, an intracellular product of the gamma-glutamyl cycle, stimulates the sodium-dependent systems A and Bo,+ by 70 and 20%, respectively. Study of system A showed that 2 mM oxoproline increased the affinity for its specific substrate N-methylaminoisobutyrate by 50%. This relationship between the activity of the gamma-glutamyl cycle and system A transport may provide a short term regulatory mechanism by which the entry of potentially deleterious amino acids (i.e. neurotransmitters or their precursors) may be retarded and their removal from brain accelerated.

A sodium- and energy-dependent glucose transporter with similarities to SGLT1–2 is expressed in bovine cortical vessels

In order to investigate glucose transport at the blood-brain barrier (BBB), glucose transport properties were studied pharmacologically with a novel model system of inverted bovine brain cortical arteries. These vessels displayed glucose transport characteristics of facilitative glucose transporters (GLUT1–5) and of sodium- and energy-dependent glucose transporters (SGLT1–2). So far, glucose transport in the central nervous system (CNS) has only been shown to be achieved by facilitative transporters, in particular by GLUT1 at the blood-brain barrier and in glial cells and by GLUT3 in a subset of neurons. We report here that a SGLT-like transporter might partake in glucose transport at the bovine BBB, as indicated by immunocytochemical analysis and by Western immunoblot analysis of cultured bovine brain endothelial cells. A RNA protection assay revealed the presence of a SGLT-like gene fragment in rabbit cortex.

Molecular cloning of the bovine blood-brain barrier glucose transporter cDNA and demonstration of phylogenetic conservation of the 5′-untranslated region

The brain-type glucose transporter (GLUT-1) is a blood-brain barrier (BBB)-specific gene. The isolation of BBB-specific genes from total brain cDNA libraries is difficult because the brain capillary endothelium, which makes up the BBB in vivo, constitutes <0.2% of brain volume. Therefore, the present studies describe the preparation of a bovine brain capillary cDNA library in the λgt11 vector and the cloning of the BBB GLUT-1 cDNA. The cDNA sequenced was full length, as confirmed by primer extension analysis. The 5′-untranslated region (UTR) of the bovine GLUT-1 was 67 and 75% homologous with 5′-UTR sequences of the rabbit and human GLUT-1 through nucleotide overlaps of 116 and 159, respectively. A unique proline-rich sequence near the N-terminus of the bovine GLUT-1 was not found in other species and this correlated with marked immunoreactivity of the bovine, but not the human or rat, BBB GLUT-1 protein with an antiserum directed against the 15 amino acids at the N-terminus. In conclusion, these studies describe the cloning of the GLUT-1 cDNA from a BBB cDNA library. The extensive phylogenetic conservation of the 5′-UTR suggests the GLUT-1 gene may be subject to translational control in the regulation of BBB glucose transport.

Brain-type glucose transporter (GLUT-1) is selectively localized to the blood-brain barrier. Studies with quantitative western blotting and in situ hybridization.

The hypothesis that the GLUT-1 glucose transporter isoform is expressed selectively in brain at the capillary endothelium, i.e. the blood-brain barrier (BBB), was tested by using quantitative Western blotting, cytochalasin B binding, and in situ hybridization in bovine brain cortex. Purified human red cell glucose transporter was used as the standard for quantitative Western blots, because the mobility of the human erythrocyte and BBB glucose transporters in electrophoretic gels was identical. The concentration of immunoreactive glucose transporter in bovine BBB plasma membranes was 10.8 +/- 0.9 pmol/mgp (mean +/- S.E., n = 6). This value was not statistically different from the estimate of the maximal binding sites of D-glucose-displaceable [3H]cytochalasin B binding in the BBB membrane preparations, 11.7 +/- 3.5 pmol/mgp. In situ hybridization experiments using 35S-labeled antisense and sense riboprobes corresponding to nucleotides 385-932 of the GLUT-1 cDNA showed prominent hybridization of the antisense probe over brain microvascular endothelium, but no hybridization over neuropil greater than that found with the 35S-labeled sense probe. These studies are consistent with the following conclusion: (a) essentially 100% of the glucose transporter binding sites at the BBB can be accounted for by the GLUT-1 isoform; (b) in situ hybridization studies confirm previous Northern blot analysis and indicate the GLUT-1 gene is expressed selectively in microvascular endothelium in brain with minimal, if any, expression of this gene in neurons or glial cells in vivo.

Methyl Mercury Uptake Across Bovine Brain Capillary Endothelial Cells in Vitro: The Role of Amino Acids

Previous studies in the rat in vivo have demonstrated that co-injection of methyl mercury (MeHg) with L-cysteine into the common carotid artery enhances brain Hg levels following a single capillary pass through the CNS vasculature. In order to elucidate the relationship between MeHg transport and the neutral amino acid transport carrier system, regulatory aspects of MeHg transport across the bovine blood-brain barrier were investigated in isolated brain microvessel preparations. Following 1 hour co-incubations of 203Hg-MeHgCl with 0.1 mM L-cysteine at 37 degrees, 203Hg uptake by suspended microvessels was significantly increased (P less than 0.05) compared with controls. This enhanced capillary uptake of 203Hg was abolished by co-incubations of microvessels with 0.1 mM L-cysteine-L-methionine, or 0.1 mM L-cysteine plus AT-125 (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazolacetic acid), an irreversible inhibitor of gamma-glutamyl-transpeptidase. One hr co-incubations of bovine capillaries with 203Hg-MeHgCl and 0.1 mM D-cysteine at 37 degrees or 0.1 mM L-cysteine at 0 degrees did not increase rat of 203Hg uptake compare with controls. These results indicate that L-cysteine enhances the rate of capillary MeHg uptake. The accumulation of 203Hg in the bovine microvessels appears to be a carrier-mediated process. It is inhibited by L-methionine, a competitive substrate for neutral amino acid transport, and by AT-125. Capillary uptake of 203Hg is stereospecific to the L-enantiomorph of cysteine, suggesting selective uptake of MeHg across the blold-brain barrier. The data emphasize the relationship between the L-enantiomorph neutral amino acid carrier system and MeHg transport across the capillaries.

Relationship of octanol/buffer and octanol/water partition coefficients to transcellular diffusion across brain microvessel endothelial cell monolayers

The importance of lipid solubility on the rate of passage of solutes across the bovine blood-brain barrier (BBB) was studied with an in vitro BBB model. The model consisted of primary cultured bovine brain microvessel endothelial cell monolayers mounted in a side-by-side diffusion cell. Nine compounds (sucrose, mannitol, urea, glycerol, butyrate, thiourea, caffeine, antipyrine, propanolol) with different octanol/water partition coefficients were selected in order to provide a wide range of lipid solubility. Time- and concentration-dependence, and the effect of bovine serum albumin on the rate of transport of these compounds across the monolayers was determined. Consistent with in vivo studies, a significant positive correlation between transcellular diffusion and octanol/buffer or octanol/water partition coefficients was observed. The passage of these compounds across the monolayers was also concentration-dependent, non-saturable, and reduced in the presence of bovine serum albumin. Results demonstrate that these compounds undergo passive diffusion across the monolayers in which the rate of transfer across the monolayers depends directly on their lipid solubility and indirectly upon molecular size. In addition, serum albumin is an apparent factor in determining the passive permeability of the monolayers.