Bovine Brain Microvessel Endothelial Cells Research Articles

Infection of bovine brain microvessel endothelial cells with Cowdria ruminantium elicits IL-1 beta, -6, and -8 mRNA production and expression of an unusual MHC class II DQ alpha transcript.

Cowdria ruminantium is a bacterial parasite that infects ruminants, causing an acute and often fatal disease. These obligate intracellular Gram-negative bacteria preferentially infect neutrophils and vascular endothelial cells, especially in the brain. The present study was performed with bovine brain microvessel endothelial cells in culture, infected by C. ruminantium in the presence or absence of IFN-gamma. Infection induced the production of IL-1 beta, -6, and -8 mRNAs, and this effect was potentiated by IFN-gamma. A semi-quantitative PCR analysis indicated that similar amounts of IL-1 beta and IL-6 mRNAs were produced in response to C. ruminantium infection and to treatment with 30 to 40 ng/ml LPS. In addition, although IFN-gamma induced the synthesis of an MHC class II DQ alpha transcript (1.3 kb), an unusual transcript (1.5 kb) was induced by infection and not after LPS treatment. Infection did not affect MHC class I, class II DQ beta, and invariant chain mRNA levels. The present results suggest that C. ruminantium infection raises the immune activity of brain endothelial cells in vitro and that only part of this response can be attributed to LPS. One can hypothesize that cerebral endothelium in vivo efficiently contributes, by MHC Ag expression and production of ILs, to the activation and/or recruitment of leukocytes to the brain and thus plays an active role in the pathogenesis of cowdriosis and in the immune response to this pathogen.

培養牛脳微小血管内皮細胞を用いたｉｎ ｖｉｔｒｏ ＢＢＢモデルにおけるｌｉｐｏｓｏｍｅ封入ｃｉｓｐｌａｔｉｎ透過性の基礎的検討

Liposomes have been proved to be effective as carriers of water-soluble drugs into the central nervous system, but the mechanisms by which water-soluble drugs encapsulated in liposomes permeate the blood-brain barrier(BBB) are not clear. In order to elucidate the mechanisms, the authors investigated the permeability of liposome encapsulated cisplatin [cis-diamminedichloroplatinum (II), CDDP] across in vitro BBB model compared with that of free CDDP. This in vitro BBB model is consisted of primarily cultured bovine brain microvessel endothelial cell (BMEC) monolayer onto the collagen-coated polycarbonate membrane which is put in side by side diffusion cells. This in vitro BBB model matches in vitro BBB in morphology and enzymatic activity, and has been used in many transport studies. The result showed that liposome encapsulated CDDP permeated across BMEC monolayers and the permeability was significantly attenuated by metabolic inhibitor, 2-deoxyglucose treatment (p<0.05 by Student's t-test). Our findings confirmed that CDDP encapsulated in liposomes could be transported through BBB by way of energy-dependent transcellular pathway.

Leucine Enkephalin Effects on Paracellular and Transcellular Permeation Pathways Across Brain Microvessel Endothelial Cell Monolayers

Leucine enkephalin (YGGFL) effects on markers for transcellular and paracellular permeation across the blood-brain barrier (BBB) were investigated in vitro with bovine brain microvessel endothelial cell (BMEC) monolayers in primary culture. Intact YGGFL, but not metabolites of YGGFL, stimulated BMEC uptake of lucifer yellow (LY), a marker for fluid-phase endocytosis, in a concentration-dependent manner. However, D-[Ala2]-leucine enkephalin (YAGFL), a YGGFL analogue that altered BMEC monolayer permeability, had no effect on LY uptake. In part, these results suggested that YGGFL's effects on fluid-phase uptake might not relate directly to enhanced BMEC transcellular permeability in the presence of the peptide. The measurement of the fluorescence anisotropy of membrane-bound diphenyl-hexatriene probes did not show substantial peptide-induced changes in membrane lipid packing order (i.e., membrane fluidity) and indicated a limited role for membrane perturbations in YGGFL-induced changes in BMEC monolayer permeability. Conversely, the apparent permeability coefficients showed size-dependent YGGL-induced alterations for passage of membrane-impermeant substances across BMEC monolayers. The apparent permeability coefficients of low-molecular-weight (low-mol-wt) molecules (mannitol, sucrose, and fluorescein) were increased on exposure to YGGFL. The apparent permeability coefficients for high-mol-wt molecules, FITC dextran conjugates (4, 20, and 71.6 Kd), were not affected by exposure to YGGFL. Transmission electron micrographs of lanthanum (Stoke's radius, 10 A) exclusion from BMEC intercellular junctions supported these observations. Collectively, results from this study suggest that YGGFL enhanced BMEC permeability either by altering paracellular openings or through formation of a small pore in the monolayers to allow preferential penetration of low-mol-wt or small molecular size (< 10 A) substances.

Identification and distribution of insulin receptors on cultured bovine brain microvessel endothelial cells: possible function in insulin processing in the blood-brain barrier.

The binding of 125I-insulin to primary cultures of bovine brain microvessel endothelial cells was examined. Insulin binding was both time and temperature dependent and inhibited by excess unlabeled insulin. Furthermore, the specific binding of insulin was polarized to the apical side of the cell monolayers. Upon binding, the labeled insulin was internalized, with approximately 70% resistant to acid wash over a 90-min period. The inhibition of insulin internalization observed with cell monolayers exposed to either phenylarsine oxide or unlabeled insulin suggests a receptor-mediated endocytic process. Furthermore, the ability of chloroquine to reduce the metabolism of insulin indicates a significant portion of the peptide is processed through a lysosomal pathway. In contrast to the fluid-phase endocytosis marker, Lucifer yellow, as much as 65% of internalized insulin undergoes apical to basolateral trancytosis in brain microvessel endothelial cells. While most of the effluxed insulin was degraded, as assessed by trichloroacetic acid precipitation, the results of the present study suggest insulin receptors within the brain microvasculature may be involved in the processing and transport of blood-borne insulin.

Changes in brain microvessel endothelial cell monolayer permeability induced by adrenergic drugs

Brain microvessel endothelial cell monolayers have been shown to be a suitable blood-brain barrier in vitro system to study adrenergic regulation of permeability. We tested adrenergic drugs on bovine brain microvessel endothelial cell monolayer permeability to a biomembrane impermeant molecule, sodium fluorescein. Endonenous catecholamines noradrenaline and adrenaline were tested as well as the α-adrenoceptor agonist phenylephrine, the β-adrenoceptor agonist clenbuterol and the α-adrenoceptor antagonist prazosin. Results showed an α-adrenoceptor mediated increase and a β-adrenoceptor mediated decrease in monolayer permeability. Both α- and β-adrenoceptor mediated changes in permeability were abolished by inhibiting fluid-phase pinocytosis, either by vincristine or by avoiding bovine brain microvessel endothelial cell's energy utilization. The reverse transport (i.e., from brain to blood side) was also influenced by adrenergic drugs; α- or β-adrenoceptor stimulation induced a permeability-reducing effect. We conclude that α-adrenoceptor stimulation increases bovine brain microvessel endothelial cell monolayer permeability and that β-adrenoceptor stimulation has the opposite effect. Reverse transport results obtained with β-adrenoceptor stimulation seem controversial and deserve further study. These results also support in vivo findings that demonstrated adrenergic influences on blood-brain barrier permeability.

Uptake of surfactant-coated poly(methyl methacrylate)-nanoparticles by bovine brain microvessel endothelial cell monolayers

The objective of this study was to determine the influence of coating of radiolabeled poly(methylmethacrylate) nanoparticles (PMMA nanoparticles) with surfactants on their uptake by bovine microvessel endothelial cell cultures (BMEC cultures). For this purpose, BMEC cultures were grown in 24-well culture plates, where they formed confluent monolayers 10–12 days after seeding. The nanoparticle suspensions were then incubated with the cell cultures at 37°C, and the radioactivity within the cell cultures and the supernatant was measured after 30 min, 2 h and 6 h. The rates of uptake of the coated nanoparticles and of a control group of uncoated nanoparticles were compared.

Expression of a widely distributed, novel sulfated membrane glycoprotein by bovine endothelia and certain transporting epithelia.

We generated monoclonal antibodies (MAbs) against cultured bovine brain microvessel endothelial cells (BMEC) for use as probes to study membrane protein traffic and polarity. One MAb recognized a heterogeneous family of acidic sulfoglycoproteins called gp4A4 with molecular weights of 50-65 KD and 85 KD. Gp4A4 is a long-lived integral membrane protein which resides mostly at the plasma membrane, and a portion appears to be in equilibrium with an intracellular pool via endocytosis. Gp4A4 is expressed by many endothelial cells, except for fenestrated capillaries in choroid plexus, and specific epithelial cells in bile duct, kidney, and choroid plexus. A comparison of indirect immunoperoxidase and immunofluorescence detection using semi-thin cryosections gave contrasting results on the apparent distribution of gp4A4 on the apical and basolateral membranes of cerebral endothelia and choroid plexus epithelia. Immunogold labeling of ultra-thin cryosections showed that gp4A4 was expressed by the apical and basolateral membrane domains of BMEC and choroid plexus epithelia. This was consistent with the results using indirect immunofluorescence microscopy. On an average, gp4A4 expression by cerebral endothelia was not asymmetric and was considerably variable between capillaries. These results emphasize the need to compare several different techniques in assessing polarized expression of cell surface antigens in vivo.

Leucine-enkephalin metabolism in brain microvessel endothelial cells

The metabolism of leucine-enkephalin was investigated in primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. Leucine-enkephalin was hydrolyzed slowly with less than 40% of the peptide metabolized following a 5-h incubation with intact BMEC monolayers at 37°C. Following separation and extraction of BMEC enzymes into cytosolic and membrane-bound fractions, leucine-enkephalin was observed to be labile in the presence of both cytosolic and membrane-associated enzymes. A much greater concentration of enkephalin-hydrolyzing enzyme was associated with the cytosolic fraction. Resulting metabolite profiles for leucine-enkephalin appeared to be the result of interactions of the peptide with primarily aminopeptidases and angiotensin-converting enzyme. Our results suggested that extensive metabolism of leucine-enkephalin soley by BMECs in the cerebrovasculature would require internalization by the cells and presentation to cytosolic enzymes.

Hydrogen bonding potential as a determinant of the in vitro and in situ blood-brain barrier permeability of peptides.

With the exception of various central nervous system (CNS)-required nutrients for which specific, saturable transport systems exist, the passage of most water-soluble solutes through the blood-brain barrier (BBB) is believed to depend largely on the lipid solubility of the solutes. Most peptides, therefore, do not enter the CNS because of their hydrophilic character. Recently, utilizing homologous series of model peptides and Caco-2 cell monolayers as a model of the intestinal mucosa, it was concluded that the principal determinant of peptide transport across the intestinal cellular membrane is the energy required to desolvate the polar amide bonds in the peptide (P. S. Burton et al., adv. Drug Deliv. Rev. 7:365, 1991). To determine whether this correlation can be extended to the BBB, the permeabilities of the same peptides were determined using an in vitro as well as an in situ BBB model. The peptides, blocked on the N- and C-terminal ends, consisted of D-phenylalanine (F) residues: AcFNH2, AcF2NH2, AcF3NH2, AcF2(NMeF)NH2, AcF(NMeF)2NH2, Ac(NMeF)3NH2, and Ac(NMeF)3NHMe. A good correlation among the permeabilities of these model peptides across the bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the BBB, and their permeabilities across the BBB in situ was observed (r = 0.928, P < 0.05). The permeabilities of these peptides did not correlate with the octanol-buffer partition coefficients of the peptides (r = 0.389 in vitro and r = 0.155 in situ; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin fragments as a carrier for peptide delivery across the blood-brain barrier.

The possibility of using insulin (INS), which is transported into the brain by receptor-mediated transcytosis, as a peptide carrier for delivery across the blood-brain barrier (BBB) was investigated. After mice received an i.v. injection of horseradish peroxidase (HRP, M.W., 40,000) conjugated with INS, the HRP activity in the brain was higher than that after HRP injection. Since INS-HRP lowered the blood glucose level, we prepared insulin fragments by chemical and enzymatic procedures in an effort to find a carrier with no hypoglycemic activity. Seven fragments were synthesized taking the binding regions into consideration, but none showed any receptor binding affinity in cultures of bovine brain microvessel endothelial cells (BMEC). However, the fragment (F007) obtained by trypsin digestion showed high affinity and scarcely any hypoglycemic activity in mice even at a dose ten times the effective dose of insulin. These results suggest that this fragment may be useful as a carrier to transport therapeutic peptides across the BBB.

Specific Uptake of Succinylated Proteins Via a Scavenger Receptor-Mediated Mechanism in Cultured Brain Microvessel Endothelial Cells

Cellular uptake of succinylated catalase (Suc-CAT; Mw 227 kDa), bovine serum albumin (Suc-BSA; Mw 70 kDa), superoxide dismutase (Suc-SOD; Mw 34 kDa) and soybean trypsin inhibitor (Suc-STI; Mw 21 kDa) was studied usin9 primary cultures of bovine brain microvessel endothelial cells (BMECs) developed as an in vitro model of the blood-brain barrier. Large succinylated proteins (Suc-CAT, Suc-BSA) were taken up by BMECs whereas significant uptake was not observed for native proteins and small succinylated proteins (Suc-SOD, Suc-STI). Uptake of Suc-BSA was significantly inhibited at 4°C and in the presence of endocytosis inhibitors. Large succinylated proteins, maleylated BSA and dextran sulfate also showed competitive inhibition against Suc-BSA uptake while small succinylated proteins and carboxymethyl dextran did not show any effect. These results indicate that microvessel endothelial cells obtained from the brain endocytose succinylated proteins via a scavenger receptor-mediated mechanism for polyanions, and in addition, the importance of molecular weight or total numbers of anionic charges per one molecule of proteins is suggested. Usefulness of direct succinylation of proteins for their delivery to the brain capillary endothelium is thus demonstrated.

Catecholamines stimulate MHC class I, class II, and invariant chain gene expression in brain endothelium through different mechanisms.

Bovine brain microvessel endothelial cells in primary culture retain a differentiated phenotype reminiscent of the in vivo blood-brain barrier endothelium. The IFN-gamma-induced surface expression of MHC class II molecules on those cells is stimulated by catecholamines through a cAMP-independent mechanism. We report that both the induction of MHC class II molecule expression by IFN-gamma and its potentiation by isoproterenol, a catecholamine analog, are preceded by increases of steady-state levels of the corresponding mRNA. Similar results were obtained for the regulation of invariant chain expression. In addition, isoproterenol alone is able to increase class I mRNA levels, but, in contrast to what is observed for class II expression, this effect appears to be mainly mediated by cAMP. These studies demonstrate that, in bovine brain microvessel endothelial cells, catecholamines up-regulate MHC class I and class II Ag, as well as invariant chain with different kinetics and through different mechanisms and suggest that these regulatory effects occur at the transcriptional level.

Comparison of Fluvastatin and Lovastatin Blood-Brain Barrier Transfer Using In Vitro and In Vivo Methods

We measured the permeability of fluvastatin, lovastatin, and its pharmacologically active hydroxy acid (HA) across the blood-brain barrier (BBB) with three different techniques. The brain uptake index (BUI) technique yielded values for fluvastatin and HA below the limit of detection of this method; in contrast, brain extraction of lovastatin was high: 57 and 7% with addition of buffer and human plasma, respectively. Brain perfusion studies in rats indicated that when fluvastatin and HA were exposed for a longer time to the BBB, small but measurable brain uptake occurred, with permeability coefficients of 2.5 x 10(-4) and 1.4 x 10(-4) cm/min, for fluvastatin and HA, respectively, whereas the permeability coefficient of lovastatin was 2.3 x 10(-2) cm/min. An in vitro BBB model consisting of a primary culture of confluent monolayers of bovine brain microvessel endothelial cells yielded very similar values: 7.2 x 10(-4) cm/min for fluvastatin and 1.3 x 10(-3) cm/min for HA and a permeability coefficient of 1.1 x 10(-2) cm/min for lovastatin. The results of the different methods show that lovastatin crosses the BBB to a much greater extent than does fluvastatin or HA. Although lovastatin undergoes hydrolytic conversion to HA, it subsists in blood for sufficient time to cross the BBB. Therefore, lovastatin and fluvastatin are expected to be different in their central side effect profile, as was observed in patients treated for hypercholesterolemia. This finding supports the hypothesis that sleep disturbances due to some cholesterol biosynthesis inhibitors might be correlated with their abilities to cross the BBB.

Biotin uptake and transport across bovine brain microvessel endothelial cell monolayers.

Primary cultures of bovine brain microvessel endothelial cells (BMECs) were used to characterize blood-brain barrier (BBB) uptake and transport of biotin. Both the uptake and the transcellular transport of either radiolabeled or fluorescein-conjugated biotin by confluent monolayers of BMECs were measured. Biotin uptake (Km = 123 microM) and bidirectional transport across BMEC monolayers was a saturable process and could be competed for by unlabeled biotin, biocytin, and biotinmethyl ester. Pantothenic and nonanoic acid were found not to be effective competitors for either biotin uptake or transport. The metabolic inhibitor, 2-deoxyglucose, had only small effects on the saturable apical-to-basolateral transport and apical uptake of biotin by BMECs. In contrast, basolateral-to-apical transport of biotin was substantially attenuated by 2-deoxyglucose pretreatment. Results supported the existence of specific and saturable uptake and efflux carrier systems for biotin in BMEC monolayers. The function of these systems was dependent to some degree on the metabolic status of the BMECs. Our findings confirm the existence of a biotin uptake system at the BBB in vivo and provide the first indication of an efflux system for biotin in BMECs.

Characterization of Dextromethorphan and Dextrorphan Uptake by a Putative Glutamic Acid Carrier and Passive Diffusion across Brain Microvessel Endothelium

AbstractThe mechanisms of uptake and transcellular passage of dextromethorphan (DM) and its major metabolite dextrorphan (DX) across the endothelial component of the blood–brain barrier have been investigated with primary cultures of bovine brain microvessel endothelial cells (BMECs). The uptake of [14C]DM and [14C]DX by BMECs was observed to be temperature-sensitive and saturable, with approximate Km's of 0.12 and 0.29 mM and Vmax's of 9.2 and 11.0 pmol/mg/min, respectively. The BMEC uptake of [14C] DM was inhibited half-maximally by approximately 0.57 mM L-glutamic acid, 0.71 mM N-methyl-d-asparatate (NMDA), and 0.99 mM DL-threo-β-hydroxyaspartic acid. The BMEC uptake of [14C]DX was inhibited half-maximally by approximately 0.48 mM L-glutamic acid, 1.50 mM NMDA, and 0.69 mM DL-threo-β-hydroxyaspartic acid. Conversely, the bidirectional passage of DM and DX across confluent BMEC monolayers occurred at a faster rate but was neither saturable nor inhibited by high concentrations of glutamic acid, NMDA, or ...

Application of cultured endothelial cells of the brain microvasculature in the study of the blood-brain barrier

The endothelial cells of the brain microvasculature provide both physical and enzymatic barriers to the exchange of molecules between the extracellular fluid environment of the brain and the systemic circulation. To better understand these barrier properties and the factors that influence them at the cellular level, an in vitro model of the blood-brain barrier has been developed using primary cultures of bovine brain microvessel endothelial cells. The focus of the present paper is on the procedures associated with the isolation, culturing, and maintenance of bovine brain microvessel endothelial cells. Experimental applications of the cell culture model in studying the blood-brain barrier and efforts directed at improving the current cell culture model are also discussed.

The effect of protein binding on ivermectin uptake by bovine brain microvessel endothelial cells.

The effect of albumin binding on ivermectin uptake and transfer across the endothelial component of the blood-brain barrier (BBB) was determined with an in vitro model comprised of bovine brain microvessel endothelial cell (BMEC) monolayers. Cellular uptake of ivermectin was limited in the absence of albumin and 90% inhibited in the presence of 10% albumin. Cell membrane association of ivermectin, as followed by fluorescent probe labelling, was observed only at high (micromolar) concentrations of the drug. Membrane association was about 75% inhibited in the presence of albumin. Similarly, transfer across BMEC monolayers was restricted, equivalent to that of BBB impermeant markers. Unlike the uptake studies, however, albumin had little effect on the transfer of ivermectin across BMEC monolayers. These results support recent in vivo findings on the distribution of ivermectin into the brain and suggest that ivermectin has only a limited affinity for the endothelial component of the normal BBB.

Permeability of bovine brain microvessel endothelial cells in vitro: Barrier tightening by a factor released from astroglioma cells

It has been shown both in vivo and in culture that astrocytes communicate with brain microvessel endothelial cells (BMECs) to induce many of the blood—brain barrier characteristics attributed to these unique cells. However, the results using cultured cells are conflicting as to whether this communication is dependent upon cell-cell contact. In this study we used primary cultures of bovine BMECs grown as monolayers on polycarbonate filters to study the formation of the barrier in vitro and examine its modulation by rat C 6 glioma cells. Effects were examined by treating postconfluent BMEC monolayers with medium conditioned continually by C 6 cells from the basolateral side to mimic the in vivo orientation. Cell monolayer integrity was assessed using electrical resistance and by measuring diffusion of uncharged molecules. BMEC monolayers form a functionally polarized and leaky barrier, with maximal resistance of 160 Ω · cm 2 and significant flux of molecules of molecular weight less than 350 Da. Treatment with rat or human astroglioma cells rather than pericytoma cells or transformed fibroblasts results in a concentration-dependent 200–440% increase in electrical resistance and a coincident 50% decrease in permeability to sucrose and dextran (70 kDa). The decrease in passive diffusion is most likely due to a change in tight junctions and not to transcellular vesicular traffic. The findings support that astroglioma cells release one or more signals that are required for cultured BMECs to express a “differentiated” phenotype associated with a tighter barrier, increased γ-glutamyl transpeptidase activity, and decreased pinocytic activity. The relative ease and quickness of this culture system makes it amenable to studies on cell-cell interaction and regulation of barrier maintenance.

Adhesion and Migration of Human Polymorphonuclear Leukocytes Across Cultured Bovine Brain Microvessel Endothelial Cells

Adhesion and migration of human polymorphonuclear leukocytes (PMN) across cerebral endothelium were studied in an in vitro model consisting of monolayers of bovine brain microvessel endothelial cells (BBMEC) grown on amniotic stroma or collagen membranes. Polymorphonuclear leukocytes were stimulated to adhere to and migrate across confluent BBMEC monolayers in response to chemotactic gradients produced by formyl-methionyl-leucyl phenylalanine (fMLP), leukotriene B4 (LTB4) or acetyl-glyceryl-ether-phosphorylcholine (AGEPC) placed below the cultures. Under these conditions, PMN adherence to endothelium was 2-10-fold greater than that observed in the absence of chemoattractants or in the presence of equal concentrations of chemoattractants below and above the cultures. Transendothelial migration of PMN occurred rapidly and at focal points across the monolayers. Scanning and electron microscopic studies revealed that stimulated PMN migrated across the monolayers by first adhering to the apical surface of the endothelium and then moving between adjacent endothelial cells. Following their migration, PMN accumulated beneath the endothelium. The overlying endothelial monolayers showed no evidence of disruption and the interendothelial junctions appeared intact at the end of the migration period. We conclude that this in vitro system reproduces the endothelial cell-leukocyte interactions occurring during acute inflammation in vivo and should provide a useful in vitro model for studying the molecular mechanisms underlying these interactions in inflammatory diseases of the central nervous system.

Hexose uptake in primary cultures of bovine brain microvessel endothetial cells. I. Basic characteristics and effects of d-glucose and insulin

The basic characteristics of hexose uptake and regulation of the glucose transporter (GLUT1) by d-glucose and insulin were studied in primary cultures of bovine brain microvessel endothelial cells (BMECs). A non-metabolizable glucose analog, 3- O-[ 3H]methyl- d-glucose ([ 3H]3MG), was used as a model substrate, and the uptake was studied using BMECs grown in tissue culture plates. Uptake of [ 3H]3MG was equilibrative, temperature-dependent, and independent of sodium. The uptake also decreased gradually with culture age from 7 to 13 days. Saturation kinetics were observed for [ 3H]3MG uptake and the apparent K m and V max values were determined to be 13.2 mM and 169 nmol/mg per min, respectively. Pre-incubation with high concentrations of d-glucose and 3MG accelerated [ 3H]3MG uptake by BMECs by a counter-transport mechanism. d-Glucose, 2-deoxy- d-glucose, d-mannose, d-xylose, d-galactose and d-ribose showed significant competitive inhibition with (3H]3MG, whereas l-glucose, d-fructose, and sucrose did not affect [ 3H]3MG uptake by BMECs. [ 3H]3MG uptake was inhibited significantly by cytochalasin B and phloretin but not by phlorizin, 2,4-dinitrophenol, or ouabain. d-Glucose starvation of BMECs by incubation with d-glucose-free media for 24 h resulted in a significant increase (40–70%) in uptake of [ 3H]3MG compared with control conditions (7.3 mM d-glucose). Low d-glucose treatments (2.43 and 1.83 mM) for 7 days induced a slight but significant increase (20%) in [ 3H]3MG uptake, while long-term high glucose treatments (25 mM) showed no significant effect on [ 3H]3MG uptake irrespective of exposure time. The increase in [ 3H]3MG accumulation following d-glucose starvation was dependent upon starvation time (12 to 48 hr) and protein synthesis. Refeeding of d-glucose (7.3 mM) to d-glucose-starved BMECs resulted in a return of [ 3H]3MG uptake to control levels in 48 h. The d-glucose-starvation-induced increase in [ 3H]3MG uptake was shown to result from an increase in V max; the K m remained constant. In addition, d-glucose-starved BMECs were shown to have an increased level of GLUT1 using an antibody against human GLUT1 and an enzyme-linked immunosorbent assay (ELISA). The increased uptake following d-glucose starvation was not significantly affected by the presence of l-glucose, was partially impaired by the presence of d-galactose, d-fructose, and d-xylose, and was completely inhibited by the presence of d-mannose and 3MG. Furthermore, preincubation of BMECs with insulin (10 μg/ml) for 20 min did not affect the uptake of [ 3H]3MG or 2-deoxy d-[ 3H]glucose ([ 3H]2DG). The present study demonstrated that hexoses can be taken up by cultured bovine BMECs by a carrier-mediated, facilitated diffusio mechanism, similar to the mechanism observed in the blood-brain barrier (BBB) and that the uptake by bovine BMECs was regulated by low levels of d-glucose but not by high levels of d-glucose or by insulin.