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

Lee, W. J.; Peterson, Darryl R.; Sukowski, E. J.; Hawkins, Richard A.

doi:10.1152/ajpcell.1997.272.5.c1552

Cited by 43 publications

(20 citation statements)

References 11 publications

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“…The proportion of luminal versus abluminal membrane was determined in each fraction by measurement of ␥-glutamyltranspeptidase activity (luminal) and System A amino acid transport activity (abluminal) as previously described (8). B, glucose transport activity was determined by equilibrium exchange (11). C, the five fractions (10 g of membrane protein/lane) were analyzed for GLUT1 protein by Western blot using three antibodies: 1) rabbit polyclonal anti-C-terminal peptide (20 amino acids, 472-492) (29); 2) rabbit polyclonal antibody raised against the purified human erythrocyte GLUT1 glucose transporter, H-sera (19,20); and 3) rabbit polyclonal antibody raised against amino acids in the intracellular loop, kindly provided by Dr. Baldwin.…”

Section: Resultsmentioning

confidence: 99%

“…The antibodies used were: 1) C, rabbit polyclonal anti-C-terminal peptide (20 amino acids, 472-492) (17); 2) H, rabbit polyclonal antibody raised against the purified human erythrocyte GLUT1 glucose transporter (H-sera (19,20)); and 3) loop, a rabbit polyclonal antibody raised against residues 231-246 of the central cytoplasmic loop of GLUT1 (21), kindly provided by Dr. Steve Baldwin, Leeds University, Leeds, United Kingdom. Glucose transport was measured as described by Lee et al (11).…”

Section: Methodsmentioning

confidence: 99%

“…It has generally been agreed that GLUT1 is the sole mediator of glucose transport across the mammalian BBB and that the polarity of glucose transport has been derived from the observed asymmetry in the distribution of transporter numbers in the two membranes (12). However, actual measurements of transport activities in isolated luminal and abluminal membrane vesicles suggested equivalent levels of transport (11). Because the latter study did not include any specific measure of GLUT1 concentration in these vesicles, we undertook a comprehensive characterization of the glucose transport properties of these membrane vesicles by a variety of techniques.…”

Section: Blood-brain Barrier Glut1mentioning

confidence: 99%

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Glucose Transporter Asymmetries in the Bovine Blood-Brain Barrier

Simpson¹,

Vannucci

DeJoseph

et al. 2001

Journal of Biological Chemistry

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The transport of glucose across the mammalian bloodbrain 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 The mammalian brain depends on a continuous supply of glucose for normal function. The delivery of circulating glucose to the brain requires transport across the endothelial cells of the cerebral microvessels that constitute the blood-brain barrier (BBB).1 Glucose transport across the BBB is mediated by the highly glycosylated, 55-kDa form of the facilitative glucose transporter protein, GLUT1 (1, 2). The capacity for facilitated glucose transport across both the luminal (blood-facing) and abluminal (brain-facing) membranes of the endothelial cell was confirmed by electron microscopy immunolocalization studies, which reported GLUT1 protein in both luminal and abluminal membranes as well as in intracellular vesicles (3, 4). Most studies support an asymmetric distribution of GLUT1 among these three compartments: 11% luminal membrane, 44% abluminal membrane/vesicles, and 45% intracellular membrane/ vesicles for rat (4) with similar distributions reported for rabbit (5) and human (6). This apparent difference in GLUT1 concentrations between luminal and abluminal membranes could convey a polarity to BBB glucose transport favoring transport from the blood, across the endothelial cell, and into the brain. In addition, the existence of the intracellular pool and the potential for transporter recruitment and redistribution between membranes has been proposed as a mechanism underlying the acute modulations in brain glucose transport observed in vivo (7). The question of the rate-determining step in BBB glucose transport has been difficult to resolve because of the inability to study the transport properties of the luminal and abluminal membranes separately. Hawkins and colleagues (8 -10) developed a methodology to separate luminal and abluminal membranes to specifically address the kinetics of glucose and amino acid transport across the respective membranes. Utilizing this preparation, Lee et al. (11) ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Blood-brain Barrier Glut1mentioning

confidence: 99%

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Glucose Transporter Asymmetries in the Bovine Blood-Brain Barrier

Simpson¹,

Vannucci

DeJoseph

et al. 2001

Journal of Biological Chemistry

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“…Isolation of vesicles from these luminal and abluminal plasma membranes of brain capillary endothelial cells are used to characterize drug/substrate transport processes across BBB. These studies on drugs/substrates fulfill a number of purposes such as measuring their transport, determining initial rate of transport, distinguishing binding and transport, measuring kinetic constants and determining the distribution of transport activities (81-83). This method has the advantage of measuring the actual disposition of the substrate across the BBB membrane.…”

Section: Predictive Non-cell Based Modelsmentioning

confidence: 99%

In Vitro Cerebrovascular Modeling in the 21st Century: Current and Prospective Technologies

et al. 2014

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The blood-brain barrier (BBB) maintains the brain homeostasis and dynamically responds to events associated with systemic and/or rheological impairments (e.g., inflammation, ischemia) including the exposure to harmful xenobiotics. Thus, understanding the BBB physiology is crucial for the resolution of major central nervous system CNS) disorders challenging both health care providers and the pharmaceutical industry. These challenges include drug delivery to the brain, neurological disorders, toxicological studies, and biodefense. Studies aimed at advancing our understanding of CNS diseases and promoting the development of more effective therapeutics are primarily performed in laboratory animals. However, there are major hindering factors inherent to in vivo studies such as cost, limited throughput and translational significance to humans. These factors promoted the development of alternative in vitro strategies for studying the physiology and pathophysiology of the BBB in relation to brain disorders as well as screening tools to aid in the development of novel CNS drugs. Herein, we provide a detailed review including pros and cons of current and prospective technologies for modelling the BBB in vitro including ex situ, cell based and computational (in silico) models. A special section is dedicated to microfluidic systems including micro-BBB, BBB-on-a-chip, Neurovascular Unit-on-a-Chip and Synthetic Microvasculature Blood-Brain Barrier.

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“…*, p Ͻ 0.05, significantly different from rNaGLT1 (Student's paired t test with Bonferroni's correction). and kidney, possess activity for Na ϩ -dependent glucose transport (29,30). It remains to be clarified whether rNaGLT1 or its analog takes part in glucose transport in such tissues or not.…”

Section: Fig 8 Concentration and Namentioning

confidence: 99%

Cloning and Characterization of a Novel Na+-dependent Glucose Transporter (NaGLT1) in Rat Kidney

Horiba

Masuda

Takeuchi

et al. 2003

Journal of Biological Chemistry

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To identify novel transporters in the kidney, we have constructed an mRNA data base composed of 1000 overall clones by random sequencing of a male rat kidney cDNA library. After a BLAST search, ϳ40% of the clones were unknown and/or unannotated and were screened by measuring the uptake of various compounds using Xenopus oocytes. One clone stimulated the uptake of ␣-methyl-D-glucopyranoside and therefore was termed rat Na ؉ -dependent glucose transporter 1 (rNaGLT1). The rNaGLT1 cDNA (2173 bp) has an open reading frame encoding a 484-amino acid protein, showing <22% homology to known SGLT and GLUT glucose transporters. ␣-Methyl-D-glucopyranoside uptake by rNaGLT1 cRNAinjected oocytes showed saturability, with an apparent K m of 3.7 mM and a coupling ratio of 1:1 with Na ؉ . rNa-GLT1 mRNA was expressed predominantly in the kidney upon Northern blot analysis and reverse transcription-PCR. Reverse transcription-PCR in microdissected nephron segments revealed that rNaGLT1 mRNA was primarily localized in the proximal tubules. A clear signal corresponding to rNaGLT1 protein was recognized in the brush-border (but not basolateral) membrane fraction by immunoblot analysis. The rNaGLT1 mRNA level in the kidney was significantly higher than rat SGLT1 and SGLT2 mRNA levels. These findings suggest that rNaGLT1 is a novel Na ؉ -dependent glucose transporter with low substrate affinity that mediates tubular reabsorption of glucose.

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

Cited by 43 publications

References 11 publications

Glucose Transporter Asymmetries in the Bovine Blood-Brain Barrier

Glucose Transporter Asymmetries in the Bovine Blood-Brain Barrier

In Vitro Cerebrovascular Modeling in the 21st Century: Current and Prospective Technologies

Cloning and Characterization of a Novel Na+-dependent Glucose Transporter (NaGLT1) in Rat Kidney

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