Identification and characterization of the glucose transporter of the blood-brain barrier by cytochalasin B binding and immunological reactivity.

Abstract

ABSTRACT[3H]Cytochalasin B was used as a ligand to identify and characterize the glucose transporter in cerebral microvessels of the rat and the pig. Specific cytochalasin B binding, defined as that fraction of the total binding that is stereospecifically displaced by excess (500 mM) D-glucose, is saturable. Kinetic studies of this specific binding to cerebral microvessel preparations shQwed a dissociation constant (Kd) of 0.65-0.88 ,IM and a maximal binding (Bmax) of 60-80 pmol/mg of protein. In comparison, the Bmax of particulate fractions of the cerebral cortex was about one-tenth that of cerebral microvessels. The ability of various hexoses to displace specific cytochalasin B binding to cerebral microvessels in vitro correlated well with the capability of these hexoses to cross the blood-brain barrier in vivo. Irreversible photoaffinity labeling of the glucose transporter of cerebral microvessels with cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis labeled a polypeptide(s) with a molecular weight of about 53,000. Antibodies prepared against the glucose transporter of human erythrocytes also reacted with a polypeptide(s) with a molecular weight of about 53,000 on electrophoresed preparations of cerebral microvessels. These results indicate that cerebral microvessels are richly endowed with a glucose transporter moiety of similar molecular weight and antigenic characteristics as the glucose transporter of human erythrocytes and other mammalian tissues.Under normal conditions, the central nervous system of mammals relies on a large and uninterrupted supply of Dglucose for its oxidative metabolism. The mammalian nervous system is also isolated from the systemic circulation by a unique capillary endothelium possessing tight cell junctions, which is referred to as the blood-brain barrier (BBB) (1, 2). Because this barrier is poorly permeable to polar molecules, Crone (3) suggested the existence of a carrier-mediated facilitated transport system iti brain capillary endothelium that enables D-glucose to cross the BBB. In recent years, a variety of studies using in vivo (4-7) and in vitro (8, 9) techniques has confirmed Crone's original ideas and has established that the transport of glucose by the endothelial cells of brain capillaries is saturable, stereospecific, nonconcentrative, nonenergy dependent, and not influenced by insulin. This subject has recently been extensively reviewed (10-12).The glucose transporter in human erythrocytes has been characterized by D-glucose-displaceable specific cytochalasin B binding [see review by Jones and Nickson (13)]. Similar techniques have been used to characterize glucose transporters in a variety of mammalian tissues (14-16). More recently, photoaffinity covalent labeling of cytochalasin B to the glucose transporter (17) has allowed further identification of the transport polypeptide (18,19). Immunological labeling with antiserum against the erythrocyte glucose transporter has also established the antigenic similarity of the glucose trans...

Glucose transporters in chromaffin cells: Subcellular distribution and characterization

“…Our results indicate the presence of a single class of saturable binding sites with a similar dissociation constant for each subcellular fraction. The maximal binding capacity of the P 10000 x g fraction present in chromaffin tissue correlates well with similar fractions from cerebral cortex and cerebellum from pig and rat brain [10,11]. When more enriched fractions from neuroblastoma and astrocytes were compared, the values reported were also very similar to that found in chromaffin tissue membrane preparations (12-16 pmol/mg protein) [25].…”

“…In all of the brain membranes the affinity constants are close to 0.3/zM [10]. Our results indicate the presence of a single class of saturable binding sites with a similar dissociation constant for each subcellular fraction.…”

“…The glucose transport and transporters in neural tissues have been characterized [10,11], even for chromaffin cells [12], and the stimulatory effects of secretagogues and insulin established [13].…”

Glucose-targeted niosomes deliver vasoactive intestinal peptide (VIP) to the brain

“…Umezawa and Eto (1988) reported that liposomes bearing aminophenyl mannoside were efficiently taken up into the mouse brain, suggesting that mannose could be recognized by the cells of the BBB. The BBB cells are known to express high levels of the glucose transporter GLUT1 (Dick et al, 1984). Therefore, an active targeting based on GLUT1 ligands could be a useful strategy for drug delivery in the brain.…”

“…Indeed, the large and uninterrupted energetic demand of the brain is provided almost exclusively by ␤-d-glucose. Furthermore, the glucose consumption of the brain amounts to about 30% of the total body glucose consumption (Dick et al, 1984;La Manna and Harik, 1985). This high level of cerebral glucose uptake suggests that the facilitative ␤-d-glucose transporter GLUT1 might be a useful carrier for efficient and selective glucose-targeted drug delivery to the brain.…”