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...
We used [3H]cytochalasin B as a specific ligand to study the glucose transporter of the following tissue preparations: (a) microvessels derived from the cerebral cortex and cerebellum of the rat and pig, (b) particulate fractions of the cerebral cortex and cerebellum of the rat and pig, (c) lateral, third, and fourth ventricular choroid plexus of the pig, and (d) synaptosomes from the pig cerebral cortex. Specific, D-glucose-displaceable binding of [3H]cytochalasin B was present in all the preparations studied. This binding was saturable and displayed the kinetics of a single class of binding sites, similar to the glucose transporter found in other mammalian tissues. The density of the glucose transporter was much higher in cerebral and cerebellar microvessels and choroid plexus than either in crude particulate fractions of the cerebrum and cerebellum or in cerebral synaptosomes. These findings agree with the physiologic function of brain microvessels that transport glucose, not only for their own use, but also for the much greater mass of the entire brain. In the pig, the density of the glucose transporter in cerebral microvessels was significantly higher than in cerebellar microvessels. Irreversible photoaffinity labeling of the glucose transporter of synaptosomal membranes with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis demonstrated a single region of radioactivity that corresponded to a molecular mass of 60,000-64,000 daltons.
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