LOCALIZATION OF GLUCOSE-TRANSPORTER GLUT-3 IN BRAIN - COMPARISON OF RODENT AND DOG USING SPECIES-SPECIFIC CARBOXYL-TERMINAL ANTISERA

The carboxyl-terminal amino acid sequences of the canine and gerbil gl ucose transporter GLUT3 were determined and compared to the published rat sequence. Eleven of 16 amino acids comprising the carboxyl terminu s of GLUT3 were found to be identical in rat and dog. However, the can ine sequence ''ATV'' substitutes for the rat sequence ''PGNA'' at the end of the molecule. The gerbil sequence has 12 of 16 amino acids iden tical to the rat, including the PGNA terminus. Based on these sequence s, four peptides were synthesized, and two polyclonal antisera (one to the canine sequence and one to the rat sequence) were raised to exami ne the distribution of GLUT3 in canine and rodent brain. Immunoblots o f brain membrane preparations showed that both antisera identified pep tide-inhibitable protein bands of molecular weight 45,000-50,000. Immu nocytochemical studies demonstrated that binding sites for these antis era were abundantly distributed in neuropil in all brain regions. Area s rich in synapses and areas surrounding microvessels exhibited especi ally high reactivity. GLUT3 reactivity was similarly distributed in ca nine and rodent brain, except at the blood-brain barrier. GLUT3 was no t detected in the blood-brain barrier in gerbil and rat but was presen t in many canine cerebral endothelial cells, particularly in cerebellu m and brain stem. The carboxyl-terminal antisera employed in this stud y exhibited high degrees of species specificity, indicating that the t hree or four terminal amino acids of the immunizing peptides (ATV and PGNA) are important epitopes for binding the polyclonal antibodies. Th ese antisera exhibited only minimal binding to brain tissue of non-tar get species, yet yielded similar staining patterns in neuropil of rode nt and canine brain. This finding provides strong evidence that the ob served staining patterns accurately reflect the distribution of GLUT3 in brain. In addition, the presence;of vascular GLUT3 in dog brain sug gests that the canine blood-brain barrier may be preferable to that of the rat as a model for studies of glucose transport relevant to human brain.