Brain-type glucose transporter (GLUT-1) is selectively localized to the blood-brain barrier. Studies with quantitative western blotting and in situ hybridization.
Brain-type glucose transporter (GLUT-1) is selectively localized to the blood-brain barrier. Studies with quantitative western blotting and in situ hybridization.
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DOI：
10.1016/s0021-9258(18)38267-x
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发表时间：
1990-10
期刊：
影响因子：
--
通讯作者：
W. Pardridge;R. Boado;Christine R. Farrell
中科院分区：
文献类型：
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作者：
W. Pardridge;R. Boado;Christine R. Farrell
通过定量Western blotting、细胞松弛素B结合和牛大脑皮层原位杂交，验证了GLUT-1葡萄糖转运体亚型在脑内毛细血管内皮细胞即血脑屏障(BBB)选择性表达的假说。纯化的人红细胞葡萄糖转运蛋白被用作定量Western blotting的标准，因为人红细胞和血脑屏障葡萄糖转运蛋白在电泳胶中的流动性是相同的。牛血脑屏障质膜上葡萄糖转运蛋白的浓度为10.8+/-0.9pmol/mgp(平均值+/-S.E.，n=6)。这个值与血脑屏障膜制剂中D-葡萄糖可置换的[~3H]细胞松弛素B结合的最大结合部位的估计值11.7+/-3.5pmol/mgp没有统计学差异。与GLUT-1基因385-932核苷酸对应的35S标记的反义和正义核糖核酸探针的原位杂交实验表明，反义探针与脑微血管内皮细胞有显著的杂交，但在神经束上的杂交没有大于35S标记的正义探针的杂交。这些研究结果与以下结论一致：(A)血脑屏障上的葡萄糖转运蛋白结合位点基本上100%可以由GLUT-1亚型解释；(B)原位杂交研究证实了先前的Northern印迹分析，并表明GLUT-1基因选择性地表达于脑内微血管内皮细胞，在活体神经元或神经胶质细胞中表达极少。
The hypothesis that the GLUT-1 glucose transporter isoform is expressed selectively in brain at the capillary endothelium, i.e. the blood-brain barrier (BBB), was tested by using quantitative Western blotting, cytochalasin B binding, and in situ hybridization in bovine brain cortex. Purified human red cell glucose transporter was used as the standard for quantitative Western blots, because the mobility of the human erythrocyte and BBB glucose transporters in electrophoretic gels was identical. The concentration of immunoreactive glucose transporter in bovine BBB plasma membranes was 10.8 +/- 0.9 pmol/mgp (mean +/- S.E., n = 6). This value was not statistically different from the estimate of the maximal binding sites of D-glucose-displaceable [3H]cytochalasin B binding in the BBB membrane preparations, 11.7 +/- 3.5 pmol/mgp. In situ hybridization experiments using 35S-labeled antisense and sense riboprobes corresponding to nucleotides 385-932 of the GLUT-1 cDNA showed prominent hybridization of the antisense probe over brain microvascular endothelium, but no hybridization over neuropil greater than that found with the 35S-labeled sense probe. These studies are consistent with the following conclusion: (a) essentially 100% of the glucose transporter binding sites at the BBB can be accounted for by the GLUT-1 isoform; (b) in situ hybridization studies confirm previous Northern blot analysis and indicate the GLUT-1 gene is expressed selectively in microvascular endothelium in brain with minimal, if any, expression of this gene in neurons or glial cells in vivo.