Qian Y, Tiffany-Castiglioni E, Welsh J, Harris E D
Departments of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
J Nutr. 1998 Aug;128(8):1276-82. doi: 10.1093/jn/128.8.1276.
Previously, we showed that the transport of Cu by PC12 pheochromocytoma cells and C6 glioma cells correlated with the expression of a Cu-transporting ATPase (Atp7a) that has been linked to Menkes disease. Here, we show that cerebrovascular endothelial (CVE) cells that comprise the blood-brain barrier (BBB) also express the gene for the Cu-ATPase. By using reverse transcription-polymerase chain reaction (RT-PCR) and primers designed from mouse Atp7a cDNA, we amplified a 925-bp and a 760-bp cDNA fragment from two extreme regions of Atp7a mRNA from murine CVE cells; 777 bp of the 925-bp fragment and 677 bp of the 760-bp fragment had a 99.7 and 100% sequence homology, respectively, with mouse Atp7a cDNA. The 777-bp sequences covered the heavy metal binding (Hmb) domain and the 677-bp fragment coded for residues at the -COOH terminus of Atp7a. A functional analysis showed that Cu efflux was blocked by the sulfhydryl reagent p-chloromercuribenzoate (p-CMB), a potential inhibitor of Atp7a function. This study provides strong evidence that a Cu-ATPase in the BBB controls the penetration of Cu into the brain and that lesions to the Cu-ATPase in CVE cells are a primary cause of low brain Cu levels in Menkes disease.
此前，我们发现PC12嗜铬细胞瘤细胞和C6胶质瘤细胞对铜的转运与一种与门克斯病相关的铜转运ATP酶（Atp7a）的表达有关。在此，我们发现构成血脑屏障（BBB）的脑血管内皮（CVE）细胞也表达铜ATP酶基因。通过逆转录聚合酶链反应（RT-PCR）并使用从小鼠Atp7a cDNA设计的引物，我们从小鼠CVE细胞的Atp7a mRNA的两个极端区域扩增出一个925 bp和一个760 bp的cDNA片段；925 bp片段中的777 bp和760 bp片段中的677 bp分别与小鼠Atp7a cDNA具有99.7%和100%的序列同源性。777 bp序列覆盖重金属结合（Hmb）结构域，677 bp片段编码Atp7a羧基末端的残基。功能分析表明，铜外流被巯基试剂对氯汞苯甲酸（p-CMB）阻断，p-CMB是Atp7a功能的潜在抑制剂。这项研究提供了强有力的证据，表明血脑屏障中的铜ATP酶控制着铜进入大脑的渗透，并且CVE细胞中铜ATP酶的损伤是门克斯病患者脑铜水平低的主要原因。
