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      Copper efflux from murine microvascular cells requires expression of the menkes disease Cu-ATPase.

      The Journal of Nutrition
      Adenosine Triphosphatases, chemistry, genetics, metabolism, Amino Acid Sequence, Animals, Base Sequence, Biological Transport, Blood-Brain Barrier, Brain, blood supply, Carrier Proteins, Cation Transport Proteins, Copper, DNA, Complementary, Endothelium, Vascular, enzymology, Gene Expression, Menkes Kinky Hair Syndrome, Mice, Microcirculation, Molecular Sequence Data, Polymerase Chain Reaction, RNA-Directed DNA Polymerase, Recombinant Fusion Proteins, Sequence Homology

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          Abstract

          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.

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