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.

Summary The effects of access to hay and of restricted feeding on the pharmacokinetics of flunixin administered orally to six healthy ponies were compared in a cross‐over study. No access to feed for a few hours before and after flunixin administration resulted in rapid absorption with a mean peak plasma concentration of 2.84 ± 0.28 μg/ml attained in an average time of 0.76 ± 0.18 h, followed by an exponential decline in plasma concentration. A lower peak plasma concentration was obtained when ponies had free access to hay before and after drug dosing. The mean maximum concentration (Cmax) was 1.30 ± 0.23 μug/ml and maximum time (tmax) was prolonged to a mean time of 7.66 ± 1.74 h. Free access to hay reduced and delayed the peak plasma concentration resulting in two or three separate concentration peaks in some ponies. The mean area under the plasma concentration‐time curve was not significantly different for the two feeding regimens.

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