Direct uptake of copper from ceruloplasmin by cells with and without CTR1, and potential involvement of a copper reductase

Abstract

The blood plasma protein, ceruloplasmin (Cp), is well known for its role in enhancing efflux of iron from certain cells; but it also has other functions, and its copper enters cells all over the mammalian organism, as shown by i.v. infusion of 67Cu‐labeled Cp into rats. In current studies, the ability of Cp to directly donate its Cu to cultured cells was investigated. 64Cu‐labeled human Cp secreted by HepG2 cells was purified and incubated in serum‐free medium with polarized human mammary epithelial cell monolayers with tight junctions (PMC42 cells), applied to the basolateral (“blood” side). Most of the radioactivity entered the cells and 30–50% of what entered was released into the apical (“milk”) fluid. Larger amounts of 64Cu‐Cp were produced in mice and purified for incubation with mouse embryonic fibroblasts (MEF) that did and did not express Ctr1 (kindly provided by Dennis Thiele, Duke University). 64Cu from Cp accumulated in both types of MEFs at a linear rate over 3 h, and was internalized, most being present in the cytosol. Uptake was somewhat less in Ctr1−/− MEFs. Uptake of Cp‐64Cu (which is not exchangeable) was inhibited by an excess of Cu(I) and Cu(II)‐histidine; and the presence of external Cu(II) reductase activity was verified. We conclude that Ctr1 and another (unknown) transporter take up Cu from Cp, and that this is mediated by a Cu reductase. Supported in part by US PHS Grant HD46949.