Secretion and uptake of copper via a small copper carrier in blood fluid.

Studies with Wilson disease model mice that accumulate excessive copper, due to a dysfunctional ATP7B "copper pump" resulting in decreased biliary excretion, showed that the compensatory increase in urinary copper loss was due to a small copper carrier (∼1 kDa) (SCC). We show here that SCC is also present in the blood plasma of normal and Wilson disease model mice and dogs, as determined by ultrafiltration and size exclusion chromatography (SEC). It is secreted by cultured hepatic and enterocytic cells, as determined by pretreatment with 67Cu nitrilotriacetate (NTA) or nonradioactive 5-10 µM Cu-NTA, and collecting and examining 3 kDa ultrafiltrates of the conditioned media, where a single major copper peak is detected by SEC. Four different cultured cell types exposed to the radiolabeled SCC all took up the 67Cu at various rates. Rates differed somewhat when uptake was from Cu-NTA. Uptake of SCC-67Cu was inhibited by excess nonradioactive Cu(I) or Ag(I) ions, suggesting competition for uptake by copper transporter 1 (CTR1). Knockout of CTR1 in fibroblasts reduced uptake rates by 60%, confirming its participation, but also involvement of other transporters. Inhibitors of endocytosis, or an excess of metal ions taken up by divalent metal transporter 1, did not decrease SCC-67Cu uptake. The results imply that SCC may play a significant role in copper transport and homeostasis, transferring copper particularly from the liver (but also intestinal cells) to other cells within the mammalian organism, as well as spilling excess into the urine in copper overload-as an alternative means of copper excretion.

Mobilization of iron from ferritin: new steps and details.

Much evidence indicates that iron stored in ferritin is mobilized through protein degradation in lysosomes, but concerns about this process have lingered, and the mechanistic details of its aspects are lacking. In the studies presented here, 59Fe-labeled ferritin was induced by preloading hepatic (HepG2) cells with radiolabeled Fe. Placing these cells in a medium containing desferrioxamine resulted in the loss of ferritin-59Fe, but adding high concentrations of reducing agents or modulating the internal GSH concentration failed to alter the rates of ferritin-59Fe release. Confocal microscopy showed that Fe deprivation increased the movement of ferritin into lysosomes and hyperaccumulation was observed when lysosomal proteolysis was inhibited. It also resulted in the rapid movement of DMT1 to lysosomes, which was inhibited by bafilomycin. Ferrihydrite crystals isolated from purified rat liver/spleen ferritin were solubilized at pH 5 and 7 by GSH, ascorbate, citrate and lysosomal fluids obtained from livers and J774a.1 macrophages. The inhibition of DMT1/Nramp2 and siRNA knockdown of Nramp1 each reduced the transfer of 59Fe from lysosomes to the cytosol; and hepatocyte-specific knockout of DMT1 in mice prevented the release of Fe from the liver responding to EPO treatment, but did not inhibit lysosomal ferritin degradation. We conclude that ferritin-Fe mobilization does not occur through changes in cellular concentrations of reducing/chelating agents but by the coordinated movement of ferritin and DMT1 to lysosomes, where the ferrihydrite crystals exposed by ferritin degradation dissolve in the lysosomal fluid, and the reduced iron is transported back to the cytosol via DMT1 in hepatocytes, and by both DMT1 and Nramp1 in macrophages, prior to release into the blood or storage in ferritin.