Uptake of copper from hemopexin (HPX) in the absence and presence of heme by hepatic and mammary epithelial cells in culture

Abstract

Hemopexin (HPX) captures heme units in the blood in conditions such as hemolysis. The resulting heme‐HPX complex is transported mainly to hepatocytes, taken up by receptor mediated endocytosis, and degraded, the heme degradation being initiated by heme oxygenases that release the iron for recycling. This process also provides a safe way to dispose of heme, which would otherwise be toxic [1]. Uptake of the heme‐HPX complex also brings copper into cells, and that copper appears to mediate a coordinate response resulting in induction of heme oxygenase and metallothionein [1]. We recently isolated HPX from porcine blood plasma, and found copper bound to it very tightly (that could not be removed by extensive dialysis with 100 uM histidine (pH 7). EPR analysis showed that Cu(II) was bound to oxygen and nitrogen ligands in HPX. The UV‐Vis spectrum of the same sample showed a typical heme absorbance peak. Treatment with chelating Sepharose however removed not only the copper but also the heme. These findings led us to wonder whether perhaps HPX might normally have a role in the distribution and transport of copper to cells from the circulation. To examine this, we obtained human HPX purified from blood plasma by Athens Research and Technology (Georgia), dialyzed it at pH 5 to remove any heme, added traces of radioactive 67Cu (as the NTA complex), and applied it to size exclusion chromatography on Sephadex G100 equilibrated with MOPS buffer pH 7.4, containing electrolytes to make it isotonic for tissue culture studies. The 67Cu eluted with the HPX protein peak, and no low molecular weight 67Cu was detected, indicating it had all stuck to the HPX. The peak fractions were pooled and incubated with human hepatic (HepG2) or mammary epithelial cells (PMC42) for various lengths of time. 67Cu radioactivity in the cells (washed with medium containing EDTA) was then determined, and rates of uptake (percent dose/h/mg cell protein) were calculated. For both cell types, the uptake was linear over several hours, but were 2–3 times faster with the hepatic cells. In subsequent experiments, the effects of heme on copper uptake were also measured. When heme was added to the 67Cu‐prelabeled HPX at a molar ratio of 1.1:1.0 (heme:HPX), we expected the rate of copper uptake to increase stimulated, since adding heme to HPX would allow receptor‐mediated endocytosis of the complex. Instead, heme addition markedly reduced copper uptake. The potential effects of three different endocytosis inhibitors were also examined with mixed results. However, some significant inhibition of uptake was observed, including when HPX with 67Cu but no added heme was administered. These findings indicate that copper attached to HPX can be delivered to hepatic and non‐hepatic cells even in the absence of heme and suggest that copper may have more than one role in the function of this plasma protein.