Intestinal uptake of Cu by processes that do not involve copper transporter 1 (CTR1), as determined with Caco2 cell monolayers

Abstract

Dietary copper absorption across the apical brush border of intestinal cells involves several uptake systems, yet in mammals, only one gene product (CTR1) has as yet been identified as specific for copper uptake across the cell plasma membrane of intestinal and other cells. Some studies have suggested that the important iron uptake system, divalent metal transporter 1 (DMT1), is also involved, and yet another, uncharacterized chloride-dependent system has been implicated. CTR1 takes up Cu(I), and it is generally accepted that dietary copper is first reduced by cell surface reductases before entering the enterocyte. Uptake of Cu(I) by CTR1 is inhibited by Ag(I), while Fe(III) and Cu(II) compete for reduction by the reductases in the brush border. Using Caco2 cell monolayers with tight junctions grown in bicameral chambers, we determined that, whether given as Cu(I) or Cu(II), only about one third of copper uptake was inhibited by an excess of Ag(I), implying that CTR1 accounts for only a portion of intestinal copper uptake. Rates of Cu uptake were measured by following accumulation of radioactive Cu over 60 min in cells and the basal medium (equivalent to blood fluid) after application of 67Cu-labeled Cu(II)-nitrilotriacetate (5 uM) to the apical surface, and were calculated in terms of % dose/h/mg cell protein. Our finding implied that CTR1 does not account for most of the uptake carried out by the enterocytes. Measuring uptake of Cu(II) in the presence of an excess of Fe(III) – to prevent reduction of Cu(II), failed to inhibit copper uptake, implying that not all copper needs to be reduced prior to uptake. To further investigate the chloride-dependent process, reported by Zimnicka et al.[1]) the effects of substituting sulfate for chloride in the culture medium was examined. Chloride-dependent uptake was measured by the fall in uptake rate when sulfate was substituted for chloride, and the effects of substituting with other halides was also examined. We found that substitution of sulfate for chloride reduced rates of copper uptake about 30%, and that the effects of Ag(I) and sulfate substitution were additive. Substituting bromide for chloride made no difference, while iodide substitution actually doubled uptake rates. Thus, a halide dependent system may also be prominent in mediating intestinal copper absorption. Effects of altering the nutritional iron status of the Caco2 cell monolayer was also examined. Pretreatment with excess iron (3 days with 5 uM ferric ammonium citrate) reduced expression of CTR1 and DMT1 mRNAs (determined by qPCR relative to 18S rRNA) but had little effect on rates of copper uptake. However, 48 h depletion of iron with desferrioxamine increased rates of copper uptake. As expected, this also increased expression of mRNA for DMT1. However, the iron deficiency also enhanced expression of CTR1. We conclude that at least in this intestinal model, CTR1 and a halide-dependent uptake process each account for about one third of Cu uptake; that CTR1 expression is enhanced by iron deficiency – which would explain increased uptake of copper in that condition; and that a Cu(II) uptake system may also be present. [1]Zimnicka et al. (2011) Am J Physiol Cell Physiol 300: C588–C599.