Masters Thesis

Mechanisms of Intestinal Copper Transport

Copper Transporter 1 has been known to be the main route of dietary copper uptake while the involvement of Divalent Metal Transporter 1 is still disputed. in human colorectal adenocarcinoma cells (Caco2), knockdown of DMT1 mRNA has been shown to reduce rates of both Cu(I) and Fe(II) uptake [18]. Over-expression of mouse or rat DMT1 mRNA in HEK293 (human embryonic kidney) cells has increased Cu(II) uptake [19, 26]. Using divalent ions known to inhibit DMT1, we measured uptake of 5µm radiolabeled 67Cu(I) in Caco2 cells. Neither 50 or 200 uM of Ni(II) or Mn(II) inhibited Cu(I) uptake. CTR1 and a potential Cl dependent transporter were also examined in Caco2 [20]. Silver inhibition (Ag(I)) can be used to determine contribution of CTR1 and substitution of 150 mM Na2SO4 for NaCl was used to assess Cl- dependent uptake. Ag(I) inhibited Cu(I) uptake (30%, p < 0.01). Substitution of 150 mM SO42- for Cl- decreased uptake (~30%, p < 0.01). in the presence of SO42- and Ag(I), ~50% reduction of the total rate resulted suggesting that the effects are additive. Pretreatment with extra Cu failed to change uptake rates significantly; iron status decreased rates significantly by ~60% (absolute rate). to conclude, Cl- dependent transporters and CTR1 make up approximately 60% of the total Cu(I) uptake rate and that DMT1 is unlikely to be involved in Caco2 cells. the remaining 40-50% reveals that there are still other Cu(I) transporters still not accounted for.

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