Mechanistic characterization of gastric copper transport in rainbow trout
An in vitro gut-sac technique and 64Cu as a radiotracer were used to characterize gastric copper (Cu) transport. Cu transport was stimulated by low luminal pH (4.0 vs. 7.4), to a greater extent than explained by the increased availability of the free Cu2+ ion. At pH = 4.0, uptake kinetics were indicative of a low affinity (Km = 525 μmol L−1), saturable carrier-mediated component superimposed on a large linear (diffusive and/or convective) component, with about 50% occurring by each pathway at Cu = 50 μmol L−1. Osmotic gradient experiments showed that solvent drag via fluid transport may play a role in Cu uptake via the stomach, in contrast to the intestine. Also unlike the intestine, neither the Na+ gradient, high Ag, nor phenamil had any influence on gastric Cu transport, and a tenfold excess of Fe and Zn failed to inhibit Cu uptake. These findings indicate that neither Na+-dependent pathways nor DMT1 are likely candidates for carrier-mediated Cu transport in the stomach. We have cloned a partial cDNA sequence for the copper transporter Ctr1, and show its mRNA expression in all segments of the trout gastrointestinal tract, including the stomach. Based on the fact that this transporter is functional at low pH conventionally found in the stomach lumen, we suggest Ctr1 is a pathway for gastric Cu transport in trout. Extreme hypoxia inhibited Cu uptake. High $$ P_{{{\text{CO}}_{2} }} $$ levels (7.5 torr) increased Cu uptake and acetazolamide (100 μmol L−1) significantly inhibited Cu uptake, indicating carbonic anhydrase activity was involved in gastric Cu transport. Transport of Cu was insensitive to bafilomycin (10 μmol L−1) suggesting a V-ATPase did not play a direct role in the process. Expression (mRNA) of H+, K+-ATPase, carbonic anhydrase 2, and the α-3 isoform of Na+–K+-ATPase were observed in the stomach. We suggest these enzymes facilitate Cu transport in the stomach indirectly as part of a physiological mechanism exporting H+ to the cell exterior. However, pre-treatment with the H+, K+-ATPase proton pump blocker omeprazole did not affect gastric Cu transport, suggesting that other mechanisms must also be involved.