Apb

Fig. 6. Effect of different copper chelates on tight-junction permeability in Caco-2 cells. (A) Caco-2 cells were treated in the AP compartment for 3 h at pH 6.0 with either CuCl2, Cu(His)2, or Cu(Gly)2 in the concentration range 10-100 pM, and the TEER was measured at the end of the treatment. Data, expressed as the percentage of control, are the means ± SD of triplicate filters from a representative experiment. (B) Following copper treatment as detailed in (A), copper was removed and the cells were transferred to buffered culture medium and TEER was measured after 24 h. Data, expressed as the percentage of control, are the means ± SD of (continued)

Fig. 6. Effect of different copper chelates on tight-junction permeability in Caco-2 cells. (A) Caco-2 cells were treated in the AP compartment for 3 h at pH 6.0 with either CuCl2, Cu(His)2, or Cu(Gly)2 in the concentration range 10-100 pM, and the TEER was measured at the end of the treatment. Data, expressed as the percentage of control, are the means ± SD of triplicate filters from a representative experiment. (B) Following copper treatment as detailed in (A), copper was removed and the cells were transferred to buffered culture medium and TEER was measured after 24 h. Data, expressed as the percentage of control, are the means ± SD of (continued)

leave the cell (40), suggest that copper acts intracellularly on protein(s) that are involved in the maintenance of tight-junction closure, resulting in an increase in the permeability of the junctions. During the long recovery period in which mRNA transcription and subsequent protein synthesis are required to restore tight-junction functionality, copper does not leave the cell but redistributes to other intrac-ellular ligands, including metallothionein and Cu,Zn-superoxide dismutase (55).

When copper was presented to the Caco-2 cell monolayer complexed with L-histidine [Cu(His)2] or complexed with glycine, as the copper Chelazome Cu(Gly)2 the effect on TEER were markedly different (Fig. 6A). Copper in the form of Cu(HiS)2 had no effect on tight-junction permeability: Between 20 and 100 pM Cu(His)2 did not, in fact, produce the decrease in TEER observed with corresponding concentrations of CuCl2. Conversely, copper in the form of Cu(Gly)2 reduced TEER as a function of the concentration to the same extent as CuCl2. The effects of Cu(Gly)2 were fully reversible after 23 h in complete culture medium, similar to what was observed with CuCl2 at the same concentration (Fig. 6B). In addition, the effect of 30 pM Cu(Gly)2 for 3 h on TEER was not modified by the presence of an excess of dipeptides (10 mM Gly-Gly or Gly-L-Leu) in the AP medium (Fig. 6C). These results indicate that Cu(His)2 at the concentrations employed in the present study does not induce changes to tight-junction permeability and this may be related to the reduced AP uptake of this copper complex compared to CuCl2 previously reported in Caco-2 cells (40).

The copper Chelazome may enter the intestinal cells intact via the intestinal dipeptide transporters (56,57) and then be hydrolyzed intracellularly. However, this is unlikely, as an excess Gly-Gly or Gly-L-Leu did not reduce the effect of Cu(Gly)2 on tight-junction permeability. Conversely, the similar effect on tight junctions exerted both by ionic copper and by copper Chelazome indirectly suggest that this complex may be dissociated before entering the intestinal cell. Similarly, the bioavailability of zinc Chelazome in rats fed marginal levels of zinc was found to be the same as that of zinc carbonate (58).

0 0

Post a comment