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Fig. 11. Effect of PKC inhibition on the copper decrease in TEER and subsequent recovery following copper removal. (A) Caco-2 cells were treated with 30 MCuCl2 in the AP compartment at pH 6.0 with or without the addition of 5.2 |J,M chelerythrine chloride (CHT) an inhibitor of PKC, in the AP and BL media. TEER was monitored during the treatments. Data, expressed as a percentage of their respective control, are the means ± SD from a representative experiment performed in triplicate. (B) Following the treatment described in (A), copper was removed and the cells were transferred to the complete culture medium for recovery, and the TEER was measured after 24 h. Data, expressed as a percentage of their respective control, are the means ± SD from a representative experiment performed in triplicate.

hydroxyl radical were able to decrease paracellular permeability in Caco-2 cells (73). Because the hydroxyl radical is the main radical species produced by copper ions via the Fenton reaction, we therefore suggest that, under the conditons used in our experiments, copper ions are unlikely to produce alterations in tight-junction permeability via an oxidant-induced mechanism.

5.2. Protein Kinase C

The assembly of tight junctions is tightly regulated by a network of signal transduction pathways that include, among others, protein kinase C (PKC) (74). The role of PKC in tight-junction formation, however, appears to be multifaceted, and this may explain some conflicting results reported in the literature on the effects of PKC activators and inhibitors on tight-junction permeability. PKC activation by phorbol esters has been reported to decrease TEER in Caco-2 cells (75). Similarly, in renal LLCPK1 cells, activation of protein kinase by phorbol esters led to a decrease in TEER associated with occludin dephosphorylation and delocalization from the tight junctions to the cytoplasm (76). An opposite effect of phorbol esters on tight-junction permeability has recently been reported in Caco-2 cells: PKC inhibition prevented the increase in TEER observed upon activation of PKC, suggesting a role of PKC in the regulation of TEER via decreased phosphorylation of the regulatory light chain of myosin II (77).

In order to investigate a possible role of PKC in the copper-induced changes in tight-junction permeability and during the recovery period, Caco-2 cells were treated with 30 |M CuCl2 at pH 6.0 for 3 h with or without the PKC inhibitor chelerythrine chloride (Sigma-Aldrich Srl) (78) in the AP and BL compartments, and TEER was monitored during treatment. Chelerythrine chloride was used at 5.2 |M in HBSS/HEPES at pH 7.4 in the BL and in HBSS/MES at pH 6.0 in the AP compartment. After copper removal, cells were transferred to complete cell culture medium with or without 5.2 |M chelerythrine in both compartments, and the TEER was measured after 24 h. As shown in Fig. 11, inhibition of PKC activity by chelerythrine did not markedly modify the effect of copper on TEER (Fig. 11A), but it prevented the restoration of monolayer permeability after 24 h of recovery (Fig. 11B).

The lack of effect of PKC inhibition on the copper-induced increase in tight-junction permeability does not point to an involvement of copper in the phosophorylation events controlling tight-junction permeability. Inhibition of PKC had no effect on control cells, confirming that its activity is not required for the maintenance of already established tight monolayers (79). Conversely, PKC inhibition prevented the recovery of TEER following copper removal, suggesting a role of PKC in the reassembly of the F-actin cytoskeleton as reported in T84 and IEC6 intestinal cells (80-82).

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