Cadmium uptake from apical membrane of LLC-PK1 cells via inorganic anion exchanger

Effects of Na+, Cl-, OH- and HCO3-, and an inorganic anion exchange inhibitor (HCO3-/Cl-), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, on Cd uptake from the apical membrane of LLC-PK1 cells were investigated to elucidate the mechanism of Cd uptake by these cells. Furthermore, the initial uptake of Cd incubated at different temperatures was analyzed by using the Arrhenius plot to test if Cd is taken up by the cells via the carrier-mediated process. The cells were incubated with 1 mu M CdCl2 for 30 min, at 37 degrees in phosphate buffer containing NaCl at pH 5.5 or 7.4. Cd accumulation by the cells at pH 7.4 was about 5 times higher than at pH 5.5. Replacement of NaCl in the phosphate buffer with KCl or mannitol decreased the Cd accumulation at pH 7.4 about 80%, but had no effect at pH 5.5. The addition of 2 mM NaHCO3 to the pH 7.4 buffer containing NaCl significantly increased the Cd accumulation, and pretreatment of the cells with the inorganic anion exchange inhibitor abolished this effect of NaHCO3 on Cd accumulation. The cells were incubated for 10 min. at different temperatures with 1 mu M CdCl2 in the phosphate buffer containing NaCl at pH 7.4 in the presence or absence of 2 mM NaHCO3 or at pH 5.5 in the absence of NaHCO3. In all cases, the Arrhenius plots of Cd accumulation were nonlinear. The breakpoint was observed at about 30 degrees in the Cd accumulation at pH 7.4 in the presence of NaHCO3, suggesting the involvement of a carrier-mediated process. This breakpoint was not as clear in the Cd accumulation at pH 7.4 in the absence of NaHCO3 and not observed at all at pH 5.5. These findings suggested that Cd is partially taken up from the apical membrane of LLC-PK1 cells via the inorganic anion exchanger and the uptake of Cd is more efficient at alkaline pH and in the presence of Na+, Cl- and HCO3-.