Further analysis of cadmium uptake from apical membrane of LLC-PK1 cells via inorganic anion exchanger

This study was undertaken to analyze Cd uptake via the inorganic anion exchanger (HCO3-/Cl-) by LLC-PK1 cells cultured on permeable membranes. LLC-PK1 cells were incubated at 37 degrees with 1 microM CdCl2 added to the apical medium, and Cd accumulation in the cells was fractionated into a membrane-bound (non-internalized) Cd fraction and an internalized Cd fraction using ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraace tic acid (EGTA), a chelating agent for Cd. Incubation at a lower temperature (4 degrees) significantly decreased the membrane-bound Cd fraction, and drastically decreased the internalized Cd fraction. Addition of NaHCO3 (a stimulator of Cd uptake via inorganic anion exchanger) to the apical medium significantly increased both membrane-bound and internalized Cd fractions, and this increase was greater for the internalized fraction. Pretreatment of cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a specific inhibitor of inorganic anion exchangers, significantly decreased the internalized Cd fraction without changing the membrane-bound Cd fraction. Addition of NaHCO3 did not effect both Cd fractions in DIDS-pretreated cells. These results suggest that most of Cd binds non-specifically to the apical membrane surface and then is internalized via simple diffusion and some Cd specifically binds to the inorganic anion exchanger and is internalized in a metabolism-dependent manner.