Uptake of Cd-metallothionein into LLC-PK1 cells: a comparative study with CdCl2

The uptake of the Cd-metallothionein complex (CdMT) into LLC-PK1 cells was investigated and compared with that of CdCl2. The cells were incubated at 37 degrees C for up to 45 min with 1 microM Cd, as either CdMT or CdCl2 at pH 5.5, 6.4, or 7.4. Under all the experimental conditions described below, the accumulation of Cd from CdMT was markedly lower than that from CdCl2. Cd accumulation at pH 7.4 from CdMT increased linearly with the time of incubation, whereas Cd accumulation from CdCl2 was saturable. Metabolic inhibitors, 2,4-dinitrophenol (DNP) and carbonylcyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP), and incubation at low temperature significantly decreased Cd accumulation from both Cd compounds. Coincubation with 30 microM ZnCl2, an antagonist of CdCl2 uptake, slightly decreased Cd accumulation from CdMT, but it markedly decreased that from CdCl2. Cd accumulation from CdMT at pH 5.5 was significantly higher than at pH 6.4 or 7.4, whereas Cd accumulation from CdCl2 at pH 5.5 and 6.4 was significantly lower than at pH 7.4. Although Cd accumulation from CdCl2 at pH 7.4 was significantly decreased by coincubation with 100 microM cysteine or glutathione (GSH), this decrease was not observed at pH 5.5 or 6.4. A small amount of Cd was removed, by the chelating agent EGTA, from the cell membranes after incubation with CdMT at pH 6.4 and 7.4, whereas a considerable amount of Cd was removed by EGTA after incubation with CdMT at pH 5.5 and with CdCl2 at three pHs. It appears that the CdMT complex is taken up into LLC-PK1 cells partially via an energy-dependent process, and the increase in Cd accumulation at low pH is due to the liberation of Cd. High stability and molecular size of the CdMT complex explains why it is not taken up readily into LLC-PK1 cells.