Different mechanisms mediate uptake of lead in a rat astroglial cell line

The mechanism by which lead (Pb) enters astrocytes was examined in a rat astroglial cell line in order to characterize specific pathways for transport. Pb uptake was saturable at pH 5.5 and 7.4, although quantitative differences existed in the Michaelis-Menten constants. At pH 7.4, the Vmax and Km were 2700 fmoles/mg protein/min and 13.4 μM, respectively, whereas the Vmax and Km were 329 fmoles/mg and 8.2 μM in the buffer at pH 5.5, respectively. The presence of extracellular iron inhibited uptake in a buffer at pH 5.5 but not at pH 7.4. Cells treated with the iron chelator deferoxamine displayed higher levels of the iron transporter divalent metal transporter 1 (DMT1) mRNA and protein, and consistent with increased DMT1 expression, the treated cells displayed greater uptake of Pb in the buffer at pH 5.5 but not at pH 7.4. Alternatively, at pH 7.4, the transport of Pb was blocked by the anion transporter inhibitor 4,4′-diisothiocyanatodihydrostilbene-2,2′-disulfonic acid (DIDS), which bound to cell surface proteins at concentrations that were similar to those that blocked Pb uptake. DIDS did not inhibit uptake of Pb in the buffer at pH 5.5. Greater uptake of Pb was observed in a buffer containing sodium bicarbonate, which was abrogated in the presence of DIDS. In summary, the astroglial cell line displays two distinct pH-sensitive transport mechanisms for Pb.