Figure 5.

Characterization of iodide uptake by tRA-treated MCF-7 cells. Cells were treated with 1 μM of tRA for 48 h before assay. (a) Time course of iodide uptake in tRA-treated MCF-7 cells. Iodide uptake was initiated by incubating MCF-7 cells with 500 μl of HBSS containing ≈0.1 μCi Na¹²⁵I and 10 μM NaI. The reactions were terminated at the indicated times, and the content of iodide in the cells was determined. Values are means ± SD (n = 3). (b) Inhibition of RA-induced iodide uptake by ouabain and KClO₄. (Upper) Indicated concentration of ouabain was added to the growth medium containing RA 30 min before the assay. In iodide uptake assay, cells were incubated for 30 min at 37°C with 500 μl of HBSS containing ≈0.1 μCi Na¹²⁵I, 10 μM NaI, and the various concentrations of ouabain. (Lower) Inhibition of RA-induced iodide uptake by KClO₄. Iodide uptake assay was performed by 30-min incubation with ≈0.1 μCi Na¹²⁵I, 10 μM NaI, and indicated concentration of KClO₄. (c and d) Dependency of initial velocity of iodide uptake (5 min) on the extracellular iodide concentration in tRA-treated MCF-7 cells (c), and comparison of the kinetics of iodide uptake between MCF-7 cells and FRTL-5 rat thyroid cells (d). Cells were incubated at 37°C for 5 min with 500 μl of HBSS containing 20 mCi/mmol ¹²⁵I^- and indicated concentration of NaI. Trapped ¹²⁵I^- was measured by γ-counter and normalized to the cell number. Nonspecific binding of ¹²⁵I^- was determined in duplicate assays in the presence of 30 μM KClO₄, and this value was normalized to the cell number and subtracted from the values measured (36). (c) The subtracted data (net uptake velocity) are expressed as means ± SD (n = 3). (d) The data of 3–300 μM NaI, which were chosen according to previous studies (33, 36, 41), are graphed as Lineweaver-Burk plot. All points of triplicate wells are plotted. ●, MCF-7 cells; ○, FRTL-5 cells.