Preferential Inhibition of Cytoplasmic T 3 Binding Is Associated with Reduced Nuclear Binding in Cultured Cells

Previous studies from our laboratory have suggested that the nonsteroidal antiinflammatory drug, diclofenac (DCF), is a more potent competitor for T₃ binding sites in cytoplasm than for those in the nucleus. In the present study we have examined the competitive potency for DCF and its effect on nuclear binding of T₃ in cultured cells. DCF was a weak competitor for T₃ binding sites in cytosol and nuclear extracts prepared from HepG2 cells with a potency of 21 and 295 μM, respectively. When expressed relative to T₃, DCF was 135-fold more potent in cytosol than in nuclear extract. In intact cells, T₃ was bound by nuclei with an affinity, K _d of 0.22 ± 0.07 nM whereas in nuclear extract the affinity was 0.60 ± 0.21 nM. DCF was a competitive inhibitor in both preparations but reduced the apparent affinity 4-fold in intact cells but only 2-fold in nuclear extract. In wholecell experiments, DCF increased the rate of dissociation of T₃ from cells prelabeled with hormone for 30 min. When these prelabeled cells were incubated with DCF, 0.1 mM, cell-associated T₃ was significantly lower at 30 and 60 min than in cells reincubated without the drug. These data show that cellular transport mechanisms precede nuclear binding by T₃ and suggest that there is a critical role for nonnuclear binding proteins in thyroid hormone action.