Preferential Inhibition of Cytoplasmic T3 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, Kd 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...