PREFERENTIAL INHIBITION OF CYTOPLASMIC T-3 BINDING IS ASSOCIATED WITHREDUCED NUCLEAR-BINDING IN CULTURED-CELLS

Previous studies from our laboratory have suggested that the nonsteroi dal antiinflammatory drug, diclofenac (DCF), is a more potent competit or for T-3 binding sites in cytoplasm than for those in the nucleus. I n the present study we have examined the competitive potency for DCF a nd its effect on nuclear binding of T-3 in cultured cells. DCF was a w eak competitor for T-3 binding sites in cytosol and nuclear extracts p repared from HepC2 cells with a potency of 21 and 295 mu M, respective ly. When expressed relative to T-3, DCF was 135-fold more potent in cy tosol than in nuclear extract. In intact cells, T-3 was bound by nucle i 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 whole-cell experiments, D CF increased the rate of dissociation of T-3 from cells prelabeled wit h hormone for 30 min. when these prelabeled cells were incubated with DCF, 0.1 mM, cell-associated T-3 was significantly lower at 30 and 60 min than in cells reincubated without the drug. These data show that c ellular transport mechanisms precede nuclear binding hy T-3 and sugges t that there is a critical role for nonnuclear binding proteins in thy roid hormone action.