To study the effect of 5,5'-diphenylhydantoin (DPH) on cellular and specific nuclear T3 binding, we have studied the effect of DPH on binding of T3 by rat anterior pituitary in vivo and in cultured GC cells, a rat pituitary tumor cell line that secretes GH. As determined by in vivo displacement techniques, DPH (10 mg/100 g BW daily for 3 days or as a single dose of 20 mg/100 g BW) resulted in a significant decrease in T3 binding by cytosol and specific nuclear sites in rat anterior pituitary. In cultured GC cells, the addition of 400 µM DPH resulted in a 70-80% decrease in specific nuclear T3 binding and a 40-50% decrease in cytosol T3 binding without affecting the rate of T3 equilibration. The effect of DPH was dose dependent between 10 and 400µM. Similar effects, but of a smaller magnitude, were noted in studies of isolated GC cell nuclei. The maximum percent decrease in specific T3 binding in isolated nuclei was 22% at 400 µM DPH. Scatchard analysis suggested that the DPH-induced decrease in specific nuclear T3 binding was competitive when measured in intact cells; the interaction between DPH and T3 appeared noncompetitive in studies of isolated GC cell nuclei. Other experiments indicated that DPH did not affect the exit rate of T3 from either the cytosol or specific nuclear compartments. The present studies suggest that DPH may decrease the fractional rate of entry of T3 into cultured cells and also inhibit binding of T3 by nuclear receptors. The similarity of effects observed in cultured GC cells and in rat anterior pituitary in in vivo studies suggests that studies in GC cells should be useful for examination of biological changes that result from the interaction between DPH and T3 at nuclear binding sites.
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