Direct Radioimmunoassay of Nuclear 3,5,3′ Triiodothyronine in Rat Anterior Pituitary

Previous tracer studies have suggested that 5'-monodeiodination of l-thyroxine (T(4)) in anterior pituitary may contribute a substantial portion of specifically bound nuclear 3,5,3' l-triiodothyronine (T(3)) in this tissue in rats. To evaluate this possibility, a radioimmunoassay for nuclear T(3) in individual anterior pituitaries was developed. Animals received (125)IT(3) 60 min before removal of the anterior pituitary and isolation of the nuclei by differential centrifugation. This allowed calculation of the nuclear:serum T(3) ratio and comparison of expected with measured T(3). T(3) was extracted in ethanol, dried, and reconstituted in assay buffer. In untreated hypothyroid rats, anterior pituitary nuclear T(3) was 0.18 +/- 0.06 pg/mug DNA which was 0.13 pg/mug DNA greater than expected from the serum T(3) concentration and the pituitary nuclear:serum (125)IT(3) ratio. In 10 hypothyroid rats given a single bolus of 400 ng T(3)/100 g body wt., the nuclear T(3) by radioimmunoassay was 1.0 +/- 0.06 pg/mug DNA, whereas that expected from the T(3) specific activity calculations was 0.85 pg/mug DNA (P < 0.025). Serum T(4) concentrations in these rats were < 0.25 mug/dl but the nuclear T(3) derived from as little as 0.2 mug/dl T(4) could explain a large portion of these small discrepancies between observed and measured nuclear T(3). In 29 normal rats, anterior pituitary nuclear T(3) was 0.63+/-0.04 pg/mug DNA, whereas that expected from the serum T(3) concentration (55+/-2 ng/dl) was 0.23+/-0.02 pg/mug DNA (P < 0.001). Total pituitary T(3) based on this measurement was 92+/-6 pg. Because the maximal nuclear binding capacity for T(3) in rat anterior pituitary is 0.77 pg/mug DNA, these results suggest there is 82% occupancy of these nuclear receptors. The requirement for normal serum concentrations of both T(4) and T(3) to achieve normal nuclear T(3) saturation in anterior pituitary is in marked contrast to the situation in liver, kidney, and heart muscle which appear to require only a normal serum T(3). As a consequence, the anterior pituitary can monitor both serum T(4) and T(3) and respond appropriately to changes in their concentrations.