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

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Abstract

Previous tracer studies have suggested that 5′-monodeiodination of l-thyroxine (T4) in anterior pituitary may contribute a substantial portion of specifically bound nuclear 3,5,3′ l-triiodothyronine (T3) in this tissue in rats. To evaluate this possibility, a radioimmunoassay for nuclear T3 in individual anterior pituitaries was developed. Animals received [125I]T3 60 min before removal of the anterior pituitary and isolation of the nuclei by differential centrifugation. This allowed calculation of the nuclear:serum T3 ratio and comparison of expected with measured T3. T3 was extracted in ethanol, dried, and reconstituted in assay buffer. In untreated hypothyroid rats, anterior pituitary nuclear T3 was 0.18 ± 0.06 pg/μg DNA which was 0.13 pg/μg DNA greater than expected from the serum T3 concentration and the pituitary nuclear:serum [125I]T3 ratio. In 10 hypothyroid rats given a single bolus of 400 ng T3/100 g body wt., the nuclear T3 by radioimmunoassay was 1.0 ± 0.06 pg/μg DNA, whereas that expected from the T3 specific activity calculations was 0.85 pg/μg DNA (P < 0.025). Serum T4 concentrations in these rats were < 0.25 μg/dl but the nuclear T3 derived from as little as 0.2 μg/dl T4 could explain a large portion of these small discrepancies between observed and measured nuclear T3. In 29 normal rats, anterior pituitary nuclear T3 was 0.63±0.04 pg/μg DNA, whereas that expected from the serum T3 concentration (55±2 ng/dl) was 0.23±0.02 pg/μg DNA (P < 0.001). Total pituitary T3 based on this measurement was 92±6 pg. Because the maximal nuclear binding capacity for T3 in rat anterior pituitary is 0.77 pg/μg DNA, these results suggest there is 82% occupancy of these nuclear receptors. The requirement for normal serum concentrations of both T4 and T3 to achieve normal nuclear T3 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 T3. As a consequence, the anterior pituitary can monitor both serum T4 and T3 and respond appropriately to changes in their concentrations.

Authors

P. R. Larsen, S. Z. Bavli, M. Castonguay, R. Jove