Evidence for Two Pathways of Iodothyronine 5′-Deiodination in Rat Pituitary That Differ in Kinetics, Propylthiouracil Sensitivity, and Response to Hypothyroidism

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Abstract
We have studied 5′-deiodination of thyroxine (T4) and 3,3′,5′-triiodothyronine (rT3) in rat pituitary tissue in vitro, with respect to substrate specificity, reaction kinetics, effects of 6-n-propyl-2-thiouracil (PTU), and the time course of effects of thyroid hormone depletion and repletion. Removal of one phenolic iodine or both tyrosyl iodines from the T4 molecule resulted in compounds that were not deiodinated, but alterations in the alanine side chain had little effect. 5′-Deiodination of 2 nM rT3 by pituitary microsomes from euthyroid rats was inhibited >90% by 1 mM PTU, but was inhibited 4. The apparent Michaelis constant (Km) and maximum velocity (Vmax) for rT3 at 20 mM dithiothreitol (DTT) were 33 nM and 84 pmol/mg protein per h. This reaction followed ping-pong type reaction kinetics when concentrations of DTT were varied. PTU inhibition was competitive with DTT and uncompetitive with rT3. In contrast, when pituitary microsomes from hypothyroid rats (21 d postthyroidectomy) were used, deiodination of 2 nM rT3 was inhibited only 20% by 1 mM PTU and up to 80% by 100 nM T4. At 20 mM DTT, the apparent Km and Vmax in hypothyroid microsomes were 4.7 nM rT3 and 16 pmol/mg protein per h. T4 was a competitive inhibitor of PTU-insensitive rT3 5′-deiodination (Ki = 1.3 nM). T4 5′-deiodination by hypothyroid microsomes was not affected by PTU, was competitively inhibited by rT3 (Ki, 1.7 nM), and exhibited sequential type reaction kinetics with DTT as cosubstrate. When T4 5′-deiodination was measured in euthyroid and hypothyroid microsomes, respectively, the apparent Km and Vmax for T4 at 20 mM DTT, were 0.9 nM and 0.55 pmol/mg protein per h (euthyroid), and 0.8 nM and 6.9 pmol/mg protein per h (hypothyroid). The T4 5′-deiodination rate and the PTU-insensitive, but not total, rT3 5′-deiodination rate (i.e. measured in the presence and the absence of 1 mM PTU, respectively) in pituitary homogenates were significantly elevated 24 h after thyroidectomy. PTU-insensitive activity continued to increase until at ≥30 d after thyroidectomy it was 11 times the PTU-insensitive activity in controls. At the latter time, PTU-sensitive rT3 5′-deiodinase activity appeared to be decreased. The increase in PTU-insensitive T4 and rT3 5′-deiodination observed 48 h after thyroidectomy was prevented by replacement doses of T4 or T3. The PTU-insensitive activity of long term hypothyroid pituitaries was decreased by 71% and ≥84% 4 h after injection of 20 and 200 μg T3, respectively, with no change in PTU-sensitive rT3 deiodination. These data show that rat pituitary tissue contains two distinct iodothyronine 5′-deiodinating pathways that differ with respect to substrate specificity, PTU sensitivity, reaction kinetics, and regulation by thyroid hormone. One of these resembles the 5′-deiodinase of liver and kidney, and predominates in euthyroid pituitary tissue in vitro. The other, also found in rat brain, predominates in hypothyroid pituitary tissue, is rapidly responsive to changes in thyroid hormone availability, and, as judged by previous, in vivo studies, appears to account for all the T3 produced locally in the pituitary and, thereby, 50% of the intracellular T3 in this tissue.

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