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

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 (K_m) and maximum velocity (V_max) 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 K_m and V_max 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 (K_i = 1.3 nM). T₄ 5′-deiodination by hypothyroid microsomes was not affected by PTU, was competitively inhibited by rT₃ (K_i, 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 K_m and V_max 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.