Regulation of iodothyronine 5'-deiodinases: effects of thiol blockers and altered substrate levels in vivo and in vitro.

To study the physiological regulation of the iodothyronine 5'-deiodinases (I-5'D), we have examined the effects of some thiol blockers and of thyroid status on I-5'D activities both in vitro and in vivo. At low (less than 5 mM) concentrations of dithiothreitol, propylthiouracil (PTU) inhibited I-5'D in the brain, pituitary, and brown adipose tissue (BAT) of hypothyroid rats (which contain predominantly the type II activity); the patterns of inhibition in these tissues were essentially similar, with a Ki of about 174 microM at 250 microM dithiothreitol. Hydroxyethyldisulfide was a strong inhibitor of the type II enzyme, with relatively little effect on the renal enzyme at both high concentrations (micromolar) of T4, i.e. predominantly type I activity, and low concentrations (nanomolar) to T4, i.e. both type I and low Km activity. Preincubation of cerebral microsomes with PTU, followed by removal of excess PTU, resulted in 70% inhibition of I-5'D activity in cerebral microsomes at 5 mM dithiothreitol; the corresponding inhibitions of the renal enzyme at high and low substrate concentrations were 66% and 48%, respectively. Specific binding of PTU to renal and cerebral microsomes was diminished by hydroxyethyldisulfide, but not by T4, suggesting that PTU binding was not dependent on substrate interaction. Administration of PTU in vivo (1 mg/100 g BW, ip; 1 h before killing) resulted in approximately 80% inhibition of I-5'D activity in renal microsomes at high T4, and 50-70% inhibition in pituitary, BAT, and renal microsomes at low T4, but no inhibition was observed in brain microsomes. HPLC analyses revealed a PTU content of 35-65 nmol/g wet wt in the pituitary, BAT, liver, and kidney, but no PTU was detected in the brain, suggesting that PTU may be excluded by the blood-brain barrier. Maintaining hypothyroid rats on 1 microgram T4/100 g BW.day for 5 days enhanced renal type I and low Km I-5'D with restoration of serum T3 to normal levels, although the type II I-5'Ds from all sources were severely depressed. A supraphysiological dose of T4 depressed all three I-5'Ds. The data indicate that the I-5'Ds are regulated in a qualitatively similar fashion.