Phenolic and Tyrosyl Ring Deiodination of Iodothyronines in Rat Brain Homogenates

Abstract

Conversion of thyroxine (T₄) to 3,5,3′-triiodothyronine (T₃) in rat brain has recently been shown in in vivo studies. This process contributes a substantial fraction of endogenous nuclear T₃ in the rat cerebral cortex and cerebellum. Production of T₄ metabolites besides T₃ in the brain has also been suggested. To determine the nature of these reactions, we studied metabolism of 0.2-1.0 nM [¹²⁵I]T₄ and 0.1-0.3 nM [¹³¹I]T₃ in whole homogenates and subcellular fractions of rat cerebral cortex and cerebellum. Dithiothreitol (DTT) was required for detectable metabolic reactions: 100 mM DTT was routinely used. Ethanol extracts of incubation mixtures were analyzed by paper chromatography in t-amyl alcohol:hexane:ammonia and in 1-butanol:acetic acid. Rates of production of iodothyronines from T₄ and T₃ were greater at pH 7.5 than at 6.4 or 8.6 and greater at 37°C than at 22° or 4°C. Lowering the pH, reducing the protein or DTT concentrations, and preheating homogenates to 100°C all increased excess I⁻ production but reduced iodothyronine production. In cerebral cortical homogenates from normal rats, products of T₄ degradation were as follows (percent added T₄±SEM in nine experiments): T₃, 1.9±0.5%; 3,3′,5′-triiodothyronine (rT₃), 34.0±2.4%; 3,3′-diiodothyronine (3,3′-T₂), 5.8±1.6%; 3′-iodothyronine (3′-T₁), ≤2.5%; and excess I⁻, 4.7±1.2%. In the same experiments, products of T₃ degradation were 3,3′-T₂, 63.3±5.5%, and 3′-T₁, 12.6±1.4%. Cerebral cortical homogenates from hyperthyroid rats and normals were similar in regard to T₄ to T₃ deiodination. In contrast, in cerebral cortical homogenates from hypothyroid rats, phenolic ring deiodination rates were increased and tyrosyl ring deiodination rates were decreased compared with normals. T₄ to T₃ conversion rates in cerebellar homogenates were greater than rates in cerebral cortical homogenates from the same normal rats and less than rates in cerebellar homogenates from hypothyroid rats. T₄ and T₃ tyrosyl ring deiodination rates were greatly diminished in cerebellar homogenates compared with cerebral cortical homogenates in normal and hypothyroid rats. High-speed (1,000-160,000 g) pellets from cerebral cortical homogenates were enriched in phenolic and tyrosyl ring deiodinating activities relative to cytosol. Fractional conversion of T₄ to T₃ was inhibited by T₄, iopanoic acid, and rT₃, but not by T₃. Tyrosyl ring deiodination reactions were inhibited by T₃, T₄, and iopanoic acid, but not by rT₃. These studies demonstrate separate phenolic and tyrosyl ring iodothyronine deiodinase enzymes in rat brain. The brain phenolic ring deiodinase serves in vivo as a T₄ 5′-deiodinase and closely resembles anterior pituitary T₄ 5′-deiodinase in physiological and biochemical characteristics. The physiological significance of the tyrosyl ring iodothyronine deiodinase enzyme is unclear; it shares several properties with rat hepatic T₄ 5-deiodinase.