The Ca²⁺- and Reduced Nicotinamide Adenine Dinucleotide Phosphate-Dependent Hydrogen Peroxide Generating System Is Induced by Thyrotropin in Porcine Thyroid Cells

Hydrogen peroxide (H₂O₂) is an essential electron acceptor for thyroid peroxidase-catalyzed iodination and coupling reactions. In the presence of iodide, its production is a limiting step in thyroid hormone biosynthesis. Several studies have demonstrated that the thyroid particulate fraction contains a Ca²⁺- and NADPH-dependent H₂O₂ generator (NADPH-O₂:oxidoreductase), the so-called thyroid NADPH-oxidase. It has recently been demonstrated that cellular H₂O₂ release is under the tonic control of TSH in primary cultures of dog thyrocytes. The present study evaluates the effect of TSH on the thyroid NADPH-oxidase and cytochrome c reductase activities, two enzymes believed to be involved on H₂O₂ generation in the thyroid gland. There was almost no detectable NADPH-dependent H₂O₂ generator in the membranes of cells grown for 18 h without TSH. But cells grown in the presence of TSH (0.1 mU/ml) had a Ca²⁺- and NADPH-dependent H₂O₂-generating activity that increased up to the third day in culture, as did the cell iodide organification capacity. This increase was also partially blocked by 12-O-tetradecanoylphorbol 13-acetate and cycloheximide. Forskolin and 8-bromo-cAMP both reproduced the action of TSH on the Ca²⁺- and NADPH-dependent H₂O₂ generator. In contrast, the thyroid NADPH-cytochrome c reductase activity in particles from control cells was similar to that of TSH-treated cells and was unaffected by forskolin or 12-O-tetrade-canoylphorbol 13-acetate. These results suggest that NADPH-cytochrome c reductase activity is not regulated by TSH and, thus, reinforce the idea that this enzyme is not involved in thyroid H₂O₂ generation. On the other hand, the Ca²⁺- and NADPH-dependent H₂O₂ generator, so-called thyroid NADPH-oxidase, is induced by TSH through the cAMP cascade. Thus, it seems to be another marker of thyroid differentiation, in addition to thyroperoxidase and thyroglobulin, and could play a key role in thyroid hormone production.