Polyphloretin phosphate, an antagonist of thyrotropin action: Evidence for an interaction with the hormone rather than the receptor

Polyphloretin phosphate (PPP) is known to be an inhibitor of bovine TSH (bTSH)-induced stimulation of the thyroid in both in vivo and in vitro assays. The present studies were undertaken to delineate the mechanism of these effects. A high molecular weight PPP preparation strongly inhibited both the binding of ¹²⁵I-labeled bTSH ([¹²⁵I]bTSH) to human thyroid membranes and the stimulation of adenylate cyclase evoked by bTSH therein. Inhibition of bTSH-induced adenylate cyclase activity by PPP was evident both in the absence and the presence of NaCl (150 mMJ in the incubation medium. Incubation of membranes with PPP, followed by its removal, did not affect subsequent binding of [¹²⁵I]bTSH, indicating that PPP did not bind firmly to or damage the TSH receptor. Gel chromatography on Sephadex G-100 revealed that [¹²⁵I]bTSH incubated with PPP eluted earlier than [¹²⁶I]bTSH alone, indicating that PPP had formed a higher molecular weight complex with [¹²⁵I]bTSH. This effect could be prevented by the addition of an excess of unlabeled bTSH to the incubation mixture. Binding of [¹²⁵I] bTSH in the higher molecular weight peak generated by incubation with PPP was less than half that in control specimens of [¹²⁵I]bTSH. Studies with PPP were also conducted in a highly sensitive assay that employs cultured porcine thyroid cells and measures the cAMP response induced by bTSH. The inhibitory effect of PPP on bTSH-induced cAMP accumulation was also evident in this assay. However, the presence of divalent cations Ca⁺⁺ and Mg⁺⁺ in the assay medium greatly diminished the inhibitory effect of PPP. Similarly, addition of Ca⁺⁺ and Mg⁺⁺ to the incubation medium greatly reduced or abolished the inhibitory effect of PPP on [¹²⁵I]bTSH binding. Both effects of these salts to lessen the inhibitory response to PPP were overcome by increasing the PPP concentration. Gel chromatographic studies revealed that Ca⁺⁺ and Mg⁺⁺ acted by inhibiting the formation of the high molecular weight complex of bTSH and PPP. From these findings, we conclude that PPP exerts its inhibitory effect on TSH-induced stimulatory responses in the thyroid, in vivo as well as in vitro, by forming a complex with the hormone. The complex either does not bind to TSH receptors or does so with much lower affinity.