Amir S M, Kasagi K, Corretti M, Blank M, Ingbar S H
Endocrinology. 1985 Sep;117(3):860-8. doi: 10.1210/endo-117-3-860.
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 mM) 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 [125I]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.
聚磷酸根皮素（PPP）在体内和体外试验中均被认为是牛促甲状腺激素（bTSH）诱导的甲状腺刺激的抑制剂。本研究旨在阐明这些作用的机制。一种高分子量的PPP制剂强烈抑制125I标记的bTSH（[125I]bTSH）与人甲状腺膜的结合以及bTSH在其中引起的腺苷酸环化酶的刺激。在孵育培养基中不存在和存在NaCl（150 mM）的情况下，PPP对bTSH诱导的腺苷酸环化酶活性的抑制作用均很明显。用PPP孵育膜，然后去除PPP，并不影响随后[125I]bTSH的结合，这表明PPP没有牢固地结合到TSH受体上或对其造成损害。在Sephadex G - 100上进行凝胶过滤显示，与PPP一起孵育的[125I]bTSH比单独的[125I]bTSH洗脱得更早，这表明PPP与[125I]bTSH形成了更高分子量的复合物。向孵育混合物中加入过量的未标记bTSH可以阻止这种效应。与PPP孵育产生的较高分子量峰中[125I]bTSH的结合量不到[125I]bTSH对照样品的一半。还在一种高度敏感的试验中对PPP进行了研究，该试验使用培养的猪甲状腺细胞并测量bTSH诱导的cAMP反应。在该试验中，PPP对bTSH诱导的cAMP积累的抑制作用也很明显。然而，试验培养基中存在二价阳离子Ca++和Mg++会大大降低PPP的抑制作用。同样，向孵育培养基中加入Ca++和Mg++会大大降低或消除PPP对[125I]bTSH结合的抑制作用。通过增加PPP浓度可以克服这些盐减少对PPP抑制反应的两种作用。凝胶色谱研究表明，Ca++和Mg++通过抑制bTSH和PPP的高分子量复合物的形成而起作用。从这些发现中，我们得出结论，PPP通过与激素形成复合物，在体内和体外对TSH诱导的甲状腺刺激反应发挥抑制作用。该复合物要么不与TSH受体结合，要么以低得多的亲和力结合。
