Gore S D, Behrman H R
Endocrinology. 1984 Jun;114(6):2020-31. doi: 10.1210/endo-114-6-2020.
To evaluate the role of transmembrane ion flux on the acute response of luteal cells to LH, the effects of two drugs, ouabain and monensin, and changes in ion composition of the medium were examined. Ouabain, an inhibitor of the Na+ extrusion pump, and monensin, a selective Na+ ionophore, would be expected to increase the intracellular level of Na+. Both drugs produced a highly significant, dose-related and Na+-dependent inhibition of LH-stimulated cAMP accumulation and progesterone secretion. The IC50 values for ouabain and monensin for both responses to LH were about 50 and 0.1 microM, respectively, and inhibition of cAMP accumulation was competitive [inhibitory constant (Ki) = 20 and 0.06 microM, respectively]. Both drugs showed inhibition only in the intact cell, and no nonspecific cytotoxic effects were evident. No effect on the specific binding of gonadotropin or on LH-stimulated adenylate cyclase activity in membranes was seen, nor was inhibition reduced by coincubation of the cells with isobutyl methylxanthine, a cAMP phosphodiesterase inhibitor. Both drugs inhibited (Bu)2cAMP-stimulated progesterone accumulation. No effect of ouabain was seen on cholera toxin- or forskolin-stimulated cAMP accumulation, whereas monensin significantly inhibited this response to both agonists. Preincubation of cells with LH protected against inhibition of cAMP accumulation by ouabain and monensin. Removal of extracellular Na+ completely prevented inhibition by both drugs and slightly blunted the response to LH alone. However, reduction of extracellular Na+ from 128 to 32 mM, treatment of cells with tetrodotoxin (a sodium channel blocker), or increasing extracellular K+ from 5.4 to 66 mM had no effect on the stimulation of cAMP accumulation by LH. On the other hand, removal of extracellular Ca2+ completely blocked inhibition of LH-stimulated cAMP accumulation by ouabain or monensin. It is concluded that ouabain and monensin inhibit the acute stimulation of cAMP accumulation by LH probably as a result of an influx of Na+ into the luteal cell. The increase in intracellular Na+ does not appear to directly inhibit LH-sensitive adenylate cyclase activity, but induces a secondary influx of extracellular Ca2+ which inhibits activation of adenylate cyclase by LH at a site involved in coupling of the receptor to the enzyme. Luteal regression may be initiated by mechanisms similar to that caused by ouabain and monensin, because the characteristics of inhibition by these drugs is identical to that caused by prostaglandin F2 alpha, a physiological luteolysin.(ABSTRACT TRUNCATED AT 400 WORDS)
为评估跨膜离子通量在黄体细胞对促黄体生成素（LH）急性反应中的作用，研究了两种药物哇巴因和莫能菌素的效应以及培养基离子组成的变化。哇巴因是一种钠泵抑制剂，莫能菌素是一种选择性钠离子载体，预计两者均会增加细胞内钠离子水平。两种药物均对LH刺激的环磷酸腺苷（cAMP）积累和孕酮分泌产生高度显著、剂量相关且依赖钠离子的抑制作用。哇巴因和莫能菌素对LH这两种反应的半数抑制浓度（IC50）值分别约为50和0.1微摩尔，且对cAMP积累的抑制具有竞争性[抑制常数（Ki）分别为20和0.06微摩尔]。两种药物仅在完整细胞中表现出抑制作用，且未观察到非特异性细胞毒性效应。未发现对促性腺激素特异性结合或对细胞膜中LH刺激的腺苷酸环化酶活性有影响，细胞与cAMP磷酸二酯酶抑制剂异丁基甲基黄嘌呤共同孵育也未降低抑制作用。两种药物均抑制（丁酰）2cAMP刺激的孕酮积累。未观察到哇巴因对霍乱毒素或福斯高林刺激的cAMP积累有影响，而莫能菌素显著抑制对这两种激动剂的反应。用LH预孵育细胞可防止哇巴因和莫能菌素对cAMP积累的抑制。去除细胞外钠离子完全阻止了两种药物的抑制作用，并略微减弱单独对LH的反应。然而，将细胞外钠离子从128毫摩尔降至32毫摩尔、用河豚毒素（一种钠通道阻滞剂）处理细胞或使细胞外钾离子从5.4毫摩尔增加至66毫摩尔，对LH刺激的cAMP积累均无影响。另一方面，去除细胞外钙离子完全阻断了哇巴因或莫能菌素对LH刺激的cAMP积累的抑制作用。得出结论，哇巴因和莫能菌素抑制LH对cAMP积累的急性刺激，可能是由于钠离子流入黄体细胞所致。细胞内钠离子增加似乎并非直接抑制LH敏感的腺苷酸环化酶活性，而是诱导细胞外钙离子的继发性流入，后者在受体与酶偶联相关位点抑制LH对腺苷酸环化酶的激活。黄体退化可能由类似于哇巴因和莫能菌素引起的机制启动，因为这些药物的抑制特性与生理性黄体溶解素前列腺素F2α引起的抑制特性相同。（摘要截于400字）
