Davis J S, Weakland L L, Farese R V, West L A
J Biol Chem. 1987 Jun 25;262(18):8515-21.
The present studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases intracellular cAMP, also increases "second messengers" derived from inositol phospholipid hydrolysis in isolated bovine luteal cells. In luteal cells prelabeled with 32PO4, LH provoked increases in labeling of phosphatidic acid, phosphatidylinositol, and polyphosphatidylinositol (PIP). No reductions in 32P-prelabeled PIP and PIP2 were observed in LH-treated cells. In luteal cells prelabeled with myo-[2-3H]inositol, LH provoked rapid (10-30 s) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (IP, IP2, and IP3, respectively. IP3 was formed more rapidly than IP2 or IP following LH treatment. In addition, LH increased (50%) levels of [3H]inositol phospholipids in 30-min incubations. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH. Maximal increases in IP3 occurred at 1-10 micrograms/ml of LH. Similar temporal and dose-response relationships were observed for LH-stimulated IP3 and cAMP accumulation. However, exogenous cAMP (8-bromo-cAMP, 5 mM) and forskolin (10 microM) had no effect on inositol phosphate synthesis. The initial (1 min) effects of LH on IP3 and cAMP were independent of extracellular calcium concentrations, whereas the sustained (5 min) effect of LH on IP3, but not cAMP, was dependent on a source of extracellular calcium. LH-stimulated progesterone synthesis was also dependent on the presence of extracellular calcium. LH induced rapid and concentration-dependent increases in [Ca2+]i as measured by Quin 2 fluorescence. The LH-induced increases in [Ca2+]i were maximal within 30 s (approximately 2-fold) and remained elevated for at least 10 min. In Ca2+-free media containing 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, LH was still able to increase [Ca2+]i, but the increase was slightly less in magnitude and of shorter duration (2-4 min). These findings demonstrate that LH can rapidly raise levels of IP3 and [Ca2+]i, as well as, cAMP in bovine luteal cells. These findings suggest that at least two second messenger systems exist to mediate the action of LH in the corpus luteum.
本研究旨在确定促黄体生成素（LH）这种能增加细胞内cAMP的激素，是否也会增加分离的牛黄体细胞中由肌醇磷脂水解产生的“第二信使”。在用32PO4预标记的黄体细胞中，LH促使磷脂酸、磷脂酰肌醇和多磷酸磷脂酰肌醇（PIP）的标记增加。在LH处理的细胞中未观察到32P预标记的PIP和PIP2减少。在用肌醇-[2-3H]预标记的黄体细胞中，LH促使肌醇单磷酸、双磷酸和三磷酸（分别为IP、IP2和IP3）水平迅速（10 - 30秒）且持续（长达60分钟）增加。LH处理后，IP3的形成比IP2或IP更快。此外，在30分钟的孵育中，LH使[3H]肌醇磷脂水平增加了50%。LiCl（10 mM）增强了对LH的肌醇磷酸积累反应。IP3的最大增加出现在1 - 10微克/毫升的LH浓度下。LH刺激的IP3和cAMP积累观察到类似的时间和剂量反应关系。然而，外源性cAMP（8-溴-cAMP，5 mM）和福斯可林（10 microM）对肌醇磷酸合成没有影响。LH对IP3和cAMP的初始（1分钟）作用与细胞外钙浓度无关，而LH对IP3的持续（5分钟）作用而非对cAMP的作用，依赖于细胞外钙源。LH刺激的孕酮合成也依赖于细胞外钙的存在。通过喹啉2荧光测量，LH诱导[Ca2+]i迅速且浓度依赖性增加。LH诱导的[Ca2+]i增加在30秒内达到最大值（约2倍），并至少持续升高10分钟。在含有2 mM [乙二胺双（氧乙基腈）]四乙酸的无钙培养基中，LH仍能增加[Ca2+]i，但增加幅度略小且持续时间较短（2 - 4分钟）。这些发现表明，LH能迅速提高牛黄体细胞中IP3、[Ca2+]i以及cAMP的水平。这些发现表明，至少存在两种第二信使系统来介导LH在黄体中的作用。
