SECOND MESSENGER CONTROL OF LUTEAL STEROIDOGENESIS

哺乳动物黄体产生的黄体酮是 维持妊娠。因此，适当的黄体功能是必不可少的。 为了繁殖。了解黄体功能的一个强有力的方法是 观察不同类固醇生成黄体细胞类型的功能。这个 绵羊黄体含有两种功能不同的黄体 类固醇生成细胞，大小不一。小鼠的孕酮分泌 细胞似乎受到黄体生成素通过cAMP机制的调节，而在 大细胞不受黄体生成素或细胞内cAMP升高的刺激 级别。然而，大细胞对前列腺素E2的反应是增强的 黄体酮的分泌。这种影响似乎并不是由 夏令营。独立的证据表明，两个第二信使 涉及蛋白质磷酸化的机制可能介导激素 黄体中类固醇合成的调节。一种机制是 由cAMP激活cAMP依赖的蛋白激酶(A 激酶)。另一种是由二酰甘油激活的 钙，磷脂依赖的蛋白激酶(C激酶)。这项建议 概述了确定这种蛋白质磷酸化作用的实验。 在调节大小悬浮绵羊孕酮分泌中的作用 黄体细胞(通过淋洗分离)。1)A和C的比较 每种细胞类型内和不同细胞类型之间的激酶活性将是 制造。2)促黄体生成素刺激特定蛋白的磷酸化。 小细胞与(P32)PO4孵育(通过SDS-PAGE监测)将被 与孕酮分泌相关。3)小细胞和大细胞将 与(P32)PO4和A的激活剂(DBcAMP、霍乱毒素和 Forskolin)和C(佛波醇酯)激酶。这些代理人有能力 模拟促黄体生成素促进蛋白质磷酸化和孕酮分泌 在较小的细胞中将被确定。4)确定参与的蛋白质 在调节两种细胞类型的类固醇生成方面的作用将在 它们作为A和/或C的特定磷酸蛋白底物的能力 细胞组分中的激酶。这种方法将利用MG(GammaP32)ATP 和纯化的催化亚基(A激酶)或Ca+2，磷脂酰肌醇 和佛波酯(C激酶激活剂)。从上面的观察来看， 蛋白磷酸化在促黄体生成素刺激的小鼠类固醇合成中的作用 将描述单元格。此外，一种机制来解释缺乏 大细胞中cAMP对类固醇合成的调节将被提出。

Progesterone produced by the mammalian corpus luteum is required for maintenance of pregnancy. Therefore, proper luteal function is essential to reproduction. A powerful approach to understanding luteal function is to observe how different steroidogenic luteal cell types function. The ovine corpus luteum contains two functionally distinct types of steroidogenic cells, small and large. Secretion of progesterone in small cells appears to be regulated by LH via a cAMP mechanism while that in large cells is not stimulated by LH or increased intracellular cAMP levels. Large cells, however, respond to prostaglandin E2 with enhanced secretion of progesterone. This effect does not seem to be mediated by cAMP. Independent lines of evidence suggest that two second messenger mechanisms involving protein phosphorylation may mediate hormonal regulation of steroidogenesis in the corpus luteum. One mechanism is initiated by cAMP activation of the cAMP-dependent protein kinase (A kinase). The other is initiated by diacylglycerol activation of the calcium, phospholipid-dependent protein kinase (C kinase). This proposal outlines experiments to determine the role of such protein phosphorylations in regulating secretion of progesterone in suspended ovine small and large luteal cells (as separated by elutriation). 1) A comparison of A and C kinase activities within each cell type and between cell types will be made. 2) Stimulation by LH of phosphorylation of specific proteins in small cells incubated with (P32)PO4 (as monitored by SDS-PAGE) will be correlated with secretion of progesterone. 3) Small and large cells will be incubated with (P32)PO4 and activators of A (dbcAMP, cholera toxin and forskolin) and C (phorbol esters) kinase. An ability of these agents to mimic LH enhancement of protein phosphorylation and progesterone secretion in small cells will be determined. 4) Proteins determined to be involved in the regulation of steroidogenesis in both cell types will be assessed in their ability to serve as specific phosphoprotein substrates for A and/or C kinase in cellular fractions. This approach will utilize Mg(gammaP32)ATP and purified catalytic subunit (A kinase) or Ca+2, phosphatidyl inositol and phorbol esters (C kinase activators). From the above observations, the role of protein phosphorylation in LH-stimulated steroidogenesis in small cells will be described. Further, a mechanism to explain the lack of regulation of steroidogenesis by cAMP in large cells will be proposed.