SECOND MESSENGER CONTROL OF LUTEAL STEROIDOGENESIS

哺乳动物黄体产生的孕酮是 维持妊娠。 因此，适当的黄体功能是必不可少的 到繁殖。 了解黄体功能的一个有效方法是 观察不同类型的类固醇黄体细胞的功能。 的 绵羊黄体含有两种功能不同的类型， 类固醇生成细胞，大小不一。 孕激素的分泌 细胞似乎通过cAMP机制由LH调节，而在 大细胞不受LH或细胞内cAMP增加的刺激 程度. 然而，大细胞对前列腺素E2的反应增强， 孕酮的分泌。 这种效应似乎并不通过 营 独立的证据表明，两个第二信使 蛋白质磷酸化机制可能介导激素 调节黄体中的类固醇生成。 一种机制是 由cAMP激活cAMP依赖性蛋白激酶（A 激酶）。 另一种是由甘油二酯激活 钙，磷脂依赖性蛋白激酶（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 在调节孕酮分泌中的作用 黄体细胞（通过淘洗分离）。 1)A和C的比较 每种细胞类型内和细胞类型之间的激酶活性将被 进行了 2)LH刺激特定蛋白质磷酸化， 将用（P32）PO 4孵育的小细胞（通过SDS-PAGE监测） 与孕酮的分泌有关。 3)小型和大型细胞将 与（P32）PO 4和A（dbcAMP，霍乱毒素和 毛喉素）和C（佛波醇酯）激酶。 这些代理人有能力 模拟LH增强蛋白磷酸化和孕酮分泌 在小的细胞将被确定。 4)确定参与的蛋白质 在两种细胞类型中调节类固醇生成的作用将在 它们作为A和/或C的特异性磷蛋白底物的能力 细胞组分中的激酶。 该方法将利用Mg（gammaP 32）ATP 和纯化的催化亚基（A激酶）或Ca+2、磷脂酰肌醇 和佛波醇酯（C激酶激活剂）。 从上述观察， 蛋白磷酸化在LH刺激的小细胞类固醇生成中的作用 将描述细胞。 此外，一种解释缺乏 将提出在大细胞中cAMP对类固醇生成的调节。

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.