THE ROLE OF RNA SYNTHESIS IN EARLY ESTROGEN ACTION.
THE ROLE OF RNA SYNTHESIS IN EARLY ESTROGEN ACTION.
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RNA 合成在早期雌激素作用中的作用。
DOI：
10.1073/pnas.50.2.256
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发表时间：
1963
影响因子：
11.1
通讯作者：
G. Mueller
中科院分区：
文献类型：
--
作者：
Hiroshi Ui;G. Mueller
本实验室以前的研究表明，在大鼠子宫中，对雌二醇的早期反应的特征是合成反应迅速加速，导致磷脂、核糖核酸和蛋白质的积累。阻断蛋白质合成的嘌呤霉素水平阻止了这些反应，这表明该激素的主要作用是加速这些合成代谢途径的限速酶的合成。7由于在其他生物系统中已经证明特定蛋白质的合成取决于新RNA种类的先行合成，8-”研究早期雌激素反应是否需要合成新的RNA是很有趣的。许多研究者最近的研究已经证明了放线菌素D阻断细胞中和分离的酶系统中RNA的DNA依赖性合成的能力。本文将描述使用该试剂来阻止完整大鼠子宫中的RNA合成。结果表明，在放线菌素D阻断新RNA合成的条件下，磷脂和蛋白质合成的早期加速，以及雌二醇体内作用引起的水吸收的主要部分，未能发生。新的RNA的合成在早期雌激素反应的要求进行了讨论。方法。通过背侧入路切除相同年龄和体重约180 gm的雌性Holtzman大鼠的卵巢，并在实验前维持Purina狗饲料饮食至少3周。仅在同一天切除卵巢的大鼠用于任何一项实验。如图所示，将大鼠腹膜内注射放线菌素D（375 μ g，在0.5ml的0.154 Ml NaCl中）或单独注射盐水。在0小时，通过尾静脉注射10 μ g雌二醇-17的1.0 ml含1%乙醇的缓冲盐水溶液或缓冲盐水和乙醇的对照溶液。为了评估RNA、蛋白质和磷脂的体内合成，将溶解在0.5 ml盐水中的25 uc尿苷-H3（比活性= 1.0 mc/0.036 mg）和6 Jc甘氨酸-2C '4（比活性= 1 mc/12.4 mg）的组合在2和3小时时腹腔内注射。在某些实验中，仅给予甘氨酸-2CT'。在注射激素后4小时通过断头处死大鼠，取出子宫，剥离副脂肪，并在Roller-Smith扭力天平上称重。然后将子宫在冷蒸馏水中均质化，并将所得分散体用4%高氯酸（PCA）处理以除去酸溶性部分。沉淀的组织残留物用4%PCA洗涤两次，然后依次用90%乙醇、无水乙醇和无水乙醚萃取两次。在液体闪烁计数器中对构成粗脂质级分的合并的乙醇-醚提取物进行计数。将乙醇-乙醚提取后剩余的组织残留物悬浮于pH 7.4的0.01 M Tris（三羟甲基氨基甲烷）缓冲液中，并在37 ℃下孵育。C下用IOOyg RNA酶孵育30分钟。用4%PCA处理反应混合物以获得RNA酶释放的物质;在液体闪烁计数器中对该级分的H3含量进行计数。结果表示为每个子宫的每分钟计数（CPM）。将PCA不溶性残余物依次用90%乙醇、无水乙醇洗涤，并用无水乙醚洗涤两次。借助于少量甲酸将残留的蛋白质涂布在铝片上，干燥后在气流计数器256中计数
Previous studies from this laboratory have demonstrated that in the rat uterus the early response to estradiol is characterized by a rapid acceleration of synthetic reactions leading to the accumulation of phospholipid, ribonucleic acid, and protein.'-6 Levels of puromycin which blocked protein synthesis prevented these responses, suggesting that the primary action of this hornmone was to accelerate synthesis of rate-limiting enzymes for these anabolic pathways.7 Since it has been demonstrated in other biological systems that the synthesis of specific proteins depends on the antecedent synthesis of new RNA species,8-" it was of interest to inquire whether or not the early estrogenic response required the synthesis of new RNA. The recent studies of a number of investigators have documented the ability of actinomycin D to block the DNA-dependent synthesis of RNA in cells and in isolated enzyme systemns.l-"4 This paper will describe the use of this agent to prevent RNA synthesis in the uterus of the intact rat. It will be shown that under conditions in which the synthesis of new RNA was blocked by actinomycin D, the early acceleration of phospholipid and protein synthesis, as well as a major portion of the imbibition of water resulting from in vivo action of estradiol, failed to occur. The requirement for the synthesis of new RNA in the early estrogenic response is discussed. Methods.-Female Holtzman rats of the same age and weighing approximately 180 gm were ovariectomized through the dorsal approach and maintained on a diet of Purina dog chow for at least 3 weeks prior to experimentation. Only rats which were ovariectomized on the same day were used in any one experiment. The rats were injected intraperitoneally with actinomycin D (375 pg in 0.5 ml of 0.154 Ml NaCI) or with saline alone as indicated in the figures. At zero hr 10 pg of estradiol-17, in 1.0 ml of buffered saline containing 1% ethanol' or the control solution of buffered saline and ethanol was injected via the tail vein. To assess the in vivo synthesis of RNA, protein, and phospholipid, a combination of 25 uc of uridine-H3 (specific activity = 1.0 mc/0.036 mg) and 6 Jc of glycine-2C"4 (specific activity = I mc/12.4 mg) dissolved in 0.5 ml of saline was injected intraperitoneally at 2 and 3 hr. In certain experiments only glycine-2CT" was administered. The rats were killed 4 hr after the injection of hormone by decapitation, and the uteri were removed, stripped of accessory fat, and weighed on a Roller-Smith torsion balance. The uteri were then homogenized in cold distilled water, and the resulting dispersion was treated with 4%0 perchloric acid (PCA) to remove the acid-soluble fraction. The sedimented tissue residue was washed twice with 4% PCA and then was extracted successively with 90% ethanol, absolute ethanol, and twice with anhydrous ethyl ether. The combined ethanol-ether extracts, making up the crude lipid fraction, were counted in a liquid scintillation counter. The tissue residue remaining after ethanol-ether extraction was suspended in 0.01 M Tris (trishydroxymethylaminomethane) buffer at pH 7.4 and incubated at 37?C for 30 min with 100 pg of RNAse. The reaction mixture was treated with 4% PCA to obtain the RNAse-released material; this fraction was counted for H3 content in a liquid scintillation counter. The results were expressed as counts per min (CPM) per uterus. The PCA-insoluble residue was washed successively with 90% ethanol, absolute ethanol, and twice with anhydrous ethyl ether. The residual protein was spread on aluminum planchets with the aid of a small amount of formic acid, and after drying it was counted in a gas flow counter 256