THE ROLE OF RNA SYNTHESIS IN EARLY ESTROGEN ACTION

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