Darbon J M, Knecht M, Ranta T, Dufau M L, Catt K J
J Biol Chem. 1984 Dec 10;259(23):14778-82.
The hormonal regulation of cAMP-dependent protein kinase was examined in granulosa cells from diethylstilbestrol-implanted immature rats. Follicle-stimulating hormone (FSH) increased the number of available cAMP-binding sites in a dose- and time-dependent manner, with a maximum 4-6-fold increase at 50-100 ng/ml between 6 and 48 h of culture after a transient decrease in available sites during the first 6 h. The potent gonadotropin-releasing hormone (GnRH) agonist [D - Ala6]des - Gly10 - GnRH - N - ethylamide (GnRHa) reduced the FSH-induced increase in cAMP-binding sites by approximately 50% at 24 and 48 h of culture. Photoaffinity labeling with 8-azido-[32P] cAMP revealed the existence of one major cAMP-binding protein (Mr = 55,000 +/- 400) which appeared to be the regulatory (R) subunit of type II cAMP-dependent protein kinase. While FSH induced a 5-10-fold increase in the labeling of R II both in vivo and in vitro, GnRHa reduced the amount of R II induced by FSH in granulosa cells cultured for 48 h. The large increase in R II subunit was not accompanied by a corresponding increase in protein kinase activity, which was only enhanced by 50% after 48 h of culture with FSH. Fractionation of granulosa cell cytosol from FSH-treated ovaries on DEAE-cellulose showed a single peak of cAMP-dependent phosphokinase activity with the elution properties of a type II protein kinase. However, the peak of cAMP binding activity (eluted at 0.20 M KCl) was not coincident with the protein kinase activity. FSH transiently stimulated cAMP-dependent protein kinase activity during the first 10-30 min of culture. GnRHa impaired the FSH-induced early increase in protein kinase activity, causing a delay in activation until 60 min. These findings suggest that a large dose- and time-dependent increase in the content of cAMP-binding sites may be a major factor in cAMP-mediated differentiation of granulosa cells. The inhibitory effect of GnRHa on both FSH-induced protein kinase activation during the first minutes of culture and on FSH-induced R II synthesis during the subsequent 48 h of culture could be crucial events in the prevention of granulosa cell maturation by GnRH agonists.
在己烯雌酚植入的未成熟大鼠的颗粒细胞中研究了环磷酸腺苷（cAMP）依赖性蛋白激酶的激素调节。促卵泡激素（FSH）以剂量和时间依赖性方式增加了可利用的cAMP结合位点的数量，在培养的前6小时可利用位点短暂减少后，在培养6至48小时期间，在50 - 100 ng/ml时最大增加4 - 6倍。强效促性腺激素释放激素（GnRH）激动剂[D - Ala6]去 - Gly10 - GnRH - N - 乙酰胺（GnRHa）在培养24和48小时时使FSH诱导的cAMP结合位点增加减少了约50%。用8 - 叠氮基 - [32P]cAMP进行光亲和标记揭示存在一种主要的cAMP结合蛋白（Mr = 55,000 ± 400），其似乎是II型cAMP依赖性蛋白激酶的调节（R）亚基。虽然FSH在体内和体外均诱导R II标记增加5 - 10倍，但GnRHa降低了在培养48小时的颗粒细胞中FSH诱导的R II量。R II亚基的大量增加并未伴随着蛋白激酶活性的相应增加，在用FSH培养48小时后蛋白激酶活性仅增强了50%。用DEAE - 纤维素对FSH处理的卵巢的颗粒细胞胞质溶胶进行分级分离显示出一个cAMP依赖性磷酸激酶活性峰，其具有II型蛋白激酶的洗脱特性。然而，cAMP结合活性峰（在0.20 M KCl处洗脱）与蛋白激酶活性不一致。FSH在培养的前10 - 30分钟短暂刺激cAMP依赖性蛋白激酶活性。GnRHa损害了FSH诱导的蛋白激酶活性早期增加，导致激活延迟至60分钟。这些发现表明，cAMP结合位点含量的大量剂量和时间依赖性增加可能是cAMP介导的颗粒细胞分化的主要因素。GnRHa在培养的最初几分钟对FSH诱导的蛋白激酶激活以及在随后48小时对FSH诱导的R II合成的抑制作用可能是GnRH激动剂预防颗粒细胞成熟的关键事件。
