Azhar S, Menon K M
Biochem J. 1979 Apr 15;180(1):201-11. doi: 10.1042/bj1800201.
The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4-5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50-2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose-response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [(3)H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [(3)H]AMP, the radioactivity associated with the cells increased almost linearly up to 250mum-cyclic [(3)H]AMP concentration in the incubation medium. The (3)H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.
在分离的大鼠卵巢细胞中,研究了环核苷酸磷酸二酯酶和环磷酸腺苷(cAMP)代谢在促性腺激素与孕酮生成关系中的调节作用。当在不存在磷酸二酯酶抑制剂的情况下进行实验时,低浓度的绒毛膜促性腺激素(0.4 - 5ng/ml)可增加类固醇生成,而cAMP没有任何可检测到的增加。最大程度刺激孕酮合成的绒毛膜促性腺激素浓度(10ng/ml)导致cAMP积累和绒毛膜促性腺激素结合的最小增加。然而,浓度为10ng/ml及更高的绒毛膜促性腺激素显著刺激蛋白激酶活性,在激素浓度为250至1000ng/ml时达到最大值。更高浓度(50 - 2500ng/ml)的绒毛膜促性腺激素导致内源性cAMP增加,且这种增加先于类固醇合成的增加。当在3 - 异丁基 - 1 - 甲基黄嘌呤存在下进行实验时,促性腺激素刺激的cAMP积累、孕酮生成和蛋白激酶活性的剂量 - 反应关系分析显示,在很宽的浓度范围内这些反应之间存在相关性。磷酸二酯酶抑制剂罂粟碱、茶碱和3 - 异丁基 - 1 - 甲基黄嘌呤均以剂量依赖性方式刺激类固醇生成。用二丁酰环磷酸腺苷或8 - 溴环磷酸腺苷孵育卵巢细胞模拟了促性腺激素的类固醇生成作用,且这种作用取决于孵育时间和核苷酸浓度。用2mM二丁酰环磷酸腺苷和8 - 溴环磷酸腺苷可获得最大刺激,这种增加接近最大刺激剂量的绒毛膜促性腺激素所产生的增加。其他8 - 取代衍生物,如8 - 羟基环磷酸腺苷和8 - 异丙硫基环磷酸腺苷,它们对磷酸二酯酶作用的敏感性较低,也有效地刺激了类固醇生成。还研究了卵巢细胞中环[³H]AMP的摄取和代谢与类固醇生成的关系。当卵巢细胞在存在浓度不断增加的环[³H]AMP的情况下孵育2小时时,与细胞相关的放射性在孵育培养基中环[³H]AMP浓度达到250μM之前几乎呈线性增加。细胞提取物中的³H标记主要以ATP、ADP、AMP、腺苷和肌苷的形式回收,环磷酸腺苷占总组织放射性的不到1%。在体外将环磷酸腺苷与卵巢细胞一起孵育导致培养基中的核苷酸迅速分解。培养基中的降解产物已鉴定为AMP、腺苷和肌苷。磷酸二酯酶对环磷酸腺苷的快速降解使得难以将环磷酸腺苷浓度的变化与类固醇生成相关联。因此,这些观察结果为先前观察到的在低剂量绒毛膜促性腺激素刺激类固醇生成而环磷酸腺苷积累没有任何可检测变化的情况下缺乏环磷酸腺苷积累提供了解释。