Stein P, Téllez-Iñón M T, Tesone M
Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina.
Mol Reprod Dev. 1996 Sep;45(1):43-7. doi: 10.1002/(SICI)1098-2795(199609)45:1<43::AID-MRD6>3.0.CO;2-S.
The present studies were carried out to characterize the cAMP-phosphodiesterase enzyme (PDE) in luteal cells recovered from pseudopregnant rats with streptozotocin-induced diabetes. A significant increase in the specific activity of the enzyme was detected in luteal cells from diabetic rats (Group D) with respect to control rats (Group C). This increase could not be prevented by insulin therapy (Group I). Luteal cells from Groups C and D rats responded in vitro to insulin by increasing their PDE activity (% of stimulus of specific activity: C = 75%, D = 110%). However, in cells isolated from Group I, the hormone caused an inhibition of PDE activity (% of inhibition of specific activity: 48%). When cytosolic fractions from Groups C, D and I were submitted to ion exchange chromatography, two PDE activity peaks could be observed and the activity of the different fractions was increased in the presence of Ca2+ and calmodulin. Nevertheless, the Ca(2+)-calmodulin effect was much lower in the extracts from Groups D and I than for controls. Kinetic studies of luteal PDE showed nonlinear Lineweaver-Burk graphs with two apparent ATP hydrolysis sites. Similar K(m) values were found for PDE from groups C, D, and I, whereas the Vmax2 for the enzyme was higher in Groups D and I. The endogenous concentration of cAMP, measured by RIA, showed no significant differences among Groups C, D, and I. On the basis of these results, we conclude that the specific activity of PDE is significantly increased in luteal cells from streptozotocin-induced diabetic animals, which could explain the previously described reduction in LH-stimulated progesterone production by luteal cells in diabetic rats.
本研究旨在表征从链脲佐菌素诱导糖尿病的假孕大鼠中回收的黄体细胞中的环磷酸腺苷磷酸二酯酶（PDE）。与对照大鼠（C组）相比，在糖尿病大鼠（D组）的黄体细胞中检测到该酶的比活性显著增加。胰岛素治疗（I组）无法阻止这种增加。C组和D组大鼠的黄体细胞在体外对胰岛素有反应，通过增加其PDE活性（比活性刺激百分比：C组=75%，D组=110%）。然而，在从I组分离的细胞中，该激素导致PDE活性受到抑制（比活性抑制百分比：48%）。当C组、D组和I组的胞质组分进行离子交换色谱分析时，可以观察到两个PDE活性峰，并且在Ca2+和钙调蛋白存在的情况下，不同组分的活性增加。然而，D组和I组提取物中的Ca(2+)-钙调蛋白效应远低于对照组。黄体PDE的动力学研究显示，Lineweaver-Burk图呈非线性，有两个明显的ATP水解位点。C组、D组和I组的PDE的K(m)值相似，而该酶的Vmax2在D组和I组中较高。通过放射免疫分析测量的cAMP内源性浓度在C组、D组和I组之间没有显著差异。基于这些结果，我们得出结论，链脲佐菌素诱导的糖尿病动物的黄体细胞中PDE的比活性显著增加，这可以解释先前描述的糖尿病大鼠黄体细胞中LH刺激的孕酮产生减少的现象。
