Sodium Pump Inhibitors In Blood Pressure Regulation

细胞色素450 CYP11A1对胆固醇侧链的切割（即甾体生成的经典途径）与MBG的生物合成无关，而内源性瓦巴因则与此有关。先前对蟾蜍的研究表明，已知会产生蟾二烯内酯的蟾蜍，给予标记的胆固醇和胆酸，而不是黄体酮（胆固醇侧链裂解的产物），会导致蟾二烯内酯分子中含有放射性标记。由于胆汁酸的肝外合成最近被描述，我们假设在胎盘中，在子痫前期检测到高水平的MBG，蟾油二烯内酯可以由胆汁酸合成。在人滋养层JEG-3细胞中，我们检测了两个基因转录后沉默对MBG和黄体酮产生的影响，这两个基因分别编码甾醇27-水解酶（CYP27A1）和CYP11A1，前者是一种从胆固醇合成胆酸的起始酶，后者是一种控制胆固醇侧切成孕烯醇酮的酶。在细胞培养基中测量类固醇水平。在JEG-3细胞中，CYP11A1的沉默导致蛋白量减少90% (Western blot)， mRNA减少80% (qPCR)，孕酮的产生减少77%，但与未转染和模拟转染的细胞相比，不影响MBG的产生。反之，CYP27A1沉默，CYP27A1蛋白减少75%，mRNA水平减少65%，不影响黄体酮的产生，但与未转染和模拟转染的细胞相比，MBG的产生抑制了80%。因此，在人滋养细胞中，由胆固醇经胆汁酸途径合成丁二烯内酯类促心类固醇MBG，代表了一种新的激素生物合成途径。

Side-chain cleavage of cholesterol by cytochrome 450 CYP11A1 (i.e., classical pathway of steroidogenesis) is not implicated in the biosynthesis of MBG, as it is for endogenous ouabain. Previous studies in toads, known to produce bufadienolides, demonstrated that administration of labeled cholesterol and cholanic acid, but not of progesterone, the product of cholesterol side-chain cleavage, resulted in the incorporation of a radioactive label into bufadienolide molecules. Because the extra-hepatic synthesis of bile acids has recently been described, we hypothesized that in placenta, in which high levels of MBG are detected in preeclampsia, bufadienolides could be synthesized from bile acids. In human trophoblast JEG-3 cells, we examined the impact of post-transcriptional silencing of two genes, encoding sterol 27-hydrolase (CYP27A1), an initiating enzyme for synthesis of cholanic acid from cholesterol, and CYP11A1, an enzyme which controls side-cleavage of cholesterol into pregnenolone, on production of MBG and progesterone. Levels of steroids were measured in cell culture media. In JEG-3 cells, silencing of CYP11A1 resulted in 90% decrease of the protein amount (Western blot), in 80% decrease of mRNA (qPCR), and reduced production of progesterone by 77%, but did not affect production of MBG as compared to non-transfected and mock-transfected cells. Silencing of CYP27A1, accompanied by 75% reduction in CYP27A1 protein, and by 65% reduction in mRNA level, conversely, did not affect production of progesterone, but suppressed production of MBG by 80% vs. that in non-transfected and mock-transfected cells. Thus, in human trophoblast cells, bufadienolide cardiotonic steroid MBG is synthesized from cholesterol via bile acid pathway which represents a novel pathway of hormone biosynthesis.