IDENTIFICATION OF ENDOGENOUS DIGITALIS AND THE ROLE IN WATER-ELETROLYTES BALANCES AND BLOOD PRESSURE REGULATION

乌巴因样因子（OLF）被认为是一种类固醇，可能在肾上腺中产生。因此，黄体酮可能是黄韧带骨化的底物。然而，没有证据表明黄韧带骨化是在哺乳动物细胞内源性产生的。我们通过免疫组织化学发现肾上腺髓质有瓦巴因样免疫反应性，通过酶联免疫吸附法发现嗜铬细胞瘤组织、肾上腺皮质和髓质有瓦巴因样免疫反应性。由于PC12细胞来源于大鼠嗜铬细胞瘤，我们探索了通过在培养基中添加黄体酮，利用培养的PC12细胞产生黄韧带黄细胞的可能性。黄体酮增加PC12细胞的瓦拜样免疫反应性呈剂量依赖性。采用高效液相色谱法（HPLC）对培养基中的OLI进行纯化。利用高效液相色谱/质谱（LC/MS）分析其分子结构，发现其与正宗瓦巴因相同。这是第一个直接证据，表明哺乳动物细胞产生与植物来源相同的瓦巴因，因为培养系统显然没有受到植物来源的瓦巴因的污染。这可能是一个新的发现，神经元来源的细胞可以产生生物活性类固醇。此外，我们正在尝试开发一种敏感和选择性的检测方法，采用LC/MS与选定的离子模式。到目前为止，已经可以从5ml血浆中测量黄韧带骨化和地高辛样因子。为了探索其他洋地黄样因子的可能存在，我们正在开发一种新的生物检测系统，通过测量内皮细胞内钙离子来检测洋地黄样生物活性。

Ouabainlike factor (OLF) has been suggested to be a steroid, and may be produced in adrenals. Thereby, progesterone could be a substrate for the OLF.However, there is no evidence that OLF is endogenously produced in mammalian cells. We have found Ouabainlike immunoreactivity in the adrenal medulla by immunohistochemistry, the pheochromocytoma tissue, adrenal cortex and medulla by enzyme-linked immunosorbent assay for ouabain. Since PC12 cells are of rat pheochromocytoma origin, we explored the possible production of OLF by using cultured PC12 cells by adding progesterone in the culture medium. Progesterone increased Ouabainlike immunoreactivity in the culture medium of PC12 cells dose-dependently. OLI in the culture medium was purified by 5 steps of high-performance liquid chromatography (HPLC). The final fraction of OLI was applied to HPLC/mass spectrometry (LC/MS) to analyze the molecular structure, and the OLI was found to be identical to authentic ouabain. This is the first direct evidence to show that mammalian cells produce ouabain, identical to that of plant origin, because the culture system is apparently out of contamination of ouabain of plant origin. It may be a novel finding that cells of neuronal origin can produce biologically active steroids.Furthermore, we are trying to develop a sensitive and selective assay method by using LC/MS with a selected ion mode. So far, it became possible to measure OLF and a digoxinlike factor from 5ml of plasma. To explore the possible existence of other digitalislike factors, we are developing a novel biological assay system for digitalislike bioactivity by measuring intracellular calcium ions in endothelial cells.