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

哇巴因样因子（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OLF的底物。然而，没有证据表明OLF是哺乳动物细胞内源性产生的。免疫组化法在肾上腺髓质中发现哇巴因样免疫反应阳性，酶联免疫吸附试验在嗜铬细胞瘤组织、肾上腺皮质和髓质中发现哇巴因样免疫反应阳性。由于PC 12细胞来源于大鼠嗜铬细胞瘤，我们通过在培养基中加入孕酮来使用培养的PC 12细胞来探索OLF的可能产生。Progestine呈剂量依赖性地增加PC 12细胞培养液中哇巴因样免疫反应性。通过5步高效液相色谱（HPLC）纯化培养基中的OLI。将OLI的最终级分应用于HPLC/MS（LC/MS）以分析分子结构，发现OLI与真实的哇巴因相同。这是表明哺乳动物细胞产生与植物来源的哇巴因相同的哇巴因的第一个直接证据，因为培养系统显然没有受到植物来源的哇巴因的污染。神经元来源的细胞能够产生具有生物活性的甾体化合物，这可能是一个新的发现。此外，我们正在尝试建立一种灵敏、选择性的离子模式LC/MS分析方法。到目前为止，可以从5 ml血浆中测量OLF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测定系统洋地黄样生物活性，通过测量细胞内钙离子在内皮细胞。

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.