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，可在肾上腺产生。因此，黄体酮可能是OLF的底物。然而，没有证据表明OLF是在哺乳动物细胞中内源性产生的。我们用免疫组织化学方法在肾上腺髓质、嗜铬细胞瘤组织、肾上腺皮质和髓质中发现哇巴因免疫反应阳性。由于PC12细胞起源于大鼠嗜铬细胞瘤，我们探讨了在培养的PC12细胞中加入孕激素产生OLF的可能性。孕酮呈剂量依赖性地增强PC12细胞培养液中哇巴因样免疫反应。经5步高效液相色谱分离纯化培养基中的OLI。用高效液相色谱/质谱仪(LC/MS)对产物进行了分子结构分析，得到的产物与道地哇巴因完全一致。这是第一个表明哺乳动物细胞产生哇巴因的直接证据，与植物来源的哇巴因完全相同，因为培养系统显然是来自植物来源的哇巴因污染。神经元来源的细胞可以产生具有生物活性的类固醇，这可能是一个新的发现。此外，我们还试图通过选择离子模式的LC/MS来开发一种灵敏和选择性的分析方法。到目前为止，可以从5毫升的血浆中测量OLF和一种类似地高辛的因子。为了探索其他类数字因子的可能存在，我们正在开发一种新的生物测定系统，通过测量内皮细胞内的钙离子来检测类数字生物活性。

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.