Human adrenal cells in culture produce both ouabain-like and dihydroouabain-like factors.
Human adrenal cells in culture produce both ouabain-like and dihydroouabain-like factors.
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DOI：
10.1093/clinchem/48.10.1720
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发表时间：
2002-10
影响因子：
9.3
通讯作者：
M. A. El-Masri;B. Clark;H. Qazzaz;R. Valdes
中科院分区：
文献类型：
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作者：
M. A. El-Masri;B. Clark;H. Qazzaz;R. Valdes
背景哇巴因样因子（OLF）及其新发现的还原型二氢哇巴因样因子（Dh-OLF）是哺乳动物的强心内酯，其结构和功能特征与植物源化合物哇巴因和二氢哇巴因相似。这些内源性化合物被认为是由肾上腺产生的，并构成激素轴的一部分，该激素轴可以调节Na（+），K（+）-ATP酶的α-亚基的催化活性。我们开发了特异性足以区分OLF和Dh-OLF的抗体，并在这项研究中证明了OLF和Dh-OLF从培养的人H295 R-1肾上腺皮质细胞的选择性分泌。方法采用反相高效液相色谱法、Na（+），K（+）-ATP酶催化活性抑制法和两种酶免疫分析法，对哇巴因和二氢哇巴因的特异性抗体进行纯化和鉴定。纯化的抗血清具有高滴度（哇巴因为1 × 10（6），二氢哇巴因为8 × 10（5）），并对其相应的抗原具有特异性。结果在无血清培养基中生长的人H295 R-1细胞在24 h内每10（6）个细胞分泌0.18 +/- 0.03 pmol OLF和0.39 +/- 0.04 pmol Dh-OLF。OLF和Dh-OLF均抑制钠泵对哇巴因敏感的催化活性（0.03 μ mol/L OLF抑制29%，0.07 μ mol/L Dh-OLF抑制17%）。通过二丁基cAMP刺激细胞培养物使Dh-OLF的分泌比对照（未刺激）增加50%，而OLF的分泌没有显著增加。结论OLF和Dh-OLF由人肾上腺细胞分泌，以植物源性OLF为抗原，可制备出特异性抗体。Dh-OLF的分泌响应于cAMP依赖性刺激机制，而OLF则不是。我们的数据表明，无论是分泌或生物合成途径的生产这两种化合物的人肾上腺细胞可能有不同的控制机制，或者它们可能是通过一个受体产品的关系。
BACKGROUND Ouabain-like factor (OLF) and its newly discovered reduced species, dihydroouabain-like factor (Dh-OLF), are mammalian cardenolides whose structural and functional characteristics are similar to the plant-derived compounds ouabain and dihydroouabain. These endogenous compounds are believed to be produced by the adrenals and to constitute part of an hormonal axis that may regulate the catalytic activity of the alpha-subunit of Na(+),K(+)-ATPase. We developed antibodies sufficiently specific to distinguish between OLF and Dh-OLF, and in this study demonstrate the selective secretion of OLF and Dh-OLF from human H295R-1 adrenocortical cells in culture. METHODS We used reversed-phase HPLC, inhibition of Na(+),K(+)-ATPase catalytic activity, and two enzyme immunoassays developed with antibodies specific to ouabain and dihydroouabain to purify and characterize the secretion of these two compounds by human adrenal cells in culture. Purified antisera had high titers (1 x 10(6) for ouabain and 8 x10(5) for dihydroouabain) and were specific to their corresponding antigens. RESULTS Human H295R-1 cells grown in serum-free medium secreted 0.18 +/- 0.03 pmol of OLF and 0.39 +/- 0.04 pmol of Dh-OLF per 10(6) cells in 24 h. Both OLF and Dh-OLF inhibited the ouabain-sensitive catalytic activity of the sodium pump (0.03 micro mol/L OLF inhibited 29% of the catalytic activity; 0.07 micro mol/L Dh-OLF inhibited 17%). Stimulation of the cell culture by dibutryl cAMP increased the secretion of Dh-OLF 50% over control (unstimulated), whereas the secretion of OLF did not increase significantly. CONCLUSIONS OLF and Dh-OLF are secreted by human adrenal cells, and antibodies specific to these two compounds can be developed, using the plant-derived counterparts as antigens. The secretion of Dh-OLF is responsive to a cAMP-dependent stimulation mechanism, whereas OLF is not. Our data suggest that either the secretory or biosynthetic pathways for production of these two compounds by human adrenal cells may have different control mechanisms or that they may be linked via a precursor-product relationship.