Ouabain production by cultured adrenal cells.
Ouabain production by cultured adrenal cells.
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DOI：
10.1210/endo.137.2.8593799
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发表时间：
1996-02
期刊：
影响因子：
4.8
通讯作者：
P. Doris;A. Hayward-Lester;D. Bourne;D. Stocco
中科院分区：
文献类型：
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作者：
P. Doris;A. Hayward-Lester;D. Bourne;D. Stocco
肾上腺皮质释放钠泵抑制剂。本研究通过1)研究长期培养的肾上腺皮质细胞中产生的瓦阿因，2)寻找证据证明肾上腺皮质细胞可能从外源性来源摄取瓦阿因，3)检查携带外源性瓦阿因的肾上腺皮质细胞的释放，4)尝试刺激培养的肾上腺皮质细胞中的瓦阿因甾体生成，以验证这种物质是否内源性且与瓦阿因相同。5)进一步对培养的肾上腺皮质细胞释放的乌阿巴因免疫反应性进行化学分析。我们的研究结果表明，在小鼠Y-1肾上腺皮质培养物和原代牛肾上腺皮质细胞（BAC）培养物的条件培养基中都存在瓦巴因的免疫反应性。我们还发现BACs结合并内化了[3H] abain。结合[3H]瓦巴因通过受体解离和内化[3H]瓦巴因的胞质释放从BACs中释放。只有膜钠的一种异构体，钾-腺苷三磷酸酶α 1在肾上腺中表达。真正的瓦巴因在膜结合或存在于细胞内时不会代谢。用22r -羟基胆固醇和25-羟基胆固醇刺激Y-1和BAC的类固醇生成，并证实类固醇生成增加；然而，对沃巴因的免疫反应性含量和释放量没有影响。比较培养BAC、肝癌细胞和3T3成纤维细胞的瓦阿因结合密度，提示肾上腺皮质细胞具有较高的瓦阿因结合能力。高效液相色谱法研究了牛肾上腺皮质细胞释放的瓦阿因免疫反应性，表明基本上没有真正的瓦阿因分泌。目前的研究证实，BAC和Y-1培养物都释放出一种类似瓦巴因的物质，这种物质的结构与真正的植物瓦巴因不同，并且不是胆固醇侧链断裂的产物。
The adrenal cortex releases a sodium pump inhibitor. The present studies tested whether this material was endogenous and identical to ouabain by 1) studying the production of ouabain in long term cultures of adrenocortical cells, 2) seeking evidence that ouabain might be taken up from exogenous sources by adrenocortical cells, 3) examining the release of adrenocortical cells loaded with exogenous ouabain, 4) attempting to stimulate ouabain steroidogenesis in cultured adrenocortical cells, and 5) performing further chemical analysis on ouabain immunoreactivity released by cultured adrenocortical cells. Our results indicate that ouabain immunoreactivity is present in conditioned medium from both murine Y-1 adrenocortical cultures and primary bovine adrenocortical cell (BAC) cultures. We also found that BACs bind and internalize [3H]ouabain. Bound [3H]ouabain is released from BACs by both receptor dissociation and cytoplasmic release of internalized [3H]ouabain. Only one isoform of membrane sodium, potassium-adenosine triphosphatase, alpha 1, was expressed in the adrenal. Authentic ouabain was not metabolized during membrane binding or while present intracellularly. Stimulation of steroidogenesis in Y-1 and BAC with 22R-hydroxycholesterol and 25-hydroxycholesterol was performed and confirmed increased steroidogenesis; however, there was no effect on ouabain immunoreactivity content or release. Comparison of the ouabain binding density in cultured BAC, hepatoma cells, and 3T3 fibroblasts indicated that adrenocortical cells have a high ouabain-binding capacity. HPLC studies of the ouabain immunoreactivity released by bovine adrenocortical cells indicated that essentially no authentic ouabain was secreted. The present studies confirm that both BAC and Y-1 cultures release a ouabain-like material that differs in structure from authentic plant ouabain and is not a product of cholesterol side-chain cleavage.