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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Aldosterone signaling pathway across the nuclear envelope
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Aldosterone signaling pathway across the nuclear envelope

机译：醛固酮穿越核膜的信号通路

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We describe the route by which aldosterone-triggered macromol-ecules enter and exit the cell nucleus of Xenopus laevis oocyte. Oocytes were microinjected with 50 fmol aldosterone and then enucleated 2-30 min after injection. After isolation, nuclear envelope electrical resistance (NEER) was measured in the intact cell nuclei by using the nuclear hourglass technique. We observed three NEER stages: an early peak 2 min after injection, a sustained depression after 5-15 min, and a final late peak 20 min after injection. Because NEER reflects the passive electrical permeability of nuclear pores, we investigated with atomic force microscopy aldosterone-induced conformational changes of individual nuclear pore complexes (NPCs). At the early peak we observed small (≈100 kDa) molecules (flags) attached to the NPC surface. At the sustained depression NPCs were found free of flags. At the late peak large (≈800 kDa) molecules (plugs) were detected inside the central channels. Ribonuclease or actinomycin D treatment prevented the late NEER peak. Coinjection of aldosterone (50 fmol) and its competitive inhibitor spironolactone (500 fmol) eliminated the electrical changes as well as flag and plug formation. We conclude: (ⅰ) The genomic response of aldosterone can be electrically measured in intact oocyte nuclei. (ⅱ) Flags represent aldosterone receptors on their way into the cell nucleus whereas plugs represent ribonucleoproteins carrying aldosterone-induced mRNA from the nucleoplasm into the cytoplasm. (ⅲ) Because plugs can be mechanically harvested with the atomic force microscopy stylus, oocytes could serve as a bioassay system for identifying aldosterone-induced early genes.
机译：我们描述了醛固酮触发的大分子进入和退出非洲爪蟾卵母细胞的细胞核的途径。卵母细胞用50 fmol醛固酮微注射，然后在注射后2-30分钟摘出去核。分离后，通过使用核沙漏技术在完整的细胞核中测量核被膜电阻（NEER）。我们观察到三个NEER阶段：注射后2分钟的早期高峰，注射5-15分钟后的持续抑郁和注射后20分钟的最终晚期高峰。因为NEER反映了核孔的被动电导率，所以我们用原子力显微镜研究了醛固酮诱导的单个核孔复合物（NPC）的构象变化。在早期峰值时，我们观察到附着在NPC表面的小分子（约100 kDa）（标志）。在持续的低迷时期，发现NPC没有国旗。在峰值后期，在中央通道内检测到大分子（约800 kDa）。核糖核酸酶或放线菌素D处理可防止晚期NEER高峰。醛固酮（50 fmol）与其竞争性抑制剂螺内酯（500 fmol）的共同注射消除了电学变化以及标志和栓塞的形成。我们得出以下结论：（ⅰ）可以在完整的卵母细胞核中用电法测定醛固酮的基因组反应。 （ⅱ）标记代表醛固酮受体进入细胞核的过程，而栓塞则代表核糖核蛋白，其携带醛固酮诱导的mRNA从核质进入细胞质。 （ⅲ）因为可以用原子力显微镜测针机械地收获栓子，所以卵母细胞可以用作鉴定醛固酮诱导的早期基因的生物测定系统。

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