Edinger Robert S, Watkins Simon C, Pearce David, Johnson John P
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
Am J Physiol Renal Physiol. 2002 Aug;283(2):F254-61. doi: 10.1152/ajprenal.00337.2001.
Immunosuppressive agents such as FK-506 and rapamycin inhibit aldosterone- stimulated Na+ transport in A6 cells. Concentration dependence is consistent with the known affinities of these agents for immunophilins. The inhibition was also dependent on time, requiring preincubation with FK-506 or rapamycin before inhibition was seen. The present studies were designed to determine whether this inhibition was pretranscriptional and whether it was due to an effect on either receptor translocation or nuclear accumulation. Because transport effects of steroids in A6 cells are mediated by glucocorticoid receptors (GRs), we examined the transcriptional response of GR-regulated reporters transfected into these cells. Preincubation of cells with FK-506 and rapamycin completely blocked reporter gene activation, whereas preincubation with cyclosporin A partially inhibited this activation. A minimum of 8 h of preincubation was required before the effect was seen. Using a transiently transfected green fluorescent protein-GR construct, we examined the effect of FK-506 and rapamycin on GR translocation. GR translocation induced by dexamethasone was extremely rapid (<5 min) and was largely unaffected by FK-506 or rapamycin but was completely blocked by geldanamycin. Digital deconvolutions revealed a punctate nuclear accumulation of GR, which was still seen after preincubation with immunosuppressive agents. These agents clearly inhibit steroid action by blocking GR-stimulated gene transcription, but this effect is not mediated by altered translocation or nuclear accumulation of receptors. Inhibition of steroid-regulated gene transcription by immunosuppressive agents may explain the electrolyte abnormalities seen in patients receiving these drugs.
免疫抑制剂如FK-506和雷帕霉素可抑制A6细胞中醛固酮刺激的Na+转运。浓度依赖性与这些药物对免疫亲和蛋白的已知亲和力一致。这种抑制作用也取决于时间，在观察到抑制作用之前需要用FK-506或雷帕霉素进行预孵育。本研究旨在确定这种抑制作用是否发生在转录前，以及是否是由于对受体易位或核积累的影响。由于A6细胞中类固醇的转运效应是由糖皮质激素受体（GRs）介导的，我们检测了转染到这些细胞中的GR调节报告基因的转录反应。用FK-506和雷帕霉素对细胞进行预孵育可完全阻断报告基因的激活，而用环孢素A预孵育则部分抑制这种激活。在观察到这种效应之前，至少需要8小时的预孵育。使用瞬时转染的绿色荧光蛋白-GR构建体，我们检测了FK-506和雷帕霉素对GR易位的影响。地塞米松诱导的GR易位非常迅速（<5分钟），并且在很大程度上不受FK-506或雷帕霉素的影响，但被格尔德霉素完全阻断。数字去卷积显示GR在细胞核中有斑点状积累，在用免疫抑制剂预孵育后仍然可见。这些药物显然通过阻断GR刺激的基因转录来抑制类固醇作用，但这种效应不是由受体易位或核积累的改变介导的。免疫抑制剂对类固醇调节基因转录的抑制作用可能解释了接受这些药物的患者出现的电解质异常。
