Aldosterone signaling pathway across the nuclear envelope

Abstract

We describe the route by which aldosterone-triggered macromolecules 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 (Х100 kDa) molecules (flags) attached to the NPC surface. At the sustained depression NPCs were found free of flags. At the late peak large (Х800 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.nuclear pore complex ͉ atomic force microscopy ͉ RNA export ͉ mineralocorticoid receptor S ignals such as an increase in intracellular calcium concentration or intracellular alkalinization are the early responses of target cells to the mineralocorticoid hormone aldosterone. The intracellular calcium signal can be detected a few seconds after hormone exposure (1-3), whereas the pH signal occurs later but usually lasts longer (4). Although there are no doubts about the existence of these early hormone responses, the physiological relevance is still unknown. For almost 40 years it has been postulated that inorganic ions could play a crucial role in steroid hormone-induced gene activation (5). This suggestion has been supported by more recent findings in our own laboratory (6, 7). The signaling pathway of steroid hormones involving intracellular macromolecules is well established. Aldosterone is known to bind to mineralocorticoid receptors located in the cytosol. Hormone binding changes protein conformation and translocates the receptor into the nucleus where it attaches to specific DNA. Transcription occurs and finally mRNA is exported to be translated into aldosterone-induced proteins at the ribosomes. Although this macromolecular signal pathway has been described in detail, there are no data available that directly show both functionally and structural...

Glucocorticoids remodel nuclear envelope structure and permeability

“…Oocytes injection, isolation of the nuclei and preparation of nuclear envelopes Oocytes were collected as previously described (Schäfer et al, 2002). For the triamcinolone acetonide (TA) experiments, oocytes were injected with 50 nl solution containing TA (TA concentration in the injection fluid: 2ϫ10 -5 mol l -1 in water; TA stock solution dissolved in ethanol: 10 -2 mol l -1 ).…”

“…Oberleithner et al (Oberleithner et al, 1994) showed in kidney cells that the NE responds to aldosterone by increasing both, NPCs density and NE permeability within 6 hours of hormone exposure. Using a nano-imaging technique, atomic force microscopy (AFM) (Binnig and Quate, 1986), we could visualize some of the endogeneous macromolecules on their route through the NPCs following aldosterone injection into X. laevis oocyte (Schäfer et al, 2002). Changes in NPC conformation were paralleled by sharp changes in NE electrical conductivity (Danker et al, 1999).…”

Aldosterone: Refreshing a Slow Hormone by Swift Action

“…Third, MR antagonists such as spironolactone or canrenone usually do not block rapid aldosterone effects, which indicates that these effects are not mediated by the MR but by an unrelated receptor. However, a few cases of rapid MR antagonist-sensitive aldosterone effects have been reported (13,18). These findings may suggest that at least some rapid effects are mediated via the classical intracellular MR.…”

Nongenomic Vascular Action of Aldosterone in the Glomerular Microcirculation

“…Then vasoconstrictor actions of Aldo were examined as in protocol 1. It has been demonstrated that this concentration of actinomycin D or cycloheximide prevents Aldoinduced genomic nuclear signaling (16) or de novo protein synthesis in rat aorta (17), respectively. We used same vehicle for actinomycin D or cycloheximide with that for spironolactone.…”