Aldosterone signaling pathway across the nuclear envelope

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 ( congruent with 100 kDa) molecules (flags) attached to the NPC surface. At the sustained depression NPCs were found free of flags. At the late peak large ( congruent with 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.