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The action of glucocorticoids on vascular permeability is well established. However, little is known about the action of mineralocorticoids on the structure and function of blood vessels. As endothelial cells are targets for both glucocorticoids and mineralocorticoids, we exposed human umbilical vein endothelial cells to both types of steroids. Aldosterone (mineralocorticoid) and dexamethasone (glucocorticoid) were applied for 3 days in culture before measurements of transendothelial ion and macromolecule permeability, apical cell surface and cell stiffness were taken. Transendothelial ion permeability was measured with electrical cell impedance sensing, macromolecule permeability with fluorescence-labeled dextran and apical cell membrane surface by three-dimensional AFM imaging. Cell stiffness was measured using the AFM scanning tip as a mechanical nanosensor. We found that aldosterone increased both apical cell surface and apical cell stiffness significantly, while transendothelial permeability remained unaffected. By contrast, dexamethasone significantly decreased ion and macromolecule permeability, while apical cell surface and cell stiffness did not change. Specific receptor antagonists for dexamethasone (RU486) and aldosterone (spironolactone) prevented the observed responses. We conclude that glucocorticoids strengthen cell-to-cell contacts (;peripheral action'), whereas mineralocorticoids enlarge and stiffen cells (;central action'). This could explain the dexamethasone-mediated retention of fluid in the vascular system, and endothelial dysfunction in states of hyperaldosteronism.

The mineralocorticoid hormone aldosterone controls fluid and electrolyte transport in target cells of the kidney and the cardiovascular system. Classic genomic aldosterone action involves the activation of cytosolic mineralocorticoid receptors and translocation into the cell nucleus where specific transcription processes are initiated. A key barrier of the intracellular signalling pathway is the nuclear envelope, which physically separates the nucleoplasm from the cytoplasm. It was shown recently that aldosterone changes ion conductivity of the nuclear envelope mediated by nuclear pore complexes. The latter are supramolecular nanomachines responsible for import and export of inorganic ions and macromolecules. The aim of the present study was to test whether aldosterone changes the macromolecule permeability of the nuclear envelope. Aldosterone-responsive Xenopus laevis oocytes were used as a model system. We isolated the cell nuclei at defined times after hormone injection. By means of confocal fluorescence microscopy and fluorescence-labelled dextrans we evaluated passive macromolecule import and export in isolated nuclei. 10 minutes after aldosterone injection nuclear envelope permeability of 10 kD dextran was found sharply increased. At the same time cell nuclei were found swollen by about 28%. Changes in nuclear volume and nuclear envelope permeability lasted 5 to 15 minutes and could be inhibited by the mineralocorticoid receptor blocker spironolactone. We conclude that aldosterone transiently changes the barrier function of the nuclear envelope. This short-lasting permeability change signals the start of a sustained transcription process that follows in response to steroids.

Aldosterone has long been known to control water and electrolyte balance by acting on mineralocorticoid receptors in kidney. However, recent studies demonstrated the presence of these receptors in nonclassical locations, including the cardiovascular system. We tested the hypothesis whether endothelial cells respond to aldosterone with changes in cell volume, a measure for ion-mediated water movement across the cell membrane. By means of atomic force microscopy in fluid, we measured volume of adherent human umbilical venous endothelial cells exposed for 72 hours to 10 nmol/L aldosterone. Over this period of time, cells swell by approximately 18%. Aldosterone-induced swelling is prevented by 100 nmol/L of the mineralocorticoid receptor antagonist spironolactone, added to the primary endothelial cell culture. Aldosterone-treated cells dramatically shrink when 1 micromol/L of the diuretic amiloride is applied. Cells deprived of aldosterone do not respond to amiloride. Our conclusions are: (1) aldosterone leads to sustained cell swelling inhibited by administration of spironolactone or the sodium channel blocker amiloride; (2) cells respond to amiloride after aldosterone exposure; (3) renal diuretics act on endothelial cells; and (4) both amiloride and spironolactone could be useful for medical applications to prevent aldosterone-mediated endothelial dysfunction.

Transport of salt and water in various tissues is under control of the mineralocorticoid hormone aldosterone. As a liphophilic hormone, aldosterone diffuses through the plasma membrane and, then, binds to cytosolic mineralocorticoid receptors in the target cells. After binding to nuclear pore complexes, the activated receptor is translocated to the nucleus where transcription processes are initiated. After a lag period of about 20 minutes hormone-specific early mRNA transcripts leave the nucleus through nuclear pores. Some of the steps in this cascade can be followed by electrophysiology in Xenopus laevis oocyte nuclei. In addition to the genomic pathway, aldosterone exerts a rapid pre-genomic response that involves an increase in intracellular calcium. In this study, we tested for the potential role of Ca(2+) in the genomic response of the hormone. We measured the electrical resistance across the nuclear envelope in response to aldosterone, in presence and absence of intracellular Ca(2+). Nuclear envelope electrical resistance reflects receptor binding to the nuclear pore complexes ("early" resistance peak, 2 minutes after aldosterone), ongoing transcription ("transient" resistance drop, 5-15 minutes after aldosterone) and mRNA export ("late" resistance peak, 20 minutes after aldosterone). Pre-injection of the Ca(2+) chelator EGTA eliminated all electrical responses evoked by aldosterone. The transient resistance drop and the late resistance peak, induced by the hormone, were prevented by the transcription inhibitor actinomycin D, coinjected with aldosterone, while the early resistance peak remained unaffected. We conclude that (i). the presence of intracellular Ca(2+) is a prerequisite for the genomic action of aldosterone. (ii). Intracellular calcium plays a role early in the signaling cascade, either in agonist-receptor interaction, or receptor transport/docking to the nuclear pore complexes.

There is accumulating evidence that mineralocorticoids not only act on kidney but also on the cardiovascular system. We investigated the response of human umbilical venous endothelial cells (HUVECs) to aldosterone at a time scale of 20 minutes in absence and presence of the aldosterone antagonist spironolactone or other transport inhibitors. We applied atomic force microscopy (AFM), which measures cell volume and volume shifts between cytosol and cell nucleus. We observed an immediate cell volume increase (about 10%) approximately 1 min after addition of aldosterone (0.1 micromol/l), approaching a maximum (about 18%) 10 min after aldosterone treatment. Cell volume returned to normal 20 min after hormone exposure. Spironolactone (1 micromol/l) or amiloride (1 micromol/l) prevented the late aldosterone-induced volume changes but not the immediate change observed 1 min after hormone exposure. AFM revealed nuclear swelling 5 min after aldosterone addition, followed by nuclear shrinkage 15 min later. The Na(+)/H(+) exchange blocker cariporide (10 micromol/l) was ineffective. We conclude: (i). Aldosterone induces immediate (1 min) swelling independently of plasma membrane Na(+) channels and intracellular mineralocorticoid receptors followed by late mineralocorticoid receptor- and Na(+)-channel-dependent swelling. (ii). Intracellular macromolecule shifts cause the changes in cell volume. (iii). Both amiloride and spironolactone may be useful for medical applications to prevent aldosterone-induced vasculopathies.

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.