Background-Life-threatening cardiac arrhythmia is a major source of mortality worldwide. Besides rare inherited monogenic diseases such as long-QT or Brugada syndromes, which reflect abnormalities in ion fluxes across cardiac ion channels as a final common pathway, arrhythmias are most frequently acquired and associated with heart disease. The mineralocorticoid hormone aldosterone is an important contributor to morbidity and mortality in heart failure, but its mechanisms of action are incompletely understood. Methods and Results-To specifically assess the role of the mineralocorticoid receptor (MR) in the heart, in the absence of changes in aldosteronemia, we generated a transgenic mouse model with conditional cardiac-specific overexpression of the human MR. Mice exhibit a high rate of death prevented by spironolactone, an MR antagonist used in human therapy. Cardiac MR overexpression led to ion channel remodeling, resulting in prolonged ventricular repolarization at both the cellular and integrated levels and in severe ventricular arrhythmias. Conclusions-Our

The presence of mineralocorticoid receptors (MRs) and their physicochemical characteristics were investigated in the heart and blood vessels of rabbits. Immunohistochemical methods using the monoclonal anti-idiotypic antibody H10E, which interacts with the steroid binding domain of MRs, revealed the presence of immunoreactive material in the heart and large blood vessels. In the heart, a positive staining was observed in myocytes and endothelial cells of atria and ventricles. In vessels, MRs were detected in the aorta and pulmonary artery. They were localized in endothelial and vascular smooth muscle cells. No staining was present in the small vascular bed, arterioles, and capillaries. In all these studies, the mineralocorticoid specificity of the staining was assessed by in situ competition experiments with aldosterone and RU486, a glucocorticoid antagonist. The presence of MRs in the heart and vessels was further demonstrated by specific aldosterone binding to one class of high affinity binding sites in the cytosol of the adrenalectomized rabbit heart (Kd, 0.25 nM; maximum MR concentration, 15-20 fmol/mg protein), whose mineralocorticoid specificity has been clearly established by competition studies. Sedimentation gradient analyses revealed that the cardiovascular MR is an 8.5S hetero-oligomer that includes the heat shock protein 90. The physicochemical characteristics of the cardiovascular MRs are virtually identical to those of the renal MRs. Altogether, our results clearly demonstrate the presence of MRs in the cardiovascular system. This supports the possibility of direct aldosterone actions in the heart and blood vessels.

The localization of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) was determined in the rabbit kidney by immunohistochemistry with the use of a monoclonal, anti-GR antibody and a monoclonal, anti-idiotypic, anti-MR antibody. Immunostaining was performed on serial histological sections from normal and adrenalectomized rabbits. The specificity of immunostaining was assessed for MR by in situ competition studies with steroids and for GR by presaturation of the antibody with GR preparation. Immunostaining by both the anti-MR and the anti-GR antibodies was present in all parts of the distal nephron (beyond proximal tubule) and absent in the glomerulus and proximal tubule. The absence of staining by the anti-GR antibody in the proximal tubule suggests that the effects of glucocorticoids in this structure involve either a GR different from that of distal structures or a non-receptor mediated mechanism of action. MR immunostaining predominates in the distal and all along the collecting tubule in its cortical, medullary, and papillary portions. GR immunostaining was most abundant in the medullary ascending limb and distal tubule. Immunostaining by both antibodies was present in papillary interstitial cells and cells of the epithelium lining the papilla. Fifteen to twenty percent of the cells of the cortical collecting tubule, presumably intercalated cells, were devoid of MR and GR immunostaining. Immunostaining was present in both nuclear and cytoplasmic cell compartments. No clear difference was observed between normal and adrenalectomized rabbits. This study is the first report on renal immunolocalization of GR compared with MR. In addition, we show evidence for new targets for corticosteroid hormones such as papillary interstitial cells and papillary epithelium.

A monoclonal antibody (H1OE), generated by an auto-anti-idiotypic procedure and directed at the aldosterone-binding site of mineralocorticoid receptor (MR), was used in immunohistochemical studies to localize MR in rabbit kidney preparations. In agreement with earlier physiological and biochemical observations, MR was detected in connecting and cortical collecting tubules. Additionally, MR was detected in the distal tubules, the medullary and papillary collecting ducts, and in the epithelial cells lining the papilla. MATERIALS AND METHODSReagents. Aldosterone, dexamethasone, and estradiol were from Sigma. Deoxycorticosterone acetate and RU 486 were from Roussel-Uclaf (Romainville, France). SC 9420 was from Searle.Tissue Preparation. New Zealand White female rabbits weighing 1.5 kg were used in this study. Bilateral adrenalectomy was performed under Fluothan anesthesia. The animals received Syncortyl (1.9 mg/kg; deoxycorticosterone acetate) i.p. at the time of operation and the 2 following days and were given 0.9% saline to drink ad libitum. These animals were then used 5 to 7 days after surgery. Aldosterone-treated rabbits received an i.p. injection of 1.5 mg of aldosterone 45 min before the study.Normal, adrenalectomized, or aldosterone-treated rabbits were anesthetized with 25 to 50 mg of Nembutal and perfused through the abdominal aorta with 500 ml of Zamboni's solution [2% (wt/vol) paraformaldehyde/15% (vol/vol) saturated picric acid solution/300 mM (85%, vol/vol) sodium phosphate buffer, pH 7.4] at 37°C. The perfusion procedure was done as described (3). At the end of perfusion, the kidneys were removed, cut into slices and pyramids (-2 mm thick), and postfixed for 24 hr in the Zamboni fixative. The kidney slices and pyramids were washed in 70o (vol/vol) ethanol, dehydrated in graded ethanol, cleared in 1-butanol, and embedded in Paraplast. Sections (7 ,um) were cut, mounted on histological slides, and processed for immunohistochemistry.Immunohistochemical Technique. A routine procedure of indirect immunostaining was used (4). Briefly, after deparaffinization and rehydration, sections were incubated with 3% normal horse serum in phosphate-buffered saline (PBS) solution. The anti-idiotypic mAb H1OE, a mouse IgG1 immunoglobulin, was used as diluted ascites fluid (1). A nonimmune preparation of mouse IgG (prepared in the laboratory) was used as control. The mAb was used at -1-5 ,g/ml, followed by a horse biotinylated anti-mouse IgG antibody (Vector Laboratories). The avidin-biotin-peroxidase complex (ABC-Elite from Vector Laboratories) was used as a detection system. After each incubation step, the slides were rinsed in PBS. Peroxidase activity was revealed by diamiAbbreviations: MR, mineralocorticoid receptor; mAb, monoclonal antibody.§To whom reprint requests should be addressed. 1086The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Although the mineralocorticosteroid receptor (MR) belongs to the superfamily of hormone-dependent transcription factors, the molecular mechanism by which it regulates gene expression is poorly understood. Binding of the MR to target gene promoters has never been characterized, and specific mineralocorticosteroid response elements (MREs) remain to be identified. The human MR (hMR) was overexpressed in Sf21 insect cells using the baculovirus system. The high degree of similarity between the glucocorticosteroid receptor (GR) and the MR prompted us to examine the DNA-binding properties of the recombinant MR with glucocorticosteroid-regulated genes. Gel shift mobility assays demonstrated that the recombinant receptor interacted with oligonucleotides containing perfect and imperfect palindromic sequences of GRE. A monoclonal anti-hMR antibody (FD4) induced a supershift of protein-DNA complexes and identified the MR in Western blot analysis. In vitro DNAse I protection assays with the hormone-regulated murine mammary tumour virus promoter showed that recombinant hMR generated four footprints whose limits encompassed the GRE motifs. By means of these two complementary approaches, no difference between the interaction of free, agonist- or antagonist-bound MR and DNA was detected. We provide evidence that hMR functions as a sequence-specific DNA-binding protein.

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The A6 cell line is derived from the kidney of Xenopus laevis. Aldosterone increases sodium transport across A6 cell epithelia. In the present study, aldosterone binding characteristics were studied in A6 cell cytosol. Both type I (mineralocorticoid) and type II (glucocorticoid) receptors are present in the cytosolic fraction of these cells. Aldosterone and corticosterone had a high affinity for type I sites (Kd = 1.25 and 0.16 nM, respectively) and a lower affinity for type II sites (Kd = 39 and 10 nM, respectively). Testosterone and estradiol did not compete for aldosterone binding. RU 26988, a highly specific glucocorticoid agonist, competed with aldosterone for type II but not for type I sites. Hydrodynamic parameters of both type I and type II corticosterone receptor complexes were identical. Their Stokes radius was approximately 6 nm, as estimated by high-performance size-exclusion chromatography, and their sedimentation coefficient determined by ultracentrifugation on glycerol gradients was approximately 9s. The molecular mass calculated from these parameters was approximately 200 kDa, a value that is very close to the value estimated for nontransformed mineralocorticoid and glucocorticoid receptors of other species. The [3H]aldosterone labeling of intact A6 cells was examined by autohistoradiography. At every concentration tested (2, 20, and 50 nM), all cells were found to be specifically labeled in both cytoplasm and nucleus. At 20 nM, in the presence of an excess of RU 26988, labeling was also detected. At every concentration the labeling data was compatible with a Gaussian distribution, indicating that A6 cells correspond to a homogeneous population with regard to aldosterone binding and that probably both type I and type II sites are present in the same cells.

: 11beta-HSD2 appears to play two important roles in the epithelial target tissues, kidney and toad bladder. The first is to protect GC access to MR, and the second involves the product of the enzyme to regulate the magnitude of aldosterone-induced Na+ retention.