Mineralocorticoid receptor gene expression in the gastrointestinal tract: distribution and ontogeny

The gastrointestinal tract is a well characterized target tissue for aldosterone, where it regulates electrolyte transport, particularly in the descending colon. Previous studies have demonstrated the presence of aldosterone receptors in gastrointestinal tissues. We have used specific cRNA probes for the rat mineralocorticoid receptor to explore both the distribution and ontogeny of mineralocorticoid receptor gene expression in the gastrointestinal tract. Mineralocorticoid receptor gene expression is found throughout the small and large intestine, but is absent from the stomach. The highest levels are observed in the distal colon, and significant expression is found in the duodenum; in both tissues levels of expression are higher than those in kidney. In both the developing duodenum and colon, mineralocorticoid receptor gene expression precedes the development of the full physiological response to aldosterone. These findings emphasise the colon as an important target tissue for aldosterone, and raise the question of potential roles for aldosterone in the duodenum.     

Mineralocorticoid effector mechanism in human mononuclear leukocytes

Mineralocorticoid receptors and mineralocorticoid effector mechanism were determined in mononuclear leukocytes (MNL) from normal subjects. The hierarchy of affinities of competitors for the receptor was similar to that described in other non-classical target tissues for aldosterone. In spite of the relative high affinity of cortisol for the receptor, these binding sites are occupied in vivo by aldosterone and play a mineralocorticoid effect in terms of electrolyte content of the cells. The effect of aldosterone is to prevent the loss of electrolytes due to incubation in medium alone and this action is reversed by addition of actinomycin D. In addition, the incubation of the MNL with aldosterone plus human alpha ANP leads to complete block of the action of aldosterone alone. This effect is not mediated by binding of alpha ANP to mineralocorticoid receptors but is probably related to a some postereceptorial effect of aldosterone at the level of plasma membrane. We conclude that the model of MNL is a good tool for studying mineralocorticoid receptors regulation and consequent effector mechanism in humans.     

Elevated cardiac tissue level of aldosterone and mineralocorticoid receptor in diastolic heart failure: Beneficial effects of mineralocorticoid receptor blocker

Cardiac aldosterone levels have not been evaluated in diastolic heart failure (DHF), and its roles in this type of heart failure remain unclear. This study aimed to detect cardiac aldosterone by use of a liquid chromatographic-mass spectrometric method and to assess the effects of mineralocorticoid receptor blockade on hypertensive DHF. Dahl salt-sensitive rats fed 8% NaCl diet from 7 wk (hypertensive DHF model) were divided at 13 wk into three groups: those treated with subdepressor doses of eplerenone (12.5 or 40 mg x kg(-1) x day(-1)) and an untreated group. Dahl salt-sensitive rats fed 0.3% NaCl diet served as controls. Cardiac aldosterone was detected in the DHF rats but not in the control rats, with increased ventricular levels of mineralocorticoid receptor. Cardiac levels of 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone were not different between the control and DHF rats, but the tissue level of corticosterone that has an affinity to mineralocorticoid receptor was 1,000 times as high as that of aldosterone. Aldosterone synthase activity and CYP11B2 mRNA were undetectable in the ventricular tissue of the DHF rats. Administration of eplerenone attenuated ventricular hypertrophy, ventricular fibrosis, myocardial stiffening, and relaxation abnormality, leading to the prevention of overt DHF. In summary, the myocardial aldosterone level increased in the DHF rats. However, its value was extremely low compared with corticosterone, and no evidence for enhancement of intrinsic myocardial aldosterone production was found. The upregulation of mineralocorticoid receptor may play a central role in the pathogenesis of DHF, and blockade of mineralocorticoid receptor is likely an effective therapeutic regimen of DHF.     

Aldosterone modifies hemostasis via upregulation of the protein-C receptor in human vascular endothelium

In human bone marrow endothelial cell (HBMEC) exposed for 8 h to aldosterone, the microarray screening revealed an upregulation of the mRNAs for six genes and downregulation of mRNAs for four genes, all implicated in hemostasis. In HBMEC, immunocytochemistry revealed the presence of the membrane-bound endothelial protein C receptor (EPCR) whereas the mineralocorticoid receptor (MCR) was present as a nucleo-cytoplasmic. In HBMEC treated with aldosterone the induction of EPCR protein was evident by both FACS analysis and dot blot procedure. When aldosterone-treated HBMEC were incubated with the activated protein C (APC), the partial thromboplastin clotting time (aPTT) increased 2.5-fold over control, from 10 to 25 s. The MCR antagonists aldactone and eplerenone reduced the basal coagulation time in untreated cells to 33.5% and 42% of the control, respectively. These data add an entirely new dimension to delineating the receptor-mediated action of mineralocorticoid hormones.     

Nongenomic effects of aldosterone: cellular aspects and clinical implications

Nongenomic action of aldosterone has been observed in many cell types which often are different from the classic target tissues for mineralocorticoid action, such as vascular cells. As judged from their time scale and insensitivity toward inhibitors of protein synthesis, effects are not mediated by the classic mineralocorticoid receptor pathway. Here we summarize studies on rapid in vitro aldosterone effects, e.g. ion fluxes, and second messengers involved therein. Furthermore, several clinical studies on in vivo aldosterone action have shown rapid effects on cardiovascular parameters, among them baroreflex and vascular resistance. Taken together with the beneficial effect of aldosterone antagonism in heart failure patients that was demonstrated in the Randomized Aldactone Evaluation Study (RALES), aldosterone may be an equally important factor of the renin-angiotensin-aldosterone system in cardiovascular pathogenesis.     

Comparison of aldosterone binding in aortic cells from Dahl salt-susceptible and salt-resistant rats

The Dahl salt-resistant substrain of Sprague-Dawley rats represents a uniform population of animals that are resistant to salt and mineralocorticoid induced hypertension. Aldosterone binding in the aortae of these rats is contrasted to that of age- and sex-matched rats of the Dahl salt-susceptible strain in an effort to identify a mechanism for resistance to salt induced hypertension. Cultured smooth muscle cells of both substrains contain two classes of aldosterone binding sites: corticoid receptor I with high affinity and low capacity, and corticoid receptor II with low affinity and high capacity. No differences were found between the two substrains in the affinities or binding capacities of these receptors. Both groups of Sprague-Dawley rats had a significantly higher corticoid receptor I affinity than the salt resistant Fischer 344 rats, a strain with a two-fold lower affinity than salt sensitive strains. These results indicate that an intrinsic defect in mineralocorticoid binding in aortic smooth muscle cells could not account for the resistance to salt and mineralocorticoid induced hypertension seen in Sprague-Dawley rats and that the biochemical abnormality underlying salt resistance is likely to be different from that of Fischer 344 rats.     

Antimineralocorticoids.

A number of chemical modifications in the spironolactone molecule were attempted over the last decade to synthesize ligands with high affinity for the mineralocorticoid receptor (MCR), and for possible use in the clinical control of the hypertensive disease. ZK 91587 has been commercialized as the 'ideal' ligand for the MCR, replacing the natural hormone aldosterone. None of the derivatives was retained for possible clinical use as an improvement over Canrenone or Spironolactone. No apparent correlation could be drawn between affinity for the MCR in vitro and biological potency in vivo. Such considerations challenge classical notions regarding the receptor mediated hormone action.     

The mineralocorticoid signaling pathway throughout development: Expression,regulation and pathophysiological implications

The mineralocorticoid signaling pathway has gained interest over the past few years, considering not only its implication in numerous pathologies but also its emerging role in physiological processes during kidney, brain, heart and lung development. This review aims at describing the setting and regulation of aldosterone biosynthesis and the expression of the mineralocorticoid receptor (MR), a nuclear receptor mediating aldosterone action in target tissues, during the perinatal period. Specificities concerning MR expression and regulation during the development of several major organs are highlighted. We provide evidence that MR expression is tightly controlled in a tissue-specific manner during development, which could have major pathophysiological implications in the neonatal period.     

Corticosteroids mediate fast feedback of the rat hypothalamic-pituitary-adrenal axis via the mineralocorticoid receptor

The aim of this study was to investigate fast corticosteroid feedback of the hypothalamic-pituitary-adrenal (HPA) axis under basal conditions, in particular the role of the mineralocorticoid receptor. Blood samples were collected every 5 min from conscious rats at the diurnal peak, using an automated blood sampling system, and assayed for corticosterone. Feedback inhibition by rapidly increasing concentrations of ligand was achieved with an intravenous bolus of exogenous corticosteroid. This resulted in a significant reduction in plasma corticosterone concentrations within 23 min of the aldosterone bolus and 28 min of methylprednisolone. Evaluation of the pulsatile secretion of corticosterone revealed that the secretory event in progress at the time of administration of exogenous steroid was unaffected, whereas the next secretory event was inhibited by both aldosterone and methylprednisolone. The inhibitory effect of aldosterone was limited in duration (1 secretory event only), whereas that of methylprednisolone persisted for 4-5 h. Intravenous administration of canrenoate (a mineralocorticoid receptor antagonist) also had rapid effects on the HPA axis, with an elevation of ACTH within 10 min and corticosterone within 20 min. The inhibitory effect of aldosterone was unaffected by pretreatment with the glucocorticoid receptor antagonist RU-38486 but blocked by the canrenoate. These data imply an important role for the mineralocorticoid receptor in fast feedback of basal HPA activity and suggest that mineralocorticoids can dynamically regulate basal corticosterone concentrations during the diurnal peak, a time of day when there is already a high level of occupancy of the cytoplasmic mineralocorticoid receptor.     

A new affinity matrix for mineralocorticoid receptors

The behavior of mineralocorticoid and glucocorticoid receptors of rabbit kidney cytosol was investigated on two affinity gels: a new affinity matrix prepared with a 3-O-derivative of carboxymethyloxime deoxycorticosterone (deoxycorticosterone gel) and a gel linked to a 17 beta-dexamethasone derivative (dexamethasone gel). Deoxycorticosterone gel was highly specific, since it retained mineralocorticoid but not glucocorticoid receptors, and dexamethasone gel exhibited high selectivity for glucocorticoid receptors since it did not bind mineralocorticoid receptors. The use of these two matrices allowed separation of mineralocorticoid and glucocorticoid receptors and further characterization of each type of cytosolic receptors after its isolation. Cytosolic mineralocorticoid and glucocorticoid receptors stabilized by tungstate were found to have a Stokes radius of approximately 6 nm, as determined by high performance size exclusion chromatography and a sedimentation coefficient of approximately 9 S, determined on a glycerol density gradient containing tungstate, under either high or low salt conditions. The hydrodynamic parameters, binding characteristics, and specificity of mineralocorticoid receptors were the same in the untreated and dexamethasone gel-treated cytosol. Similarly glucocorticoid receptor characteristics remained unchanged after deoxycorticosterone gel treatment, indicating biochemical independence of cytosolic mineralocorticoid and glucocorticoid receptors. The [3H]aldosterone receptor complex eluted from deoxycorticosterone gel was recovered with a 30-40% yield and a purification factor of about 1000. Purified mineralocorticoid receptors had the same sedimentation coefficient as cytosolic mineralocorticoid receptors (9 S) but a different Stokes radius (4 versus 6 nm). The decrease in the Stokes radius of the purified mineralocorticoid receptors was probably due to the gel filtration method. These results indicate that the newly synthesized matrix specific for mineralocorticoid receptors constitutes a powerful tool for their extensive purification.     

Affinity of corticosteroids for mineralocorticoid and glucocorticoid receptors of the rabbit kidney: effect of steroid substitution

Corticosteroid derivatives coupled in the C3, C7 or C17 position with a long aliphatic chain were synthesized in order to select a suitable ligand for the preparation of a biospecific affinity adsorbent for mineralocorticoid receptor purification. The affinity of these derivatives for mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) was explored in rabbit kidney cytosol. In this model, aldosterone bound to a single class of receptors with high affinity (Kd 1 nM) and mineralocorticoid specificity. RU26988, a highly specific ligand for GR, did not compete for these sites. The C7 and C17 positions were found to be of crucial importance in the steroid's interaction with the mineralocorticoid receptors, since the linkage of a long side chain in these positions induced complete loss of affinity. Hence, deoxycorticosterone no longer bound to MR after 17 beta substitution with a 9-carbon aliphatic chain. This loss of affinity was not observed for glucocorticoids. The 17 beta nonylamide derivative of dexamethasone still competed for GR. Increasing the length of the C7 side of the spirolactone SC26304 suppressed its affinity for MR. Finally, C3 was an appropriate position for steroid substitution. The 3-nonylamide of carboxymethyloxime deoxycorticosterone bound to MR but not to GR, and therefore constitutes a suitable ligand for the preparation of a mineralocorticoid adsorbent.     

Differential regulation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene by human mineralocorticoid hormone-receptor complexes.

The brain tissues of the rat and mouse express two types of corticosteroid binding proteins, the glucocorticoid (GR) and aldosterone (MR) receptors. Unlike the type II (GR) receptor, type I receptor has a high affinity for aldosterone (ALDO) and corticosterone and is structurally similar to the kidney mineralocorticoid receptor (MR). The results reported in this study provide direct evidence for the interaction of dexamethasone (DEX), triamcinolone acetonide (TA), dexamethasone-21-mesylate (DXM) and 11-deoxycorticosterone (DOC) with human MR expressed in cells by transient co-transfection of a hMR expression vector. The interactions of hMR with DEX, TA, DXM, DOC, promegestone (R5020) and methyltrienelone (R1881) were measured by trans-activation of mouse mammary tumor virus long terminal repeat fused to bacterial chloramphenicol acetyltransferase (MMTV-tk-CAT) in gene co-transfection experiments and by cell free hormone binding assay. The incubation of various steroid hormones in the presence of [3H]ALDO in a competition assay with extracts prepared from HeLa cells co-transfected with hMR expression vector, showed that hMR expressed under these conditions has a high relative affinity for DEX which is similar to ALDO, TA and DOC. Incubation with DXM under these conditions showed very little competition, as was observed with R1881 and R5020. Incubation of the co-transfected cells with DEX, ALDO, DOC, R5020, TA, R1881 and DXM demonstrated that the level of trans-activation did not reflect the previously observed order of binding affinity for the hMR. The level of transactivation was always higher with DEX and TA compared to ALDO and DOC. Analysis of the binding of labeled glucocorticoid regulatory element (GRE) and hMR incubated with DEX, ALDO and DXM by gel shift analysis demonstrated that the trans-activation of MMTV-tk-CAT by hMR is a result of the interaction of hMR with GRE in the MMTV-LTR.     

Gain of function mutation in the mineralocorticoid receptor of the Brown Norway rat

The aim of this research was to identify the molecular bases of differences in sensitivity to corticosteroid hormones between Brown Norway and Fischer 344 rats. We previously showed an apparent insensitivity to adrenalectomy in Brown Norway rats. Based on our first hypothesis of a different activity/reactivity of the mineralocorticoid signaling pathway between the two rat strains, we sequenced Brown Norway and Fischer 344 mineralocorticoid receptor cDNA and identified a tyrosine to cysteine substitution (Y73C) in the N-terminal part of the Brown Norway mineralocorticoid receptor. As a first step, this substitution gave us a means to distinguish the Brown Norway allele from the Fischer 344 at the mineralocorticoid receptor locus in an F2 population. We showed a strong genetic linkage between the mineralocorticoid receptor genotype and sensitivity to adrenalectomy. A subsequent genome-wide linkage analysis confirmed the involvement of the mineralocorticoid receptor locus and implicated other loci, including one on chromosome 4, which collectively explain a large part of the strain differences in corticosteroid receptor responses. In vitro studies further revealed that the Y73C substitution induces greater transactivation of the mineralocorticoid receptor by aldosterone, and surprisingly by progesterone as well, which could substitute for aldosterone after adrenalectomy in Brown Norway rats. We challenged this hypothesis in vivo and showed that plasma progesterone is higher in Brown Norway male rats and partially compensates for aldosterone after adrenalectomy. This work illustrates the interest of a pluristrategic approach to explore the mineralocorticoid receptor signaling pathway and its implication in the regulation of hydroelectrolytic homeostasis and blood pressure.     

Glucocorticoid metabolism and Na+ transport in chicken intestine

The role of aldosterone in regulation of electrogenic Na+ transport is well established, though mineralocorticoid receptors bind glucocorticoids with similar binding affinity as aldosterone and plasma concentration of aldosterone is much lower than glucocorticoids. In mammals, the aldosterone specificity is conferred on the low-selective mineralocorticoid receptors by glucocorticoid inactivating enzyme 11beta-hydroxysteroid dehydrogenase (11HSD) that converts cortisol or corticosterone into metabolites (cortisone, 11-dehydrocorticosterone) with lower affinity for these receptors. The present study examined the chicken intestine, whether changes in 11HSD activity are able to modulate the effect of corticosterone on Na+ transport, and how the metabolism of this hormone is distributed within the intestinal wall. This study shows that not only aldosterone, but also corticosterone (B), was able to increase the electrogenic Na+ transport in chicken caecum in vitro. The effect of corticosterone was higher in the presence of carbenoxolone, an inhibitor of steroid dehydrogenases, and was comparable to the effect of aldosterone. The metabolism of B in the intestine was studied; results showed oxidation of this steroid to 11-dehydrocorticosterone (A) and reduction to 11-dehydro-20beta-dihydrocorticosterone (20diA) as the main metabolic products at low nanomolar concentration of the substrate. In contrast, 20beta-dihydrocorticosterone and 20diA were the major products at micromolar concentration of B. Progesterone was converted to 20beta-dihydroprogesterone. The metabolism of corticosterone was localized predominantly in the intestinal mucosa (enterocytes). In conclusion, the oxidation at position C11 and reduction at position C20 suggest that both 11HSD and 20beta-hydroxysteroid dehydrogenase (20HSD) operate in the chicken intestine and that the mucosa of avian intestine possesses a partly different system of modulation of corticosteroid signals than mammals. This system seems to protect the aldosterone target tissue against excessive concentration of corticosterone and progesterone.     

The role of 11β-hydroxysteroid dehydrogenase type 2 in human hypertension

Cortisol and aldosterone have the same in vitro affinity for the mineralocorticoid receptor (MR), although in vivo only aldosterone acts as a physiologic agonist of the MR, despite circulating levels of cortisol in humans and corticosterone in rodents being three orders of magnitude higher than aldosterone levels. In mineralocorticoid target organs the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) inactivates 11-hydroxy steroids, to their inactive keto-forms, thus protecting the nonselective MR from activation by glucocorticoids. The gene is highly expressed in all sodium-transporting epithelia, particularly in the kidney and colon, but also in human placenta and vascular wall. Mutations in the HSD11B2 gene cause a rare monogenic juvenile hypertensive syndrome called apparent mineralocorticoid excess (AME). In AME, compromised 11βHSD2 enzyme activity results in activation of the MR by cortisol, causing sodium retention, hypokalaemia, and salt-dependent hypertension. Whereas mutations or inhibition of 11βHSD2 by licorice have been clearly shown to produce a congenital or acquired syndrome of mineralocorticoid excess, the questions remaining are the extent to which subtle abnormalities in MR/11βHSD2 mechanisms may contribute to essential hypertension. Studies in patients with essential hypertension showed a prolonged half-life of cortisol and an increased ratio of urinary cortisol to cortisone metabolites, suggesting a deficient 11βHSD2 activity. These abnormalities may be genetically determined, as suggested by the association of a microsatellite flanking the HSD11B2 gene with hypertension in black patients with end-stage kidney disease and with salt sensitivity of blood pressure in healthy subjects. These findings indicate that variants of the HSD11B2 gene may contribute to the enhanced blood pressure response to salt and possibly to hypertension in humans.     

Valine 571 functions as a regional organizer in programming the glucocorticoid receptor for differential binding of glucocorticoids and mineralocorticoids.

The glucocorticoid receptor (GR) interacts specifically with glucocorticoids, whereas its closest relative, the mineralocorticoid receptor (MR), interacts with both glucocorticoids and mineralocorticoids, such as aldosterone. To investigate the mechanism underlying the glucocorticoid/mineralocorticoid specificity of the GR, we used a yeast model system to screen for GR ligand-binding domain mutants, substituted with MR residues in the segment 565-574, that can be efficiently activated by aldosterone. In all such increased activity mutants, valine 571 was replaced by methionine, even though most mutants also contained substitutions of other residues with their MR counterparts. Further analysis in yeast and COS-7 cells has revealed that the identity of residue 571 determines the behavior of other MR substituted residues in the 565-574 segment. Generally, MR substitutions in this region are only consistent with aldosterone binding if residue 571 is also replaced with methionine (MR conformation). If residue 571 is valine (GR conformation), most other MR substitution mutants drastically reduce interaction with both mineralocorticoid and glucocorticoid hormones. Based on these functional data, we hypothesize that residue 571 functions as a regional organizer involved in discriminating between glucocorticoid and mineralocorticoid hormones. We have used a molecular model of the GR ligand-binding domain in an attempt to interpret our functional data in structural terms.     

A physico-chemical comparison of aortic receptors in rat hypertension models

Rat models of genetic hypertension include spontaneous hypertension and resistance or sensitivity to mineralocorticoid and salt induced hypertension. Previously, altered aldosterone binding to corticoid receptor I was found in aortic smooth muscle cells cultured from Fischer 344 rats which are extremely resistant to steroid and salt induced hypertension. The corticoid receptor I of Fisher 344 rats had a lower affinity than that of salt sensitive Wistar-Kyoto controls, as well as spontaneously hypertensive rats and Sprague-Dawley rats. In the present study, we have used DEAE-cellulose ion exchange chromatography to compare the structure (charge properties) and steroid specificity of vascular corticoid receptor I and II sites in these same rat hypertension models. No variations in ion exchange properties of type I and II receptors were found. Together with the lower aldosterone affinity of corticoid receptor I sites in Fischer 344 rats these data suggest an altered binding domain which is not seen as a difference in charge density of the receptor protein by ion exchange chromatography.