Pharmacology and physiology of ovine corticosteroid receptors

The aim of these studies was to characterize the ovine corticosteroid receptors (MR, mineralocorticoid receptors and GR, glucocorticoid receptors) in ovine hippocampus and brainstem. Adrenal-intact and adrenalectomized ewes were studied; adrenalectomized ewes were killed 47 +/- 9 h after steroid withdrawal, when symptoms of hypotension and/or hyperkalemia became evident. RT-PCR, immunoblotting and pharmacologic studies indicated the presence of both MR and GR in hippocampus and brainstem. Competitive binding studies using 3H-cortisol in brain tissue showed that the ovine MR binds cortisol, aldosterone and progesterone with equal affinity. Differences in receptor availability in intact and adrenalectomized ewes, along with determination of the binding affinity (K(d)) of MR and GR, suggested that MR occupancy is about 90%, whereas GR occupancy is about 30%, in normal animals. There was a significant increase in protein level of MR in brainstem, and the appearance of a higher molecular weight band for MR in hippocampus following steroid withdrawal, however no significant change in mRNA was detected by semiquantitative RT-PCR for either MR or GR in hippocampus or brainstem following steroid withdrawal. These studies suggest that physiological ligands of MR in the sheep brain include progesterone and cortisol, and that, as in other species, affinity of MR for cortisol is greater than that of GR.     

Difference of in vivo and in vitro antimineralocorticoid potency of progesterone

Progesterone (P) given intramuscularly increases renal sodium excretion. We tested the in vitro capacity of P to bind to the human mineralocorticoid receptor (MR) with a reticulocyte lysate system and Ps transactivation potency in transfected CV-1 cells. Progesterone binds with higher affinity to the MR than aldosterone, but shows only low transactivation activity. This results in a very strong anti-mineralocorticoid (MC) potency of P in vitro. To test the in vivo anti-MC potency of P we infused aldosterone intravenously to hypo-MC Addison's patients, followed by increasing P infusions. During the study we measured normal aldosterone plasma concentrations and high P concentrations similar to the third trimester of pregnancy. Despite the 1000-fold higher plasma P concentrations, the in vivo anti-MC effect of P (increase of urinary sodium/potassium ratio) was rather small. We suggest that this may be due to effective MR protection mechanisms, such as conversion of P to inactive metabolites.     

Mineralocorticoid receptor-mediated changes in membrane properties of rat CA1 pyramidal neurons in vitro.

Pyramidal neurons in the rat hippocampus contain mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) to which the adrenal steroid corticosterone binds with differential affinity. We have used intracellular recording techniques to examine MR-mediated effects on membrane properties of CA1 pyramidal neurons in hippocampal slices from adrenalectomized rats. Low doses of corticosterone (1 nM) applied by perfusion for 20 min decreased the spike accommodation observed during a depolarizing current pulse (0.5 nA for 500 ms) and the amplitude of the subsequent afterhyperpolarization without affecting other membrane properties tested. The decrease became apparent ca. 15 min after steroid perfusion was started and reached its peak value 10-20 min after the steroid perfusion was terminated. The steroid effect was blocked by the MR antagonist spironolactone and mimicked by the natural MR ligand aldosterone (1 nM). Neurons recorded 30-90 min after termination of aldosterone application still displayed a decreased spike accommodation. However, 30-90 min after corticosterone application, the decrease in spike accommodation/afterhyperpolarization appeared to be reversed. Higher doses of corticosterone (greater than or equal to 30 nM) induced a significant increase in accommodation and amplitude of the afterhyperpolarization, as was previously observed for selective GR ligands. The data indicate that MR and GR activations induce opposite actions on the spike accommodation/afterhyperpolarization of CA1 pyramidal neurons, an important intrinsic mechanism of these neurons to regulate their response to excitatory input. We suggest that occupation of both MR and GR by the endogenous ligand corticosterone will result in an initial MR-mediated enhanced cellular excitability, which is gradually reversed and overridden by a GR-mediated suppression of cellular activity.     

The Ubiquitous Mineralocorticoid Receptor: Clinical Implications

Mineralocorticoid receptors (MR) exist in many tissues, in which they mediate diverse functions crucial to normal physiology, including tissue repair and electrolyte and fluid homeostasis. However, inappropriate activation of MR within these tissues, and especially in the brain, causes hypertension and pathological vascular, cardiac, and renal remodeling. MR binds aldosterone, cortisol and corticosterone with equal affinity. In aldosterone-target cells, co-expression with the 11??-hydroxysteroid dehydrogenase 2 (HSD2) allows aldosterone specifically to activate MR. Aldosterone levels are excessive in primary aldosteronism, but in conditions with increased oxidative stress, like CHF, obesity and diabetes, MR may also be inappropriately activated by glucocorticoids. Unlike thiazide diuretics, MR antagonists are diuretics that do not cause insulin resistance. Addition of MR antagonists to standard treatment for hypertension and cardiac or renal disease decreases end-organ pathology and sympathetic nerve activation (SNA), and increases quality of life indices.     

Identification of aldosterone-induced proteins in the toad's urinary bladder.

Aldosterone (the 8,11-hemiacetal 0f 11beta,21-dihydroxy-3,20-dioxo-4-pregnen-18-al) markedly stimulates sodium transport in a number of epithelial tissues. We have attempted to determine whether aldosterone induces the synthesis of specific protein(s) in the course of its action upon the toad urinary bladder. Paired hemibladders were incubated in media containing either [3H]methionine or [35S]methionine; aldosterone in physiologic concentrations was added to one bath and, after incubation, the intact "mitochondria-rich" (MR) and "granular" (G) mucosal cells were isolated. The ratio (3H/35S) was used to identify proteins whose synthesis was induced in the mucosal cells of the steroid-treated bladders. Using exclusion gel chromatography and isoelectric focusing, we identified several aldosterone-induced proteins in the supernatant (105,000 x g) fraction of the MR cell. None was evident in this fraction of the G cell. These proteins have apparent molecular weights ranging from 17,000 to 38,000 and the isoelectric point of the major component is 4.5. Corticosterone (11beta,21-dihydroxy-4-pregnene-3,20-dione) induced the synthesis of proteins in the G cells, but none of these proteins was similar in molecular weight to the aldosterone-induced proteins in the MR cell. Our findings support the hypothesis that aldosterone induces the synthesis of specific proteins and indicate that, in this tissue, these proteins are synthesized by the MR cell.     

Neues zu Aldosteron und dem Mineralokortikoidrezeptor

The role of mineralocorticoid receptors (MR) in the pathogenesis of cardiovascular and renal diseases is increasingly appreciated. Primary aldosteronism is observed in 8–13% of hypertensive patients, the prevalence being higher in patients with more resistant hypertension. MR participates in the pathogenesis of heart failure, sleep apnea, metabolic syndrome, and nephrotic syndrome. Furthermore, MR mediate aldosterone-induced endothelial dysfunction and adipogenesis. Direct effects of aldosterone on glomerular cells have been demonstrated in animal and in-vitro studies. Reactive oxygen species are important mediators of aldosterone-induced damage to target organs. MRs binds aldosterone and cortisol with similar affinity. Protection from cortisol through 11β-hydroxydehydrogenase 2-mediated inactivation varies among different tissues. Elevated cortisol levels are associated with increased cardiovascular mortality. MR blockade is an attractive treatment; however, life-threatening hyperkalemia may occur. Inhibition of aldosterone synthase may become an attractive alternative for selected patients.     

Mechanisms of ligand specificity of the mineralocorticoid receptor

The mineralocorticoid receptor (MR) differs from the other steroid receptors in that it responds to two physiological ligands, aldosterone and cortisol. In epithelial tissues, aldosterone selectivity is determined by the activity of 11β-hydroxysteroid dehydrogenase type 2, while in other tissues, including the heart and regions of the central nervous system, cortisol is the primary ligand for the MR where it may act as an antagonist. Clinical trials have demonstrated the potential of MR antagonists in the treatment of cardiovascular disease, though their use has been limited by concurrent hyperkalaemia. In order to better target the MR, an understanding of the structural determinants of tissue- and ligand-specific MR activation is needed. Interactions of the MR have been identified, which exhibit ligand discrimination and/or specificity. These interactions include those of the ligand-binding domain with ligand, with the N-terminal domain and with putative co-regulatory molecules. Agonist and antagonist binding have been characterised using chimeras between the human MR and the glucocorticoid receptor or the zebra fish MR together with molecular modelling. The interaction between the N-terminus and the C-terminus is aldosterone dependent but is unexpectedly antagonised by cortisol and deoxycorticosterone in the human MR. Nuclear receptor-mediated transactivation is critically dependent on, and modulated by, co-regulatory molecules. Proteins that interact with the MR in the presence of either aldosterone or cortisol, but not both, have been identified. The successful identification of ligand-specific interactions of the MR may provide the basis for the development of novel MR ligands with tissue specificity.     

Towards selectively modulating mineralocorticoid receptor function: lessons from other systems

Although there is clinical utility in blocking mineralocorticoid receptor (MR) action, the usefulness of available MR antagonists is limited because of cross-reactivity with the androgen and progesterone receptors (spironolactone) or possibly by low affinity for MR (eplerenone). MR binds aldosterone and physiologic glucocorticoids, such as cortisol, which both can act as MR agonists in epithelial tissues. However, in preliminary studies aldosterone and cortisol appear to induce different conformations in non-epithelial tissues; in the cardiomyocyte, cortisol usually acts as an MR antagonist, whereas in vascular smooth muscle cortisol mimics aldosterone actions if it can access MR, just as it does in the kidney. Thus, there are needs for improved MR antagonists with higher selectivity and potency and, if possible, for compounds that lock MR into specific desirable conformations. Efforts are underway to modulate selectively the action of many nuclear receptors, and insights from one nuclear receptor may be applicable to others given the similarities in structure and function. We have used traditional approaches aided by X-ray crystallography to obtain several classes of selective ligands that modulate thyroid receptor (TR) action. We describe the properties of these selective TR modulators here, and discuss the possibility that similar approaches to ligand design may yield MR interacting compounds with improved specificity and, possibly, tissue specificity.     

The Role of Aldosterone in the Metabolic Syndrome

The metabolic syndrome associates metabolic abnormalities such as insulin resistance and dyslipidemia with increased waist circumference and hypertension. It is a major public health concern, as its prevalence could soon reach 30% to 50% in developed countries. Aldosterone, a mineralocorticoid hormone classically involved in sodium balance regulation, is increased in patients with metabolic syndrome. Besides its classic actions, aldosterone and mineralocorticoid receptor (MR) activation affect glucose metabolism, inducing insulin resistance through various mechanisms that involve oxidative stress, inflammation, and downregulation of proteins involved in insulin signaling pathways. Aldosterone and MR signaling exert deleterious effects on the cardiovascular system and the kidney that influence the cardiovascular risk associated with metabolic syndrome. Salt load plays a major role in cardiovascular injury induced by aldosterone and MR signaling. Large multicenter, randomized clinical trials testing the beneficial effects of MR antagonists on cardiovascular events and mortality in patients with metabolic syndrome are needed.     

Mineralocorticoid resistance: pseudohypoaldosteronism type 1

Pseudohypoaldosteronism type 1 (PHA1) is a rare genetic disease characterized by neonatal renal salt wasting, vomiting, dehydration and failure to thrive. Affected patients present hyponatremia, hyperkalemia, associated with high levels of plasma renin and aldosterone resulting from a renal or systemic resistance to aldosterone. The systemic form of PHA1 results in a severe phenotype, and high doses of salt supplementation are necessary. The symptoms are life-long recurrent. This form is associated with autosomal recessive transmission. Homozygous or compound heterozygous loss of function mutations in the genes coding for the epithelial sodium channel (ENaC) subunities are responsible for this disease. The renal form of PHA1 results in a mild phenotype. Low doses of salt supplementation are required and usually the symptoms remit at the end of the first year of life. Heterozygous loss-of-function mutations in the mineralocorticoid receptor (MR) gene are associated with the renal form of PHA1 in the majority of the affected families but sporadic cases have been reported. In this review the mechanisms of aldosterone action and its effects are discussed. Additionally, clinical and molecular findings of a Brazilian family with the renal form of PHA1 caused by a nonsense mutation (R947X) in the MR gene are presented.     

Mineralocorticoid Actions in the Brain and Hypertension

Mineralocorticoid receptors (MR) and epithelial sodium channels (ENaC) in the brain mediate central aldosterone-induced sympathetic hyperactivity and hypertension. Enzymes for biosynthesis of aldosterone are present in the brain, and aldosterone can be produced locally in the brain. Hypothalamic aldosterone levels increase in Dahl salt-sensitive rats on high-salt diet, and in Wistar rats with chronic central infusion of sodium-rich artificial cerebrospinal fluid (CSF) or with subcutaneous infusion of angiotensin II. Functional studies using antagonists of MR, ENaC, and ouabain-like compounds (“ouabain”), as well as specific aldosterone synthase inhibitors, suggest that an increase in local synthesis of aldosterone via MR and ENaC in the brain increases “ouabain” and thereby causes enhanced AT₁ receptor stimulation, leading to sympathoexcitation and hypertension. An increase in CSF sodium or an increase in angiotensinergic output from circumventricular organs such as the subfornical organ projecting to hypothalamic nuclei may increase local production of aldosterone and “ouabain” in magnocellular neurons in the supraoptic nucleus and paraventricular nucleus. This aldosterone-“ouabain” neuromodulatory mechanism appears to play a major role in salt-induced or angiotensin II–induced hypertension.     

Aldosterone-induced cardiac damage: focus on blood pressure independent effects

Mineralocorticoid receptor (MR) antagonists have been used as potassium-sparing diuretics in hypertension. However, in addition to their diuretic and secondary blood pressure (BP)-lowering effects, there exists strong evidence from clinical and experimental studies that they prevent aldosterone-induced myocardial fibrosis independent of their effect on BP. Sustained elevation of aldosterone levels and increased sodium intake in animal models has been found to induce myocardial fibrosis. Fibrosis of the right ventricle, the atria, and the pulmonary artery supports the concept that these effects are BP independent, corroborated by the finding that spironolactone in a dosage not sufficient to lower BP prevents myocardial fibrosis. Patients suffering from primary hyperaldosteronism or Conn's adenoma show more myocardial fibrosis, as assessed by echocardiography, than essential hypertensive patients. Several mechanisms have been proposed to mediate the profibrotic effects of aldosterone, including the possibility of local aldosterone production in the heart, an increase of myocardial AT(1)-receptor density, and enhanced local angiotensin converting enzyme expression. Furthermore, aldosterone increases endothelin receptor expression, which also might cause myocardial fibrosis. Because of the pivotal importance of aldosterone binding to the MR, MR antagonists have emerged as attractive compounds that provide specific end organ protection beyond solely their antihypertensive effects.     

Role of cardiovascular aldosterone in hypertension

Aldosterone plays an important role in the pathogenesis of cardiovascular disease. We have reported that aldosterone is synthesized in cardiovascular tissues and local aldosterone synthesis plays important roles for hypertension and cardiac hypertrophy. High sodium intake develops and accelerates vascular injury and cardiac hypertrophy in SHRSP. Plasma aldosterone concentrations and PRA were decreased by high salt intake in SHRSP. Aldosterone production, the expression of CYP11B2 mRNA and angiotensin II receptor AT1R mRNA in blood vessels were significantly increased by high salt intake. These results suggest that high salt intake increases aldosterone production and expression of the AT1R mRNA in the vascular tissue in SHRSP, which may contribute to the development of malignant hypertension in salt-loaded SHRSP. However, there are several reports of conflicting data. Mineralocorticoid receptor (MR) binding is tightly regulated by the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) which selectively metabolizes glucocorticoids to inactive metabolites, thus allowing for MR activation by aldosterone. We have reported that decreased 11beta-HSD2 in blood vessels in Dahl salt-sensitive (DS) rats, a model for salt-sensitive hypertension. Local aldosterone excess may play a significant role in the salt sensitivity and development of hypertension. High sodium intake decreased circulating rennin-angiotensin-aldosterone system and increased blood pressure and cardiac hypertrophy in DS rats, which were prevented by the treatment with a selective MR antagonist, eplerenone. Eplerenone also improved endothelial nitric oxide synthase (eNOS) activity and eNOS mRNA expression in blood vessels in DS rats. These results further suggest that not only circulating aldosterone but also local aldosterone is of critical importance in the pathophysiology of cardiovascular disorders.