Apparent mineralocorticoid excess, pseudohypoaldosteronism, and urinary electrolyte excretion: toward a redefinition of mineralocorticoid action

Patients with apparent mineralocorticoid excess (AME) have low or absent activity of the enzyme 11 beta OH steroid dehydrogenase (11SD), and inappropriately high intrarenal levels of cortisol resulting in Na+ retention and hypertension. Pseudohypoaldosteronism (PHA), in contrast, is characterized by salt wasting despite hyperaldosteronemia, reflecting low or absent mineralocorticoid receptors (MR). Although AME is presumed to reflect inappropriate cortisol occupancy of MR, several features also suggest inappropriate occupancy of glucocorticoid receptors (GR). To test this possibility, we administered carbenoxolone, which is known to block 11SD, to four patients with PHA, and observed marked mineralocorticoid effects, e.g., antinatriuresis and elevated plasma bicarbonate. To further test the possibility that occupancy of renal GR may induce a classical mineralocorticoid response, we administered the highly specific glucocorticoid RU 28362 to adrenalectomized rats and showed that it has profound antinatriuretic effects. Finally, by selectively blocking MR with RU 28318 or GR with RU 38486, we have shown that corticosterone, the physiologic glucocorticoid in rats, has an antinatriuretic effect in adrenalectomized rats via either MR or GR occupancy. Previous studies have clearly shown that MR are inherently nonselective and have equivalent intrinsic affinity for aldosterone, corticosterone, and cortisol. The present studies suggest that this nonselectivity includes the nuclear response element to which either MR or GR may bind to elicit a mineralocorticoid effect, and further underscore the importance of the enzyme 11SD in the specific mineralocorticoid action of aldosterone.     

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.     

Modulators of the glucocorticoid receptor also regulate mineralocorticoid receptor function.

Modulators are proposed to be novel ether aminophosphoglycerides that stabilize unoccupied and occupied glucocorticoid receptor steroid binding and inhibit glucocorticoid receptor complex activation. Two isoforms, modulator 1 and modulator 2, have been purified from rat liver cytosol [Bodine, P.V., & Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554]. Since the mineralocorticoid receptor is relatively resistant to activation, modulator's effect on rat distal colon mineralocorticoid receptor function was examined. Warming of unoccupied receptor decreased residual specific [3H]aldosterone binding by 86 +/- 2%. Both modulator isoforms completely prevented this destabilization with Km's of 2 +/- 1 microM modulator 1 and 24 +/- 5 microM modulator 2. Warming of occupied mineralocorticoid receptors decreased [3H]aldosterone binding by 56 +/- 3%. Modulator only partially stabilized occupied receptor binding with Km's of 10 +/- 2 microM modulator 1 and 68 +/- 8 microM modulator 2. Modulator inhibited receptor activation with Km's of 3 +/- 1 microM modulator 1 and 33 +/- 10 microM modulator 2. Double-reciprocal analysis showed linear kinetics, and mixing modulator isoforms together had additive effects on unoccupied and occupied receptor steroid binding stabilization and activation inhibition. Colon cytosol contained a low molecular weight, heat-stable factor(s) which inhibited receptor activation and stabilized occupied receptor steroid binding. Molybdate completely stabilized unoccupied mineralocorticoid receptor steroid binding and inhibited activation with half-maximal effects at 3-4 mM but only stabilized occupied receptor binding by approximately 40%. These data indicate that (i) apparent physiologic concentrations of modulator stabilize mineralocorticoid receptor steroid binding and inhibit receptor activation, (ii) an aldosterone-responsive tissue contains a modulator-like activity, and (iii) molybdate mimics the effects of modulator.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Influence of morphine treatment in pregnant rats on the mineralocorticoid activity of the adrenals in their neonates

Exposure of pregnant rats to morphine, from day 11 to day 18 of gestation, was previously reported to induce both an adrenal atrophy and hypoactivity of the glucocorticoid function in newborns at term, but did not affect, in vitro, the responsiveness of those glands to adrenocorticotrophin hormone (ACTH) concerning corticosterone release. Moreover, these effects were mediated by maternal hormones from the adrenal glands. In the present work, we investigated the effects of a prenatal morphine exposure on the mineralocorticoid activity of the adrenals in neonates. The first aim of the present study was to determine in these newborns 1) the adrenal and plasma aldosterone concentrations at birth time and during the early postnatal period 2) the plasma levels of Na+ and K+ at birth time, 3) the in vitro responsiveness of the newborn adrenals to angiotensin II (A(II)) and ACTH. The second aim of our study was to investigate the mineralocorticoid activity of the adrenals in newborns from adrenalectomized mothers treated with morphine during gestation. According to present data morphine given to intact mothers induced in newborns a severe adrenal atrophy but increased adrenal aldosterone content and plasma aldosterone level. However, prenatal morphine was unable to affect significantly Na+/K+ ratio in both mothers and newborns. In vitro, the adrenals of neonates from morphine-treated mothers were unresponsive to An and ACTH for promoting aldosterone release; in contrast, aldosterone secretion was significantly stimulated by high potassium levels (55 mEq). Maternal adrenalectomy performed one day before the beginning of morphine treatment prevented morphine-induced adrenal atrophy but was unable to affect significantly the adrenal mineralocorticoid function of the offspring. Such data suggest that a prenatal morphine exposure stimulated both aldosterone synthesis and release in neonates. However, this basal hyperfunction did not appear to be coupled with an enhanced adrenal responsivity to AII or ACTH. Prenatal morphine-induced hyperactivity of the mineralocorticoid function of the newborn adrenals, which drastically contrast with hypoactivity of the glucocorticoid one, was independent of adrenal factors from maternal origin.     

Induction of 5-aminolevulinate synthase by activators of steroid biosynthesis

Different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group. We previously reported that ACTH, an activator of glucocorticoid biosynthesis in adrenal, requires heme biosynthesis for a maximal response. In the present study, we investigated the effect of ACTH, and the effect of two activators of the adrenal mineralocorticoid synthesis, endothelin-1 and low sodium diet on 5-aminolevulinate-synthase (ALA-s) mRNA. ALA-s is the rate-limiting enzyme in heme biosynthesis. It was found that infusion of rats with ACTH for 1 h caused an increase of adrenal ALA-s mRNA and activity accompanied by an increase in plasma corticosterone. CYP21, a cytochrome involved in the synthesis of both corticosterone and aldosterone, was not modified at the RNA level in adrenal glands by 1 h of ACTH infusion. Consistently, infusion of endothelin-1 for 1 h increased ALA-s mRNA and aldosterone content in adrenal gland without modifying CYP21 mRNA levels. To study if ALA-s is also regulated by the main physiological stimuli that increase adrenal mineralocorticoid secretion, we fed rats with low salt diet for 2 or 15 days. Low salt diet treatment increased adrenal gland ALA-s mRNA levels. On the other hand, the rapid stimulation of ALA-s mRNA by ACTH which acts through cyclic AMP was confirmed in H295R human adrenocortical cells, the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells. Our findings suggest that the acute activation of adrenal steroidogenic cytochromes by trophic hormones involves an increase in heme biosynthesis which will favor the production of active cytochromes.     

Equivalent affinity of aldosterone and corticosterone for type I receptors in kidney and hippocampus: direct binding studies

In studies from several laboratories evidence has been adduced that renal Type I (mineralocorticoid) receptors and hippocampal "corticosterone-preferring" high affinity glucocorticoid receptors have similar high affinity for both aldosterone and corticosterone. In all these studies the evidence for renal mineralocorticoid receptors is indirect, inasmuch as the high concentrations of transcortin (CBG) in renal cytosol make studies with [3H]corticosterone as a probe difficult to interpret, given its high affinity for CBG. We here report direct binding studies, with [3H]aldosterone and [3H]corticosterone as probes, on hippocampal and renal cytosols from adrenalectomized rats, in which tracer was excluded from Type II dexamethasone binding glucocorticoid receptors with excess RU26988, and from CBG by excess cortisol 17 beta acid. In addition, we have compared the binding of [3H]aldosterone and [3H]corticosterone in renal cytosols from 10-day old rats, in which CBG levels in plasma and kidney are extremely low. Under conditions where neither tracer binds to type II sites or CBG, they label an equal number of sites (kidney 30-50 fmol/mg protein, hippocampus approximately 200 fmol/mg protein) with equal, high affinity (Kd 4 degrees C 0.3-0.5 nM). Thus direct tracer binding studies support the identity of renal Type I mineralocorticoid receptors and hippocampal Type I (high affinity, corticosterone preferring) glucocorticoid receptors.     

Antagonistic effects of aldosterone on corticosterone-mediated changes in exploratory behavior of adrenalectomized rats

The effect of aldosterone administration on exploratory activity of chronic adrenalectomized (10 days) male rats was investigated. Aldosterone (30 μg/100 g body wt sc) administered 1 hr or 30 min prior to the behavioral test failed to normalize disturbed exploratory activity of adrenalectomized rats, in contrast to the restoration observed after corticosterone, the naturally occurring glucocorticoid of the rat. Administration of the mineralocorticoid 30 min prior to corticosterone prevented the normalization of the behavioral response by the latter steroid. Administration of the same dose of aldosterone 30 min prior to a tracer amount of [³H]corticosterone effectively blocked cell nuclear uptake of radioactive-labeled hormone in the hippocampus. The specific action of corticosterone on exploratory behavior corresponds with the stringent specificity of the neuronal hippocampal corticosterone receptor system. Mineralocorticoid receptors do not seem to be involved in effects on this behavior. The antagonistic action of aldosterone is probably exerted by competitive binding to the corticosterone receptor.     

Crystal Structure of the Mineralocorticoid Receptor DNA Binding Domain in Complex with DNA

The steroid hormone receptors regulate important physiological functions such as reproduction, metabolism, immunity, and electrolyte balance. Mutations within steroid receptors result in endocrine disorders and can often drive cancer formation and progression. Despite the conserved three-dimensional structure shared among members of the steroid receptor family and their overlapping DNA binding preference, activation of individual steroid receptors drive unique effects on gene expression. Here, we present the first structure of the human mineralocorticoid receptor DNA binding domain, in complex with a canonical DNA response element. The overall structure is similar to the glucocorticoid receptor DNA binding domain, but small changes in the mode of DNA binding and lever arm conformation may begin to explain the differential effects on gene regulation by the mineralocorticoid and glucocorticoid receptors. In addition, we explore the structural effects of mineralocorticoid receptor DNA binding domain mutations found in type I pseudohypoaldosteronism and multiple types of cancer.     

Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species

The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross-reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRG1 could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity.     

Localization of mineralocorticoid receptors at mammalian synapses

In the brain, membrane associated nongenomic steroid receptors can induce fast-acting responses to ion conductance and second messenger systems of neurons. Emerging data suggest that membrane associated glucocorticoid and mineralocorticoid receptors may directly regulate synaptic excitability during times of stress when adrenal hormones are elevated. As the key neuron signaling interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. The lateral amygdala is a key site for synaptic plasticity underlying conditioned fear, which can both trigger and be coincident with the stress response. A large body of electrophysiological data shows rapid regulation of neuronal excitability by steroid hormone receptors. Despite the importance of these receptors, to date, only the glucocorticoid receptor has been anatomically localized to the membrane. We investigated the subcellular sites of mineralocorticoid receptors in the lateral amygdala of the Sprague-Dawley rat. Immunoblot analysis revealed the presence of mineralocorticoid receptors in the amygdala. Using electron microscopy, we found mineralocorticoid receptors expressed at both nuclear including: glutamatergic and GABAergic neurons and extra nuclear sites including: presynaptic terminals, neuronal dendrites, and dendritic spines. Importantly we also observed mineralocorticoid receptors at postsynaptic membrane densities of excitatory synapses. These data provide direct anatomical evidence supporting the concept that, at some synapses, synaptic transmission is regulated by mineralocorticoid receptors. Thus part of the stress signaling response in the brain is a direct modulation of the synapse itself by adrenal steroids.     

Effect of adrenalectomy and aldosterone on the modulation of mineralocorticoid receptors in rat kidney

Adrenalectomized rat kidney is commonly used for the study of mineralocorticoid mechanism of action in mammals. In this model, aldosterone is known to bind to two classes of binding sites: type I (mineralocorticoid) and type II (glucocorticoid). The study of the aldosterone binding in normal rat kidney requires the elimination of endogenous hormones bound to each type of receptor. Thus, a suitable technique was developed using in situ perfusion of the kidneys. The efficacy of this method was of about 85 to 90% at the level of both cytoplasm and nucleus. Aldosterone binding capacity was checked in normal rat kidney after in situ perfusion and was found to be 300 to 500% lower than in adrenalectomized rat kidney, both in cytoplasm and nuclei. Computer analysis of aldosterone binding parameters in the cytoplasm (30,000 X g supernatant) of rat kidney suggested that adrenalectomy might induce an important rise in the number of mineralocorticoid receptors (congruent to 260%). An increase in the number of glucocorticoid receptors was also observed but appeared to be lower. Aldosterone, when perfused during 24 h in adrenalectomized rats, lowered the number of type I sites to the same level as observed in normal rat kidney. This effect was fully reversible after interruption of aldosterone perfusion. These results suggested an aldosterone-induced down regulation of mineralocorticoid receptors.     

Activation of pregnane X receptor disrupts glucocorticoid and mineralocorticoid homeostasis

The pregnane X receptor (PXR) was isolated as a xenobiotic receptor that regulates responses to various xenobiotic agents. In this study, we show that PXR plays an important endobiotic role in adrenal steroid homeostasis. Activation of PXR by genetic (transgene) or pharmacological (ligand, such as rifampicin) markedly increased plasma concentrations of corticosterone and aldosterone, the respective primary glucocorticoid and mineralocorticoid in rodents. The increased levels of corticosterone and aldosterone were associated with activation of adrenal steroidogenic enzymes, including cytochrome P450 (CYP)11a1, CYP11b1, CYP11b2, and 3beta-hydroxysteroid dehydrogenase. The PXR-activating transgenic mice also exhibited hypertrophy of the adrenal cortex, loss of glucocorticoid circadian rhythm, and lack of glucocorticoid responses to psychogenic stress. Interestingly, the transgenic mice had normal pituitary secretion of ACTH and the corticosterone-suppressing effect of dexamethasone was intact, suggesting a functional hypothalamus-pituitary-adrenal axis despite a severe disruption of adrenal steroid homeostasis. The ACTH-independent hypercortisolism in the PXR-activating transgenic mice is reminiscent of the pseudo-Cushing's syndrome in patients. The glucocorticoid effect appears to be PXR specific, as the activation of constitutive androstane receptor in transgenic mice had little effect. We propose that PXR is a potential endocrine disrupting factor that may have broad implications in steroid homeostasis and drug-hormone interactions.     

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.     

Adrenocorticoid receptors in C6 glioma cells: Effects on cell growth

Rat C6 glioma cells contain two receptors for adrenocorticoids—the predominant glucocorticoid receptor and low densities of the Type I corticosteroid (mineralocorticoid) receptor. Nanomolar concentrations of deoxycorticosterone, corticosterone and aldosteceptor. Nanomolar concentrations of deoxycorticosterone, corticosterone and aldosterone, which fully occupy Type I receptors, produced a slight stimulatory effect on C6 cell growth in serum-free media. However, spironolactone, a Type I receptor antagonist, and pregnenolone, which does not bind to Type I receptors, had similar effects. Therefore, the slight growth stimulation produced by low steroid concentrations is not mediated by Type I or glucocorticoid receptors, but may be due to an effect on cell membrane properties or other receptor-independent action. Occupation of glucocorticoid receptors by higher concentrations of corticosteroids inhibited C6 cell growth.     

Binding of steroids in nuclear extracts and cytosol of rat pituitary and estrogen-induced pituitary tumors

We have determined binding sites for estrogen, progestin, androgen and glucocorticoid in anterior pituitaries from Sprague-Dawley rats, a strain with low estrogen sensitivity, and in diethylstilbestrol-induced pituitary tumors in Fischer 344 rats, a strain with high estrogen sensitivity. Binding sites differ in their quantity and subcellular distribution. Cytosolic sites for [3H]estradiol in normal pituitaries from untreated rats were high prevailing over sites for other hormones, but they were depleted in the tumors due to their retention in nuclei under the influence of estrogen. Unoccupied nuclear sites for estrogen in normal glands also prevailed over sites for other steroids, and were similar to those in tumors. Second, the progestin site labeled with [3H]R 5020 was concentrated 5.7-fold in cytosol and 8.5-fold in nuclei of the tumors over the values found in glands from normal males estrogenized for 3 days. Third, glucocorticoid receptors labeled with [3H]dexamethasone were predominantly cytosolic in normal glands, but very low in cytosol and more evident in nuclear extracts from the tumors, the reverse of the profile found in normal pituitaries. Last, limited and comparable amounts of androgen receptors were measured in the subcellular fractions of both tissues. It is suggested that the subcellular distribution of some steroid receptors may be controlled in part by the cell population of the tissue and its degree of genetic activity.