Aldosterone Binding Sites Research Articles

Is GPR30 the Membrane Aldosterone Receptor Postulated 20 Years Ago?

HomeHypertensionVol. 57, No. 5Is GPR30 the Membrane Aldosterone Receptor Postulated 20 Years Ago? Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBIs GPR30 the Membrane Aldosterone Receptor Postulated 20 Years Ago? Alexandra Wendler and Martin Wehling Alexandra WendlerAlexandra Wendler Search for more papers by this author and Martin WehlingMartin Wehling Search for more papers by this author Originally published21 Mar 2011https://doi.org/10.1161/HYPERTENSIONAHA.111.170977Hypertension. 2011;57:e16Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2011: Previous Version 1 To the Editor:Gros et al1 describe the involvement of GPR30 in rapid actions of aldosterone. The hypothesis that a structure other than the mineralocorticoid receptor (MR) may mediate rapid actions of aldosterone was postulated many years ago. Wehling et al2 showed in 1991 that the rapid effect of aldosterone on the sodium-proton exchanger in human mononuclear leukocytes and vascular smooth muscle cells was independent of classic aldosterone antagonists like canrenoate or canrenone.3 Grossmann et al4 showed almost 15 years later that cytosolic Ca2+ was increased by aldosterone in mock- and in human MR-transfected cells to the same extent, and spironolactone did not block this effect, pointing to MR-independent mechanisms. Most recently, aldosterone binding sites were detected at the membrane of human endothelial cells using atomic force microscopy. Aldosterone binding to these sites was spironolactone and dexamethasone insensitive.5 These examples are just a few of those pointing to MR-independent rapid aldosterone actions, and this interpretation is mainly based on their insensitivity to MR antagonists.The functions of GPR30, especially with respect to estrogen actions, are a matter of ongoing debate, and the new findings on its involvement in rapid aldosterone actions add an important facet to this discussion.Gros et al1 postulate that eplerenone and spironolactone are partial antagonists of GPR30. This needs further clarification. Analyzing Figure 2 of the article by Gros et al1 in detail, it is tempting to assume that, in vascular smooth muscle cells which have been transfected with the control construct (green fluorescent protein), extracellular signal–regulated kinase phosphorylation by aldosterone reflects basic MR expression, because it is completely inhibited by eplerenone. When MR is overexpressed in those cells, the effect of aldosterone is roughly doubled, but it remains completely (=100%) eplerenone sensitive. In GPR30-transfected cells, extracellular signal–regulated kinase phosphorylation by aldosterone is also roughly doubled. However, the added effect is not eplerenone sensitive, because eplerenone decreases extracellular signal–regulated kinase phosphorylation only by exactly the same amount seen in cells transfected with the control construct (green fluorescent protein). Therefore, the effect of eplerenone in the GPR30-transfected cells seems to be fully explained by the inhibition of the assumed endogenous MR activity. This is underlined by numerically identical results in Figure 7A through 7C of the article by Gros et al,1 which show identical relations for apoptosis as readout or by Figure 9B in the article by Gros et al1 for myosin light chain phosphorylation as readout. Figure 3 in the article by Gros et al1 demonstrates an incomplete inhibition by eplerenone of G1-stimulated responses, as seen in Figure 5A of this article for endothelial cells and aldosterone as an agonist. This interpretation is only flawed by more complete effects of G15 on aldosterone responses in GPR30-transfected cells (Figure 9C of the article by Gros et al1), which have to remain unexplained. If correct, these interpretations underscore the fact that eplerenone and spironolactone are not antagonists for the rapid action of aldosterone mediated through GPR30.This interpretation would identify GPR30 as an almost perfect candidate for the membrane aldosterone receptor, which we characterized mainly by its landmark property of resistance to classic MR antagonists more than 2 decades ago. Further studies need to validate GPR30 as an aldosterone receptor and to corroborate our interpretation of the impressive data by Gros et al.1Alexandra WendlerMartin Wehling University of Heidelberg Clinical Pharmacology Mannheim Mannheim, GermanyDisclosuresM.W. received consulting and lecture fees from Pfizer, Roche, Novartis, Lilly, PAION, MorphoSys, 4D-Biomedical, and Daiichi-Sankyo. He is a former employee of AstraZeneca (2004–2006).FootnotesLetters to the Editor will be published, if suitable, as space permits. They should not exceed 1000 words (typed double-spaced) in length and may be subject to editing or abridgment.

Aldosterone- and progesterone-membrane-binding proteins: new concepts of nongenomic steroid action.

n the classical theory of steroid action steroids penetrate into cells and bind to intracellular receptors resulting in modulation of nuclear transcription and protein synthesis within hours. In addition, rapid actions of steroids have been identified, which are incompatible with the classic model of steroid action. Specific binding sites for aldosterone and progesterone have been reported in membrane preparations of liver, vascular smooth muscle cells and kidney. These sites are discussed to be involved in rapid nongenomic steroid actions, such as the rapid activation of the Na(+)/H(+) exchanger and elevation of [Ca(2+)]i in vascular smooth muscle cells by aldosterone. In addition, rapid progesterone-induced increases of [Ca(2+)]i have been reported in spermatozoa. A high affinity progesterone-membrane binding protein from porcine liver has been identified and cloned. The derived amino acid sequence showed no significant identity with any functional protein suggesting a binding site completely different to classic progesterone receptors. These binding sites are possibly involved in rapidly induced meiotic maturation of amphibian oocytes and the spermatozoan acrosome reactions as evidenced by recent studies, where the progesterone induced acrosome reactions and calcium signaling was blocked by a specific antibody raised against the membrane binding site for progesterone. In addition to data on specific steroid binding and rapid steroid signaling in vitro, results of nongenomic steroid effects in vivo are presented and their physiological relevance are discussed in the review.

Rapid Nongenomic Effects of Aldosterone in Mineralocorticoid-Receptor-Knockout Mice

In addition to genomic effects of aldosterone, rapid nongenomic effects of steroids have been reported in various tissues that were clearly incompatible with a genomic action of aldosterone. Rapid effects of aldosterone involve second messengers such as calcium and cAMP. Specific high affinity binding sites for aldosterone have been characterized in membranes for different cells, which probably transmit those rapid steroid effects. To date, it is unclear if these binding sites are modified classical mineralocorticoid receptors (MR) or if they represent an unrelated receptor protein. The aim of the present study was to investigate whether rapid aldosterone action still occurs in the absence of the classical MR. For this purpose we used the model of MR knockout mice. Rapid effects were analyzed in skin cells, measuring intracellular calcium and cAMP levels after stimulation with aldosterone. We found that rapid effects are not only present in MR knockout mice, but that the effects are even larger than in wild-type mice cells. The results of the present study demonstrate that the classic MR is dispensable for rapid aldosterone action. The study, thus, proves that a receptor different from the classic intracellular receptor is involved in rapid aldosterone signaling.

Coexpression of mineralocorticoid receptors and 11beta-hydroxysteroid dehydrogenase 2 in human gastric mucosa.

The role of mineralocorticoids in human gastrointestinal tract is well established. In the stomach, aldosterone is thought to regulate electrolyte transport associated with gastric acid secretion. In mineralocorticoid target organs, the action of the glucocorticoid inactivating enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) facilitates aldosterone binding to a nonselective mineralocorticoid receptor (MR) in the face of high levels of circulating glucocorticoids. In the present study, we examined 25 specimens of human stomach for the presence of MR and 11beta-HSD2 using a [3H]aldosterone binding assay, Northern blot analysis, RT-PCR, and immunohistochemistry. Specific [3H]aldosterone binding sites were detected in gastric fundic mucosa, but not in the antrum. In fundic mucosa the Kd was 0.72+/-0.05 nmol/L (mean +/- SE), and Bmax was 6.0+/-1.4 fmol per milligram of protein. Northern blot analysis demonstrated a faint band for MR mRNA at 6.0 kb, although message for 11beta-HSD2 was undetectable. However, RT-PCR demonstrated specific PCR products for both MR and 11beta-HSD2. Immunohistochemistry demonstrated the colocalization of MR and 11beta-HSD2 only in parietal cells. MR-positive cells were further characterized by electron microscopy, confirming the identity of parietal cells. This study shows that parietal cells contain both MR and 11beta-HSD2, suggesting that the human stomach is a novel target organ for mineralocorticoids. Aldosterone may, therefore, regulate biological functions of parietal cells including gastric acid secretion.

Characterization and Solubilization of Novel Aldosterone‐Binding Proteins in Porcine Liver Microsomes

Using the radioligand [1,2,6,7‐3H]aldosterone ([3H]aldosterone), specific binding sites for aldosterone were identified and characterized in microsomal preparations from porcine liver. The maximum binding capacity is approximately 700 fmol · mg‐1 microsomal protein. The reversible binding of [3H]aldosterone was saturable and Scatchard analysis revealed two apparent dissociation constants (Kd), Kd1≤ 11 nM and Kd2= 118 nM. Binding was optimal at pH 7.2, thermolabile, and was reduced by more than 70% when membrane vesicles were pretreated with trypsin. Binding was selective for aldosterone with cortisol being a weak agonist at 1000‐fold higher concentrations only. Among those detergents tested to optimize conditions for solubilization, n ‐octylglucoside (75 mM) was most favorable and solubilized 25% of the radio‐ligand‐binding protein complex in the undissociated form. These binding sites have unique pharmacological properties, which are similar to those found for aldosterone membrane binding in human lymphocytes and pig kidney, and for rapid aldosterone effects on sodium‐proton exchange.

Characterization and solubilization of novel aldosterone-binding proteins in porcine liver microsomes.

Using the radioligand [1,2,6,7-3H]aldosterone ([3H]aldosterone), specific binding sites for aldosterone were identified and characterized in microsomal preparations from porcine liver. The maximum binding capacity is approximately 700 fmol x mg-1 microsomal protein. The reversible binding of [3H]aldosterone was saturable and Scatchard analysis revealed two apparent dissociation constants (Kd), Kd1 < or = 11 nM and Kd2 = 118 nM. Binding was optimal at pH 7.2, thermolabile, and was reduced by more than 70% when membrane vesicles were pretreated with trypsin. Binding was selective for aldosterone with cortisol being a weak agonist at 1000-fold higher concentrations only. Among those detergents tested to optimize conditions for solubilization, n-octylglucoside (75 mM) was most favorable and solubilized 25% of the radioligand-binding protein complex in the undissociated form. These binding sites have unique pharmacological properties, which are similar to those found for aldosterone membrane binding in human lymphocytes and pig kidney, and for rapid aldosterone effects on sodium-proton exchange.

3H]-aldosterone binding sites (type I receptors) in the lateral wall of the cochlea: distribution assessment by quantitative autoradiography.

Mineralocorticoids, such as aldosterone, are steroids that enhance Na+ retention and K+ excretion in ion-transporting epithelial tissues through regulation of Na, K-ATPase. Previous studies suggest that aldosterone may regulate labyrinthine ion transport through up-regulation of Na, K-ATPase sites, a process mediated by high-affinity aldosterone (type I) receptors. In the present study, information concerning density and distribution of aldosterone binding sites in cochlear lateral wall tissues was determined by quantitative autoradiography using [3H]-aldosterone and RU-28362, a glucocorticoid agonist that blocks low affinity binding to glucocorticoid (type II) receptors. The results revealed that the distribution of aldosterone binding sites differs among the individual cochlear regions of the lateral wall. The highest level of binding was associated with the stria vascularis and epithelial cells of the spiral prominence. Elevated levels of binding were also observed in stromal cells of the spiral prominence, and to a lesser extent in the spiral ligament. The differential distribution of aldosterone binding sites in the lateral wall resembles the pattern of localization of Na, K-ATPase sites observed in previous studies and is compatible with the idea that mineralocorticoids play a role in the regulation of cochlear cation transport.

Immunofluorescence of mineralocorticoid receptors in peripheral lymphocytes: Presence of receptor-like activity in patients with the autosomal dominant form of pseudohypoaldosteronism, and its absence in the recessive form

Pseudohypoaldosteronism is a syndrome characterized by salt wasting and a failure to thrive due to the resistance towards the action of aldosterone. Aldosterone levels and plasma renin activity are extremely elevated and aldosterone binding sites in peripheral mononuclear leukocytes have regularly shown to be reduced or absent. Sporadic as well as familial cases have been identified and an autosomal dominant as well as an autosomal recessive mode of inheritance has been described. A defect in the aldosterone receptor has been postulated, however, molecular genetic analysis in selected patients has not revealed a mutation in the sequence of the coding region of the cDNA of the mineralocorticoid receptor gene. In the present study we have used a fluorescence-labeled antibody to detect possible receptor expression in monocytes from patients with various clinical forms of pseudohypoaldosteronism. Patients with the sporadic as well as with the autosomal dominant form were clearly immunopositive despite being negative in terms of aldosterone receptor binding. In contrast in two patients with the autosomal recessive form there was no detectable receptor protein, consistent with the results obtained in the aldosterone binding studies. These results suggest that the pathogenesis of pseudohypoaldosteronism is heterogeneous not only regarding the mode of inheritance but also in terms of receptor binding. Thus, in a subgroup of patients the inability of the receptor to bind ligand may be due to a defect involving other, probably cellular factors rather than a deficiency or a defect in the mineralocorticoid receptor system itself.

High affinity aldosterone binding sites (Type I receptors) in the mammalian inner ear

The presence of aldosterone (Type 1) binding sites in the mammalian inner ear has been previously suggested by an increase in inner ear Na, K-ATPase ouabain binding sites in response to the administration of aldosterone in vivo (Pitovski et al., 1993). Type I binding sites have now been identified and characterized in the lateral wall of the basal turn of the cochlea and in the ampullae of the semicircular canals of the guinea pig. In the presence of RU 28362, which blocks low-affinity binding of the labeled hormone to Type II sites, [ 3H]-aldosterone binds to a single class of high-affinity (Type I) sites with K d values of 34.7 nM in lateral wall of the basal turn of the cochlea and 31.3 nM in the ampullae of the semicircular canals. B max is 17.1 fmol/mg dry tissue for the cochlear sample and 17.4 fmol/mg dry tissue for the ampullae, comparable to reported values in renal tissue (17–31 fmol/mg protein). Thus, the results of receptor-binding experimental protocols with [ 3H]-aldosterone clearly suggest that these inner ear tissues are a target site of mineralocorticoid action.

Aldosterone stimulated differentiation of mouse 3T3-L1 cells into adipocytes.

We find that 1-10 nM aldosterone can induce differentiation of 3T3-L1 cells into adipose cells as evaluated by microscopic accumulation of fat droplets and quantitative measurement of triglycerides and of glycerol-3-phosphate dehydrogenase, an enzyme specific for adipocyte differentiation. Moreover, the aldosterone antagonist ZK91587 inhibits aldosterone-but not glucocorticoid-mediated differentiation of 3T3-L1 cells. Steroid binding assays with 3T3-L1 cells indicate the presence of specific binding sites for aldosterone. We conclude that there is an aldosterone receptor-mediated pathway for terminal differentiation of 3T3-L1 cells into adipose cells. Receptors for aldosterone have also been found in a variety of cells that do not function to regulate sodium and potassium transport. The aldosterone receptor may have a role in regulation expression of genes involved in differentiation of these cells.

11 beta-Hydroxysteroid dehydrogenase mRNA expression in rat kidney

11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) protects nonspecific renal mineralocorticoid receptors from exposure to circulating glucocorticoid in vivo by catalyzing the conversion of corticosterone to inactive 11-dehydrocorticosterone. Although 11 beta-OHSD bioactivity and aldosterone binding sites are found in distal tubular cells, mineralocorticoid receptor and 11 beta-OHSD immunoreactivities are not colocalized. However, there are several kidney isoforms of 11 beta-OHSD, not all of which may be immunoreactive, whereas only a single mRNA species has been described. Using in situ hybridization we found 11 beta-OHSD mRNA is highly expressed in all renal tubular epithelia in the rat. It is therefore likely that 11 beta-OHSD is colocalized with mineralocorticoid receptors in distal tubular cells.

Rapid effects of mineralocorticoids on sodium-proton exchanger: genomic or nongenomic pathway?

High-affinity aldosterone binding sites have been described in human mononuclear leukocytes (HML), and in vitro effects of aldosterone on intracellular sodium, potassium, and calcium concentrations and cell volume have been shown in HML. In the present paper, the response of the sodium-proton exchanger of the cell membrane to agonists and antagonists was studied by determining the kinetics of HML swelling in isotonic sodium propionate. Within 1-2 min of incubation, aldosterone significantly stimulated propionate-induced swelling by an additional 30-50% at concentrations as low as 0.07 nM. This effect was not blocked by the classical aldosterone antagonists potassium canrenoate or canrenone at concentrations of 140 or 700 nM. Hydrocortisone and dexamethasone were effective agonists only at much higher concentrations (4,000 nM). These data are not well explained by a mechanism of steroid action involving the interaction of a steroid receptor complex with nuclear DNA, since the effect on the sodium-proton exchanger is too fast. The findings could indicate distinct membrane receptors with a high affinity for aldosterone, but not hydrocortisone, and rapid direct membrane effects of aldosterone. These putative novel receptors may in turn bind novel aldosterone antagonists with possible diuretic and vasodilator effects distinct from those of spironolactones.

Extrarenal receptor-effector-mechanisms for aldosterone: The sequence of effects on the cellular electrolyte transport in human lymphocytes and their implications for disorders of the water and electrolyte balances

High affinity aldosterone binding sites have not only been described in the classic target tissues such as the renal tubules, but also in non-classic target tissues such as the hippocampus, mammary gland, endothelial cells and, recently, human mononuclear leukocytes. An in vitro effect of aldosterone on intracellular sodium, potassium and calcium concentrations and cell volume was shown in human mononuclear leukocytes. In the absence of aldosterone, the intracellular Na+, K+ and Ca2+ concentrations and the cell volume decreased significantly, but remained constant when aldosterone (1.4 nmol/l) was added to the incubation medium. These effects of aldosterone were blocked by the aldosterone antagonist canrenone (140 nmol/l). The sodium/proton exchanger of the cell membrane could be identified as the primary target of the aldosterone action, possibly non-genomically mediated through membrane receptors. The clinical significance of this model was underlined by the demonstration of absent or a decreased number of mineralocorticoid receptors and the lack of electrolyte response to aldosterone in human mononuclear leukocytes of patients with pseudohypoaldosteronism and aldosteronism. Additionally, an abnormal effector mechanism could be demonstrated in human mononuclear leukocytes from essential hypertensives. These studies are the first to demonstrate the significance of extrarenal, nonepithelial mineralocorticoid receptors and the related effector mechanism in different disorders of the water and electrolyte balance in man.

Aldosterone binding sites along nephron of Xenopus and rabbit

Distal segment of several amphibians exhibits aldosterone-modulated ion transport properties. On the other hand, A6 cells, derived from Xenopus laevis (XL) kidney, are aldosterone sensitive. We examined the distribution of aldosterone binding sites in isolated tubules of XL compared with rabbit. After incubation with 2 nM [3H]aldosterone, microdissected tubular segments from proximal (PT), distal straight segment (DST), and flask cell collecting (CT) tubules from XL and from rabbit cortical thick ascending limb (CTAL), connecting (CNT), and collecting (CCD) tubules were processed for dry film autoradiography. In XL, specific nuclear labeling of type I (mineralocorticoid) sites was restricted to DST. Labeling of type II (glucocorticoid) sites was present all along the tubule. No specific cytoplasmic labeling was observed, except for type II sites in PT. In the rabbit, aldosterone binds to both type I and type II sites in the three tubular segments studied. In these segments, the binding was about fourfold higher than in DST of XL. These results bring direct evidence in designating the distal tubule of amphibians as a target epithelium for aldosterone. In addition, they suggest that A6 cell line may derive from DST of the Xenopus nephron.

High affinity aldosterone binding sites (type I receptors) in rat heart.

1. The use of the receptor stabilizing agent sodium molybdate, and of RU26988 to exclude [3H]-aldosterone binding from Type II glucocorticoid receptors, has enabled the characterization of high affinity Type I aldosterone binding sites in rat atrial and ventricular cytosols. 2. In adult male and female rats the affinity of binding (Kd 4 degrees C) is approximately 1-2 nmol/l for both atria and ventricles; specificity of binding is similar to that for Type I sites in classical aldosterone target tissues (aldosterone = corticosterone much greater than dexamethasone). 3. Levels of atrial Type I sites are higher than the corresponding levels in ventricle in both males and females, whereas for Type II (classical glucocorticoid) receptors the reverse is the case; levels of both Type I and Type II sites fall over the age range examined (40 days-6 months). 4. The physiological function(s) of cardiac Type I sites, and their in vivo mineralocorticoid or glucocorticoid selectivity, remain to be explored.

Aldosterone and dexamethasone binding in human arterial smooth muscle cells.

Regulation of blood pressure by direct action of corticosteroids on blood vessel walls was first hypothesized in 1952 [1]. The presence of receptors specific for these hormones within the peripheral vessel walls is a pre-requisite of this hypothesis. This report documents specific binding of both aldosterone (AL) and dexamethasone (DM) in cultured human arterial smooth muscle cells. After the arterial smooth muscle cultures were incubated with tritiated AL or DM at 37 degrees C for 30-60 min, the cells were sonicated and the protein bound steroid fraction isolated on a Sephadex G25 column. Using ion exchange chromatography of this fraction, each corticosteroid receptor complex displayed a distinct, reproducible elution pattern. Aldosterone showed a single peak at 0.096 +/- 0.005 mol/l sodium phosphate and DM had two peaks at 0.029 +/- 0.003 and 0.050 +/- 0.004 mol/l sodium phosphate. The Scatchard plots of specific binding from AL saturation curves are linear and revealed mean +/- s.d. steady state binding parameters of dissociation constant (Kd) = 0.35 +/- 0.15 nmol/l and maximum binding capacity (Bmax) = 98 +/- 53 X 10(-18) mol/micrograms DNA. Similarly, the mean +/- s.d. steady state binding parameters for DM are Kd = 4.4 +/- 2.0 nmol and Bmax = 3031 +/- 1385 X 10(-18) mol/micrograms DNA. Therefore, there are approximately 1150 AL binding sites and 30000 DM binding sites per cell. The Kd and Bmax values are similar to those previously described for corticoid receptors in rat aortic smooth muscle cells and are consistent with physiological steroid concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo competitive autoradiographic study of [ 3H]corticosterone and [ 3H]aldosterone binding sites within mouse brain hippocampus

The binding sites for [ 3H]corticosterone ( 3HB) and [ 3H]aldosterone ( 3HA) within the hippocampal area of the mouse brain have been studied by autoradiography in competition experiments. Excess unlabelled aldosterone (A) or corticosterone (B) both abolished the nuclear accumulation of radioactivity within neurons observed after injection of either 3HA or 3HB. Experiments where a subcutaneous injection of a “pure glucocorticoid” RU26988 was given before injection of 3HA alone showed a marked accumulation of radioactivity within neuronal nuclei of the hippocampus suggesting the presence of 3HA binding sites distinct from classical type II glucocorticoid receptors. In addition, when RU26988 was given before the injection of 3HA associated with a 30- or 100-fold excess of either A or B, the cell nuclear accumulation of radioactivity was no longer observed. These results showed that in our in vivo experimental conditions, B displayed the same ability as A to occupy 3HA binding sites, supporting the view that in mouse hippocampal neuronal nuclei, the aldosterone-binding and corticosterone-preferring sites represent the same molecular entity.

High-affinity aldosterone binding in rat liver--a re-evaluation.

The use of sodium molybdate as a stabilizing agent, and a RU26988 to exclude [3H]aldosterone from Type II glucocorticoid receptors, has enabled us to characterize high affinity Type I aldosterone binding sites in rat liver cytosol. In liver cytosols from male rats aldosterone bound with an affinity (Kd-22 degrees C) of 0.6 nmol/l (range 0.3-0.8 nmol/l), and Nmax 1.7 fm/mg protein (s.e.m. = 0.4); specificity of binding was similar to that for Type I sites in classical aldosterone target tissues (aldosterone greater than or equal to corticosterone greater than dexamethasone). Hepatic Type I receptor levels were relatively constant in both male and female rats aged 30-120 days, with levels significantly higher in females. Parallel studies on hepatoma H4 cells showed levels of Type I sites similar to those in normal liver, suggesting a general distribution of such sites throughout liver parenchyma, rather than a concentration in a specific cell type. The function of such Type I sites, and whether or not they are aldosterone-selective in vivo and can thus act as mineralocorticoid receptors, remains to be determined.

Glycyrrhizic acid and its hydrolysate as mineralocorticoid agonist

Mineralocorticoid activity of glycyrrhetinic acid (GR) was studied in vivo (electrical potential difference in rat rectum) and in vitro (brush border Mg 2+-HCO − 3 ATPase in rat small intestine, kidney cytosol binding of GR with and without RU-28362, anti-glucocorticoid compound) in order to clarify the mechanism of mineralocorticoid-like activity of GR. Scatchard analysis of [ 3H]aldosterone showed that K d of higher affinity site (type I) 6.0 × 10 −9 M, B max 1.0 × 10 −14 mol/mg protein, and K d of lower affinity site (type II) 1.6 × 10 −7M, B max 7.5 × 10 −14 mol/mg protein. GR competed for [ 3H]aldosterone binding sites in kidney cytosol at the concentration of 10 4 times as that of unlabeled aldosterone. RU-28362 displaced aldosterone binding curve, whereas GR binding kinetic was not affected by this compound. Adrenalectomy caused a significant fall in brush border Mg 2+-HCO − 3 ATPase activity (75% reduction compared with the initial level) which was not restored by GR administration. Electrical potential differences in the adrenalecomized rats were significantly lower than those in the control rats, which did not increase after GR administration.

Nuclear localization of type 1 aldosterone binding sites in steroid-unexposed GH3 cells.

The nuclear localization of unoccupied oestrogen receptors has been demonstrated in MCF 7 (human breast tumour) cells by immunocytochemistry, and in GH3 (rat pituitary tumour) cells by enucleation techniques. The present study shows, by similar enucleation techniques, that high affinity (Type 1) aldosterone binding sites are similarly located in the nucleus of steroid-unexposed pituitary GH3 cells. It is, therefore, suggested that such high-affinity Type 1 sites, which are mineralocorticoid receptors in the kidney and glucocorticoid receptors in the hippocampus, are nuclear-associated proteins in the absence of steroid and are found in the cytosol compartment only upon cell disruption.