Specific Binding Of Aldosterone Research Articles

Aldosterone Nongenomically Produces NADPH Oxidase−Dependent Reactive Oxygen Species and Induces Myocyte Apoptosis

The roles of aldosterone in the progression of heart failure have not been fully elucidated. This study examined whether aldosterone nongenomically activates reactive oxygen species (ROS) production, causing myocyte apoptosis. Addition of aldosterone to neonatal rat cardiac myocytes caused the activation of NADPH oxidase and intracellular ROS production in a dose-dependent manner (10-(9)-10(-7) mol/L). NADPH oxidase activation was evident as soon as 5 min after aldosterone treatment. Neither an inhibitor for nuclear transcription (actinomycin D) nor an inhibitor of new protein synthesis (cycloheximide) blocked this rapid activation, and specific binding of aldosterone to plasma membrane fraction was inhibited by eplerenone, suggesting a nongenomic mechanism. Aldosterone did not affect the mRNA or protein levels of NOX2, which is a major subunit of NADPH oxidase in myocytes, after 48 h. Nuclear staining with DAPI showed that aldosterone (10(-7) mol/L) increased the myocyte apoptosis (2.3 fold, p<0.001), coincident with the activation of caspase-3 (1.4 fold, p<0.05), compared with the serum-deprived control after 48 h. Aldosterone also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1). These effects of aldosterone on myocyte ROS accumulation, ASK1 activation, and apoptosis were abolished by eplerenone, a mineralocorticoid receptor (MR) antagonist, apocynin, an inhibitor of NADPH oxidase activation, and tempol, a free radical scavenger, but by neither RU486, a glucocorticoid receptor antagonist, nor butylated hydroxyanisol (BHA), a mitochondrial ROS scavenger. In conclusion, aldosterone-mediated ROS production is blocked by eplerenone and induced by the nongenomic activation of NADPH oxidase, leading to myocyte apoptosis associated with ASK1 activation. These proapoptotic actions of aldosterone may play a role in the progression of heart failure.

Expression and Functional State of the Corticosteroid Receptors and 11β-Hydroxysteroid Dehydrogenase Type 2 in Schwann Cells

To investigate the role of steroid receptors in mediating the reported effects of steroids on Schwann cell (SC) myelination and growth, we determined mRNA contents and transcriptional activities of the corticosteroid (glucocorticosteroid and mineralocorticosteroid) receptors (GR and MR) and sex steroid (progesterone, androgen, and estrogen alpha and beta) receptors in rat SC cultured under proliferative (in the presence of insulin and forskolin, which induces a high intracellular cAMP content) and quiescent conditions. We found no or very low expression and activity of the sex steroid receptors, as shown by mRNA concentrations determined with real-time PCR and transcriptional activities using transient expression of reporter plasmids in SC. These data and binding studies in SC lines demonstrated that the levels of the sex steroid receptors were the limiting factors. GR was clearly expressed (approximately 8000 sequences/ng total RNA) and functional. No significant modification in GR mRNA levels was observed, but an increase in transcriptional efficiency was recorded in proliferating cells compared with quiescent cells. MR was also significantly expressed at the mRNA level (approximately 450 sequences/ng total RNA) under the two culture conditions. No MR transcriptional activity was observed in SC, but a low specific binding of aldosterone was detected in SC lines. 11 beta-Hydroxysteroid-dehydrogenase type 2 (HSD2), an enzyme that inactivates glucocorticoids, was strongly expressed and active in quiescent SC, although in proliferating cells, HSD2 exhibited a strong decrease in activity and mRNA concentration. These data support a physiological role for HSD2 regulation of glucocorticosteroid concentrations in nerve SC.

Binding of Aldosterone by Epidermal Cytosol in the Tail of Bullfrog Larvae

Characteristics of aldosterone binding by epidermal cytosol in the tail of the larval bullfrog were investigated. Temperature-dependent binding experiments showed that the specific binding of [3H]aldosterone to the tail cytosol was thermolabile. A 3-hr incubation at 0° was the optimum assay condition required for reaching equilibrium. Separation of bound and free hormone was performed using a hydroxylapatite method. Specific binding of aldosterone was observed in the tail epidermis but not in the tail mesenchyme. Saturation analysis revealed that specific binding of [3H]aldosterone to the epidermal cytosol reached maximum between 20 and 40 nM. Scatchard analysis for the cytosol of the tall epidermis yielded a straight line with a dissociation constant (Kd) of 7.0 nM and the maximum number of binding sites (Bmax) of 56.4 fmol/mg protein. Sucrose density gradient analysis of crude cytosol revealed a specific peak of radioactivity in the 8-9 S area. Steroid-binding specificity revealed a significant displacement of the [3H]aldosterone by radioinert aldosterone and, to a lesser extent, by cortisol, whereas 17β-estradiol and testosterone competed very poorly. However, corticosterone and dexamethasone, glucocorticoids in mammals, were better competitors of the aldosterone binding, whereas ZK91587, a selective synthetic antagonist for mineralocorticoid receptor in mammals, scarcely inhibited the aldosterone binding. These results suggest that the aldosterone-binding site may also be a corticosterone receptor and that there may be no other receptors specific for aldosterone at least in the tail epidermis.

Immunohistochemical and biochemical evidence for a cardiovascular mineralocorticoid receptor.

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

Characterization of the rat colonic aldosterone receptor and its activation process.

Aldosterone increases sodium absorption, short circuit current, and transmural potential difference in rat colon. We studied the rat colonic aldosterone receptor using the synthetic glucocorticoid, 11 beta, 17 beta-dihydroxy-17 alpha-propynylandrosta-1,4,6-triene-3-one, to prevent binding to the glucocorticoid receptor. Specific aldosterone binding was found in proximal and distal colon. Heating to 25 degrees C decreased binding within 15 min, but the protease inhibitor, phenylmethylsulfonyl fluoride, stabilized binding. Binding was highest in terminal distal colon. Competitive binding assay showed aldosterone specificity compared to other competitors was greater at 30 than at 4 degrees C, suggesting temperature-sensitive changes in receptor specificity. Scatchard analysis revealed a straight line with a KD of 2.5 nM at 0 degrees C and 4.1 nM at 30 degrees C. Bmax was higher in distal than in proximal colon (30 degrees C, 156 +/- 33 versus 65 +/- 9 fmol/mg protein) and increased by 36% in proximal and 180% in distal colon at 30 degrees C compared to 0 degrees C. DEAE-cellulose chromatography of unactivated receptor demonstrated a single peak eluting at 200-250 mM KCl. Heat, ATP, and gel filtration did not activate the receptor, whereas increasing cytosolic salt concentration to 300 mM KCl, raising the pH to 8, or adding EGTA and EDTA caused increased DNA-cellulose binding and a new peak eluting at 30-80 mM KCl on DEAE-cellulose chromatography. There is a specific aldosterone receptor in colon with increasing number of binding sites from proximal to most distal segments paralleling aldosterone's physiological effects. Absence of receptor activation with heat, gel filtration, or ATP suggests differences between activation of the aldosterone receptor and other steroid hormone receptors.

Aldosterone binding in isolated tubules II. An autoradiographic study of concentration dependency in the rabbit nephron.

Specific binding of aldosterone along the rabbit nephron was investigated using an autoradiographic method on microdissected tubules within a large range of [3H]aldosterone ([3H]A) concentrations (3 X 10(-10) to 2 X 10(-8) M). Paired incubations with [3H]A in the presence or absence of unlabeled A (100-fold excess) were done to determine the specific binding. In addition, competition studies (10 times excess) with A, dexamethasone (Dex), and estradiol (E) were performed. No specific nuclear binding was detected in the proximal convoluted tubule and pars recta. In the cortical collecting tubule (CCT), a high specific labeling occurred at concentrations of A as low as 3 X 10(-10) M that plateaued at 1.5 X 10(-9) M. Aldosterone was the best competitor. In the bright and granular distal tubule, the binding resembled that of CCT, but Dex was nearly as potent a competitor as A. All along the loop of Henle and in the medullary collecting duct, the binding of A was weak at low concentrations and gradually rose with concentration without reaching a plateau. An almost equivalent displacement was obtained by A and Dex in 10-fold excess. We conclude that aldosterone binds mostly to mineralocorticoid sites in the collecting tubule, whereas the binding appears to be to both mineralo- and glucocorticoid sites in the distal tubule, in the different parts of the loop of Henle, and in the medullary collecting tubule.

Inhibition by protease inhibitors of binding of adrenal and sex steroid hormones.

Binding of steroid hormones is inhibited by protease inhibitors and substrates. The protease inhibitors phenylmethyl sulphonylfluoride, tosyl-lysine chloromethyl ketone, and tosylamide-phenylethyl-chloromethyl ketone and the protease substrates tosyl arginine methyl ester and tryptophan methyl ester eliminate specific binding of aldosterone, dexamethasone, dihydrotestosterone, estrogen, and progesterone to their respective receptors. These protease inhibitors and substrates also inhibit binding of progesterone to the 20,000 molecular weight mero-receptor formed from the progesterone receptor in chick oviduct. The binding of estradiol to rat alpha-fetoprotein is inhibited by the protease inhibitors and substrates but not by tryptophan or tryptophan amide, indicating the importance of an ester structure in the inhibition of steroid binding. Our results suggest that all steroid hormone receptors have a site with both common structural features and a role in the regulation of steroid hormone binding.

Aldosterone: Its occurrence, metabolism and binding in the rabbit eye

Aldosterone levels in rabbit aqueous humour and blood plasma were determined by radioimmunoassay and circulating aldosterone was identified radiochemically. The concentration of aldosterone was 22·9±14·2 pg/ml in aqueous and 87·1±26·6 pg/ml in plasma. There was no detectable transformation of aldosterone in the aqueous and very little in the cornea in vitro. In the ciliary body+iris aldosterone was transformed to several metabolites of which only tetrahydroaldosterone could be characterized and this only in small amounts. A small but definitive specific aldosterone binding in the ciliary body+iris was established. The apparent association constant ( K as) was about 10 13 mg of tissue/mol and the concentration of binding sites varied from 0·22 to 0·58 fmol/mg of wet tissue. The binding could be displaced by spirolactone but not by dexamethasone.

Genetic Studies on the Mechanism of Action of Aldosterone in Mice1

Aldosterone reduced the urinary sodium:creatinine ratio and increased the potassium:creatinine ratio in a CBA strain of mice. In the Peru strain, under the same conditions, aldosterone had no detectable effect on the sodium:creatinine ratio but had an even more marked effect on the potassium:creatinine ratio. Thus, the effect of aldosterone on potassium excretion is separable from and is not simply a secondary consequence of the effect on sodium retention. Specific binding of 3H-aldosterone to kidney was reduced in Peru mice compared to CBA. However the correlation between 3H-aldosterone binding and physiological effect of the hormone on sodium excretion disappeared totally in a second generation cross between the strains. A possible explanation is that the nuclear binding of 3H-aldosterone as measured here was heterogeneous. In this same second generation cross, the ratio of bound nuclear 3H-aldosterone extracted by low-ionic strength Tris, to the residue of nuclear 3H-aldosterone resistant to extraction by 0.4M KCl, was strongly correlated to the ratio of the effect of aldosterone on potassium excretion to that on sodium excretion. Thus in these experiments the type of nuclear binding was correlated to the type of physiological effect. These results imply that the specific binding of aldosterone to renal tissue was, in some way at least, related to the mechanism of action of the hormone; and that the heterogeneity of this binding had functional significance in terms of the separable effects of aldosterone on renal sodium retention and potassium excretion in these two strains of mice.

The binding of tritiated aldosterone in the rat liver cytosol.

Physiological target tissues for various steroids generally show a much higher concentration of the appropriate cytoplasmic receptor than do non target tissues. Previously the specific binding of aldosterone has been demonstrated in a. number of tissues—including liver—which are not customarily considered mineralocorticoid target tissues. In the present paper the binding of tritiated aldosterone in liver cytosol from adrenalectomized rats is shown to be exclusively to sites having the affinity characteristics of glucocorticoid receptors. (Endocrinology 94: 575, 1974)

Glucocorticoid and mineralocorticoid receptors for aldosterone

Specific binding of aldosterone and dexamethasone by rat kidney and hepatoma tissue culture (HTC) cell cytosol has been studied. In cytosol of HTC cells, aldosterone and dexamethasone bind to a single class of sites with affinities that correspond with their potencies as inducers of tyrosine aminotransferase. Kidney cytosol, however, contains two classes of specific aldosterone binding sites. The higher affinity sites bind aldosterone with an affinity (equilibrium, dissociation, constant) which is similar to the plasma concentration of aldosterone required for antinatriuresis. The lower affinity aldosterone-binding sites are present at a higher concentration than the higher affinity sites and also bind dexamethasone with a high affinity. We have tentatively identified these two classes of binding sites in renal cytosol as “mineraloeonicoid” and “glucocorticoid” receptors, respectively.

Specific aldosterone binding in rat kidney and parotid

The specific intracellular binding of [ 3H]-aldosterone was studied in tissue slices of kidney and parotid from adrenalectomized rats. Specific aldosterone binding proteins were isolated from (1) cytosol by G-50 Sephadex chromatography, (2) nuclei by an initial osmotic shock procedure (2.2 M sucrose followed by extraction with 0.1 M tris-3 mM CaCl 2 and 50% (NH 4) 2SO 4 precipitation = “soluble nuclear”) and (3) nuclei by a subsequent 04 M KCl-3 mM CaCl 2 extraction and 50% (NH 4) 2SO 4 precipitation (“chromatin bound”). In both tissues, the time course of uptake into the three intracellular compartments was studied by incubation at 25°C for 0–4 h with 5.2 × 10 −9 m [ 3H]-aldosterone. Both kidney and parotid show the same three-step time sequence of specific intracellular binding—first cytosol, then soluble nuclear, and then chromatin bound. The time course and extent of [ 3H]-aldosterone binding in kidney slices was unaffected by concentrations of cycloheximide sufficient to lower protein synthesis by 67%. Cytosol binding proteins in kidney and parotid have an identical affinity for aldosterone but their concentration per g wet weight tissue in the kidney is twice that in the parotid. Despite this difference in cytosol donor concentration, and the presumed identity of active sites, levels of intranuclear [ 3H]-aldosterone-protein complexes are considerably higher in parotid than in kidney (soluble nuclear × 2, chromatin bound × 15).