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(Hypertension. 2000;35:1099.)
© 2000 American Heart Association, Inc.

Scientific Contributions

Role of 11ß-Hydroxysteroid Dehydrogenase in Nongenomic Aldosterone Effects in Human Arteries

Rodrigo Alzamora; Luis Michea; Elisa T. Marusic

From the Laboratory of Cellular and Molecular Physiology, Faculty of Medicine, University of Los Andes, Las Condes, Santiago, Chile.

Correspondence to Dr Elisa T. Marusic, Laboratory of Cellular and Molecular Physiology, Faculty of Medicine, University of Los Andes, San Carlos Apoquindo 2200, Las Condes, Santiago 6782468, Chile. E-mail emarusic{at}uandes.cl

	Abstract

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Abstract—The aim of the present study was to demonstrate rapid effects of aldosterone on the Na⁺-H⁺ exchanger in strips of human vascular vessels and to determine whether 11ß-hydroxysteroid dehydrogenase enzyme (11ß-HSD) could play a protective role in this response, such as that described for the classic type I mineralocorticoid receptor (MR). The activity of 11ß-HSD isoforms 1 and 2 were measured in fetal and adult arteries. Both isoforms are present in adult and fetal vessels. However, a significant difference in the proportion of each isoform was found. Isoform 1 activity (in pmol · min^-1 · 100 mg^-1 protein) was 42±5 in fetal vessels and 29±2 in adult arteries, and isoform 2 activity was 78±7 in fetal and 12±2 in adult tissue. The nongenomic effect of aldosterone on Na⁺-H⁺ exchanger activity was measured in strips of chorionic and radial uterine arteries loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. Recordings of intracellular pH (pH_i) were made by videofluorescence microscopy. Aldosterone (0.5 nmol/L) rapidly increased pH_i, with a half-maximal effect between 2 and 3 nmol/L in both fetal and adult vessels. Ethylisopropylamiloride, a specific inhibitor of the Na⁺-H⁺ exchanger, inhibited this effect. The hormone-mediated increase in pH_i was unaffected by spironolactone, a classic antagonist of MR, but was completely blocked by RU28318. Cortisol (up to 1 µmol/L) had no effect on pH_i, but when applied in the presence of carbenoxolone, a dramatic increase in Na⁺-H⁺ exchanger activity was evident. The increments on pH_i for each cortisol concentration were similar to those observed for aldosterone. These findings suggest that vascular 11ß-HSD plays an active role in maintaining the specificity of the rapid effects of aldosterone.

Key Words: nongenomic • human • muscle, smooth, vascular • sodium-hydrogen antiporter • aldosterone • 11ß-hydroxysteroid dehydrogenase • cortisol

	Introduction

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Blood vessels are target organs for glucocorticoids and mineralocorticoids and are essential for the maintenance of vascular tone and potentiation of vascular response to a number of pressor hormones.^{1 2} The concentration of cortisol, the physiological glucocorticoid in human, exceeds the circulating levels of the mineralocorticoid aldosterone by 1000-fold. The mineralocorticoid target tissues metabolize glucocorticoids, but not aldosterone, to less active compounds by the enzyme 11ß-hydroxysteroid dehydrogenase (11ß-HSD),^{3 4} thus protecting the cytosolic mineralocorticoid receptor (MR). Considering that human type I MR has equal binding affinity for cortisol and aldosterone,⁵ the inhibition of 11ß-HSD may result in classic aldosterone actions by flooding MR with cortisol.

Several recent publications have described defective 11ß-HSD enzyme activity in hypertension,^{6 7 8 9} and inhibition of the enzyme may cause hypertension.^{10 11} Inhibitory compounds include glycyrrhizic and glycyrrhetinic acid, found in licorice and chewing tobacco, and carbenoxolone, a derivative of the above acids.⁹ Inhibition of 11ß-HSD may result in activation of MRs by cortisol in the vascular tissue, eliciting genomic and/or nongenomic (rapid) responses similar to those observed in the kidney.⁹

Recently, rapid effects of aldosterone on intracellular electrolyte concentration, cell volume,¹² protein kinase C activity,¹³ and potassium channels have been described.¹⁴ In vascular smooth muscle cells, aldosterone mediates a rapid increase of Na⁺-H⁺ exchanger activity^{15 16} and increases inositol triphosphate production¹⁵ and intracellular calcium concentration.¹⁷ Similar effects have been reported in human mononuclear leukocytes,^{18 19} endothelial cells,¹⁹ isolated colonic crypts,^{13 20} and kidney cells.²¹

In the study described here, we examined the involvement of 11ß-HSD in the nongenomic effects of aldosterone in intact segments of human vascular tissue, using Na⁺-H⁺ exchanger activity as a marker of rapid aldosterone action. The results indicate that 11ß-HSD is involved in the rapid activation of the Na⁺-H⁺ exchanger by aldosterone and suggest a regulatory role of the enzyme in vascular tissue.

	Methods

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Uterine and Chorionic Vessels
Radial branches of uterine artery were obtained immediately after surgical hysterectomy. Chorionic arteries were obtained from healthy women after elective cesarean section at term (38.5 to 41 weeks, normal pregnancies). All tissues were rapidly placed on ice, and tissue processing began within half an hour after surgery. Tissues were carefully dissected to isolate blood vessels from surrounding fat and connective tissue. Vessels (8x2 mm) were split longitudinally, and endothelium was gently removed. The Ethics Committee of the Faculty of Medicine approved the protocols, and informed consent was obtained.

11ß-HSD Activity Assay
11-Dehydrogenase activity of both 11ß-HSD isoforms was determined by measuring the rate of conversion of corticosterone (B) to 11-dehydrocorticosterone (A) as described by Stewart et al.²² In brief, vessels were homogenized in ice-cold KCl buffer (0.154 mol/L, pH 7.6) using a Polytron homogenizer (Kinematica). Homogenates were centrifuged for 10 minutes at 1000g, and protein concentration in the supernatant was determined (Bradford, Bio-Rad). Homogenates (0.5 mg protein/mL) were incubated in 0.5 mL of phosphate buffer (0.1 mol/L, pH 7.6), which contained 50 000 cpm of 1,2,6,7-[³H]corticosterone (specific activity 88 Ci/mmol; DuPont-New England Nuclear), 0.1 or 2.5 µmol/L B, and 400 µmol/L oxidized nicotinamide adenine dinucleotide (NAD⁺) or nicotinamide adenine dinucleotide phosphate (NADP⁺), for 20 minutes at 37°C. On the basis of previous studies, assays with 0.1 µmol/L B and NAD⁺ were designed to detect 11ß-HSD2 activity and assays with 2.5 µmol/L B with NADP⁺, to detect 11ß-HSD1. Aliquots were extracted into 1:10 chloroform (vol/vol), and steroids were separated by thin-layer chromatography using acetone/chloroform (18:82) as a mobile phase. Areas corresponding to steroids were identified under UV light and scraped off, and radioactivity was counted in a liquid scintillation analyzer (Packard Instrument Co). Activity was expressed as picomoles of product per minute per 100 mg of protein for each tissue.

Fluorometric Determination of Intracellular pH
pH_i was determined by monitoring the fluorescence of acetoxymethyl ester of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM; Texas Fluorescence Laboratories) as described by Foster et al.²³ Chorionic and uterine radial arteries were prepared in oxygenated physiological salt solution (PSS) containing (in mmol/L) 140 NaCl, 4 KCl, 10 d-glucose, 20 HEPES, 1.8 CaCl₂, and 1.0 MgCl₂, adjusted to pH 7.35 to 7.40. Vascular strips were incubated for 1.5 hours with 10 µmol/L BCECF-AM in HEPES-PSS with gentle agitation at 35°C. After the dye was loaded, tissue segments were mounted in a thermostatically controlled 2-mL chamber slide. Temperature in the chamber was maintained at 37°C (thermoregulated platform, FryerCo). At the start of each experiment, tissues were perfused with HEPES-PSS for 40 minutes to remove any extracellular dye (flow rate 5 mL/min). Fluorescent measurements were made with a dual-excitation wavelength imaging system using an Eclipse E400 epifluorescence microscope with a Fluor x10 water immersion objective (Nikon) attached to an optical filter changer (Lambda 10-2, Sutter Instrument Co). Emitted fluorescence was acquired by an Intensified CCD video camera (IC-100, Photon Technology International) and processed by scientific imaging software (IPLab Spectrum, Scanalytics). Wavelengths for excitation were 495 and 440 nm; emission wavelength was 530 nm. The ratio of the 495/440-nm fluorescence values was used to estimate pH_i. At the end of each experiment, tissue fluorescence values were calibrated to pH_i using the nigericin (K⁺-H⁺ ionophore) high-K⁺ protocol.²⁴

Materials
Aldosterone, cortisol, spironolactone, ethylisopropylamiloride (EIPA), amiloride, and nigericin were purchased from Sigma Chemical Co; BCECF-AM, from Texas Fluorescence Laboratories; and 1,2,6,7[³H]corticosterone, from DuPont-NEN. Stock solutions of hormones and BCECF-AM were prepared in DMSO and diluted before use in the respective buffer; final concentration of DMSO was 0.1%. Solvent controls were run in each condition.

Statistical Analysis
All values are reported as mean±SEM with the number of observations in parentheses. Differences between mean values were assessed by Student’s t test (unpaired).

	Results

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11ß-HSD Activity in Fetal and Adult Vascular Tissues
The response to aldosterone in mineralocorticoid target tissues depends not only on the number of receptors and the circulating hormone concentration but also on the 11ß-HSD oxidative and reductive activities.^{3 4 9} Therefore, we measured 11ß-HSD1 and 11ß-HSD2 in whole homogenates of human vascular tissue. Activities of both isoforms (pmol · min^-1 · 100 mg^-1 protein) were present in adult uterine arteries (28.8±2.0 for isoform 1 and 12.4±2.0 for isoform 2, n=7). Activities were significantly higher in fetal chorionic vessels (42.0±5.3 for isoform 1 and 77.9±7.2 for isoform 2; n=12), as shown in Figure 1. The 11ß-HSD1/11ß-HSD2 ratio was 2.4±0.3 and 0.5±0.1 for adult and fetal arteries, respectively.

View larger version (13K): [in this window] [in a new window]   Figure 1. Activity of 11ß-HSD isoforms in whole homogenates of human uterine radial arteries (adult, n=7) and chorionic vessels (fetal, n=12) obtained at term by elective cesarean. 11ß-HSD1 was measured in the presence of NADP+ (black bars) and 11ß-HSD2 with NAD+ as cofactor (white bars), as indicated in Methods. Values are mean±SEM of triplicate experiments. **P<0.01, *P<0.05.

Rapid Aldosterone Effect on Na⁺-H⁺ Exchanger in Segments of Vascular Smooth Muscle
The mean pH_i of strips of radial uterine arteries calculated from BCECF fluorescence was 7.17±0.02 (n=7) in HEPES-PSS (pH 7.35 to 7.40), and that of chorionic arteries was 7.18±0.01 (n=12). Perfusion with 10 nmol/L aldosterone produced rapid alkalinization in both tissues (Figure 2). The increment in pH_i was 0.17±0.01 U in uterine arteries and 0.16±0.01 U in fetal vessels (P<0.001, n=20). When amiloride or the analog EIPA was added 10 minutes before aldosterone, the effect of the hormone was completely inhibited. Furthermore, addition of amiloride or EIPA at maximal alkalinization rapidly reversed the aldosterone-mediated increment in pH_i, as shown in Figure 2. Thus, the inhibitory effect of amiloride derivatives demonstrates that aldosterone alkalinization is due to activation of the Na⁺-H⁺ exchanger, as shown in vascular smooth muscle cells of rats.^{15 16}

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of aldosterone on Na+-H+ exchanger. Recording of pHi in uterine (A) and chorionic (B) vessels by epifluorescence videomicroscopy. Segments were equilibrated for 40 minutes at 37°C with 20 mmol/L PSS-HEPES, pH 7.35 to 7.40, as indicated in Methods, and perfused with 10 nmol/L aldosterone. In both tissues, a Na+-H+ exchanger inhibitor (amiloride or EIPA) was added to a parallel sample after the maximal aldosterone-mediated increment in pHi was obtained. Representative tracings from 3 chorionic and 4 uterine arteries.

Dose-response curves for aldosterone in adult and fetal tissues are shown in Figure 3. Aldosterone concentrations of 0.5 nmol/L cause significant increments of pH_i in intact tissue, 0.05±0.01 U in uterine arteries and 0.03±0.01 U in fetal vessels (P<0.05, n=6). The maximal effect was evident at 10 nmol/L aldosterone. There were no significant differences in dose-response curves between fetal and adult human tissues, which showed half-maximal effect at 2 and 3 nmol/L aldosterone, respectively.

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of aldosterone concentration on pHi in vascular strips. Recording of pHi as indicated in Figure 2. Where indicated, perfusing buffer (PSS) was exchanged with PSS solution containing increasing concentration of aldosterone. Tracings are representative of 6 experiments with similar results. A, Chorionic vessels; B, uterine arteries.

Effect of Aldosterone Antagonists
Spironolactone (10 µmol/L), the classic mineralocorticoid antagonist, did not block the effect of 10 nmol/L aldosterone (1000-fold lower concentration) when added 10 minutes before aldosterone and maintained throughout the study (Figure 4A). Mean pH_i in the presence of both aldosterone and spironolactone was 7.29±0.02 versus 7.32±0.02 in the absence of the antagonist (NS, n=4). These results indicate that spironolactone had minimal or no effect on rapid aldosterone-mediated alkalinization.

View larger version (20K): [in this window] [in a new window]   Figure 4. Effect of aldosterone antagonist on hormone-mediated increment in Na+-H+ exchanger activity. Spironolactone or RU28318 was added to the perfusion medium 10 minutes before aldosterone and was maintained throughout the experiment. Parallel samples were run in the presence of aldosterone alone. Determinations of pHi were made in chorionic arteries as indicated in Methods (n=4 experiments for each antagonist).

A series of RU compounds have been used as antagonists of the glucocorticoid receptor (GR; RU28362) or the MR (RU26752). A molecule closely related to RU26752, such as RU28318, the potassium salt of the free acid form of this compound, is a weak competitor for cytosolic GR or MR.^{25 26} We tested the effect of RU28318 on the rapid aldosterone-mediated stimulation of Na⁺-H⁺ exchanger activity. As shown in Figure 4B, RU28318 at 10 µmol/L completely blocked the alkalinizing effect of 10 nmol/L aldosterone when given 10 minutes before stimulation with the hormone. The inhibitory effect was observed in both uterine and chorionic arteries.

Effect of Cortisol on Na⁺-H⁺ Exchanger in Vascular Tissue
Cortisol had no effect on pH_i at doses up to 1 µmol/L. However, when 11ß-HSD was inhibited by carbenoxolone, cortisol was as effective as aldosterone in raising pH_i (Figure 5A and 5B). Similar results were observed in fetal and adult vessels. Cortisol (0.5 nmol/L) caused an increase in pH_i of 0.04 U in both tissues (n=3). A maximal effect was observed with 10 nmol cortisol, 0.17±0.01 U for uterine arteries and 0.16±0.01 for fetal arteries (P<0.01, n=10). Amiloride or EIPA suppressed cortisol enhancement of pH_i when added to the incubation medium 10 minutes before the hormone. Addition of EIPA after maximal cortisol stimulation induced a rapid decrease of pH_i (Figure 5A). Finally, the rapid effect of cortisol on Na⁺-H⁺ exchanger activity in the presence of carbenoxolone was suppressed by 10 µmol/L RU28318, as shown in Figure 5C.

View larger version (17K): [in this window] [in a new window]   Figure 5. Cortisol effect on Na+-H+ exchanger in human radial uterine artery. A, Representative tracing demonstrating that cortisol by itself had no effect on pHi (n=10). However, in the presence of carbenoxolone, a rapid increment in pHi was observed. The effect was blocked by EIPA. B, Representative tracing of a dose-response curve for cortisol in the presence of carbenoxolone (n=3). C, Representative tracing demonstrating blockage of cortisol effect by RU28318 (n=4).

	Discussion

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In this study, we determined Na⁺-H⁺ exchanger activity as a measure of nongenomic hormonal effects. The exchanger has an essential role in vascular tone and blood pressure regulation. Indeed, increased Na⁺-H⁺ exchanger activity is one of the most consistent defects present in both human essential hypertension^{27 28} and in experimental rat models.^{29 30} Moreover, rapid in vitro effects of aldosterone on Na⁺-H⁺ exchanger activity have been described in vascular smooth muscle cell lines,^{15 16 19} human mononuclear leukocytes,¹⁹ colon,^{14 20} and kidney.^{19 21} Consistent with these data, we demonstrated aldosterone-mediated activation of the Na⁺-H⁺ exchanger at physiological hormone concentrations in human strips of uterine and chorionic arteries, which suggests a physiological role in vivo.

Interestingly, cortisol by itself was not able to modify exchanger activity even when its concentration was raised to 10 times that of normal plasma levels. However, in the presence of carbenoxolone, cortisol activation of the Na⁺-H⁺ exchanger closely resembled that of aldosterone, indicating a protective role of 11ß-HSD on aldosterone rapid effects.

It is known that under in vitro conditions, cortisol and aldosterone have similar affinities for type I MR.⁵ However, despite higher circulating levels in vivo, cortisol is prevented from binding to the type I receptor in mineralocorticoid target tissues by 11ß-HSD. This enzyme is responsible for the conversion of active glucocorticoids to their inactive 11-keto metabolites.^{3 4 9} In humans, two isoforms of 11ß-HSD (1 and 2) have been cloned and kinetically characterized.⁹ Type 1 is NADP⁺(H)-preferring and has been shown to have both dehydrogenase and reductase activity. In contrast, 11ß-HSD2 has been identified only in a limited range of tissues; it has a high affinity for cortisol, is NAD⁺-dependent, and appears to show only dehydrogenase activity. Immunohistochemical studies have demonstrated colocalization of 11ß-HSD2 with the cytosolic MR.^{31 32} The findings of the present study indicate that both isoforms are active in human adult and fetal vessels.

Previous studies in experimental animals, and our present results in human vessels, confirm that activity of 11ß-HSD2 is low in blood vessels as compared with renal tissue.^{6 33} Nevertheless, as indicated by our results, a putative protective action on MR is present in the vascular tissue. The protective role of 11ß-HSD on the nongenomic effects of aldosterone has not been described previously. The clinical implications of this finding are clear, because several recent publications have claimed defective 11ß-HSD activity in hypertension.^{6 7 8 9} Also, decreased 11ß-HSD activity in Cushing’s syndrome has been described,³⁴ which may in part explain the hypertension observed in these patients. Also, congenital 11ß-HSD deficiency has been related to hypertension.^{6 8 9} Therefore, this enzyme could modulate the access of glucocorticoids to vascular receptors and influence vascular tone.

Although few data exist on the pharmacology of human corticosteroid receptors,³⁵ human studies indicate that after agonist exposure, both the human MR and GR display nuclear localization.⁵ The same is true for spironolactone-MR complexes, which also show an exclusive nuclear localization indistinguishable from that of agonist-receptor complexes.⁵ This fact supports the idea of an antagonistic interference with transcriptional activation at later steps, possibly by inducing a different structure of the ligand-binding domain.^{36 37} Our results with spironolactone indicate that there is no involvement of the nuclear receptors in the rapid effect of aldosterone. This conclusion is in agreement with the data of Wehling et al,¹⁷ who found that a 1000-fold higher concentration of this mineralocorticoid antagonist did not block the aldosterone effect on the Na⁺-H⁺ exchanger. Canrenone, a highly water-soluble mineralocorticoid antagonist, also failed to block rapid aldosterone effects.^{18 19} However, RU28318, a weak competitor for either cytosolic MR or GR,²⁵ completely blocked the rapid effects of aldosterone.

Little is known about the mechanism of action of RU28318. Other compounds of the RU series indicate that RU-receptor complexes show exclusive nuclear localization.⁵ Its binding affinity for classic human MR is 11.5 nmol/L, and the k_I (inhibition constant) of spironolactone is 5.7 nmol/L. The present finding of the inhibitory effect of RU28318 on the rapid effects of aldosterone provides a new tool to study the nongenomic action of corticosteroids.

In agreement with the present data, Li et al³⁸ recently showed that neither spironolactone nor RU486 modified adrenocorticotrophin-induced hypertension in rats despite demonstrable antimineralocorticoid and antiglucocorticoid actions. Ectopic adrenocorticotropic hormone (ACTH) syndrome is characterized by defective 11ß-HSD activity.³⁹ Moreover, exogenous ACTH infusion in healthy humans indicates that ACTH inhibits 11ß-HSD activity. Together, the above observations and present results indicate an important role of vascular 11ß-HSD in the pathophysiology of hypertension.

In summary, we showed that the rapid effects of cortisol on the human vascular Na⁺-H⁺ exchanger could be modulated by the protective action of 11ß-HSD. The present results support the idea that 11ß-HSD is a key enzyme not only for the classic genomic effects of aldosterone but also for rapid actions of the hormone.

	Acknowledgments

 
This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) grant 197-0696. We thank Dr Alejandro de Nicola, Laboratory of Neuroendocrine Biochemistry, Institute of Experimental Biology and Medicine, University of Buenos Aires, Argentina, for kindly providing RU28318.

Received October 15, 1999; first decision November 27, 1999; accepted December 16, 1999.

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