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11ß-Hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex

^a Department of Anatomy, University of Padua, 35121 Padua, Italy
^b Department of Clinical and Experimental Medicine, University of Padua, 35121 Padua, Italy
^c Department of Histology and Embryology, School of Medicine, 60781 Poznan, Poland

	ABSTRACT

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Although oxidation of cortisol or corticosterone by 11ß-hydroxysteroid dehydrogenase (11ß-HSD) represents the physiological mechanism conferring specificity for aldosterone on the mineralocorticoid receptor in mineralocorticoid target tissues, little attention has been paid until now to the expression and activity of this enzyme in human adrenals. We have shown that human adrenal cortex expresses 11ß-HSD type 2 (11ß-HSD2) gene, and found a marked 11ß-HSD2 activity in microsomal preparations obtained from slices of decapsulated normal human adrenal cortices. Under basal conditions, adrenal slices secreted, in addition to cortisol and corticosterone (B), sizeable amounts of cortisone and 11-dehydrocorticosterone (DH-B), the inactive forms to which the former glucocorticoids are converted by 11ß-HSD. Addition of the 11ß-HSD inhibitor glycyrrhetinic acid elicited a moderate rise in the production of cortisol and B and suppressed that of cortisone and DH-B. ACTH and angiotensin II evoked a marked rise in the secretion of cortisol and B, but unexpectedly depressed the release of cortisone and DH-B. ACTH also lowered the capacity of adrenal slices to convert [³H]cortisol to [³H]cortisone. This last effect of ACTH was concentration-dependently abolished by both aminoglutethimide and cyanoketone, which blocks early steps of steroid synthesis, but not by metyrapone, an inhibitor of 11ß-hydroxylase. Collectively, these findings indicate that the human adrenal cortex possesses an active 11ß-HSD2 engaged in the inactivation of newly formed glucocorticoids. The activity of this enzyme is negatively modulated by the main agonists of glucocorticoid secretion through an indirect mechanism, probably involving the rise in the intra-adrenal concentration of non-11ß-hydroxylated steroid hormones.—Mazzocchi, G., Rossi, G. P., Neri, G., Malendowicz, L. K., Albertin, G., Nussdorfer, G. G. 11ß-Hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex. FASEB J. 12, 1533–1539 (1998)

Key Words: steroidogenesis • cortisone • 11-dehydrocorticosterone • ACTH • angiotensin II • aminoglutethimide • cyanoketone • metyrapone

	INTRODUCTION

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ALTHOUGH THE VARIOUS STEPS of steroidogenesis have been investigated extensively over the past decades (for review, see refs 1–3), little attention has been paid to the possibility of a fine-tuning of active vs. inactive glucocorticoid release in response to the main secretagogues. 11ß-Hydroxysteroid dehydrogenase (11ß-HSD)² catalyzes conversion of the glucocorticoid hormones cortisol and corticosterone (B) into their corresponding inactive forms, cortisone and 11-dehydrocorticosterone (DH-B), respectively (for review, see ref 4). 11ß-HSD is expressed mainly in liver and kidneys, and plays a key role in controlling the access of glucocorticoids to their intracellular receptors and protecting the nonselective mineralocorticoid receptors from glucocorticoid excess. Its congenital deficiency or licorice-induced inhibition are well known to cause arterial hypertension and apparent mineralocorticoid excess in mammals (for review, see refs 5, 6). Two different subtypes of 11ß-HSD are described and named type 1 and type 2 (11ß-HSD1 and 11ß-HSD2). 11ß-HSD1 is an NADP-dependent bidirectional enzyme with low affinity for cortisol and B (micromolar range), whereas 11ß-HSD2 is an NAD-dependent unidirectional enzyme with high affinity for glucocorticoids (nanomolar range) (4).

Evidence is now available that 11ß-HSD, especially in its type 2 isoform, is also contained in the adrenal glands of sheep (7) and rats (8–13). A preliminary report by Whorwood et al. (14) that the human adrenal gland also possesses 11ß-HSD2 activity is available, but a more recent study did not confirm this finding (15).

It therefore seemed worthwhile to investigate the gene expression, activity, and regulation of this enzyme in vitro by using slices and dispersed cells obtained from normal human adrenal cortices.

	MATERIALS AND METHODS

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Reagents
Moloney murine leukemia virus reverse transcriptase (MuLV-RT; Gene Amp RNA PCR core kit) and Taq polymerase (Ampli Taq) were obtained from Perkin-Elmer Cetus (Norwalk, Conn.). Glycyrrhetinic acid, a potent and specific inhibitor of 11ß-HSD activity (16, 17), human serum albumin (HSA), steroid–hormone standards, and other laboratory reagents were purchased from Sigma Chemical Co. (St. Louis, Mo.). Adrenocorticotropic hormone (ACTH, human) and angiotensin II (ANG-II) were obtained from Peninsula Labs (Merseyside, U.K.), medium 199 from DIFCO (Detroit, Mich.), the inhibitors of cholesterol side-chain cleavage aminoglutethimide (Elipten) and 11ß-hydroxylase metyrapone from Ciba (Origgio, Italy), the inhibitor of the 3ß-hydroxysteroid dehydrogenase cyanoketone (WIN 24540) from Sterling-Winthrop (Guilford, U.K.), and [1,2,6,7 ³H]cortisol (SA, 50–80 Ci/nmol) from Amersham Labs (Amersham, U.K.).

Preparation of adrenal specimens
Adrenal glands were obtained from consenting patients undergoing unilateral nephrectomy with ipsilateral adrenalectomy for renal cancer. Starting 2 wk before surgery, patients were kept on a normal diet; only patients not requiring medications able to alter adrenal function were recruited. Portions of the adrenal tail, which does not contain medullary chromaffin cells (18), were collected immediately after excision in the operating room, placed in Krebs-Ringer bicarbonate buffer with 0.2% glucose at 4°C, and immediately carried to our laboratory. Tail fragments were decapsulated to eliminate zona glomerulosa and then cut into slices or used to obtain dispersed adrenocortical cells by collagenase digestion and mechanical disaggregation (19). The contamination of our adrenocortical cell preparations by stromal and vascular elements, as evaluated by phase microscopy, was very low, and the viability of isolated cells, as checked by the trypan blue exclusion test, was higher than 92%. Adiacent sections of the excised adrenals underwent pathologic evaluation and were found to be histologically normal. The study protocol followed the local Ethical Committee guidelines for human studies.

11-ß HSD2 gene expression
Adrenocortical tissue and cells were immediately frozen in liquid nitrogen and stored at -195°C until they were used for nucleic acid extraction. Total RNA was isolated by guanidinium isothiocyanate methods. After isolation, quality of total RNA samples was checked by gel electrophoresis in a 1% agarose gel stained with EtBr. The purity of the extracted RNA was verified by recording the UV absorbance of each sample between 200 and 300 nm wavelength; concentrations of total RNA were then calculated by spectrophotometric measurements at 260 nm wavelength.

For use in the polymerase chain reaction (PCR), total RNA was reversely transcribed to cDNA according to Wang et al. (20) and Trapnell (21). One microgram of total RNA was dissolved in 20 µl of a mixture containing (final concentration) 1 mM of dATP, dGTP and dTTP, dCTP, 1 U/µl of RNAsin, 2.5 µM Random Hexamers, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 5 mM MgCl₂, and 2.5 U of cloned MuLV-RT. After incubation at 42°C for 15 min, the temperature was raised to 99°C for 5 min and then lowered to 5°C for 5 min. For amplification of the resulting cDNA, 10 µl of the reverse transcription (RT) mixture was used. The sample volume was increased to 50 µl with a solution containing 50 mM KCl, 10 mM Tris (pH 8.3), 2 mM MgCl₂, 0.1 µM of up- and downstream primers, and 1 U of Taq polymerase. The thermal profile used in a Delphi 1000 Thermal Cycler (Oracle Biosystems, MJ Research Inc., Watertown, Mass.) included a denaturation step at 94°C for 1 min, annealing at 60°C for 1 min, and an extension step at 72°C for 1 min for a total of 35 cycles. An additional extension step at the temperature of 72°C for 7 min was then carried out. To rule out the possibility of nonspecific amplification, two different pairs of 11ß-HSD-specific primers amplifying different regions of the gene were used: sense HSD11–5'-ACG CAG GCC ACA ATG AAG TAG-3'; and two antisense HSD11–5'-GCT CAC GGA GCC TCC TGT GC-3' and HSD11–1382–5'-GCT CAC GGA GCC TCC TGT GC-3', expected to provide PCR products of 295 and 769 bp, respectively. To rule out the possibility of amplifying genomic DNA (whose amplification products were larger for both sets of primers due to the presence of introns 1 and 1–4, respectively), in some experiments the PCR was carried out with no prior RT of the RNA. Detection of the PCR amplification products was carried out by size fractionation on 2% agarose gel electrophoresis.

11ß-HSD2 activity
Adrenal slices were homogenized at 4°C in 4 vol 100 mM potassium phosphate buffer (pH 7.4), containing 250 mM sucrose. Microsomes were prepared following the method of Mackinnon et al. (22), and 11ß-HSD2 activity was assayed according to Monder et al. (23), with few modifications. Briefly, 100 nCi of [³H]cortisol was added to 50 µg of microsomal protein in the presence of 0.25 mM NAD and 100 mM Tris (pH 8.3). Samples were incubated at 37°C for 60 min in the presence of glycyrrhetinic acid (from 10^-8 to 10^-4 M), 10^-9 M ACTH, or 10^-9 M ANG-II. The reaction was stopped and steroids were extracted with dichloromethane at 4°C, evaporated to dryness, and redissolved in 20 µl methanol. Cortisone was separated and purified by high-pressure liquid chromatography (HPLC) (see below), transferred into scintillation vials, and counted in a Beckman liquid scintillation counter. Results were expressed as percent change of cpm of [³H]cortisone formed per milligram of protein.

Steroid hormone secretion
Adrenal slices were placed in medium 199 and Krebs-Ringer bicarbonate buffer with 0.2% glucose containing 5 mg/ml HSA, and incubated (8–10 mg/ml, in replicates of three each) with 10^-5 M aminoglutethimide, 10^-6 M cyanoketone, 10^-3 M metyrapone, or 10^-5 M glycyrrhetinic acid in the presence or absence of 10^-9 M ACTH. Other slices were incubated with glycyrrhetinic acid in the presence or absence of 10^-9 M ANG-II. Steroids were extracted from the perfusion medium with dichloromethane. The extracts were washed twice with 0.1 N NaOH and 1 ml distilled water, evaporated to dryness under vacuum, and redissolved in 50 µl methanol. The recovery of steroids was 85 ± 8% (SD). Progesterone, cortisol, cortisone, 11-deoxycorticosterone (DOC), DH-B, 18-hydroxycorticosterone, and aldosterone were separated by HPLC and identified by comparison of their retention times with those of the standards ( Fig. 1). Quantification of steroid hormones was based on peak area measurement; the sensitivity of our assay system was 1 pmol/ml; the response of the detector was linear over the range of 1–1000 pmol and directly proportional to the mass of steroid hormone injected (24). Intra- and interassay variation coefficients were 6.0 and 7.5%, respectively.

View larger version (46K): [in this window] [in a new window]   Figure 1. HPLC separation of steroid standards (left panel) and an example of chromatogram of steroid hormones released by human adrenal slices (right panel). Dexamethasone is the internal standard.

[³H]Cortisone production
Other adrenal slices were incubated with 10 nCi/mg of [³H]cortisol in the presence of ACTH (10^-9 M) and/or aminoglutethimide (10^-5 M), cyanoketone (10^-6 M), or metyrapone (10^-3 M). The reaction was stopped; cortisone was extracted from the incubation medium purified by HPLC and transferred into scintillation vials (see above). [³H]Cortisone formed was expressed as cpm/mg of tissue.

In vivo studies
In six consenting Caucasian hypertensive subjects undergoing adrenal vein blood sampling for diagnostic reasons and not administered medications able to alter adrenal function ( Table 1), we measured by quantitative HPLC cortisol and cortisone plasma concentrations in the effluent from both adrenals and in the infrarenal inferior vena cava. The latter was assumed to reflect the steroid concentration in arterial blood entering the adrenal cortex.

Statistics
Data were expressed as means ± SEM of three or six separate experiments, and statistical comparison was performed by analysis of variance, followed by the multiple range test of Duncan.

	RESULTS

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The RT-PCR consistently allowed detection of the 11ß-HSD2 mRNA in all adrenal specimens examined. An example of an EtBr-stained agarose gel is shown in Fig. 2. As can be seen, amplified cDNA fragments of the expected sizes were easily detected from RNA of both normal adrenocortical tissue homogenates (lanes 2 and 8) and dispersed adrenocortical cells (lane 6). No amplification was seen in the control PCR containing no cDNA (water), thereby ruling out the possibility of false positive results.

View larger version (34K): [in this window] [in a new window]   Figure 2. 2% Agarose gel showing cDNA from human adrenocortical tissue homogenates (lanes 2 and 8) and dispersed adrenocortical cells (lane 6) amplified, with two different sets of primers amplifying different regions of the 11ß-HSD gene. An amplification product of the expected size (769 and 295 bp) was consistently obtained with both sets. No amplification with water instead of RNA, as negative control, is shown. Lane 1: DNA size marker VIII (Boehringer-Mannheim, Mannheim, Germany); lanes 3 and 7: amplification of cDNA from normal human kidney, as positive control.

11ß-HSD2 activity was elevated in microsomal preparations obtained from adrenal slices, and glycyrrhetinic acid concentration-dependently suppressed it (maximal effective concentration was 10^-5 M; 98% inhibition). ACTH or ANG-II (10^-9 M) did not affect 11ß-HSD2 activity ( Fig. 3).

View larger version (23K): [in this window] [in a new window]   Figure 3. Effect of glycyrrhetinic acid and ACTH or ANG-II (10-9 M) on 11ß-HSD activity of microsomal preparation from human adrenal slices. Values are means ±SEM of three separate experiments. *P < 0.01 vs. control value (C). Baseline value is about 2000 ± 300 cpm/mg protein.

Under basal conditions, adrenal slices secreted sizeable amounts of progesterone, cortisol, DOC, and B; due to the lack of zona glomerulosa cells, the production of 18-hydroxylated steroids was below the sensitivity of our assay system. Our preparations also released cortisone and DH-B, whose amounts represented 16 and 26% of those of cortisol and B, respectively ( Table 2). Glycyrrhetinic acid (10^-5 M) virtually abolished the production of 11ß-dehydrogenated steroids and also induced significant rises of cortisol (45%) and B (73%) in the supernatant ( Table 2). ACTH and ANG-II (10^-9 M) evoked about 2- and 1.7-fold increases in the production of progesterone, cortisol, DOC, and B by adrenal slices. In contrast, the release of cortisone and DH-B underwent a small decrease (ACTH, 45 and 30%; ANG-II, 40 and 35%, respectively), their amounts being only 4.5–7.0% and 8.3–9.7% of those of cortisol and B, respectively ( Table 2 and Table 3). In the presence of 10^-5 M glycyrrhetinic acid, the effects of ACTH and ANG-II on cortisol and B production were more intense (ACTH, 2.7- and 3.0-fold rise; ANG-II, 2.4- and 2.8-fold rise, respectively) ( Table 3and Table 4).

ACTH (10^-9 M) significantly decreased [³H]cortisone production from [³H]cortisol in adrenal slices. Aminoglutethimide and cyanoketone, but not metyrapone, at a concentration found to block ACTH secretagogue action (data not shown), increased [³H]cortisone production over the respective basal value and abolished the inhibitory effect of ACTH ( Fig. 4).

View larger version (24K): [in this window] [in a new window]   Figure 4. Effects of three inhibitors of steroidogenic enzymes on basal and ACTH-inhibited conversion of [3H]cortisol to [3H]cortisone by human adrenal slices. Values are means ± SEM of three separate experiments. *P < 0.01 vs. the respective baseline value (B); aP < 0.05 and AP < 0.01 vs. the respective control value.

A higher plasma concentration of both cortisol and cortisone was detected in adrenal vein than in infrarenal vena cava blood of all patients in whom adrenal vein sampling was selective, as shown by the fact that most values were scattered far above the identity line ( Fig. 5).

View larger version (27K): [in this window] [in a new window]   Figure 5. Scatter plot of plasma levels (pmol/ml) of cortisol and cortisone in the infrarenal inferior vena cava and right and left adrenal veins in six patients who underwent adrenal vein blood sampling for diagnostic purposes. The fact that most cortisol and cortisone values are well above the identity line indicates the selectivity of blood sampling and the existence of a net cortisone gradient across the adrenals, respectively.

	DISCUSSION

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This study has identified a novel mechanism that may be relevant for the fine-tuning of glucocorticoid secretion and that could provide a pathophysiological explanation to clinical cases of ectopic ACTH syndrome (25–27) associated with pseudohypermineralocorticoidism, possibly caused by the prolonged suppression of 11ß-HSD2 activity.

With the use of specific primers, we have been able to amplify the cDNA of 11ß-HSD2 gene after RT from total RNA by the random hexamers method. Indeed, this approach has consistently allowed amplification of cDNA fragments of expected size for the gene in all specimens of adrenocortical tissue homogenates and dispersed cells tested. The specificity of the amplification product was confirmed by using two different sets of primers amplifying different regions of the 11ß-HSD2 gene and by size identity with amplicons from normal human kidney homogenates, i.e., from a tissue known to express the 11ß-HSD2 gene.

Together, our present findings indicate that the human adrenal cortex not only expresses the gene of 11ß-HSD2, but also possesses 11ß-HSD2 activity and is able to convert cortisol and B to their inactive forms. The glycyrrhetinic acid-induced inhibition of 11ß-HSD activity and the ensuing blockade of cortisol and B metabolism may well account for the rise in the release of these hormones by adrenal slices.

ACTH evokes a marked rise in the release of the entire spectrum of steroid hormones assayed by stimulating the early rate-limiting steps of steroidogenesis, i.e., the conversion of cholesterol to pregnenolone and of pregnenolone to progesterone (for review, see refs 1, 2). ANG-II exerts effects similar to those of ACTH, an expected finding because human inner adrenocortical cells are provided with ANG-II receptors (28). However, despite the increased production of cortisol and B, the release of their 11ß-dehydrogenated inactive counterparts is significantly reduced by the peptide. This finding, coupled with the demonstration that ACTH evokes a sizeable reduction in the conversion of [³H]cortisol to [³H]cortisone by adrenal slices, suggests that ACTH exerts an inhibitory effect on 11ß-HSD2 activity. This contention is in keeping with previous findings obtained in humans and in primary cultures of bovine adrenocortical cells (25, 29).

A direct inhibitory action of ACTH on 11ß-HSD2 activity is unlikely to occur in rat adrenals (11) and human kidney slices (26), and our findings validate this contention not only for ACTH, but also for ANG-II. Our present results appear to elucidate the mechanism underlying this indirect effect of agonists in human adrenal glands. In fact, aminoglutethimide and cyanoketone, by inhibiting the early steps of steroid synthesis, markedly enhance the basal conversion of [³H]cortisol to [³H]cortisone and counteract the inhibitory effect of ACTH, whereas the blockade of 11ß-hydroxylation of steroid hormones (i.e., the conversion of DOC to B and deoxycortisol to cortisol) with metyrapone is ineffective. Evidence indicates that various progesterone derivatives are competitive inhibitors of the 11ß-HSD in vitro (30–32), and glucocorticoids were found to variously affect liver and kidney 11ß-HSD2 activity (10, 26, 33, 34). Hence, it is conceivable that the ACTH-induced abrupt rise in the intra-adrenal concentration of non-11ß-hydroxylated steroid hormones may depress 11ß-HSD2 activity and, consequently, lower the local inactivation of glucocorticoids. Obviously, our study does not exclude either a role of 11ß-HSD1 in the human adrenals or the possibility that the up-regulation of this enzyme may concur to the inhibitory effect of ACTH on intra-adrenal glucocorticoid metabolism.

To further investigate whether 11ß-HSD activity is present in vivo in human adrenals, we measured cortisol and its 11ß-dehydrogenated inactive metabolite in adrenal vein blood. According to earlier studies (35, 36), we found sizeable levels of cortisol in adrenal venous effluent. Blood from both adrenal veins had a 2.5- to 7.5-fold higher level of cortisone as compared to the infrarenal inferior vena cava, i.e., the arterial blood entering the adrenal cortex. Thus, these results strongly suggest that cortisone is generated from cortisol within the human adrenal cortex in vivo.

Our investigation allows us to draw the following conclusions: 1) human adrenal cortex expresses 11ß-HSD2 gene and possesses 11ß-HSD2 activity; 2) 11ß-HSD2 is engaged in the local inactivation of newly formed glucocorticoids, and 3) the activity of 11ß-HSD2 is negatively regulated by the intra-adrenal concentration of non-11ß-hydroxylated steroid hormones. The extent to which the ACTH-induced indirect inhibition of intra-adrenal 11ß-HSD2 activity may concur, under physiological conditions, to the glucocorticoid secretagogue action of this agonist remains to be established.

	ACKNOWLEDGMENTS

 
This study was made possible by the precious collaboration of all our Colleagues of the Departments of Urology (directed by Prof. Francesco Pagano), whom we gratefully acknowledge.

	FOOTNOTES

 
¹ Correspondence: Department of Anatomy, Via Gabelli 65, I-35121 Padova, ltaly. E-mail: ggnanat{at}ipdunidx.unipd.it

² Abbreviations: ACTH, adrenocorticotropic hormone; ANG-II, angiotensin II; B, corticosterone; DH-B, 11-dehydrocorticosterone; DOC, 11-deoxycorticosterone; HPLC, high-pressure liquid chromatography; HSA, human serum albumin; MuLV-RT, Moloney murine leukemia virus reverse transcriptase; PCR, polymerase chain reaction; RT, reverse transcription; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase.

Received for publication January 19, 1998. Revision received July 24, 1998.

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