Quantitative real-time PCR for the measurement of 11beta-HSD1 and 11beta-HSD2 mRNA levels in tissues of healthy dogs.

The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) exists in two isoforms, 11beta-HSD1 and 11beta-HSD2. 11beta-HSD1 generates active cortisol from cortisone and appears to be involved in insulin resistant states. 11beta-HSD2 protects the mineralocorticoid receptor from inappropriate activation by glucocorticoids and is important to prevent sodium retention and hypertension. The purposes of the present study were to develop two real-time PCR assays to assess 11beta-HSD1 and 11beta-HSD2 mRNA expression and to evaluate the tissue distribution of the two isoforms in dogs. Thirteen different tissues of 10 healthy dogs were evaluated. Both real-time PCR assays were highly specific, sensitive and reproducible. Highest 11beta-HSD1 mRNA expression was seen in liver, lung, and renal medulla; highest 11beta-HSD2 mRNA expression in renal cortex, adrenal gland, and renal medulla. Higher 11beta-HSD1 than 11beta-HSD2 mRNA levels were found in all tissues except adrenal gland, colon, and rectum. Our results demonstrate that the basic tissue distribution of 11beta-HSD1 and 11beta-HSD2 in dogs corresponds to that in humans and rodents. In a next step 11beta-HSD1 and 11beta-HSD2 expression should be assessed in diseases like obesity, hypercortisolism, and hypertension to improve our knowledge about 11beta-HSD activity, to evaluate the dog as a model for humans and to potentially find new therapeutic options.     

Characterisation of 11 beta-hydroxysteroid dehydrogenases in feline kidney and liver

11 Beta-hydroxysteroid dehydrogenases type 1 and 2 (11 beta-HSD1 and 11 beta-HSD2) are microsomal enzymes responsible for the interconversion of cortisol into the inactive form cortisone and vice versa. 11 beta-HSD1 is mainly present in the liver, and has predominantly reductase activity although its function has not yet been elucidated. 11 beta-HSD2, present in mineralocorticoid target tissues such as the kidney, converts cortisol into cortisone. Reduced activity due to inhibition or mutations of 11 beta-HSD2 leads to hypertension and hypokalemia resulting in the Apparent Mineralocorticoid Excess Syndrome (AMES). Like humans, cats are highly susceptible for hypertension. As large species differences exist with respect to the kinetic parameters (K(m) and V(max)) and amino acid sequences of both enzymes, we determined these characteristics in the cat. Both enzyme types were found in the kidneys. 11 beta-HSD1 in the feline kidney showed bidirectional activity with predominantly dehydrogenase activity (dehydrogenase: K(m) 1959+/-797 nM, V(max) 766+/-88 pmol/mg*min; reductase: K(m) 778+/-136 nM, V(max) 112+/-4 pmol/mg*min). 11 beta-HSD2 represents a unidirectional dehydrogenase with a higher substrate affinity (K(m) 184+/-24 nM, V(max) 74+/-3 pmol/mg*min). In the liver, only 11 beta-HSD1 is detected exerting reductase activity (K(m) 10462 nM, V(max) 840 pmol/mg*min). Sequence analysis of conserved parts of 11 beta-HSD1 and 11 beta-HSD2 revealed the highest homology of the feline enzymes with the correspondent enzymes found in man. This suggests that the cat may serve as a suitable model species for studies directed to the pathogenesis and treatment of human diseases like AMES and hypertension.     

11β—羟基类固醇脱氢酶

现已发现两型１１β－羟基因固醇脱氢酶（１１β－ＨＳＤ）：１１β－ＨＳＤ１为氧化还原酶,催化皮质醇与其代谢产物之间的相互转化;１１β－ＨＳＤ２则为专一氧化酶,只催化皮质醇的失活。１１β－ＨＳＤ１在体内分布广泛,功能目前沿不清楚。１１β－ＨＳＤ２主要存在于盐皮质激素靶器官,肾脏１１β－ＨＳＤ２通过降解糖皮质激素保护盐皮质激素受体的特异性,肾脏此酶的缺乏,可以导致严重高血压。胎盘１１β－ＨＳＤ２通过降解     

Molecular basis for hypertension in the "type II variant" of apparent mineralocorticoid excess.

The syndrome of apparent mineralocorticoid excess (AME) is a heritable form of hypertension in which cortisol acts as a potent mineralocorticoid. The type I variant results in a severe clinical and biochemical phenotype and arises because of mutations in the gene encoding the type 2 isozyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD2), an enzyme responsible for the peripheral inactivation of cortisol to cortisone. Only mild abnormalities of cortisol metabolism have been found in the type II variant of AME, suggesting that it may be a separate gene defect. In an extensive consanguineous Sardinian pedigree affected with "type II" AME, a novel homozygous point mutation (C945T) was found in the human 11beta-HSD2 gene in four affected individuals. Thirteen family members were heterozygous for the resultant R279C amino acid substitution. The LOD score of linkage of the mutation to the disease was 3.23. Expression of the 11beta-HSD2 mutant cDNA resulted in an enzyme with reduced maximum velocity, but similar substrate affinity, compared with activity of the wild-type cDNA. Affected individuals were >30 years of age and had both mineralocorticoid hypertension and evidence of impaired metabolism of cortisol to cortisone. The heterozygote state was phenotypically normal but was associated with subtle defects in cortisol metabolism. AME represents a spectrum of mineralocorticoid hypertension with severity reflecting the underlying genetic defect in the 11beta-HSD2 gene; classification into distinct subtypes is inappropriate. Hypertensive populations should be screened to identify the prevalence of milder defects in 11beta-HSD2 in patients currently labeled as having "essential" hypertension.     

Mechanism of dissociation of cortisol and adrenal androgen secretion after removal of adrenocortical adenoma in patients with Cushing's syndrome

We investigated the mechanism of dissociation of cortisol and dehydroepiandrosterone sulfate (DHEA-S) secretion by the adrenal glands after the removal of an adrenal gland containing an adrenocortical adenoma in a patient with Cushing's syndrome. After removal of the adrenocortical adenoma, the serum cortisol rapidly decreased from 24.6 +/- 6.4 micrograms/dl (mean +/- SD, n = 6) to 0.7 +/- 0.5 micrograms/dl. Serum DHEA-S levels were 15 +/- 14 micrograms/dl and 6 +/- 9 micrograms/dl before and after surgery, respectively, and significantly lower than the control values. Serum cortisol levels reverted to normal levels 1.5 to 3 years after the surgery. On the other hand, DHEA-S levels reverted to normal 5 to 7 years after the serum cortisol levels had normalized. Monolayer cultures of normal human adrenal cells obtained at adrenalectomy in patients with advanced breast cancer and atrophic adrenal cells adjacent to the adrenocortical adenoma in patients with Cushing's syndrome were used to study the mechanism of the dissociation of cortisol and DHEA-S secretion. ACTH caused significant increases in the productions of pregnenolone (P5), progesterone (P4), 17-hydroxypregnenolone (17-OH-P5), 17-hydroxyprogesterone (17-OH-P4), DHEA, DHEA-S, androstenedione (delta 4-A), and cortisol. The amounts of 17-OH-P5 and 17-OH-P4 produced by ACTH in atrophic adrenal cells were significantly greater than those in normal adrenal cells. The amounts of DHEA, DHEA-S and delta 4-A produced by ACTH in atrophic adrenal cells were significantly smaller than those of normal adrenal cells. The conversion rate of 17-OH-[3H]P5 to 17-OH-[3H]P4 and 11-deoxy-[3H] cortisol was higher in atrophic adrenal cells than in normal adrenal cells, but the conversion rate to [3H]DHEA, [3H]DHEA-S and [3H]delta 4-A was significantly lower in atrophic adrenal cells than in normal adrenal cells. These results suggest that the dissociation of cortisol from DHEA-S after the removal of adrenocortical adenoma is a probably due to diminished C17,20-lyase activity in the remaining atrophic adrenal gland.     

19-Noraldosterone in pregnancy-induced hypertension

Pregnancy-induced hypertension (PIH) is a frequent cause of maternal and neonatal morbidity and mortality. 19-Noraldosterone, which was shown to be synthesized in the human adrenal gland, exhibits potent mineralocorticoid and hypertensive activity. To examine the role of mineralocorticoids in the pathophysiology of PIH, we studied urinary 19-noraldosterone, tetrahydroaldosterone, free cortisol, and cortisone concentrations and mineralocorticoid receptor levels in peripheral blood mononuclear leukocytes, from 17 women with PIH and 16 normal pregnant women as controls. Sequence analysis of the mineralocorticoid receptor gene in PIH patients was also done. The 24-h urinary excretion of 19-noraldosterone was significantly lower in PIH (120 +/- 38 pmol/day) than in controls (358 +/- 55 pmol/day) (P < 0.05). Urinary tetrahydroaldosterone was also decreased in PIH compared with controls. Ratios of urinary free cortisol to cortisone (a measure of 11beta-hydroxysteroid dehydrogenase 2 activity) did not differ significantly between groups. Mineralocorticoid receptor density was significantly (P < 0.05) decreased in the PIH group (133 +/- 15 binding sites/cell) compared with controls (255 +/- 21 binding sites/cell). No mutations were found in the coding region of the mineralocorticoid receptor gene in PIH. These results suggest that circulating aldosterone, 19-noraldosterone, and renal 11beta-hydroxysteroid dehydrogenase2 do not contribute to the pathogenesis of PIH. Regulatory factors that cause the down-regulation of the mineralocorticoid receptor in PIH should be clarified.     

Modulation of cortisol metabolism during treatment of acromegaly is independent of body composition and insulin sensitivity

OBJECTIVES: The set point of cortisol-cortisone conversion is shifted in the direction of cortisone by the inhibition of the activity of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) during adult GH replacement and in active acromegaly. Additionally, both fat mass and insulin may modulate 11beta-HSD1 and are both influenced by changes in GH status. This study examined the relative direct contribution of GH/IGF1 in modulating cortisol metabolism. METHODS: Overall cortisol/cortisone conversion (ratio of urine 11-hydroxy-/11-oxo-cortisol metabolites; Fm/Em), insulin sensitivity (homeostatic model assessment; HOMA %S) and fat mass (DXA) were examined in parallel in 6 patients (mean age 53 years, range 42-76; 4 males, 2 females) with previously untreated active acromegaly during 6 months of therapy with Sandostatin LAR (20-30 mg i.m. 4 weekly). All but 1 patient had normal ACTH reserve. RESULTS: At baseline, Pearson correlation demonstrated an inverse relationship between serum GH (mean of a 5-point day curve) and Fm/Em (r = -0.83, p = 0.04) and a trend towards an inverse relationship between HOMA %S and Fm/Em (r = -0.79, p = 0.06) but no other patterns were evident. During the course of treatment, serum GH decreased from 9.9 +/- 6.4 (mean +/- SD) to 3.5 +/- 3.1 ng/ml (p < 0.01) and serum IGF-1 from 785 +/- 268 to 431 +/- 156 ng/ml (p < 0.005). Fm/Em increased from 0.52 +/- 0.1 to 0.75 +/- 0.08 (p < 0.03) consistent with increased 11beta-HSD1 activity. There were no significant changes in truncal fat percentage (33.0 +/- 9.0 vs. 33.0 +/- 8.2) or insulin sensitivity (HOMA %S: 37.1 +/- 8.6 vs. 52.8 +/- 33.7). CONCLUSIONS: Modulation of cortisol metabolism during treatment of active acromegaly is dependent on changes in GH/IGF-1 status and is not influenced by any individual change in body composition or insulin sensitivity.     

The role of progesterone metabolism and androgen synthesis in renal blood pressure regulation.

11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a crucial role in converting hormonally active cortisol into inactive cortisone, conferring specificity onto the human mineralocorticoid receptor (MR). Progesterone binds with even higher affinity to the MR, but acts as an MR antagonist. How aldosterone is able to keep its function as predominant MR ligand in clinical situations with high progesterone concentrations, such as pregnancy, is not clear. We have shown in vitro that the human kidney possesses an effective enzyme system that metabolizes progesterone to inactive metabolites in a process similar to the inactivation of cortisol by 11beta-HSD2. In studies on patients with adrenal insufficiency, we have shown that the in vivo anti-mineralocorticoid activity of progesterone is diminished by inactivating metabolism of progesterone, local formation of the deoxycorticosterone mineralocorticoid from progesterone, and inhibition of 11beta-HSD2 by progesterone and its metabolites resulting in decreased inactivation of cortisol and hence increased MR binding by cortisol. The enzymes involved in progesterone metabolism are also responsible for the capability of the human kidney to convert pregnenolone to DHEA and androstenedione leading to the formation of active androgens, testosterone and 5alpha-DH-testosterone. Locally produced androgens might be responsible for the observed difference in blood pressure between men and women and higher susceptibility to hypertension in men.     

Tissue localization of 11 beta-hydroxysteroid dehydrogenase and its relationship to the glucocorticoid receptor.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) dictates specificity for the mineralocorticoid receptor (MR) by converting the active steroid cortisol to cortisone in man (corticosterone to 11-dehydrocorticosterone in rodents), leaving aldosterone to occupy the MR. However cortisol is the principal circulating glucocorticoid in man and 11 beta-HSD, distributed in a tissue specific fashion, may represent a powerful mechanism in regulating exposure of active steroid to the glucocorticoid receptor (GR). A detailed localization study of 11 beta-HSD gene expression and activity in numerous rat tissues has been performed and compared with the presence of GR mRNA. 11 beta-HSD mRNA (1.4 kB) measured by hybridization to a cDNA derived from hepatic 11 beta-HSD, and enzyme activity, measured by percentage conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone by tissue homogenate, was widespread, present in all tissues studied except spleen, brain cortex and heart. There was a close correlation between tissue 11 beta-HSD mRNA levels and activity (r = 0.91, P less than 0.001) suggesting pretranslational regulation of the enzyme at a tissue level. There was also close co-localization of GR mRNA (7 kB), measured by hybridization to a rat GR cRNA probe, and enzyme mRNA/activity in every tissue studied except heart and brain cortex in which GR mRNA was found. In the mineralocorticoid target tissues kidney and colon, additional 11 beta-HSD mRNA bands were seen (kidney 1.8 kB, colon 3.4 kB), suggesting the presence of multiple dehydrogenase species. 11 beta-HSD is widely distributed and suitably placed to modulate ligand occupancy of the GR. The possibility of multiple dehydrogenase species in mineralocorticoid target tissues is consistent with the hypothesis that the ubiquitous 'native' 1.4 kB hepatic enzyme regulates the GR, and these separate dehydrogenases regulate the MR.     

Dehydroepiandrosterone 7alpha-hydroxylation in human tissues: possible interference with type 1 11beta-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7alpha-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7alpha-hydroxy-DHEA that is a substrate for 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which exists in the same tissues and carries out the inter-conversion of 7alpha- and 7beta-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11beta-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7alpha-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11beta-HSD1. Human skin was selected then and used to test its ability to produce 7alpha-hydroxy-DHEA, and to test the interference of 7alpha- and 7beta-hydroxy-DHEA and 7-oxo-DHEA with the 11beta-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11beta-HSD1 in the liver, skin and tonsils. DHEA was readily 7alpha-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11beta-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA was competitive with a Ki at 1.85+/-0.495 and 0.255+/-0.005 microM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a Ki at 1.13+/-0.15 microM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7alpha-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.     

Increased production of 11beta-hydroxysteroid dehydrogenase type 2 in the kidney microsomes of squirrel monkeys (Saimiri spp.)

In squirrel monkeys (Saimiri spp.), cortisol circulates at levels much higher than those seen in man and other Old World primates, but squirrel monkeys exhibit no physiologic signs of the mineralocorticoid effects of cortisol. These observations suggest that squirrel monkeys have mechanisms for protection of the mineralocorticoid receptor (MR) from these high levels of cortisol. We previously showed that the serum cortisol to cortisone ratio in these animals is low relative to that in human serum, suggesting that production of the MR protective enzyme, 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), is increased in squirrel monkeys. Here, we directly evaluate whether increased production of 11beta-HSD2, which inactivates cortisol to cortisone, is a mechanism for protection of MR. In vitro assays showed that 11beta-HSD2 activity in squirrel monkey kidney microsomes was 3 to 7 times higher than that seen in kidney microsomes from pig or rabbit. 11beta-HSD2 protein detected by Western blot analysis was 4 to 9 times greater in squirrel monkey microsomes than in pig or rabbit microsomes. Comparison of the effect of expression of either human or squirrel monkey 11beta-HSD2 on MR transactivation activity showed similar inhibition of MR response to cortisol by both enzymes, indicating that the intrinsic activities of the human and squirrel monkey enzymes are similar. These findings suggest that one mechanism by which squirrel monkeys protect the MR from activation by high cortisol levels in the kidney is by upregulation of 11beta-HSD2 activity through increased production of the enzyme.     

Renal 11 beta-hydroxysteroid dehydrogenase activity: effects of age, sex and altered hormonal status

The enzyme 11 beta-hydroxysteroid dehydrogenase, by converting cortisol and corticosterone to their receptor-inactive 11-keto metabolites cortisone and 11-dehydrocorticosterone, appears crucial to the aldosterone-selectivity of renal mineralocorticoid receptors. Levels of enzyme activity in the rat kidney, measured by conversion of cortisol to cortisone, are unaltered by changes in adrenal or thyroid status, or by castration in either sex; in contrast, oestrogen administration increases enzyme activity in male rats.     

Increased ratio of serum cortisol to cortisone in acute-phase response

BACKGROUND/OBJECTIVES: 11beta-Hydroxysteroid dehydrogenase (11beta-HSD) enzymes convert cortisol into inactive cortisone and vice versa. While 11beta-HSD type 2 (mainly localized in the kidney) unidirectionally inactivates cortisol to cortisone, type I isoform (mainly localized in the liver) acts bidirectionally and can thus potentially restore cortisone to active cortisol. The aim of this pilot study was to investigate whether the serum cortisol:cortisone ratio is altered during the acute-phase response, possibly due to altered modulation of 11beta-hydroxysteroid dehydrogenase isoforms. METHODS: Using liquid chromatography electrospray tandem mass spectrometry, cortisol and cortisone were measured in the serum of hospitalized patients with normal and abnormal CRP concentrations, the latter indicating acute-phase response. Fifteen unselected samples were analyzed, all with a CRP concentration within one of the following ranges to cover a wide range of CRP concentrations evenly: <5, 5-20, 21-50, 51-100, 101-200, and >200 mg/l. RESULTS: In the heterogeneous study population, increased CRP concentrations significantly correlated with an increased cortisol:cortisone ratio (p < 0.001; r = 0.65, Spearman correlation coefficient). This correlation was independent of increased serum cortisol concentrations found by multivariate regression analysis. The median ratio was 6.4 (interquartile range 5.5-7.4; n = 30) in patients with a CRP concentration < or =20 mg/l, and 11.2 (interquartile range 8.8-13.9; n = 60) in patients with CRP >20 mg/l (p < 0.01). CONCLUSION: The balance between serum cortisol and cortisone is altered during acute-phase response with a shift towards active cortisol, suggesting that 11beta-HSD isoenzymes play a role in the modulation of systemically available cortisol during acute illness.     

11beta-hydroxysteroid dehydrogenase 2 activity is elevated in severe obesity and negatively associated with insulin sensitivity

Alterations in glucocorticoid (GC) metabolism may contribute to the development of obesity and insulin resistance. We aimed to study the role of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) in human adiposity, paying special attention to the association between altered GC metabolism and insulin sensitivity. In 24-h urine samples of 72 extremely obese (mean BMI 45.5 +/- 1.1 kg/m(2)), but otherwise healthy patients urinary free cortisol (UFF), urinary free cortisone (UFE), tetrahydrocortisol (THF), 5alpha-tetrahydrocortisol (5alpha-THF), and tetrahydrocortisone (THE) were quantified by radioimmunoassay. The sum of the three major tetrahydrometabolites is an estimate for daily GC secretion, and the sum of UFF and UFE represents potentially bioactive-free-GCs. Thirty healthy lean subjects (BMI 22.3 +/- 0.3 kg/m(2)) served as controls. In obese subjects, absolute daily GC secretion and the potentially bioactive-free-GCs were significantly (P < 0.005) higher than in lean controls (11.8 +/- 0.7 vs. 8.0 +/- 0.6 mg/d; and 171.8 +/- 11.2 vs. 117.6 +/- 9.2 mug/d, respectively). However, when these values were corrected for body surface area (BSA), significant differences were no longer detectable. While enzyme activity indices for 5alpha-reductase and 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) were similar in lean and obese subjects, 11beta-HSD2 was markedly elevated in adiposity (3.7 +/- 0.2 vs. 2.1 +/- 0.1; P < 0.0001). This increase was accompanied by a significant reduction in UFF excretion corrected for BSA (16.5 +/- 1.2 vs. 21.7 +/- 2.0 mug/d/m(2); P = 0.0222). Besides, 11beta-HSD2 activity was significantly correlated with insulin sensitivity (P = 0.0262). When body size is accounted for, both adrenal GC secretion and potentially bioactive-free-GCs are indistinguishable between lean and extremely obese subjects. However in obesity, the kidney appears to intensify its supply of the direct substrate cortisone for extrarenal 11beta-HSD1, which may fuel visceral adiposity and insulin resistance.     

Human corticotropin-releasing hormone test in normal subjects and patients with hypothalamic, pituitary or adrenocortical disorders

Human corticotropin-releasing hormone (hCRH) test was performed in 57 normal volunteers and 102 patients with hypothalamic, pituitary and adrenocortical diseases. Intravenous bolus injection of synthetic hCRH, 100 micrograms for adults or 1.5 micrograms/kg for children, increased plasma ACTH and cortisol levels in about 90% of normal subjects. In 47 patients with Cushing's disease, plasma ACTH tended to show an exaggerated response to hCRH and peak ACTH was the most frequent abnormal component among the several reaction parameters. Poor responders among normal subjects and patients with Cushing's disease had significantly higher plasma cortisol levels before CRH administration. Patients with hypothalamic hypopituitarism showed exaggerated response, whereas patients with primary pituitary lesion, isolated ACTH deficiency or adrenal Cushing's syndrome showed no ACTH response. These differences in the response of patients suggest the value of the hCRH test in their differential diagnosis.     

Inhibition of cortisol production in isolated guinea-pig adrenal cells

Cortisol, added to 1 ml incubation medium containing 3-4 X 10(5) isolated guinea-pig adrenal cells, provoked a decrease in basal and ACTH (250 pg)-stimulated cortisol production, in correlation with the amounts used (50 ng-2,000 ng). A decrease in aldosterone production could be seen when cortisol concentrations reached or exceeded 1,000 ng/ml. There were no variations in either androgens (delta 4-androstenedione, dehydropiandrosterone) or 17-hydroxyprogesterone. Only 11-deoxycortisol was slightly increased. Using increasing concentrations of ACTH (50-250 pg), both in the absence and in the presence of 1,000 ng cortisol, it was noted that the inhibition induced by cortisol was of a competitive type and could be overcome by ACTH. This decrease in cortisol was concomitant with an increase in 11-deoxycortisol. Neither corticosterone nor dexamethasone reduced cortisol production. In addition, it was shown that the conversion of tritiated 11-deoxycortisol to radioactive cortisol increased significantly under the influence of 250 pg ACTH (mean relative variation of 21.7% +/- 7.7 (SEM), n = 6, P less than 0.05); but decreased significantly under the combined effect of 1,000 ng exogenous cortisol and the same dose of ACTH: (mean relative variation of 4.3% +/- 1 (SEM), n = 8, P less than 0.005). There is therefore reason to believe that the concentrations of cortisol at the adrenal level modulate the stimulation induced by ACTH and that this self-adjustment forms part of the control mechanisms involved in corticosteroidogenesis.     

The intracellular localization of the mineralocorticoid receptor is regulated by 11beta-hydroxysteroid dehydrogenase type 2

11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 has been considered to protect the mineralocorticoid receptor (MR) by converting 11beta-hydroxyglucocorticoids into their inactive 11-keto forms, thereby providing specificity to the MR for aldosterone. To investigate the functional protection of the MR by 11beta-HSD2, we coexpressed epitope-tagged MR and 11beta-HSD2 in HEK-293 cells lacking 11beta-HSD2 activity and analyzed their subcellular localization by fluorescence microscopy. When expressed alone in the absence of hormones, the MR was both cytoplasmic and nuclear. However, when coexpressed with 11beta-HSD2, the MR displayed a reticular distribution pattern, suggesting association with 11beta-HSD2 at the endoplasmic reticulum membrane. The endoplasmic reticulum membrane localization of the MR was observed upon coexpression only with 11beta-HSD2, but not with 11beta-HSD1 or other steroid-metabolizing enzymes. Aldosterone induced rapid nuclear translocation of the MR, whereas moderate cortisol concentrations (10-200 nm) did not activate the receptor, due to 11beta-HSD2-dependent oxidation to cortisone. Compromised 11beta-HSD2 activity (due to genetic mutations, the presence of inhibitors, or saturating cortisol concentrations) led to cortisol-induced nuclear accumulation of the MR. Surprisingly, the 11beta-HSD2 product cortisone blocked the aldosterone-induced MR activation by a strictly 11beta-HSD2-dependent mechanism. Our results provide evidence that 11beta-HSD2, besides inactivating 11beta-hydroxyglucocorticoids, functionally interacts with the MR and directly regulates the magnitude of aldosterone-induced MR activation.     

The 11 beta-hydroxysteroid dehydrogenase type 2 activity in human placental microsomes is inactivated by zinc and the sulfhydryl modifying reagent N-ethylmaleimide

Proper glucocorticoid exposure in utero is vital to normal fetal organ growth and maturation. The human placental 11 beta-hydroxysteroid dehydrogenase type 2 enzyme (11 beta-HSD2) catalyzes the unidirectional conversion of cortisol to its inert metabolite cortisone, thereby controlling fetal exposure to maternal cortisol. The present study examined the effect of zinc and the relatively specific sulfhydryl modifying reagent N-ethylmaleimide (NEM) on the activity of 11 beta-HSD2 in human placental microsomes. Enzyme activity, reflected by the rate of conversion of cortisol to cortisone, was inactivated by NEM (IC(50)=10 microM), while the activity was markedly increased by the sulfhydryl protecting reagent dithiothreitol (DTT; EC(50)=1 mM). Furthermore, DTT blocked the NEM-induced inhibition of 11 beta-HSD2 activity. Taken together, these results suggested that the sulfhydryl (SH) group(s) of the microsomal 11 beta-HSD2 may be critical for enzyme activity. Zn(2+) also inactivated enzyme activity (IC(50)=2.5 microM), but through a novel mechanism not involving the SH groups. In addition, prior incubation of human placental microsomes with NAD(+) (cofactor) but not cortisol (substrate) resulted in a concentration-dependent increase (EC(50)=8 microM) in 11 beta-HSD2 activity, indicating that binding of NAD(+) to the microsomal 11 beta-HSD2 facilitated the conversion of cortisol to cortisone. Thus, this finding substantiates the previously proposed concept that a compulsorily ordered ternary complex mechanism may operate for 11 beta-HSD2, with NAD(+) binding first, followed by a conformational change allowing cortisol binding with high affinity. Collectively, the present results suggest that cellular mechanisms of SH group modification and intracellular levels of Zn(2+) may play an important role in regulation of placental 11 beta-HSD2 activity.     

Molecular biology of 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase enzymes

There are two steroid 11β-hydroxylase isozymes encoded by the CYP11B1 and CYP11B2 genes on human chromosome 8q. The first is expressed at high levels in the normal adrenal gland, has 11β-hydroxylase activity and is regulated by ACTH. Mutations in the corresponding gene cause congenital adrenal hyperplasia due to 11β-hydroxylase deficiency; thus, this isozyme is required for cortisol biosynthesis. The second isozyme is expressed at low levels in the normal adrenal gland but at higher levels in aldosterone-secreting tumors, and has 11β-hydroxylase, 18-hydroxylase and 18-oxidase activities. The corresponding gene is regulated by angiotensin II, and mutations in this gene are found in persons who are unable to synthesize aldosterone due to corticosterone methyloxidase II deficiency. Thus, this isozyme is required for aldosterone biosynthesis.

Cortisol and aldosterone are both effective ligands of the “mineralocorticoid” receptor in vitro, but only aldosterone is a potent mineralocorticoid in vivo. This apparent specificity occurs because 11β-hydroxysteroid dehydrogenase in the kidney converts cortisol to cortisone, which is not a ligand for the receptor. This enzyme is a “short-chain” dehydrogenase which is encoded by a single gene on human chromosome 1. It is possible that mutations in this gene cause a form of childhood hypertension called apparent mineralocorticoid excess, in which the mineralocorticoid receptor is not protected from high concentrations of cortisol.